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Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitopes recognized by three monoclonal antibodies (MONO, 3C2, and 3E7) were mapped by expressing defined parts of the bovine G(o) alpha-cDNA in bacteria, followed by immunoblotting. All three antibodies recognize the recombinant bovine alpha o-protein, but at distinct sites. The epitopes of MONO and 3C2 were mapped between alpha o amino acids 80 and 145, and both antibodies recognize alpha o exclusively. Heterotrimeric G(o)-proteins as well as guanosine 5'-3-O-(thio)triphosphate-liganded free alpha o-subunits are readily immunoprecipitated by these monoclonal antibodies. Binding of MONO or 3C2 does not affect ADP-ribosylation of the alpha o-subunit by pertussis toxin. Apparently, the antibodies do not bind to or induce large conformational changes in regions of the alpha o-subunit that are involved in association with beta gamma-subunits or ADP-ribosylation. 3E7 behaves as an anti common alpha-subunit antibody when used in immunoblots. However, under nondenaturing conditions, 3E7 recognizes alpha o exclusively. After binding of 3E7, the pertussis toxin-dependent ADP-ribosylation of alpha o is effectively blocked, while the ADP-ribosylation of the various alpha i-subunits is not affected. The epitope of 3E7 was mapped between alpha o amino acids 13-88, a region which has been implicated in the interaction between alpha- and beta gamma-subunits. Possibly, the inhibitory effect of 3E7 on ADP-ribosylation of G(o) is secondary to the loss of beta gamma-subunits that is observed upon binding of 3E7.
Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth
of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical
stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES
activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it
can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses.
The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that
p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms
pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative
mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced
p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53−/− cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate
the p38-p53 signaling pathway among the transduction pathways that MES affects.
Two different methods were used to determine the number of Bohr protons released upon oxygenation of human hemoglobin (Hb A) and Hb A lacking beta 146 His (des-His Hb A) at the pH ranging from pH 5.0 to 9.0 in the presence of 0.1 M Cl- at 25 degrees C. One is the direct differential titration method, the other is based on the measurement of oxygen affinity as a function of pH. The results obtained for Hb A or des-His Hb A with two methods were completely mutually consistent. The number of Bohr protons released from des-His Hb A upon oxygenation at pH 7.5 was about 44% less than that from Hb A, while at pH 5.5 the number of Bohr protons taken up by des-His Hb A was 20% greater than that by Hb A. The differences in the number of Bohr protons between Hb A and des-His Hb A could not be simply ascribed to the lack of beta 146 His from Hb A. The pK alpha values, which were determined by the deuterium exchange method using 1H NMR, were 8.0 for beta 146 His of deoxy-Hb A and 6.5 for that of CO Hb A, while those of beta 143 His were 5.2 for deoxy-Hb A and 6.0 for CO Hb A. From these pK alpha values, in addition to those of alpha 1 Val proposed for the modified CO and deoxy-Hb A with carbamylated beta chains by Van Beek and De Bruin (Van Beek, G. M., and De Bruin, S. H. (1980) Eur. J. Biochem. 105, 353-360), it became evident that almost all (about 92%) of the alkaline Bohr protons released upon oxygenation of Hb A in the presence of 0.1 M Cl- could be accounted for by the protons from these 2 residues, although the involvement of other histidine residues could not be denied. About half the acid Bohr protons from Hb A, which corresponds to the higher pH part (above pH 5.0) of the acid Bohr effect, could be explained by the involvement of beta 143 His residue. The residual acid Bohr effect in the more acidic pH region was presumably contributed by an amino acid residue with pK alpha values of 4.05 and 5.95 for the deoxy- and CO Hb A, respectively, although the amino acid residue was unspecified.(ABSTRACT TRUNCATED AT 400 WORDS)
We have devised an experimental system using the T7 phage 0.3 protein to accurately quantitate in vivo errors in protein synthesis. The 0.3 protein is well suited for mistranslation studies because it is easy to purify, its precise amino acid and RNA sequences are known, and it contains no cysteine. Utilizing [35S]cysteine as a precursor we found an average of 1 cysteine residue misincorporated for every 43.5 molecules of 0.3 protein synthesized. Since there are 116 amino acids in 0.3 protein, 1 cysteine residue was misincorporated /5000 codons translated. If all 20 amino acids were misincorporated at the same frequency, the overall frequency of misincorporation of amino acids into 0.3 protein would be 4 X 10(-3)/codon translated. Parallel experiments measuring [35S]methionine incorporation into 0.3 protein supported the accuracy of our findings for cysteine misincorporation. We found an average of 5.7 methionine residues incorporated/molecule of 0.3 protein synthesized; the actual number from sequence data is known to be 6. Antibiotics which stimulate mistranslation (gentamicin and streptomycin) caused a modest increase in the number of cysteine residues misincorporated into 0.3 protein. The use of Escherichia coli strains, identical except for mutations in ribosomal protein genes known to affect the fidelity of translation, supported the contention that the errors being quantitated were mainly due to mistranslation rather than mistranscription .
