text:
Ohio – With the new federal stimulus legislation, unemployed Ohioans will soon see an extra $300 weekly supplemental payment.
The Ohio Department of Job and Family Services (ODJFS) announced today that it is expediting the payment to hundreds of thousands of Ohioans. They said the benefit will extend to March 13.
In addition, existing Pandemic Unemployment Assistance (PUA) claimants will soon be able to resume filing weekly claims for benefits, ODJFS said.
“We are committed to making the updates as quickly as we can so that we can bring much-needed relief to eligible claimants,” said ODJFS Director Kimberly Henderson. “System programming will be needed for this effort, and we appreciate everyone’s patience as that occurs. Ohio stands ready to assist those in need as soon as possible, within the bounds of the new law. Those eligible will receive all benefits to which they are entitled.”

domain: economics


text:
This weekend was a good one. A lot was accomplished and it did not rain till last night. It was a bit colder than I would have liked, but we dressed accordingly and trudged forward. Today is a beauty with the sun shining
Last weeks menus HERE served me well. But I must confess I did switch out tuesday and thursday. It really is such a help having menus planned I hope I can keep up with this throughout the summer months.
So here are this weeks menus.
Sunday......oven fried flounder, fresh collard greens and roasted onion/yellow squash/cauliflower
Monday.......BBQ pork served on a toasted Joseph's bread with coleslaw
Tuesday......grilled chicken and a chopped salad
Wednesday.....turkey veggie burgers, parsnip fries and a low carb chocolate frosty.
Thursday......tomato basil soup and a chopped salad
Friday........flat bread pizza and chopped salad
Saturday.......turkey and roasted veggies
Well now that feels like a load off my mind. All I have to do is cook it now. It seems to me to take longer to decide what we are having than to actually fix it!
Whats on your menu this week?

domain: food


text:
Check out the sexy and always looking sweet Ghanaian Actress Yvonne Nelson’s.She is getting hotter by the day.Nice curve thou!
Many people have different views about this. While majority are ..
Paul Okoye has officially come out of his shell.Unlike before, ..
I am in a dilemma over an issue and I ..
The actress, who’s celebrating her 20 years in the entertainment ..
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domain: celebrity


text:
ELLIPAL Wallet Review
Austin from Crypto Playhouse reviews the ELLIPAL Hardware Wallet. ELLIPAL operates offline, which greatly diminishes the potential for network attacks. Users are able to store their private keys in the cold wallet by importing them or they can choose to create entirely new ones. As of now, the wallet supports Bitcoin, Ethereum, Litecoin, and a variety of other altcoins.

domain: finance


“What I cannot create, I do not understand.”
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text:
Peptides (v.32, #4)
Gayle & Richard Olson prize pages (III-IV).
Editorial Board (CO2).
Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR by Lucie Catiau; Johnatan Traisnel; Véronique Delval-Dubois; Nour-Eddine Chihib; Didier Guillochon; Naïma Nedjar-Arroume (633-638).
► Peptides derived from α 107–141 were antibacterial whatever the bacterial species. ► The most active peptide is the shortest one: α 137–141. ► Shortest the sequence of the peptide is, lowest the MIC is. ► KYR sequence is the minimal antimicrobial sequence from the hemoglobin α-chain. ► These active peptides were able to interact with cellular membrane.Hemoglobin is an animal protein described as a source of biologically active peptides. Peptic digestion of bovine hemoglobin alpha-chain allowed obtaining peptide fractions with antimicrobial activity. These peptides were purified by reverse-phase High-Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry. The minimal inhibitory concentration and mode of action of these peptides were studied against five bacterial strains including Escherichia coli and Salmonella enteritidis as Gram-negative bacteria and Listeria innocua, Micrococcus luteus and Staphylococcus aureus as Gram-positive bacteria. The action aforementioned peptides were studied on artificial membranes as well. The most active peptides resulted to be the short ones. Consequently, the minimal peptidic sequence necessary for the antibacterial activity was clearly determined: KYR.
Keywords: Hemoglobin; Antimicrobial; MIC; Liposome; Minimal sequence;
Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli by BoMi Ryu; S.W.A. Himaya; Zhong-Ji Qian; Sang-Hoon Lee; Se-Kwon Kim (639-647).
► Suggesting that the administration of SPPs had a reducing effect on ROS generation. ► SPPs exerted protective effects on hydroxyl radical-induced DNA damage. ► SPPs inhibit the NF-κB pathway without altering MAP kinase activation. ► That H2O2 induced IκB phosphorylation was increased in cells treated with SPPs, where as phosphorylation of IKK is decreased. ► p38 kinase was suppressed by cotreatment with SPPs. ► SPP suppress activation of H2O2 induced NF-κB pathway.Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H2O2 stimulation.
Keywords: Seaweed pipefish; Oxidative stress; Antioxidant; Reactive oxygen species; NF-κB;
Purification and identification of a novel primitive secretory enzyme catalyzing the hydrolysis of imidazole-related dipeptides in the jawless vertebrate Lethenteron reissneri by Takahiro Oku; Seiichi Ando; Takehiko Hayakawa; Kyoko Baba; Ryuichiro Nishi; Kazuhiro Shiozaki; Shoji Yamada (648-655).
► An imidazole dipeptide-hydrolyzing enzyme is purified from brook lamprey muscle. ► The enzyme is a primitive M20A metallopeptidase distributed in jawless vertebrate. ► The lamprey secretory-type enzyme may be classified as a new peptidase of M20A.Imidazole-related dipeptides, such as carnosine and anserine, occur widely in skeletal muscles of jawed vertebrates. All of the known enzymes that catalyze the hydrolysis of these dipeptides belong to the M20A metallopeptidase subfamily; two secretory enzymes, serum carnosinase (EC 184.108.40.206) and anserinase (EC 220.127.116.11), and one non-secretory enzyme, cytosolic nonspecific dipeptidase (EC 18.104.22.168). Here we report the enzymatic characterization and molecular identification of an unidentified enzyme, which catalyzes the hydrolysis of these dipeptides, from the skeletal muscle of Far Eastern brook lamprey (Lethenteron reissneri). A 60-kDa subunit protein of the enzyme was purified to near homogeneity. We cloned two M20A genes from the skeletal muscle of Far Eastern brook lamprey; one was a secretory-type gene encoding for the 60-kD protein, and another was a non-secretory-type gene presumably encoding for cytosolic nonspecific dipeptidase. Our findings indicate that the purified enzyme is a N-glycosylated secretory M20A dipeptidase distributed specifically in the jawless vertebrate group, and may be derived from a common ancestor gene between serum carnosinase and anserinase. We propose that this dipeptidase is a novel secretory M20A enzyme and is classified as neither serum carnosinase nor anserinase.
Keywords: Anserinase; Anserine; Carnosine; Dipeptidase; Jawless vertebrate; Serum carnosinase; Lethenteron reissneri;
Purification, synthesis and characterization of AaCtx, the first chlorotoxin-like peptide from Androctonus australis scorpion venom by Ilhem Rjeibi; Kamel Mabrouk; Hend Mosrati; Caroline Berenguer; Hafedh Mejdoub; Claude Villard; Daniel Laffitte; Denis Bertin; L’Houcine Ouafik; José Luis; Mohamed ElAyeb; Najet Srairi-Abid (656-663).
► In this study we purified a new chlorotoxin-like peptide. ► We examined its effect on the U87 glioblastoma cell line. ► We find that this peptide is less active than chlorotoxin. ► The missing of acidic aminoacids is likely responsible for the lower activity. ► This will allow to design highly active anti-glioma peptides.AaCtx is the first chlorotoxin-like peptide isolated from Androctonus australis scorpion venom. Its amino acid sequence shares 70% similarity with chlorotoxin from Leiurus quinquestriatus scorpion venom, from which it differs by twelve amino acids. Due to its very low concentration in venom (0.05%), AaCtx was chemically synthesized. Both native and synthetic AaCtx were active on invasion and migration of human glioma cells. However, their activity was found to be lower than that of chlorotoxin. The molecular model of AaCtx shows that most of amino acids differing between AaCtx and chlorotoxin are localized on the N-terminal loop and the α-helix. Based on known compounds that block chloride channels, we suggest that the absence of negative charged amino acids on AaCtx structure may be responsible for its weak activity on glioma cells migration and invasion. This finding serves as a starting point for structure–function relationship studies leading to design high specific anti-glioma drugs.
Keywords: Chlorotoxin-like peptide; Human glioma; Scorpion venom; Androctonus australis; Structure–activity relationship;
Characterization of antimicrobial peptides in skin secretions from discrete populations of Lithobates chiricahuensis (Ranidae) from central and southern Arizona by J. Michael Conlon; Milena Mechkarska; Laurent Coquet; Thierry Jouenne; Jérôme Leprince; Hubert Vaudry; Jolanta Kolodziejek; Norbert Nowotny; Jay D. King (664-669).
► Characterization of six antimicrobial peptides present in skin secretions of populations of the leopard frog Lithobates chiricahuensis from distinct ranges in southern and central Arizona. ► Characterization of two peptides (brevinin-1CHc and palustrin-2CHa) present only in secretions of frogs from the southern range. ► Identification of esculentin-2CHa as a potent, broad spectrum antimicrobial peptide with therapeutic potential. ► Phylogenetic analysis of the North American leopard frogs based upon the primary structures of brevinin-1 peptides.Populations of the Chiricahua leopard frog Lithobates chiricahuensis (Ranidae) occupying regions in southern Arizona (southern range) are morphologically distinct from those from the Mogollon Rim of central Arizona (northern range) and a comparison of DNA sequences of mitochondrial genes has suggested that they may represent separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions has led to the identification of six peptides with antimicrobial activity in samples from specimens from both groups. The primary structure of the peptides (esculentin-2CHa, ranatuerin-2CHa, -CHb, and -CHc, and brevinin-1CHa and -CHb) isolated from both southern and northern range frogs are identical consistent with the proposal that the two populations are conspecific. However, palustrin-2CHa and the atypical brevinin-1CHc (FFPTIAG*****LTKLFCA ITKKC), containing a five amino acid residue deletion, were identified only in secretions from southern range specimens. Consequently, there is some support for the proposal that the two populations are closely related but separate species but this support is relatively weak. Esculentin-2CHa (GFSSIFRGVAKFASKGLG KDLAKLGVDLVACKISKQC) displayed the highest antimicrobial potency (MIC ≤ 10 μM) against a variety of microorganisms and was only moderately hemolytic (LC50 = 150 μM). Cladistic analysis based upon the primary structures of brevinin-1 peptides indicates a close phylogenetic relationship between L. chiricahuensis, L. onca, and L. yavapaiensis.
Keywords: Frog skin; Antimicrobial peptide; Brevinin-1; Esculentin-2; Ranatuerin-2; Palustrin-2; Taxonomy;
Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae by Eiko Iwakoshi-Ukena; Kazuyoshi Ukena; Aiko Okimoto; Miyuki Soga; Genya Okada; Naomi Sano; Tamotsu Fujii; Yoshiaki Sugawara; Masayuki Sumida (670-676).
▶ Nine antimicrobial peptides were isolated from the skin of the endangered frog Odorrana ishikawae. ▶These peptides belonging to five known families of antimicrobial peptides showed broad-spectrum of growth inhibitory activities against several microorganisms. ▶Each precursor protein has a common feature structure; a putative signal peptide, an N-terminal acidic spacer domain, a Lys-Arg-processing site, and an antimicrobial peptide at the C-terminus.The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.
Keywords: Antimicrobial peptide; Innate immunity; Frog skin; Odorrana; Microorganism;
Effect of proline position on the antimicrobial mechanism of buforin II by Yang Xie; Eleanor Fleming; Jessica L. Chen; Donald E. Elmore (677-682).
► First study to consider importance of proline position in buforin II mechanism. ► Sole proline is in ideal position for buforin II membrane translocation. ► Sole proline is in ideal position for bufoirn II antimicrobial activity. ► Results emphasize interplay of translocation and DNA binding in buforin II mechanism. ► Results highlight importance of considering proline position in other peptides.Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.
Keywords: Buforin II; Histone-derived antimicrobial peptide; Proline; Membrane translocation; Nucleic acid binding;
Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells by José Julián Pérez-Cordero; José Manuel Lozano; Jimena Cortés; Gabriela Delgado (683-690).
► In this study we search of new therapeutic alternatives for leishmaniasis. ► We examine antimicrobial synthetic peptides as a molecules with pharmacological potential. ► We use dendritic cells in an in vitro model to study antileishmanial peptides. ► Dermaseptin shows a relevant antileishmanial activity related with nitric oxide.Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world . The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.
Keywords: Leishmania; Antimicrobial peptides; Dendritic cells;
Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: Activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis by Samiran S. Gauri; Santi M. Mandal; Bikas R. Pati; Satyahari Dey (691-696).
► A novel antimicrobial peptide of 1676 Da was purified from venom of Bellamya bengalensis. ► Sequence of the peptide was determined by tandem mass spectrometry (MS/MS). ► The MIC and MBC values were 8 μg/ml and 16 μg/ml against Staphylococcus epidermidis resistant to ampicillin and chloramphenicol. ► Peptide increased the staphylococcal membrane permeability.Increasing tendency of clinical bacterial strains resistant to conventional antibiotics has being a great challenge to the public's health. Antimicrobial peptides, a new class of antibiotics is known to have the activity against a wide range of bacteria resistant to conventional antibiotics. An antimicrobial peptide of 1676 Da was purified from Bellamya bengalensis, a fresh water snail, using ultrafiltration and reversed phase liquid chromatography. The effect of this peptide on Staphylococcus epidermidis resistant to ampicillin and chloramphenicol was investigated; the MIC and MBC values were 8 μg/ml and 16 μg/ml, respectively. Complete sequence of the peptide was determined by tandem mass spectrometry (MS/MS). Further, peptide net charge, hydrophobicity and molecular modeling were evaluated in silico for better understanding the probable mechanisms of action. The peptide showed the specificity to bacterial membranes. Hence, this reported peptide revealed a promising candidate to contribute in the development of therapeutic agent for Staphylococcal infections.
Keywords: Antimicrobial peptides; Antibiotic resistant; Mass spectrometry; Staphylococcus epidermidis; Bellamya bengalensis;
S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis by Oscar Cirioni; Guoqiu Wu; Linxian Li; Fiorenza Orlando; Carmela Silvestri; Roberto Ghiselli; Zilong Shen; Eleonora Gabrielli; Lucia Brescini; Giovanni Lezoche; Mauro Provinciali; Mario Guerrieri; Andrea Giacometti (697-701).
► Colistin and s-thanatin showed synergy. ► Serial exposure of bacterial strains to colistin alone resulted in enrichment of resistant derivatives. ► Addition of s-thanatin in the cultures reduced the increase in colistin MIC. ► Combination between colistin and s-thanatin showed significantly lowest lethality rates in vivo.An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.
Keywords: Pseudomonas; Colistin; Antimicrobial peptides; Sepsis;
Neuropeptide FF receptor antagonist, RF9, attenuates the fever induced by central injection of LPS in mice by Yi-qing Wang; Sheng-bin Wang; Jing-lin Ma; Jia Guo; Quan Fang; Tao Sun; Yan Zhuang; Rui Wang (702-706).
▶ NPFF does not modify the fever induced by central injection of LPS in mice. ▶ Neuropeptide FF receptors antagonist, RF9, attenuates the LPS-induced fever in mice. ▶ NPFF receptors activation is required for LPS to produce fever. ▶ NPFF systems participate in the control of acute neuroinflammation in conscious animals.The endogenous opioid system has been found to be involved in the fever caused by lipopolysaccharide (LPS). Neuropeptide FF (NPFF, FLFQPQRF-NH2) is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system. Therefore, those data suggested a link between LPS-induced fever and NPFF systems. Using a model of acute neuroinflammation, we sought to determine the effects of NPFF systems on the fever induced by i.c.v. injection of LPS. Coinjected with different doses of NPFF (10 and 30 nmol), the fever of LPS (125 ng) was not modified. Interestingly, the selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it decreased the fever of LPS (125 ng) after cerebral administration in mice. These results suggest that NPFF receptors activation is required for LPS to produce fever. This interaction is the first evidence that NPFF systems participate in the control of acute neuroinflammation in conscious animals.
Keywords: Neuropeptide FF (NPFF); RF9; Lipopolysaccharide (LPS); Neuroinflammation; Fever; Mice;
The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs) by E. Fiedorowicz; B. Jarmołowska; M. Iwan; E. Kostyra; R. Obuchowicz; M. Obuchowicz (707-712).
▶ All examined peptides caused no significant changes to PBMCs proliferation measured by the enzymatic activity of the cells in the long-term incubation. ▶ Amounts of incorporated BrdU reveal a stimulation of PBMCs proliferation by bovine and human β-casomorphin-7, which indicates a short-term influence of opioid peptides on PBMCs. ▶ The μ-opioid receptor agonist peptides were found to cause a significant increase in IL-4 secretion, an insignificant increase in IL-13, and a decrease of IFN-γ secretion, which suggest an activation of the Th2 pathway.Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut–blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.
Keywords: Opioid peptides; β-Casomorphin-7; μ-Opioid receptor; Peripheral blood mononuclear cells;
Multiple 100 Hz electroacupuncture treatments produced cumulative effect on the suppression of morphine withdrawal syndrome: Central preprodynorphin mRNA and p-CREB implicated by Gui-Bin Wang; Liu-Zhen Wu; Peng Yu; Yi-Jing Li; Xing-Jie Ping; Cai-Lian Cui (713-721).
▶ Morphine withdrawal induces down-regulation of PPD mRNA and up-regulation of p-CREB. ▶ Multiple 100 Hz EA shows cumulative effect for suppressing opioid withdrawal syndrome. ▶ 100 Hz EA normalizes the PPD mRNA level by inhibiting the phosphorylation of CREB.Alleviating opiate withdrawal syndrome in addicts is a critical precondition to break away from drug and further to prevent reuse. Electroacupuncture (EA) was claimed to be effective for alleviating withdrawal syndrome, but the optimal protocol remained unclear. In the present study we found that (1) 100 Hz EA administered 12–24 h after the last morphine injection suppressed the withdrawal syndrome in rats, multiple sessions of EA were more effective than single session, with the after-effect lasting for at least 7 days. (2) A down-regulation of preprodynorphin (PPD) mRNA level was observed in spinal cord, PAG and hypothalamus 60 h after the last morphine injection, which could be reversed by multiple sessions, but not a single session of EA. (3) Accompanied with the decrease of PPD mRNA level, there was an up-regulation of p-CREB in the three CNS regions, which was abolished by 100 Hz EA treatment. The findings suggest that down-regulation of p-CREB and acceleration of dynorphin synthesis in spinal cord, PAG and hypothalamus may be implicated in the cumulative effect of multiple 100 Hz EA treatment for opioid detoxification.
Keywords: Morphine dependence; Withdrawal; Electroacupuncture; Preprodynorphin; Preproenkephalin; p-CREB;
Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid by Attila Keresztes; Erika Birkás; Annamária Páhi; Géza Tóth; Lidia Bakota; Károly Gulya; Mária Szücs (722-728).