The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.
The gene 0.3 protein of bacteriophage T7 prevents the DNA restriction system of EScherichia coli from interfering with T7 infection. A mutant strain of T7 that greatly overproduces the 0.3 protein has been constructed and used for purification of this protein. The 0.3 protein ws found to be extremely acidic and can be separated from virtually all other proteins of the infected cell by chromatography on DEAE-cellulose. Residual contaminating proteins and nucleic acids can be removed by gel filtration, but an even simpler final purification is possible, because under appropriate conditions the 0.3 protein is soluble in high concentrations of ethanol. Thus, a simple, essentially two-step purification can produce about 50 mg of pure 0.3 protein from 30 liters of culture. The purified protein appears to be a dimer of identical subunits. AS expected from its known function during infection, the purified 0.3 protein inhibits the nuclease and ATPase activities of partially purified Eco B, the DNA restriction enzyme of E. coli B, but it does not interfere with several different type II endonucleases tested. The inhibition of Eco B appears to require stoichiometric rather than catalytic amounts of 0.3 protein.
P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.
The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.
The construction, expression, and purification of an active Fv fragment of the 0.5beta monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1IIIB was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311, 315, and 318 in gp120 of HIV-1IIIB) reduce the binding free energy by 0.22 (+/- 0. 20), 4.32 (+/- 0.16), and 1.58 (+/- 0.17) kcal mol-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (+/- 0.22), -0.55 (+/- 0.18), and -0.97 (+/- 0.19) kcal mol-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.
Nucleotide sequences of 64 VH segments within the 3' 0.8-megabase region of the human immunoglobulin germ line VH locus were compared with trace evolution of human VH segments. Based on alignment of the deduced amino acid sequences of 37 functional germ line VH segments, a phylogenetic tree was generated using the neighbor-joining method. The phylogenetic tree clearly supports the previous classification of human VH segments into six families, which correlate roughly with mouse VH families with varying conservation. The human VH-III family is most homologous to mouse VH segments, suggesting that members of the VH-III family may be conserved by some functional constraint. The 5'-flanking region of each family has a family-specific structure. The sequenced 64 VH segments include 31 pseudogenes, of which 24 were highly conserved. Unidirectional transfer of segmental sequences was identified within the VH-III and VH-IV families, providing clear examples of germ line gene conversion. Such gene conversion may contribute to conserve structures of pseudo-VH segments. Comparison of the VH-IV family members indicates that recent repeated duplications and frequent gene conversions are responsible for strong conservation of this family, although functional selection is not completely excluded.
The atomic resolution structure ofLeishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations.
Also for the side chain of the catalytic glutamate, Glu167, two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of
the ligand and the catalytic glutamate. The distance between O11 of PGA and Oε2 of Glu167 is 2.61 and 2.55 Å for the major and minor conformations, respectively. In either conformation, Oε1 of Glu167 is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually
exclusive arrangements. Despite the structural disorder in the active site, the C termini of the β strands that construct
the active site display the least anisotropy compared with the rest of the protein. The loops following these β strands display
various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region
of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro168 at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.
The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.
In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from
a readily reducible disulfide formed between α-subunit residues Cys and Cys. The cross-linker S-(2-[3H]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced αCys/Cys and an amide bond with an acidic residue in the subunit (Czajkowski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NHCH2CONHCH2CH2S- is 0.9 nm long. After the disulfide bond linking -NHCH2CONHCH2CH2S- to the α subunit was reduced, -NHCH2CONHCH2CH2SH remained linked to the subunit by an amide bond. The subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation.
Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity
were obtained in the sequencing cycles corresponding to Asp, Asp, and Glu. The mutation of Asp to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either Asp, Glu, or 8 other acidic residues in the same region of decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A.(1993) Proc. Natl. Acad. Sci. U. S. A 90, 6285-6289). Because Asp both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding
site formed in the interface between the α and the subunits.
The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.
Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.