► A new tritiated endomorphin-2 analog was synthesized in which Pro2 is replaced by 2-aminocyclohexanecarboxylic acid. ► It shows high specific radioactivity, μ-opioid receptor affinity, selectivity and enzymatic resistance. ► It is a good tool to envisage the distribution of μ-opioid receptors by using receptor autoradiography. ► It may be more suitable than the parent ligand for receptor–ligand interaction and topographic studies.As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro2 is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [3H][(1S,2R)ACHC]2EM-2 (specific activity 63.49 Ci × mmol−1) bound specifically to its binding sites with high affinity (K D = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, B max of 151 ± 4 fmol × mg protein−1 in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na+ and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of [3H][(1S,2R)ACHC]2EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance , the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.
Keywords: Endomorphin; Peptide synthesis; 2-Aminocylcohexanecarboxylic acid (ACHC); Radiolabeling; Receptor binding; Autoradiography; Proteolytic stability;
Long-term peripheral infusion of nociceptin/orphanin FQ promotes hyperplasia, activation and migration of mucosal mast cells in the rat gastric fundus by Daniela Grandi; Maurizio Massi; Giuseppina Morini (729-736).
► Peripheral nociceptin/orphanin FQ (N/OFQ) increases the density of mucosal mast cells in the gastric fundus ► N/OFQ promoted the activation of mucosal mast cells. ► N/OFQ decreased the contacts between mucosal mast cells and nerve fibers. ► The antagonist of N/OFQ receptor abolishes the stress-induced hyperplasia of mucosal mast cells.The endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) modulates behavioral and gastrointestinal responses to stress. Mucosal mast cells (MMCs) are primary mediators of stress-related responses in the gastrointestinal tract. We investigated the influence of N/OFQ and of the N/OFQ peptide (NOP) receptor antagonist, UFP-101, on MMCs in the rat gastric fundus. N/OFQ was infused subcutaneously for 52 h at 0.1, 1 and 10 μg/kg/h and at 1 μg/kg/h for 4 h, 52 h, 7 days and 14 days via Alzet osmotic minipumps. Density of MMCs and connective tissue mast cells (CTMCs) was assessed histochemically and immunohistochemically. Activation and location of MMCs were assessed by transmission electron microscopy. Contacts between MMCs and nerve elements were assessed by double immunofluorescence. N/OFQ (1 μg/kg/h) and UFP-101 (10 and 30 μg/kg/h) were infused subcutaneously in the absence and presence of acute cold-restraint stress and density of MMCs was assessed. Peripheral N/OFQ dose-dependently increased the density of MMCs, while not influencing CTMCs. The increasing effect was maintained up to 14 days following continuous infusion, while after termination of the 4-h infusion, the effect declined rapidly. The peptide promoted the activation of MMCs and their migration from the lamina propria toward the epithelial layer. The association between MMCs and nerve fibers was time-dependently down-regulated following N/OFQ infusion. The stress-induced hyperplasia of MMCs was not influenced by N/OFQ and abolished by UFP-101. UFP-101 alone was ineffective. The present results suggest that endogenous N/OFQ could be considered a potential component of the circuit neuropeptides-mast cells-stress.
Keywords: Nociceptin/orphanin FQ; UFP-101; Rat; Gastric fundus; Mucosal mast cells; Nerve fibers; Cold-restraint stress;
Modulation of gastric motility by brain-gut peptides using a novel non-invasive miniaturized pressure transducer method in anesthetized rodents by Guillaume Gourcerol; David W. Adelson; Mulugeta Million; Lixin Wang; Yvette Taché (737-746).
► Validation of a novel non-invasive method to monitor gastric motility in rodents. ► Chronic overexpression of CRF prevents central vagal activation of gastric contractions in mice. ► Laparotomy and cecal manipulation decrease phasic gastric contractions in both rats and mice.Acute in vivo measurements are often the initial, most practicable approach used to investigate the effects of novel compounds or genetic manipulations on the regulation of gastric motility. Such acute methods typically involve either surgical implantation of devices or require intragastric perfusion of solutions, which can substantially alter gastric activity and may require extended periods of time to allow stabilization or recovery of the preparation. We validated a simple, non-invasive novel method to measure acutely gastric contractility, using a solid-state catheter pressure transducer inserted orally into the gastric corpus, in fasted, anesthetized rats or mice. The area under the curve of the phasic component (pAUC) of intragastric pressure (IGP) was obtained from continuous manometric recordings of basal activity and in responses to central or peripheral activation of cholinergic pathways, or to abdominal surgery. In rats, intravenous ghrelin or intracisternal injection of the thyrotropin-releasing hormone agonist, RX-77368, significantly increased pAUC while coeliotomy and cacal palpation induced a rapid onset inhibition of phasic activity lasting for the 1-h recording period. In mice, RX-77368 injected into the lateral brain ventricle induced high-amplitude contractions, and carbachol injected intraperitoneally increased pAUC significantly, while coeliotomy and cecal palpation inhibited baseline contractile activity. In wild-type mice, cold exposure (15 min) increased gastric phasic activity and tone, while there was no gastric response in corticotropin releasing factor (CRF)-overexpressing mice, a model of chronic stress. Thus, the novel solid-state manometric approach provides a simple, reliable means for acute pharmacological studies of gastric motility effects in rodents. Using this method we established in mice that the gastric motility response to central vagal activation is impaired under chronic expression of CRF.
Keywords: Cold; CRF; Ghrelin; Intragastric pressure; Surgery; TRH agonist;
Uptake, Transport and Regulation of JBP485 by PEPT1 in vitro and in vivo by Zhihao Liu; Changyuan Wang; Qi Liu; Qiang Meng; Jian Cang; Lin Mei; Taiichi Kaku; Kexin Liu (747-754).
Display Omitted► JBP485 is a substrate of PEPT1 and its absorption is actively mediated by PEPT1. ► The mRNA expression of PEPT1 is increased when treated with JBP485. ► The uptake of JBP485 could be changed when coadministered with other drugs.Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn2+ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.
Keywords: JBP485; Uptake; Transport; Regulation; PEPT1;
Chronic treatment with a stable obestatin analog significantly alters plasma triglyceride levels but fails to influence food intake; fluid intake; body weight; or body composition in rats by A. Agnew; D. Calderwood; O.P. Chevallier; B. Greer; D.J. Grieve; B.D. Green (755-762).
▶ N-terminally PEGylated obestatin is approximately 3 times more stable than native obestatin. ▶ Chronic treatment with N-terminally PEGylated obestatin reduced plasma triglycerides by 40%. ▶ Obestatin treatment did not alter food/fluid intake, body weight, or tissue morphometrics. ▶ Obestatin may play a role in modulating physiological lipid metabolism, but does not affect fat deposition.Obestatin (OB(1-23) is a 23 amino acid peptide encoded on the preproghrelin gene, originally reported to have metabolic actions related to food intake, gastric emptying and body weight. The biological instability of OB(1-23) has recently been highlighted by studies demonstrating its rapid enzymatic cleavage in a number of biological matrices. We assessed the stability of both OB(1-23) and an N-terminally PEGylated analog (PEG-OB(1-23)) before conducting chronic in vivo studies. Peptides were incubated in rat liver homogenate and degradation monitored by LC-MS. PEG-OB(1-23) was approximately 3-times more stable than OB(1-23). Following a 14 day infusion of Sprague–Dawley rats with 50 nmol/kg/day of OB(1-23) or a N-terminally PEGylated analog (PEG-OB(1-23)), we found no changes in food/fluid intake, body weight and plasma glucose or cholesterol between groups. Furthermore, morphometric liver, muscle and white adipose tissue (WAT) weights and tissue triglyceride concentrations remained unaltered between groups. However, with stabilized PEG-OB(1-23) we observed a 40% reduction in plasma triglycerides. These findings indicate that PEG-OB(1-23) is an OB(1-23) analog with significantly enhanced stability and suggest that obestatin could play a role in modulating physiological lipid metabolism, although it does not appear to be involved in regulation of food/fluid intake, body weight or fat deposition.
Keywords: Obestatin; Lipids; Peptide; Peptide analog; Triglycerides;
Repeated glucoprivation delayed hyperphagic responses while activating neuropeptide Y neurons in rats by Yoshiharu Ozawa; Hiroshi Arima; Minemori Watanabe; Hiroshi Shimizu; Yoshihiro Ito; Ryoichi Banno; Yoshihisa Sugimura; Nobuaki Ozaki; Hiroshi Nagasaki; Yutaka Oiso (763-769).
▶ Glucoprivation induced robust feeding in rats. ▶ Glucoprivation increased NPY gene expression. ▶ Repeated glucoprivation delayed feeding responses while enhancing NPY expression.It is well known that glucoprivation induces the release of counterregulatory hormones such as glucagon, and that the response is attenuated when the stimuli are repeated. Glucoprivation also activates orexigenic neurons and induces hyperphagic responses, although it remains unclear whether these responses are attenuated in repeated glucoprivation. In this study, we examined time course changes in feeding as well as activities of orexigenic neuropeptide Y (NPY) neurons in repeated glucoprivation in rats. Either 2-deoxy-d-glucose (2DG), which blocks glucose utilization, or isotonic saline (control) was injected subcutaneously to rats for 14 days, and food consumption for 1 and 2 h after injection was monitored throughout the experiment. While 2DG injection induced robust feeding responses during the first 1 h after injection, the response was gradually attenuated and the food consumption was significantly less on days 12–14 compared to that on day 1. On the other hand, food consumption during 2 h after 2DG injection was not changed significantly for 14 days. The transcriptional activities of NPY neurons in the arcuate nucleus and C1/A1 region of the hindbrain, measured by intronic in situ hybridization, were significantly enhanced after repeated 2DG injection for 14 days, while the feeding responses to intracerebroventricular injection of NPY were significantly less in the 2DG-repeated group compared to the saline-repeated group. It is thus demonstrated that repeated glucoprivation delayed hyperphagic responses while activating NPY neurons in rats. Our data also suggest that decreased feeding responses to NPY might be at least partially responsible for the delayed response.
Keywords: Repeated glucoprivation; Neuropeptide Y; Arcuate nucleus; Hindbrain; Food intake; c-fos;
Effects of glycine-extended and serine13-phosphorylated forms of peptide YY on food intake in rats by Roger Reidelberger; Alvin Haver; Prasanth Chelikani; David A. Keire; Joseph R. Reeve (770-775).
► PYY(3-36)-NH2 is significantly more potent than PYY(1-36)-NH2 in reducing food intake. ► Gly-extended forms of PYY are significantly less potent than non-extended forms. ► Ser13-phosphorylation decreases the anorexigenic potency of PYY(3-36)-NH2. ► Ser13-phosphorylation does not affect the anorexigenic potency of PYY(1-36)-NH2.The gut hormone peptide YY(3-36)-amide [PYY(3-36)-NH2] is significantly more potent than PYY(1-36)-NH2 in reducing food intake in rats and humans. Other Gly-extended and Ser13-phosphorylated PYY forms have been detected or predicted based upon known cellular processes of PYY synthesis and modification. Here we compared the effects of 3-h IV infusion of PYY(1-36)-NH2, PYY(3-36)-NH2, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser13(PO3)-PYY(1-36)-NH2, Ser13(PO3)-PYY(3-36)-NH2, Ser13(PO3)-PYY(1-36)-Gly-OH, and Ser13(PO3)-PYY(3-36)-Gly-OH during the early dark period on food intake in freely feeding rats. PYY(3-36)-NH2 and Ser13(PO3)-PYY(3-36)-NH2 reduced food intake similarly at 50 pmol/kg/min, while only PYY(3-36)-NH2 reduced food intake at 15 pmol/kg/min. PYY(1-36)-NH2 and Ser13(PO3)-PYY(1-36)-NH2 reduced food intake similarly at 50 and 150 pmol/kg/min. In contrast, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser13(PO3)-PYY(3-36)-Gly-OH, and Ser13(PO3)-PYY(1-36)-Gly-OH had no effect on food intake at doses of 50 or 150 pmol/kg/min. Taken together, these results indicate that (i) PYY(3-36)-NH2 is significantly more potent than PYY(1-36)-NH2 in reducing food intake, (ii) Gly-extended forms of PYY are significantly less potent than non-extended forms, and (iii) Ser13-phosphorylation of PYY(3-36)-NH2 decreases the anorexigenic potency PYY(3-36)-NH2, but not PYY(1-36)-NH2. Thus, PYY(3-36)-NH2 appears to be the most potent PYY form for reducing food intake in rats.
Keywords: Peptide YY(3-36); Molecular forms; Satiety;
Nitric oxide is a central component in neuropeptide regulation of appetite by John E. Morley; Susan A. Farr; Rebecca L. Sell; Stanley M. Hileman; William A. Banks (776-780).
► This study compares the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. ► Ghrelin did not increase food intake in the homozygous NOS KO mice while wild type controls did experience increased food intake. ► Nitric oxide did show to be a central mediator in food intake in this study.In recent years, there have been a large number of neuropeptides discovered that regulate food intake. Many of these peptides regulate food intake by increasing or decreasing nitric oxide (NO). In the current study, we compared the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. Satiated homozygous and heterozygous NOS KO mice and their wild type controls were administered ghrelin ICV. Food intake was measured for 2 h post injection. Ghrelin did not increase food intake in the homozygous NOS KO mice compared to vehicle treated NOS KO mice, whereas food intake was increased in the wild type controls compared to vehicle treated wild type controls. NPY was administered ICV and food intake measured for 2 h. Homozygous NOS KO mice showed no increase in food intake after NPY administration, whereas the wild type controls did. In our final study, we administered CCK intraperitoneally to homozygous and heterozygous NOS KO mice and their wild type controls after overnight food deprivation. Food intake was measured for 1 h after injection. CCK inhibited food intake in wild type mice after overnight food deprivation, however, CCK failed to inhibit food intake in the NOS KO mice. The heterozygous mice showed partial food inhibition after the CCK. The current results add further support to the theory that NO is a central mediator in food intake.
Keywords: Nitric oxide; NPY; CCK; Ghrelin; Appetite; Food intake; NOS KO mice;
Glucose-dependent insulinotropic peptide receptor expression in the hippocampus and neocortex of mesial temporal lobe epilepsy patients and rats undergoing pilocarpine induced status epilepticus by Cláudia P. Figueiredo; Victor L.S. Antunes; Eduardo L.G. Moreira; Nelson de Mello; Rodrigo Medeiros; Gabriella Di Giunta; Bruno Lobão-Soares; Marcelo Linhares; Katia Lin; Tânia L. Mazzuco; Rui D.S. Prediger; Roger Walz (781-789).
▶ GIPR present a ubiquitous distribution in the mammalian brain. ▶ The GIPR expression was inversely related to neuronal degeneration. ▶ Increase in GIPR expression was observed in rat brain after pilocarpine treatment. ▶ The increase in GIPR expression may be associated with epileptogenesis processes. ▶ The GIPR expression was found in human hippocampus and neocortex.The glucose-dependent insulinotropic peptide receptor (GIPR) has been implicated with neuroplasticity and may be related to epilepsy. GIPR expression was analyzed by immunohistochemistry in the hippocampus (HIP) and neocortex (Cx) of rats undergoing pilocarpine induced status epilepticus (Pilo-SE), and in three young male patients with left mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS) treated surgically. A combined GIPR immunohistochemistry and Fluoro-Jade staining was carried out to investigate the association between the GIPR expression and neuronal degeneration induced by Pilo-SE. GIPR was expressed in the cytoplasm of neurons from the HIP CA subfields, dentate gyrus (DG) and Cx of animals and human samples. The GIPR expression after the Pilo-SE induction increases significantly in the HIP after 1 h and 5 days, but not after 12 h or 50 days. In the Cx, the GIPR expression increases after 1 h, 12 h and 5 days, but not 50 days after the Pilo-SE. The expression of GIPR 12 h after Pilo-SE was inversely proportional to the Fluoro-Jade staining intensity. In the human tissue, GIPR expression patterns were similar to those observed in chronic Pilo-SE animals. No Fluoro-Jade stained cells were observed in the human sample. GIPR is expressed in human HIP and Cx. There was a time and region dependent increase of GIPR expression in the HIP and Cx after Pilo-SE that was inversely associated to neuronal degeneration.
Keywords: Mesial temporal lobe epilepsy; Hippocampal sclerosis; Glucose-dependent insulinotropic peptide receptor;
A chimeric peptide of intestinal trefoil factor containing cholesteryl ester transfer protein B cell epitope significantly inhibits atherosclerosis in rabbits after oral administration by Gaofu Qi; Jingjing Li; Shengying Wang; Shanshan Xin; Peng Du; Qingye Zhang; Xiuyun Zhao (790-796).
▶ The chimeric ITF containing CETP B cell epitope can form the correct trefoil structure. ▶ The chimeric ITF can resist the proteases digestion in vitro assay. ▶ The chimeric ITF can induce both of mucosal and systemic immune response in rabbits after oral administration. ▶ The anti-CETP antibodies induced by oral vaccination with cITF can inhibit CETP activity, increase HDL-C, decrease LDL-C and inhibit atherosclerosis in rabbits.Vaccination against cholesteryl ester transfer protein (CETP) is proven to be effective for inhibiting atherosclerosis in animal models. In this study, the proteases-resistant intestinal trefoil factor (TFF3) was used as a molecular vehicle to construct chimeric TFF3 (cTFF3) containing CETP B cell epitope and tetanus toxin helper T cell epitope. It was found that cTFF3 still preserved a trefoil structure, and can resist proteases digestion in vitro. After oral immunization with cTFF3, the CETP-specific IgA and IgG could be found in intestine lavage fluid and serum, and the anti-CETP antibodies could inhibit partial CETP activity to increase high-density lipoprotein cholesterol, decrease low-density lipoprotein cholesterol, and inhibit atherosclerosis in animals. Therefore, TFF3 is a potential molecular vehicle for developing oral peptide vaccines. Our research highlights a novel strategy for developing oral peptide vaccines in the future.
Keywords: Intestinal trefoil factor; Oral peptide vaccine; CETP; Atherosclerosis;
Central and peripheral forms of C-type natriuretic peptide (CNP): Evidence for differential regulation in plasma and cerebrospinal fluid by Belinda J. Schouten; Timothy C.R. Prickett; Ashley A. Hooper; Gary J. Hooper; Timothy G. Yandle; A. Mark Richards; Eric A. Espiner (797-804).
► CSF concentrations of NTproCNP are greatly elevated (>140-fold over those) those of CNP in CSF. ► NTproCNP and CNP are more closely correlated in CSF than in plasma. ► Systemic and CSF CNP are inversely correlated suggesting differential regulation. ► CSF concentrations of CNP are unrelated to neprilysin activity or cGMP in CSF.Aminoterminal proCNP (NTproCNP), a stable product of CNP gene expression and readily measured in human plasma, provides a new approach to studies of CNP which is rapidly degraded at source. CNP is detectable in human CSF but the presence and proportions of NTproCNP in CSF are unknown. Since CNP is widely expressed throughout the CNS, we hypothesized that the ratio of NTproCNP to CNP in CSF is greatly increased when compared to plasma and that CSF CNP peptides may contribute to their concentrations in the systemic circulation. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anesthesia for arranged orthopedic procedures. Elevated concentrations of NTproCNP (1045 ± 359 pmol/L), characterized by HPLC-RIA, were found in CSF and greatly exceeded those of CNP (7.9 ± 3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145 ± 55) was much higher than in plasma (31 ± 27). A significant inverse relation was found between plasma and CSF CNP concentrations (r = −0.29, p < 0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. We conclude that CNP is differentially regulated across the brain in normal health. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in the diagnosis and monitoring of subjects with central neural disorders.