A method is presented for the preparation of a "native" epidermal growth factor (EGF) receptor-kinase complex of molecular weight 170,000 from A-431 cells. Although this receptor complex is capable of binding EGF, noncovalently, in quantities similar to the previously isolated 150,000 complex (Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. 255, 4834-4842), the 170,000 preparation has 5 to 10 times the intrinsic kinase activity (autophosphorylation). However, the 170,000 kinase activity toward other proteins is lower than that of the 150,000 preparation. Both the 170,000 and 150,000 kinase activities are enhanced by EGF. The 170,000 and 150,000 proteins are also capable of forming covalent linkages to 125I-EGF, and each is precipitated by antisera directed against the 170,000 protein. We suggest the 150,000 protein is a proteolytic degradation product of the 170,000 protein. The EGF-enhanced kinase activity of the 170,000 preparation remains associated with the 125I-EGF-binding activity following EGF affinity chromatography, electrophoresis in nondenaturing gels, or immunoprecipitation with antisera directed against the sodium dodecyl sulfate (SDS) gel-purified 170,000 protein. These results indicate that the receptor, kinase, and substrate domains are linked, possibly covalently.
Members of the IclR family of transcription regulators modulate signal-dependent expression of genes involved in carbon metabolism in bacteria and archaea. The Thermotoga maritima TM0065 gene codes for a protein (TM-IclR) that is homologous to the IclR family. We have determined the crystal structure of TM-IclR at 2.2 A resolution using MAD phasing and synchrotron radiation. The protein is composed of two domains: the N-terminal DNA-binding domain contains the winged helix-turn-helix motif, and the C-terminal presumed regulatory domain is involved in binding signal molecule. In a proposed signal-binding site, a bound Zn(2+) ion was found. In the crystal, TM-IclR forms a dimer through interactions between DNA-binding domains. In the dimer, the DNA-binding domains are 2-fold related, but the dimer is asymmetric with respect to the orientation of signal-binding domains. Crystal packing analysis showed that TM-IclR dimers form a tetramer through interactions exclusively by signal-binding domains. A model is proposed for binding of IclR-like factors to DNA, and it suggests that signal-dependent transcription regulation is accomplished by affecting an oligomerization state of IclR and therefore its affinity for DNA target.
Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.
Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagen-swelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 mum to 8.8 mum after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases.
MHC class II molecules are composed of one α-chain and one β-chain whose membrane distal interface forms the peptide binding
groove. Most of the existing knowledge on MHC class II molecules comes from the cis-encoded variants where the α- and β-chain are encoded on the same chromosome. However, trans-encoded class II MHC molecules, where the α- and β-chain are encoded on opposite chromosomes, can also be expressed. We have
studied the trans-encoded class II HLA molecule DQ2.3 (DQA1*03:01/DQB1*02:01) that has received particular attention as it may explain the
increased risk of certain individuals to type 1 diabetes. We report the x-ray crystal structure of this HLA molecule complexed
with a gluten epitope at 3.05 Å resolution. The gluten epitope, which is the only known HLA-DQ2.3-restricted epitope, is preferentially
recognized in the context of the DQ2.3 molecule by T-cell clones of a DQ8/DQ2.5 heterozygous celiac disease patient. This
preferential recognition can be explained by improved HLA binding as the epitope combines the peptide-binding motif of DQ2.5
(negative charge at P4) and DQ8 (negative charge at P1). The analysis of the structure of DQ2.3 together with all other available
DQ crystal structures and sequences led us to categorize DQA1 and DQB1 genes into two groups where any α-chain and β-chain
belonging to the same group are expected to form a stable heterodimer.
The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/ H+ exchange, Na+(HCO3-)3 cotransport and Na+-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 ± 0.02 to 6.48 ± 0.02 within 3.4 ± 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 ± 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 ± 0.02 to 6.75 ± 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Naout+-dependent, Cl--independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.
Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.
Orcinol hydroxylase (EC 188.8.131.52), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized. Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone. The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm. FAD can be dissociated from the protein. Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN. The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein. K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY. The molecular activity of the crystalline enzyme is 1560 min minus 1. In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2. Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol. Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated. The latter compounds are not hydroxylated. Instead H2O2 accumulates as the only product of O2 reduction. The enzyme therefore behaves either as a hydroxylase or an oxidase. The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation. The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol. K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy. The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol. The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol. Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector. Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur. Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector. The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation.
Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G2/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent
Chk1 inhibitor that abrogates the G2/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined
the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog
SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes
in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence
of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding
interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity
of these interactions is consistent with the potency and selectivity of UCN-01.