Keywords: CSF; NTproCNP; cGMP; HPLC;
Distinct systemic distribution of salusin-α and salusin-β in the rat by Noriko Suzuki; Masayoshi Shichiri; Tae Tateno; Kengo Sato; Yukio Hirata (805-810).
▶ Immunoreactive salusin-α showed distinct distribution from salusin-β in the rat. ▶ Immunoreactive salusin-β was detected in rat urine and tissues. ▶ Salusin-α and salusin-β are conserved in the rat.Salusin-α and salusin-β are multifunctional bioactive peptides that were initially predicted using in silico analyses. These peptides should be concomitantly biosynthesized from prosalusin in humans. However, little information is available yet on the biosynthesis and mode of presence of salusin-α and salusin-β in non-human species. In the present study, we examined whether salusin-α and salusin-β are conserved in the rat and whether salusin-α and salusin-β show distinct systemic distributions. Immunohistochemical analysis of rat tissues using a specific anti-rat salusin-α antibody detected immunoreactivity extensively in neuronal cells and fibers, and abundantly in the epithelial tissues throughout the organs. This distribution contrasts sharply with that of salusin-β, which is mainly localized to the neuroendocrine and hematopoietic systems. Western blot analysis of rat spleen extracts showed the presence of cleaved fragments corresponding to putative rat salusin-α. Reverse-phase and gel filtration high performance liquid chromatography analyses coupled with radioimmunoassay detection of rat urine extracts revealed a major immunoreactive component that co-eluted with synthetic putative rat salusin-β. These data support the processing of rat prosalusin into salusin-α and salusin-β despite absent dibasic amino acids between the two.
Keywords: Salusin; Immunohistochemistry; Western blotting; Processing; High performance liquid chromatography; Rat;
Characterization of prolactin-releasing peptide: Binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor by Jana Maixnerová; Andrea Špolcová; Miroslava Pýchová; Miroslava Blechová; Tomáš Elbert; Martina Řezáčová; Blanka Železná; Lenka Maletínská (811-817).
▶ Three rodent pituitary cell lines possessed high levels of PrRP receptor. ▶ PrRP31 and PrRP20 were equipotent in binding, while PrRP13 showed lower potency. ▶ All PrRP analogs stimulated prolactin release and MAPK/ERK1/2 and CREB signaling. ▶ Only PrRP31 and PrRP20 had a significant central anorexigenic effect in mice.The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K d in the 10−9 M range and a B max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with 125I-PrRP31 with a similar K i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.
Keywords: PrRP; Pituitary cell lines; Binding; MAPK/ERK1/2; Prolactin; Food intake;
The role of neuropeptide S and neuropeptide S receptor 1 in regulation of respiratory function in mice by Hongyan Zhu; Charles Perkins; Melissa K. Mingler; Fred D. Finkelman; Marc E. Rothenberg (818-825).
▶Npsr1 does not have a dominant direct role in the development of experimental asthma. ▶ Intracerebroventricular administration of NPS increases respiratory frequency in an NPSR1-dependent manner at baseline or in response to methacholine challenge. ▶ Intracerebroventricular administration of NPS does not change airway mechanics in response to methacholine. ▶ Respiratory changes induced by intracerebroventricular administration of NPS are part of fight-or-flight response.Genome-wide screening and positional cloning have linked neuropeptide S receptor 1 (NPSR1) with asthma and airway hyperresponsiveness. However, the mechanism by which NPSR1 regulates pulmonary responses remains elusive. Because neuropeptide S and its receptor NPSR1 are expressed in brain regions that regulate respiratory rhythm, and Npsr1-deficient mice have impaired stress and anxiety responses, we aimed to investigate whether neuropeptide S and NPSR1 regulate respiratory function through a central-mediated pathway. After neuropeptide S intracerebroventricular administration, respiratory responses of wildtype and Npsr1-deficient mice were monitored by whole-body or invasive plethysmography with or without serial methacholine inhalation. Airway inflammatory and hyperresponsiveness were assessed in allergen-challenged (ovalbumin or Aspergillus fumigatus) Npsr1-deficient mice. Analysis of breathing patterns by whole-body plethysmography revealed that intracerebroventricular neuropeptide S, as compared with the artificial cerebral spinal fluid control, increased respiratory frequency and decreased tidal volume in an NPSR1-dependent manner but did not affect enhanced pause. Following serial methacholine inhalation, intracerebroventricular neuropeptide S increased respiratory frequency in wildtype mice, but not in Npsr1-deficient mice, and had no effect on tidal volume. Intracerebroventricular neuropeptide S significantly reduced airway responsiveness to methacholine as measured by whole-body plethysmography. Npsr1 deletion had no impact on airway inflammation or hyperresponsiveness in ovalbumin- or A. fumigatus-induced experimental asthma. Our results demonstrate that neuropeptide S and NPSR1 regulate respiratory function through a central nervous system-mediated pathway.
Keywords: Respiration; Brain; Neuropeptide S; Neuropeptide S receptor 1; Panting; Stress;
JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP by Inessa I. Mikhaleva; Igor A. Prudchenko; Vadim T. Ivanov; Vladislav B. Voitenkov (826-831).
► The biological origin of delta-sleep inducing peptide (DSIP) remains enigmatic. ► DSIP-like sequences were searched in nucleotide and protein databases for mammals. ► The WKGGNASGE sequence was found at 324–332 sites of Lys-specific demethylase 3B. ► This human JmjC-domain-containing histone demethylase is encoded by JMJD1B gene. WKGGNASGE and DSIP exhibited similar potency in a preliminary biological study.Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.
Keywords: Delta sleep inducing peptide (DSIP); Origin; K,N-DSIP; Protein precursors; Encoding gene; Activity; Synthesis; JmjC-domain-containing histone demethylases;
Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase by María V. Niklison-Chirou; Fernando Dupuy; Lucila Saavedra; Elvira Hebert; Claudia Banchio; Carlos Minahk; Roberto D. Morero (832-834).
► COS-7 cells undergo apoptosis upon incubation with MccJ25-Ga but not with native microcin J25. ► MccJ25-Ga displayed a strong inhibitory activity on mitochondrial RNA synthesis.MccJ25, an antimicrobial peptide, was unable to cause apoptosis of COS-7 cells in spite of inducing reactive-oxygen species overproduction as well as cytochrome c release from isolated mitochondria. Surprisingly, MccJ25-Ga, an amidated variant of MccJ25 that displays similar anti-mitochondrial effects, did induce apoptosis in COS-7. The only difference found between the activities of these peptides was the unpredicted inhibition of mitochondrial RNA synthesis by MccJ25-Ga. These results led us to hypothesize that both mitochondrial RNA polymerase and mitochondrial membrane might be the molecular targets of MccJ25-Ga in mitochondria and this combined effect may lead to apoptosis.
Keywords: Microcin; Mitochondria; RNA-polymerase; Apoptosis;
Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors by Giovanni Tulipano; Valeria Sibilia; Anna Maria Caroli; Daniela Cocchi (835-838).
► Whey proteins can reduce postprandial glucose levels and stimulate insulin release. ► Administration of whey protein was associated with higher levels of intact incretins. ► Dipeptidyl peptidase-4 (DPP-4) is the main enzyme responsible for incretin degradation. ► β-Lactoglobulin is the major whey protein found in the milk of cows and other ruminants. ► Ile-Pro-Ala (IPA) is a bioactive peptide generated by proteolysis of bovine β-lactoglobulin. ► Our results showed that IPA can be regarded as a moderate DPP-4 inhibitor.Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.
Orexins/hypocretins increase the promoter activity of selective steroidogenic enzymes by Sonja M. Kagerer; Christine Eichholz; Olaf Jöhren (839-843).
► The increased promoter activities of CYP21, HSD3B2, CYP11B1, and CYP11B2 genes in human NCI-H295R-cells after orexin A and orexin B treatment demonstrate the regulation of these genes at the transcriptional level by orexins. ► In contrast, orexin has no effect on CYP21, HSD3B2, CYP11B1, and CYP11B2 mRNA stability. ► These results substantiate a role of orexins in adrenal gland regulation of steroid synthesis.Orexins (hypocretins) regulate multiple physiological functions, including central regulation of energy homeostasis and sleep–wake behavior but also peripheral hormonal actions. Recent data suggest specific effects of orexins at adrenal glands. To further assess the mechanism by which orexins regulate steroidogenesis we analyzed the effect of orexin A and B on the transcriptional activity of the luciferase reporter gene driven by the human steroid 21-hydroxylase (CYP21), 3β-hydroxysteroid dehydrogenase (HSD3B2), 11β-hydroxylase (CYP11B1), and aldosterone synthase (CYP11B2) gene promoter regions. After transient transfection of the reporter gene constructs into human NCI H295R cells, treatment with orexin A and B for 6 and 12 h increased the promoter activity of the CYP11B2, HSD3B2 and, to a lesser extend, CYP21 genes. The activity of the CYP11B1 was increased by both orexins after 3 h of treatment. Compared to the effects of forskolin or angiotensin II, however, the effect of orexins on the transcriptional activity of the steroidogenic enzyme genes was moderate. Our results suggest that orexins increase the expression of steroidogenic enzymes at the transcriptional level and that orexins play a role in the long term regulation of adrenal steroid production.
Corrigendum to “Effects of PKA modulation on the expression of neuropeptide Y in rat amygdaloid structures during ethanol withdrawal” [Peptides 24 (9) (2003) 1397–1402] by Huaibo Zhang; Subhash C. Pandey (844).

domain: chemistry


text:
Company Chicken with Citrus Sauce and Pistachios
This company chicken with citrus, carrots, prunes & pistachios is a treat for the eyes, taste buds and cook (make-ahead). The flavors blend beautifully.
Servings (change as needed): 5
- 4 pounds (1.8kg) chicken, cut into 8 pieces
- 1 lemon, zest and juice
- 1 large orange, zest and juice (for extra sauce add juice of another 1/2 - 1 orange)
- 4 tablespoon olive oil
- 3 cloves garlic, minced
- 2 tablespoon grainy mustard
- 6 tablespoon honey
- 1 teaspoon dried thyme (or 1 tbsp fresh)
- 1/2 teaspoon chili flakes (or less, to taste)
- 2 1/2 teaspoon kosher salt
- 1 tablespoon cornstarch mixed with 1 tbsp water (optional) Note 1
- 3 cups (450g) carrots, peeled and very thinly sliced (1/4 inch thick or less)
- 1 lemon, to be cut up and blanched (optional, but great!)
- 1 medium onion, halved and thinly sliced
- 1 cup prunes, roughly chopped (or use dates, raisins or apricots)
- 1/4 cup (12g) chopped fresh parsley (or cilantro)
- 2 green onions, thinly sliced
- 1/4 cup roasted pistachios, chopped
PREPARE MARINADE: Put all marinade ingredients in a large sealed bag. Squish it around to blend ingredients.
PREPARE VEGETABLES/FRUIT: For blanched lemon: cut lemon into quarters and remove seeds. Slice each quarter into thin wedges. Place into small pot of boiling salted water for 2 minutes, then drain. Add lemon wedges, prunes, carrots and onions to the bag with the marinade.
MARINATE CHICKEN: Add chicken pieces to bag with marinade. Seal and massage to coat chicken, fruits and vegetables well. Place in fridge to marinate for 6 hours or overnight. If really short on time, marinate for 30 minutes on the counter.
ROAST CHICKEN: Preheat oven to 400F. Transfer chicken, marinade and vegetables to a rimmed pan lined with foil and sprayed with oil. Chicken should be skin side up. Roast for 35 minutes until lightly browned (if chicken is larger, you may need 40 minutes). An instant thermometer piercing the breast should read about 155-160F. Stir carrots. Turn on broiler. Leave chicken on middle rack. Broil for 3-5 minutes or until crispy, golden brown (watching carefully so it doesn't burn).
GARNISH AND SERVE: Put chicken, vegetables and sauce onto a platter. Garnish with parsley, green onions and pistachios. Serve immediately.
- To thicken Sauce (optional): If you prefer as slightly thickened sauce like I do, add a slurry of cornstarch to the marinade (1 tbsp cornstarch mixed with one tbsp. water) and mix it in well.
- Make Ahead: The whole dish can be made a day ahead (or in the morning) and popped into the oven before serving.
Calories: 750kcal | Carbohydrates: 64g | Protein: 37g | Fat: 41g | Saturated Fat: 10g | Cholesterol: 131mg | Sodium: 1416mg | Potassium: 1090mg | Fiber: 8g | Sugar: 43g | Vitamin A: 13790IU | Vitamin C: 52mg | Calcium: 111mg | Iron: 3mg


text:
Isoflurane (ISO) is known to depress cardiac contraction. Here, we hypothesized that decreasing myofilament Ca2+ responsiveness is central to ISO-induced reduction in cardiac force development. Moreover, we also tested whether the nitroxyl (HNO) donor 1-nitrosocyclohexyl acetate (NCA), acting as a myofilament Ca2+ sensitizer, restores force in the presence of ISO. Trabeculae from the right ventricles of LBN/F1 rats were superfused with Krebs-Henseleit solution at room temperature, and force and intracellular Ca2+ ([Ca2+]i) were measured. Steady-state activations were achieved by stimulating the muscles at 10 Hz in the presence of ryanodine. The same muscles were chemically skinned with 1% Triton X-100, and the force-Ca2+ relation measurements were repeated. ISO depressed force in a dose-dependent manner without significantly altering [Ca2+]i. At 1.5%, force was reduced over 50%, whereas [Ca2+]i remained unaffected. At 3%, contraction was decreased by ∼75% with [Ca2+]i reduced by only 15%. During steady-state activation, 1.5% ISO depressed maximal Ca2+-activated force (Fmax) and increased the [Ca2+]i required for 50% activation (Ca50) without affecting the Hill coefficient. After skinning, the same muscles showed similar decreases in Fmax and increases in Ca50 in the presence of ISO. NCA restored force in the presence of ISO without affecting [Ca2+]i. These results show that 1) ISO depresses cardiac force development by decreasing myofilament Ca2+ responsiveness, and 2) myofilament Ca2+ sensitization by NCA can effectively restore force development without further increases in [Ca2+]i. The present findings have potential translational value because of the efficiency and efficacy of HNO on ISO-induced myocardial contractile dysfunction.
The effects of inhalational anesthetic agents on myocardial contraction have been the focus of extensive investigations for many years, and the depression of force development by different inhalational agents has been repeatedly shown. At the cellular level, altered cardiac excitation-contraction coupling has been identified as a major vehicle of impaired cardiac performance. In fact, inhalational anesthetics have been shown to decrease sarcolemmal L-type Ca2+ channel function (Terrar and Victory, 1988), decrease sarcoplasmic reticular Ca2+ storage (Davies et al., 2000), Ca2+ release (Jiang and Julian, 1998), and decrease myofilament Ca2+ sensitivity (Bosnjak et al., 1992; Davies et al., 2000). More importantly, it appears that the impact of inhalational agents on myocardium is agent- and dose-dependent, with decreasing Ca2+ availability being viewed as the major mode of action based on previous studies (Rusy and Komai, 1987; Housmans and Murat, 1988; Housmans et al., 2000).
However, other recent studies have shown that, at clinical dosages (i.e., 0.5–1.5 minimum alveolar concentration), isoflurane (ISO) or sevoflurane differentially decrease contractile force development while producing little inhibition of Ca2+ release. This point is proven by the lack of change in the amplitudes of intracellular Ca2+ transients (Hanley and Loiselle, 1998; Bartunek and Housmans, 2000; Davies et al., 2000; Graham et al., 2005). This clearly hints at the possibility that ISO directly decreases myofilament Ca2+ responsiveness. Nonetheless, early studies that used skinned cardiac muscle preparations yielded variable results; some failed to show any effect of ISO at low concentrations (i.e., clinically relevant doses: 1–3%) (Su and Kerrick, 1980; Su and Bell, 1986), and others showed either increased or decreased myofilament Ca2+ sensitivity (Murat et al., 1988; Herland et al., 1993). The contention was that the presence of a cellular membrane system could influence the modulation of cardiac inotropy by inhalational agents (Herland et al., 1993). Thus, it remains unclear whether ISO affects myofilament function per se, i.e., independently from changes induced at the sarcolemmal level.
Nitroxyl (HNO), the protonated one-electron reduction product of nitric oxide (Tocchetti et al., 2011), augments cardiac inotropy and improves relaxation in dog hearts, both under normal and heart failure conditions (Paolocci et al., 2001, 2003). The positive inotropic and lusitropic effects of HNO are independent of β-adrenergic stimulation but are sensitive to the redox states of the cardiac cell (Tocchetti et al., 2007; Froehlich et al., 2008). Recently, we found that HNO augments cardiac contractility by increasing Ca2+ responsiveness of the myofilaments (Dai et al., 2007). This enhancement of force is not associated with an increase in diastolic tension or myofibrillar Mg-ATPase activities. Subsequently, we have confirmed that HNO, by interacting with specific, highly reactive thiols in myofilament proteins, modifies the redox states of key contractile proteins, thus promoting an increase in force production after Ca2+ binds to troponins (Tn) (Murray et al., 2009).
In this study, we tested two main hypotheses. First, we aimed to verify whether ISO is able to exert a direct effect on myofilament sensitization to Ca2+. To do so, we determined the steady-state force-Ca2+ relations of individual muscles, before and after skinning. Our second aim was to test whether the HNO donor, 1-nitrosocyclohexyl acetate (NCA) (Sha et al., 2006; Shoman et al., 2011), can restore cardiac force development in the presence of ISO by virtue of its Ca2+-sensitizing effect (Dai et al., 2007). We hope to demonstrate that HNO, donated by NCA, can restore myocardial contractility during ISO-induced anesthesia and that HNO donors are probably able to provide an inotropic counterbalancing effect in patients displaying compromised cardiac function due to inhalation of volatile anesthetics such as ISO, thus justifying the use of HNO donors for patients with heart failure undergoing inhalational anesthesia.
Materials and Methods
LBN/F1 rats (250–300 g; Harlan, Indianapolis, IN) were used in these experiments. Animal care and experimental protocols were approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine.
Trabecular Muscle Preparation.
The rats were anesthetized via intra-abdominal injection with pentobarbital (100 mg/kg); the heart was exposed by mid-sternotomy, rapidly excised, and placed in a dissection dish. The aorta was cannulated, and the heart was perfused in a retrograde fashion (∼15 ml/min) with dissecting Krebs-Henseleit (K-H) solution equilibrated with 95% O2, 5% CO2. The dissecting K-H solution was composed of 120 mM NaCl, 20 mM NaHCO3, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, 0.5 mM CaCl2, and 20 mM 2,3-butanedione monoxime, pH 7.35 to 7.45 at room temperature (21–22°C). Trabecular muscle from the right ventricle of the heart was dissected and mounted between a force transducer and a motor arm, superfused with K-H solution without 2,3-butanedione monoxime at a rate of ∼10 ml/min, and stimulated at 0.5 Hz.