A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable peptide-HLA-I complex formation required for long term presentation. Tapasin is thought to be central to this essential quality control, but the underlying mechanisms remain unknown. Here, we report that the N-terminal region of tapasin, Tpn(1-87), assisted folding of peptide-HLA-A*02:01 complexes according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database and one consisting of medium to high affinity non-SYFPEITHI ligands, were studied in the context of HLA-A*02:01 binding and stability. We show that the SYFPEITHI peptides induced more stable HLA-A*02:01 molecules than the other ligands, although affinities were similar. Remarkably, Tpn(1-87) could functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I- and peptide-specific function, together with the functions exerted by the more C-terminal parts of tapasin, are major features of tapasin-mediated HLA-I quality control. These findings are important for understanding the biogenesis of HLA-I molecules, the selection of presented T-cell epitopes, and the identification of immunogenic targets in both basic research and vaccine design.
Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade
of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have
previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein
increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational
regulation. De novo PGHS-1 synthesis, measured using [35S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To
identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3′-untranslated region from different
species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift
assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control
cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes,
one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet
of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level.
Bone sialoprotein (BSP) is a major noncollagenous, RGD-containing glycoprotein found in the extracellular matrix of bone. The RGD sequence is flanked by two tyrosine-rich regions, which fit the established consensus requirements for tyrosine sulfation. Tyrosine sulfation is suggested to be important in the regulation of protein secretion and function. The role of this post-translational modification on the cell attachment activity and secretion of a highly sulfated form of BSP isolated from a rat osteoblast-like cell line (UMR 106-01 BSP) was investigated by inhibiting sulfation with chlorate. [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to monitor biosynthetic products. Chlorate was effective in inhibiting total [35S]sulfate incorporation by 90% without altering overall protein synthesis and secretion in cultures up to 72 h under serum-free conditions. Isolated proteoglycans and purified BSP were analyzed for sulfate incorporation. Proteoglycans isolated from the medium of cells treated with chlorate displayed a difference in the hydrodynamic properties of the molecules as compared with control cultures. An increase in the specific activity of proteoglycans labeled with [3H]glucosamine isolated from chlorate-treated cells was also observed suggesting a change in hexosamine metabolism induced by chlorate. BSP purified from the medium of chlorate-treated cells contained approximately 7% of the 35S incorporation as compared with nontreated control cultures. Quantification of sulfate incorporation into glycoconjugates versus tyrosine sulfate of BSP indicates that the amount of sulfate associated with N- and O-linked oligosaccharides was reduced by approximately 97%, while that on tyrosine residues was reduced by approximately 90%. Using normal human bone cells, the cell attachment activity of the reduced sulfate form of BSP was nearly equivalent to that of the fully sulfated product.
We present a new technique for the simultaneous measurement of cell volume changes and intracellular ionic activities in single cells. The technique uses measurement of changes in the concentration of intracellularly trapped fluorescent dyes to report relative cell volume. By using pH- or Ca(2+)-sensitive dyes and recording at the ion-sensitive and -insensitive (isosbestic) wavelengths, the method can measure both cell volume changes and intracellular ionic activities. The technique was used to study the mechanisms of regulatory volume decrease (RVD) in the osteosarcoma cell line UMR-106-01 grown on cover slips. Swelling cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered hypotonic medium was followed by stable cytosolic acidification and a decrease in cell volume back toward normal. The recovery of cell volume could be blocked by depolarization, treatment with ouabain, or depletion of cell Cl-. These suggest the conductive efflux of K+ and Cl- during RVD. The cytosolic acidification that accompanied cell swelling was not blocked by amiloride, bafilomycin A, or removal of Cl- and could not be reproduced by depletion of cellular ATP. These findings exclude Na+/H+ and Cl-/HCO-3 exchange, intracellularly generated acid, or increased metabolism, respectively, as the cause of the acidification. The cell swelling-induced acidification was inhibited by depolarization, suggesting the involvement of an electrogenic pathway. The acidification, as well as RVD, was inhibited by short incubation with deoxyglucose, and these effects could not be reversed by valinomycin. Thus, the anionic pathway(s) participating in RVD and the acidification are sensitive to the cellular level of ATP. Together, these studies indicate that RVD in UMR-106-01 cells in HEPES-buffered medium is mediated by the conductive efflux of K+, Cl-, and OH-.
We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer
treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine
if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive
temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2,
were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01’s effect on the Cdc2-inhibitory
kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated
cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however,
to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action,
UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01
abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed
in these mitotic regulators are downstream consequences of UCN-01’s actions.