Force was measured by a force transducer system (KG7; Scientific Instruments GmbH, Heidelberg, Germany) and expressed in millinewtons per square millimeter of cross-sectional area. The muscles underwent isometric contractions with the resting muscle length set, such that resting force was 15% of total force development (i.e., optimal muscle length). This resting muscle length, corresponding to resting sarcomere length of 2.20 to 2.30 μm as determined by laser diffraction (Gao et al., 1996), was maintained throughout the experiments. ISO was delivered via an ISO-specific vaporizer (calibrated by a vapor analyzer) to the K-H solution along with the 95% O2, 5% CO2 gas mixture at a constant flow rate (1.0 l/min). The K-H solution was bubbled through a fine-porosity gas distribution tube with desired doses (vol %) of ISO for at least 15 min before use. Because of the volatile nature of ISO, the K-H solution was constantly bubbled with the gas mixture saturated with ISO, and the reservoir was covered to maintain the desired percentage of ISO throughout the experiments. In this design, the muscles were superfused with K-H buffer saturated with desired percentage of ISO (i.e., not via inhalation). NCA was dissolved in dimethyl sulfoxide to make a stock solution (0.06 M) and stored in −20°C until use. All experiments were performed at room temperature (20°C–22°C).
Measurement of [Ca2+]i.
Intracellular Ca2+ ([Ca2+]i) was measured by using the free acid form of fura-2 as described previously (Gao et al., 1994; Dai et al., 2007). Fura-2 potassium salt was microinjected iontophoretically into one cell and allowed to spread throughout the whole muscle (via gap junctions). The tip of the electrode (∼0.2 μm in diameter) was filled with fura-2 salt (1 mM), and the remainder of the electrode was filled with 150 mM KCl. After a successful impalement into a superficial cell in nonstimulated muscle, a hyperpolarizing current of 5 to 10 nA was passed continuously for approximately 15 min. In some muscles, multiple injections (up to 3–4) were applied at different sites, with duration of the injection limited to <10 min at each site to achieve an optimal signal-to-noise ratio. As previously established, this loading did not affect force development. Fura-2 epifluorescence was measured by excitation at 380 and 340 nm. Fluorescent light was collected at 510 nm by a photomultiplier tube (R1527; Hamamatsu Photonics, Hamamatsu City, Japan). The output of the photomultiplier was collected and digitized. [Ca2+]i was given by the following equation (after subtraction of the autofluorescence) (eq. 1): where R is the observed ratio of fluorescence (340 nm/380 nm), K′d is the apparent dissociation constant, Rmax is the ratio of 340 nm/380 nm at saturating Ca2+, and Rmin is the ratio of 340 nm/380 nm at 0 Ca2+. The values of K′d, Rmax, and Rmin were determined by in vivo calibrations as described previously (Gao et al., 1994, 1998).
Steady-State Activation of Trabeculae.
Ryanodine (1.0 μM) was used to enable steady-state activation. After the muscles were exposed to ryanodine for 15 min, different levels of tetanizations were induced briefly (∼4–8 s) by stimulating the muscles at 10 Hz at various concentrations of extracellular Ca2+ ([Ca2+]o; 0.5–20 mM). The steady-state force-[Ca2+] relations were fit with a function of the Hill equation (eq. 2): where F is the steady-state force at various Ca2+ concentrations, Fmax is the maximal Ca2+-activated force, Ca50 is the Ca2+ concentration required to achieve 50% Fmax, and n is the Hill coefficient.
After the steady-state experiments, the same trabeculae were immediately skinned by 15 to 20 min of exposure to 1% Triton X-100 in relaxing solution containing 100 mM KCl, 25 mM HEPES, 10 mM K2EGTA, 15 mM creatine phosphate sodium salt (Na2CrP), 5 mM Na2ATP, 5.15 mM MgCl2, and 0.5 mM leupeptin, pH 7.2 with KOH. Activating solution contained 10 mM Ca2+-EGTA, 100 mM KCl, 25 mM HEPES, 15 mM Na2CrP, 5 mM Na2ATP, 4.75 mM MgCl2, and 0.5 mM leupeptin, pH 7.2. Different Ca2+ concentrations were achieved by mixing the activating solution and relaxing solution at different ratios. Skinning was considered to be complete when the preparation lost its pink color and sarcomeres were visible. Diastolic sarcomere length was determined by direct visualization under 100× magnification and was set at ∼2.2 μm. Resting force was usually 10 to 15% of maximal activated force at this sarcomere length. The force-Ca2+ relationship was obtained by exposing the skinned muscles to activating solutions with various Ca2+ concentrations and were fit with the Hill equation as described above.
Paired Student's t test and multivariate ANOVA were used for statistical analysis of the data (Systat 10.2.01; Systat Software Inc., San Jose, CA). A value of P < 0.05 was considered to indicate significant differences between groups. Unless otherwise indicated, pooled data are expressed as means ± S.E.M.
Isoflurane Depresses Twitch Force without Affecting [Ca2+]i Transients.
We initially studied the effect of ISO on force development and [Ca2+]i transients in isolated intact trabecular muscles. Figure 1A depicts a representative trace of the typical raw recordings of force and [Ca2+]i transients at different ISO concentrations, whereas Fig. 1B shows the pooled data of all of the muscles tested. ISO depressed force development in a dose-dependent manner without significantly altering the amplitude of [Ca2+]i transient. Moreover, at doses between 0.5 and 1.5%, developed force was reduced by more than 50% (from 18.0 ± 1.2 to 8.6 ± 0.5 mN/mm2, P < 0.001), whereas [Ca2+]i transient amplitudes remained unaffected ([Ca2+]i = 0.58 ± 0.06 μM at 0.5% and 0.55 ± 0.06 μM at 1.5% ISO, P > 0.05). At 3% ISO, contraction was decreased by approximately 75% (to 4.9 mN/mm2), whereas [Ca2+]i was reduced by only 15% (to 0.5 ± 0.05 μm, P > 0.05). Thus, after treatment with ISO, the reduction of force seen seems to stem from changes in the properties of the myofilament (i.e., decreased myofilament Ca2+ responsiveness), rather than from decreased Ca2+ cycling/availability. In addition, relaxation of twitch force, as measured by time to half-relaxation from peak force, was faster in the presence of ISO (Table 1).
Isoflurane Decreases Myofilament Ca2+ Responsiveness.
The data presented above only indirectly supports the contention that myofilament properties change under the influence of ISO given that, during activation and contraction, peak force development and peak amplitude of the [Ca2+]i transient are not at equilibrium. Thus, to unequivocally prove that the anesthetic has a primary effect on myofilaments, we measured the steady-state force-[Ca2+]i relation before and during 1.5% ISO exposure. In the presence of ISO, Fmax decreased, whereas the [Ca2+]i, required for 50% activation (Ca50), was increased (Fig. 2A; Table 2). The Hill coefficient was not affected in these muscles. Overall, there is a significant difference between the two relationships (P < 0.001) when multivariate ANOVA was performed among the parameters of Fmax, Ca50, and Hill coefficient. Hence, the action of ISO in the intact muscle was to decrease Fmax and increase Ca50.
In the intact muscle, the myofilaments are bathed in a cellular milieu that contains many factors that modulate the steady-state force-[Ca2+]i relationship (Gao et al., 1997). ISO could potentially modify these factors, thereby decreasing myofilament Ca2+ responsiveness. Therefore, to rule out all potential confounding factors, we skinned the same trabecular muscle in which the steady-state force-[Ca2+]i relations had been obtained. This enabled us to obtain force-[Ca2+] relations devoid of potential soluble confounding factors in these muscles. Likewise, in these skinned muscles, the Fmax and Ca50 were significantly depressed (Fig. 2B; Table 2) in the presence of ISO. Multivariate ANOVA also showed significant difference between the two relationships (P < 0.001). Thus, the depressing effect of ISO on the steady-state force-Ca2+ relation persisted in skinned muscles. We also performed experiments in which trabeculae were chemically skinned immediately after stabilization and the force-Ca2+ relationship was obtained in the absence or presence of 1.5% ISO. These experiments served as internal checkpoints for the tetanization protocol performed to verify that the protocol itself did not affect the force-Ca2+ in the (same) skinned muscles afterward. Indeed, these experiments showed identical force-Ca2+ relations as in Fig. 2B, indicating that tetanization protocol had no effect on subsequent force-Ca2+ relations in skinned muscles (Fig. 3). Thus, all together these data strongly support our initial hypothesis that the ISO-induced negative effect on myocardial contraction largely stems from a direct adverse impact on the myofilament.
Reversal of the Negative Inotropic Effect of Isoflurane.
If ISO inhibits force generation by directly decreasing myofilament Ca2+ responsiveness, agents that increase myofilament Ca2+ responsiveness (so-called Ca2+ sensitizers) should be able to restore force in the presence of this anesthetic agent. We tested this hypothesis by exposing the muscles to the new HNO donor, NCA (Sha et al., 2006; DuMond and King, 2011; Shoman et al., 2011). As we learned earlier, NCA increases force development by increasing myofilament sensitivity to Ca2+ (Murray et al., 2009). At 5 μM (slightly higher than the EC50 of NCA as determined in cardiac muscle (Murray et al., 2009), NCA increased force development significantly without affecting twitch dynamics (Table 1). It is noteworthy that NCA completely restored force development in the presence of 1.5% ISO, with little increase in the amplitude of the [Ca2+]i transient (Fig. 4). To show that NCA was indeed effective, we compared force recovery and [Ca2+]i transient amplitude when [Ca2+]o was raised (Fig. 5). Although ISO-dependent force depression could be fully restored by increasing the [Ca2+]o, the [Ca2+]i more than doubled. In contrast to Ca2+, NCA produced a full recovery in force without increasing [Ca2+]i. It is noteworthy that levosimendan (Levo), another Ca2+-sensitizing agent, which binds to cardiac TnC (Haikala et al., 1995), hardly restored force in the presence of ISO (Fig. 6), although it was able to increase force development in the absence of it as expected (Sato et al., 1998). This suggests that ISO affects steps downstream of Tn activation in myofilament contraction.