An enzyme from Vibrio 01 catalyzing cis-trans isomerization of maleylacetone has been identified and purified. The enzyme has a molecular weight of about 35,000 and appears to be a single unit. Its density is low compared to other proteins. Glutathione is specifically required as a coenzyme. Treatment of the enzyme with sodium borohydride in the presence of substrate and GSH leads to increased activity while in the absence of substrate, treatment with sodium borohydride leads to inhibition. Formation of a Schiff base intermediate between substrate and enzyme appears to be unlikely. N-Ethylmaleimide leads to inactivation of enzyme and suggests the importance of one or more thiol groups near the active site. Other inhibitors and potential inhibitors have been tested. The enzyme also isomerized maleylacetoacetate at a slightly faster rate.
A checkpoint operating in the G2 phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently
undergoing clinical trials for cancer treatment, abrogates G2 checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G2 checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2.
This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1
and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on
serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of
both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited
the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1,
such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G2 checkpoint abrogation by UCN-01.
Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.
Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirty-seven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deep-sea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Site-directed mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.
7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor anticancer drug currently undergoing a phase II clinical trial. The low distribution volumes and systemic clearance of UCN-01 in human patients have been found to be caused in part by its extraordinarily high affinity binding to human alpha1-acid glycoprotein (hAGP). In the present study, we photolabeled hAGP with [3H]UCN-01 without further chemical modification. The photolabeling specificity of [3H]UCN-01 was confirmed by findings in which other hAGP binding ligands inhibited formation of covalent bonds between hAGP and [3H]UCN-01. The amino acid sequence of the photolabeled peptide was concluded to be SDVVYTDXK, corresponding to residues Ser-153 to Lys-161 of hAGP. No PTH derivatives were detected at the 8th cycle, which corresponded to the 160th Trp residue. This strongly implies that Trp-160 was photolabeled by [3H]UCN-01. Three recombinant hAGP mutants (W25A, W122A, and W160A) and wild-type recombinant hAGP were photolabeled by [3H]UCN-01. Only mutant W160A showed a marked decrease in the extent of photoincorporation. These results strongly suggest that Trp-160 plays a prominent role in the high affinity binding of [3H]UCN-01 to hAGP. A docking model of UCN-01 and hAGP around Trp-160 provided further details of the binding site topology.
Acetylpyruvate hydrolase, the terminal inducible enzyme of the pathway of orcinol catabolism in Pseudomonas putida, catalyzes the quantitative conversion of acetylpyruvate into acetate and pyruvate. The enzyme has been purified approximately 40-fold from extracts of Ps. putida grown on orcinol. Disc gel electrophoresis of the preparations show one major and one minor band of protein. The molecular weight of the enzyme is approximately 38,000 by sodium dodecyl sulfate electrophoresis. Acetylpyruvate is the only known substrate for the enzyme; maleylpyruvate, fumarylpyruvate, acetoacetate, oxalacetate, and acetylacetone are not hydrolyzed by acetylpyruvate hydrolase. Several divalent cations, includ-Mg2+, Mn2+, Co2+, Ca2+, and Zn2+, enhanced hydrolytic activity, but Cu2+ was inhibitory. The enzyme shows a sharp pH optimum at 7.4. Acetylpyruvate hydrolase has an apparent K-m of 0.1 mM for acetylpyruvate with a molecular activity of 36 min minus 1 at 25 degrees. Pyruvate, oxalacetate, and oxalate are competitive inhibitors of acetylpyruvate hydrolysis by the enzyme with K-i values of 6.0, 4.5, and 0.45 mM, respectively.
The rat osteosarcoma cell line (UMR 106-01) synthesizes and secretes relatively large amounts of a sulfated glycoprotein into its culture medium (approximately 240 ng/10(6) cells/day). This glycoprotein was purified, and amino-terminal sequence analysis identified it as bone sialoprotein (BSP). [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to label the BSP. Sulfate esters were found on N- and O-linked oligosaccharides and on tyrosine residues, with about half of the total tyrosines in the BSP being sulfated. The proportion of 35S activity in tyrosine-O-sulfate (approximately 70%) was greater than that in N-linked (approximately 20%) and O-linked (approximately 10%) oligosaccharides. From the deduced amino acid sequence for rat BSP (Oldberg, A., Franzén, A., and Heinegård, D. (1988) J. Biol. Chem. 263, 19430-19432), the results indicate that on average approximately 12 tyrosine residues, approximately 3 N-linked, and approximately 2 O-linked oligosaccharides are sulfated/molecule. The carboxyl-terminal quarter of the BSP probably contains most, if not all, of the sulfated tyrosine residues because this region of the polypeptide contains the necessary requirements for tyrosine sulfation. Oligosaccharide analyses indicated that for every N-linked oligosaccharide on the BSP, there are also approximately 2 hexa-, approximately 5 tetra-, and approximately 2 trisaccharides O-linked to serine and threonine residues. On average, the BSP synthesized by UMR 106-01 cells would contain a total of approximately 3 N-linked and approximately 25 of the above O-linked oligosaccharides. This large number of oligosaccharides is in agreement with the known carbohydrate content (approximately 50%) of the BSP.