In this study, we provide two novel findings. First, we demonstrated that ISO is able to reduce force development without affecting the intracellular Ca2+ transient. For the first time, we show that ISO decreased Fmax and increased Ca50 of the steady-state force-Ca2+ relation in individual muscles, both before and after they were chemically skinned. Second, we demonstrated the ability of HNO donors such as NCA to rescue force development in ISO-treated muscles by virtue of their sensitizing action to Ca2+ at the myofilament level. This study extended the results from previous investigations on the effect of ISO on myocardial contractility while deepening our understanding of how ISO may dampen myocardial function.
The myofilament desensitizing effect of ISO (and/or sevoflurane) has been shown by a number of prior studies in intact cardiac muscles and myocytes (Hanley and Loiselle, 1998; Jiang and Julian, 1998; Graham et al., 2005). ISO may decrease myofilament Ca2+ responsiveness by 1) directly interacting with the contractile proteins to affect activation/contraction processes and 2) modifying soluble cytoplasmic factors that may influence the behavior of the myofilaments. These pathways can be removed by skinning the muscle preparation; thus, the environment bathing the myofilaments proteins can be controlled. By equalizing the composition of solutions bathing the myofibrils, any of the effects of ISO observed is the result of a direct interaction between the agents and the contractile proteins. On the other hand, the results of previous studies using skinned muscle preparations have been variable; some have shown that myofilament Ca2+ responsiveness to inhalational anesthetics decreased (Herland et al., 1993), whereas others have shown no change or increased myofilament Ca2+ responsiveness (Su and Kerrick, 1980; Su and Bell, 1986; Murat et al., 1988). Taken together, these studies suggest that, at least in part, the myofilament desensitization may reflect changes in the soluble cytoplasmic factors. Therefore, we chose to try another approach to evaluate the effect of ISO on myofilament Ca2+ responsiveness using the same muscle before and after skinning. This is a technically challenging experimental design that allows us to neatly distinguish the relative roles of soluble cytoplasmic factors and myofilament proteins in ISO-induced force depression, directly with increased reliability. The decreased myofilament Ca2+ responsiveness induced by ISO in intact muscles was reproducible in the same skinned muscles, confirming a direct myofilament effect of ISO.
The mechanism by which ISO interacts with myofilaments to decrease force development is not known at the moment. In cardiac muscle, force development depends on this sequence of events: Ca2+ binding to TnC (Krudy et al., 1994), interactions between Tn subunits (Kleerekoper et al., 1995; Dong et al., 2003), Tn-tropomyosin-actin interactions (McKillop and Geeves, 1993; Gordon et al., 2000; Lehman et al., 2001), and cross-bridge formation to generate force (Geeves and Holmes, 2005; Fujita-Becker et al., 2006). ISO has been shown to increase TnC affinity for Ca2+ and to promote conformational changes within Tn (Breukelmann and Housmans, 2007): however, such changes would not be expected to decrease force. In in vitro motility assays, ISO does not interfere with cross-bridge formation between isolated myosin and actin (Vivien et al., 2005). The inability of Levo, another known myofilament Ca2+ sensitizer that binds TnC and stabilizes the conformation of Ca2+-Tn complex (Haikala et al., 1995), to restore force in the presence of ISO suggests that ISO probably targets process(es) after TnC activation. Thus, given the above considerations, actin-tropomyosin interactions are most probably targeted by ISO. The fact that HNO provides complete (and competitive) reversal of ISO-induced force depression without additional increases in intracellular Ca2+ provides strong support to our view that HNO increases force development by increasing myofilament Ca2+ responsiveness (Dai et al., 2007; El-Armouche et al., 2010). Recently, we have shown that HNO promotes formation of disulfide bridges between actin and tropomyosin. These disulfide bridges prime tropomyosin for exposing more force-generating cross-bridge binding sites on actin so that more force could be produced (Murray et al., 2009). However, the molecular nature of isoflurane's action on myofilament proteins is unknown. Ongoing studies in our laboratory are focusing on identifying the myofilament protein(s) that are targeted by ISO.
Our current findings have some important pragmatic implications. The negative inotropic effect of ISO is probably to depress contractility further in patients with heart failure under anesthesia. Routinely, vasoconstrictors (i.e., phenylephrine) and crystalloid are administered to restore blood pressure and maintain cardiac output. In spite of these therapies, the perioperative morbidity and mortality for patient with heart failure is surprisingly high (i.e., 11.7%) (Hernandez et al., 2004). It is conceivable that vasoconstrictors and fluid therapy may impair the capability of the heart to contract due to an increase in pre- and postload. On the other hand, positive inotropic agents are expected to be more effective because they can augment cardiac contractility but they tend to produce Ca2+ overload. Ca2+ sensitizers, which represent a new class of inotropic agents, may be an effective alternative (Kass and Solaro, 2006). Their potential is especially relevant for patients with heart failure who require anesthesia. Decreased myofilament Ca2+ responsiveness as a result of alterations in the function of myofilament regulatory proteins is a significant contributor to the severely decreased contractility of failing myocardium (Noguchi et al., 2004; VanBuren and Okada, 2005; Murphy, 2006). Thus, inotropic agents with Ca2+-sensitizing properties are more desirable and probably more efficient for restoring contractility than conventional inotropic agents that increase contractility simply by increasing the availability of Ca2+ (Brixius et al., 2005; Hasenfuss and Teerlink, 2011). In addition, because of the exaggerated Ca2+ overload, the use of conventional inotropic agents is associated with worsening morbidity and mortality in heart failure patients (Cohn et al., 1998; Cuffe et al., 2002). Another aspect that singles NCA/HNO out among other inotropes is that it does not alter ISO-induced impact on lusitropy (Table 1).
In summary, we demonstrated that, at clinically relevant doses, the inhalational anesthetic ISO directly depresses myofilament contractility by decreasing Ca2+ responsiveness. The force depression can be effectively and fully restored by the novel myofilament Ca2+ sensitizer, NCA. Ca2+ can also restore the loss in force depression, but only with concomitant doubling of [Ca2+]i. Although the molecular nature of ISO's myofilament action is still under investigation, our findings indicate that cardiac depression during anesthesia, especially in patients with heart failure, is more effectively and efficiently treated with positive inotropic agents that possess Ca2+-sensitizing properties.
Participated in research design: Ding, Li, Shen, Martin, Sivakumaran, Paolocci, and Gao.
Conducted experiments: Ding, Li, Shen, and Gao.
Contributed new reagents or analytic tools: King and Martin.
Performed data analysis: Ding, Li, Shen, and Paolocci.
Wrote or contributed to the writing of the manuscript: Ding, Li, Shen, Martin, Sivakumaran, Paolocci, and Gao.
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL091923, R01-HL075265]; and American Heart Association Mid-Atlantic [Grant 0855439E]. N.P. is a founder and stock owner at Cardioxyl Pharmaceutical, Inc.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- 1-nitrosocyclohexyl acetate
- analysis of variance
- maximal Ca2+-activated force
- Ca2+ required to achieve 50% of Fmax
- intracellular Ca2+
- extracellular Ca2+
- troponin C.
- Received June 21, 2011.
- Accepted August 23, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics

domain: biology


text:
Hype Williams’ new album out now; Dean Blunt & Inga Copeland touring separately
by Kristian Brito
Things are bustling in the world of cryptic UK experimentalists Hype Williams, the mysterious electronic project previously thought to be comprised of Dean Blunt and Inga Copeland. On July 27, the group quietly uploaded 10/10, their first release in five years, to their Bandcamp without any further details. In the following days, Copeland claimed not to know anything about the record, leaving us to wonder whether old rumors that Hype Williams is actually a curated 18-year "relay project" passed between alternating lineups was more than clever fabrication. Later that week, shady HW 'representative' Denna Frances Glass (who some have suggested is not even a real person) told Thump that Blunt/Copeland are no longer part of Hype Williams and that the project will continue with other members. As ever with these guys, the reality remains hazy and unclear. Check out the new album, which is available for purchase (but not streaming) at Bandcamp.
In the meantime, Blunt and Copeland have been busy with their respective solo work. Blunt has announced a 5-date North American tour with his Babyfather project, which kicks off in the Fall with a NYC show on 9/14 in Output's Panther Room (tickets) It'll be Blunt's second batch of US performances behind this year's Platinum Tears mixtape. See Blunt's upcoming schedule below. Also below you can check out a new Babyfather track, which samples Kate Bush's "Wuthering Heights," and the new Babyfather remix of 18+'s "Drama."
Meanwhile, Copeland will be performing a few shows across the US behind the release of her live album Live in Paris under the pseudonym Lolina. Dates include a September 10 gig at NYC's Trans-Pecos on September 10 with Giant Claw (tickets). Before hitting New York, she'll be making two US festival appearances, first on August 12 at LA's Berserktown III and then on September 9 at North Carolina's Hopscotch 2016 festival. See Copeland's full schedule below, and check out the initial video release of her Live in Paris as well.
Babyfather – 2016 Tour Dates
08.26 - Aarhus, DK Flux Festival
09.14 - New York, NY - Panther Room
09.15 - Detroit, MI - El Club
09.17 - Atlanta, GA - 787 Windsor
09.21 - Toronto, CA - The Velvet Underground
09.22 - Montreal, CA - POP Montreal
09.24 - Eupen, BE - Meakusma Festival
10.20-22 - Krakow, PL - Unsound Festival
10.28 - Manchester, UK - Soup Kitchen
10.29 - Bristol, UK - The Black Swan
10.03 - Athens, GR - Plissken Festival
Inga Copeland – 2016 Tour Dates
08-12 – Los Angeles, CA – Berserktown III
08-17 – London, UK – Echoes
08-19 – Libson, PT – Noites de Verão / Summer Nights
09-09 – Raleigh, NC – Hopscotch 2016
09-10 – New York, NY – Trans-Pecos
09-16 – Chicago, IL – TBA

domain: artistic