The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.
Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway.
Cinnamycin is a unique toxin in that its receptor, phosphatidylethanolamine (PE), resides in the inner layer of the plasma
membrane. Little is known about how the toxin recognizes PE and causes cytotoxicity. We showed that cinnamycin induced transbilayer
phospholipid movement in target cells that leads to the exposure of inner leaflet PE to the toxin. Model membrane studies
revealed that cinnamycin induced transbilayer lipid movement in a PE concentration-dependent manner. Re-orientation of phospholipids
was accompanied by an increase in the incidence of β-sheet structure in cinnamycin. When the surface concentration of PE was
high, cinnamycin induced membrane re-organization such as membrane fusion and the alteration of membrane gross morphology.
These results suggest that cinnamycin promotes its own binding to the cell and causes toxicity by inducing transbilayer lipid
Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein alpha-subunit Galpha16, which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of Galpha16 inhibited cellular growth rates. A reporter gene system was established to assess the role of Galpha16 on erythroid differentiation of MB-02 erythroleukemia cells. It is based on transient transfection with a plasmid that expresses green fluorescent protein under the control of the beta-globin promoter. Expression of Galpha16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a Galpha16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of Galpha16, Galpha16R186C, further stimulated differentiation to 195% of control values. Because the effect of Galpha16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed Galpha16 with G protein betagamma-subunits can be excluded. The corresponding mutant of Galphaq (GalphaqR182C), the phylogenetically closest family member of Galpha16, had no effect. The data define a specific role for Galpha16-dependent signal transduction in cellular differentiation: deviations from optimal levels of Galpha16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.
The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.
A diacylglycerol (DG) kinase inhibitor, R 59 022, potentiated superoxide anion (O2-) production in guinea pig polymorphonuclear leukocytes (PMNL) induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP). R 59 022 also potentiated O2- production induced by 1-oleoyl-2-acetylglycerol, a permeable DG. However, the production induced by phorbol 12-myristate 13-acetate (PMA), a direct activator for protein kinase C, was not potentiated by R 59 022. R 59 022 by itself had no significant effects on unstimulated O2- production. The potentiation of FMLP-induced O2- production by R 59 022 was correlated closely with increased formation of DG and decreased formation of phosphatidic acid, a product of DG kinase. R 59 022 had no effect on the breakdown of phosphoinositides. Phosphorylation of 46-kDa protein(s) by protein kinase C was also examined in relation to O2- production in PMNL. In coincidence with the increase in O2- production, the phosphorylation was potentiated by R 59 022 in the response to FMLP, but not in the response to PMA. In addition, staurosporine, a protein kinase C inhibitor, inhibited increases in both O2- production and phosphorylation of the 46-kDa protein(s) after PMA stimulation. Similar inhibitory effects of staurosporine were also observed upon stimulation with FMLP, irrespective of the presence of R 59 022. These results indicate that retention of DG as a result of the inhibition of further metabolism induces marked stimulation of O2- production via protein kinase C activation in PMNL. These results also provide further evidence for the close relationship between 46-kDa protein phosphorylation by protein kinase C and stimulation of O2- production in PMNL.
R 59 022 (6-[2-[4-[(4-fluorophenyl) phenylmethylene)-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-alpha] pyrimidin-5-one) was found to inhibit diacylglycerol kinase in human red blood cell membranes at concentrations where polyphosphoinositide phosphodiesterase, phosphatidylinositol kinase, and phosphatidylinositol 4-phosphate kinase activity remained unaffected. The concentration needed for half-maximal inhibition (IC50) was 2.8 +/- 1.5 X 10(-6) M for the kinase acting on endogenous diacylglycerol and 3.3 +/- 0.4 X 10(-6) M when 1-oleoyl-2-acetylglycerol (OAG) was added exogenously as substrate. In intact platelets, R 59 022 inhibits the phosphorylation of OAG to 1-oleoyl-2-acetylglyceryl-3-phosphoric acid (OAPA) (IC50: 3.8 +/- 1.2 X 10(-6) M); concomitantly the stimulation of protein kinase C activity by OAG was amplified. When in platelets inositol lipid turnover is accelerated by thrombin, further addition of R 59 022 results in a marked elevation of diacylglycerol levels, a decreased formation of phosphatidic acid and an increased protein kinase C activity as compared with the controls. It is concluded that in studies on the signal-transducing system coupled to inositol lipid metabolism R 59 022 might occupy a role comparable to cyclic AMP phosphodiesterase inhibitors, since it potentiates the effect of the putative second messenger diacylglycerol by preventing its rapid metabolism.

domain: chemistry


text:
FOR IMMEDIATE RELEASE 01/23/2013
Rep.Howard Legislation Ensures Access to Economic Stabilization Fund for States Infrastructure Needs
AUSTIN - Today, State Representative Donna Howard filed House Bill 652, which ensures that the needed infrastructure funding touted by the state's leadership will not hit a technical snag. HB 652 clarifies the original intent of the Economic Stabilization Fund (ESF) and restates in statute that appropriations from the ESF are not counted against the state's spending cap.
"This bill will clarify the intent of the ESF; namely, the constitutionally created ESF is a 'special fund' dedicated toward offsetting revenue shortfalls and, therefore, does not fall within the definition of funds to be counted toward the spending limit, i.e. ‘state tax revenues not dedicated by the constitution,’" Howard said.
"This allows the legislature to actually appropriate monies from this fund as originally intended within the constitutional constraints imposed on such appropriations by voting thresholds specified in the constitution.”
The Texas Constitution requires that certain funds be moved from the General Revenue Fund into the Economic Stabilization Fund, which was constitutionally created in 1988. By restating in statute that the ESF is a "constitutionally dedicated fund," HB 652 would ensure that proposals such as those listed as priorities by Speaker Joe Straus and Lt. Governor David Dewhurst — including water and transportation infrastructure — are able to occur without restricting the appropriations of the General Revenue Fund.

domain: finance


text:
PHELAN, CALIFORNIA — A five-year-old was honored with a plaque by the San Bernardino County Fire Department for saving his grandmother from a fire.
On Saturday, Nathaniel Stocks woke up in his grandmother’s house coughing. There was smoke in the room and the space heater in his bedroom was on fire. But instead of panicking the five-year-old just crawled down the hallway and woke up his grandmother.
Together they got out of the house safely. Firefighters soon arrived at the scene and extinguished the fire in 15 minutes. No one was injured.
The family dog was rescued from the bathroom, where she went hiding.
According to KTLA, the boy put into practice what he had learnt from a school visit to County Fire Station 10 two days earlier.
“I’m really proud of him,” Nathaniel’s grandmother, Kimberly Ratliff, was quoted by The Sun as saying. “He did everything exactly how he learned it. He came in, saved me, came in and we got out with most of the animals. Firefighters saved the rest. But he’s very good. He told me what to do and how to do it. He told me to crawl and not go back into the house.”

domain: news


text:
John Bartelson may look like an average wheat farmer. He isn't. He's one of North Dakota's new oil barons. Every month, he gets a check...
John Bartelson may look like an average wheat farmer. He isn’t. He’s one of North Dakota’s new oil barons.
Every month, he gets a check for tens of thousands of dollars from a company in Houston called EOG Resources, which drilled two oil wells on his land last year. He says the day his first royalty check arrived was one to remember.
“I smiled to beat hell, and I went to town and had a beer,” Bartelson, 65, says. His new wealth springs from the Bakken formation, a sprawling deposit of high-quality crude beneath the durum-wheat fields of North Dakota, Montana and southern Saskatchewan and Manitoba. The Bakken may give the U.S. — the world’s biggest importer of oil — a new domestic energy source.
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Unlike the tar from Canada’s oil sands, Bakken crude needs little refining. Swirl some of it in a Mason jar and it leaves a thin, honey-colored film along the sides. It’s light — almost like gasoline — and sweet, meaning it’s low in sulfur.
Best of all, the Bakken could be huge. The U.S. Geological Survey (USGS) estimated that the Bakken might hold 413 billion barrels. If so, it would dwarf Saudi Arabia’s Ghawar, the world’s biggest field, which has produced about 55 billion barrels.
The challenge is getting the oil out. Bakken crude is locked 2 miles underground in a layer of dolomite, a dense mineral that doesn’t surrender oil the way more porous limestone does. The dolomite band is narrow, too, averaging just 22 feet in North Dakota.
The USGS said in April that the Bakken holds as much as 4.3 billion barrels that can be recovered using today’s engineering techniques. That’s a fraction of the oil estimated to be there, but it’s still the largest accumulation of crude in the 48 contiguous United States.
North Dakota, where Bakken exploration is most intense now, won’t become Saudi Arabia unless technology improves.
For decades, the Bakken was the fool’s gold of the oil industry. The name describes a geological formation that looks like an Oreo cookie: two layers of black shale that bleed oil into the middle layer of dolomite. It’s named after Henry O. Bakken, the North Dakota farmer who owned the land where the first drilling rig revealed the shale layers in the 1950s.
All of the layers are thin — about 150 feet altogether — and none of them gives up oil easily. In older, vertical wells, oil would often flow for a month and then fizzle.
Now, companies like EOG are drilling horizontally. They go straight down 10,000 feet and then put a slight angle so they hit the Bakken sideways, making a horizontal tunnel as much as 4,500 feet long through the dolomite.
It eventually winds up in a pipeline that runs east to Clearbrook, Minn., and then south to Chicago.
Several billionaires are at work in the Bakken. Harold Hamm’s Continental Resources has leases on 487,000 acres in Montana and North Dakota. Hamm, who started out driving a truck, owns 73 percent of Continental, worth $7.9 billion. Philip Anschutz, 68, founder of Qwest and Regal Entertainment, is there, too.
The big winner so far has been EOG, formerly a subsidiary of bankrupt energy trader Enron. It drilled a horizontal well in western North Dakota just north of Parshall — population 1,028 — in April 2006. The well came online a month later and kicked out 1,883 barrels in the first seven days. Unlike the older vertical wells, it’s still going.
Northern Oil & Gas, a five-person company near Minneapolis, makes money without actually drilling or operating wells. It leases in promising areas like the Bakken and gets paid when someone else uses the land to drill.
The other people doing well in the Bakken are the mineral owners under the oil wells — folks like John Bartelson. Oil drillers have paid them millions for right of access to the oil deposits.
Bartelson’s checks are about to get bigger. One more EOG well just came online, he says, and another is about to. Still another has been permitted for drilling. For now, he’s farming. The oil market is fickle, he says. Previous crashes drove the rigs out of North Dakota for years, leaving only the wheat.
“It’ll crash again,” Bartelson says, sipping on a late-afternoon cup of coffee beside his tractor.
Maybe so. But with crude trading above $135 a barrel, it may be a long time before the rigs leave again, and John Bartelson is likely to be a wealthy man before they do.


Previously Scheduled for September 4, 2020 at The UTEP Don Haskins Center. Original tickets will be honored. For more information, please contact the original point of purchase.
GRAMMY Award-winning, multi-platinum powerhouse artists Enrique Iglesias and Ricky Martin are hitting the road together for the first time ever. The global superstars have built unstoppable solo careers with dozens of #1 songs and millions of albums between them, and are now joining forces live in concert this fall. Fans can expect to hear renowned hits from two of the biggest names in pop and Latin music on their historic North American arena tour. Latin music’s newest sensation Sebastián Yatra will join the outing as special guest. The GRAMMY nominee has become one of the most powerful Latin pop artists of his generation.
Produced by Live Nation, the outing will kick off September 5th in Phoenix and make stops in Houston, Los Angeles, Toronto, New York, Miami, and more before wrapping October 30th in Atlanta. Tickets go on sale to the general public starting Thursday, March 12th at LiveNation.com at 10 a.m. local time.
Citi® / AAdvantage® is the official credit card for the presale of the U.S. tour. As such, Citi / AAdvantage cardmembers will have access to purchase presale tickets beginning Tuesday, March
10th at 10:00 a.m. local time until Wednesday March 11th at 10:00 p.m. local time through the Citi Entertainment® program. For complete presale details visit www.citientertainment.com.
Both artists will continue taking care of longstanding fans with their own exclusive fan presales making sure hardcore followers have access to amazing seats as well.
A limited number of LaneOne Premium Packages will also be available, including amazing seats, transportation, preferred entrance, preshow hospitality and more.
Enrique and Ricky have created some of pop music’s most memorable songs for decades and their combined list of chart-toppers is seemingly endless. Enrique gained devoted fans around the world with songs including “Hero,” “Escape,” “Bailando,” “Tonight,” and more recent hits “Subeme La Radio” and “Duele El Corazon” with a new album to be released later this year. Ricky’s solo career exploded with “The Cup of Life" and “Livin’ La Vida Loca,” followed by “She Bangs,” “Vente Pa’ Ca,” and his most recent “Tiburones” from his upcoming 2020 studio album. The two will perform a worldclass setlist of their top hits when the tour kicks off this September.
Wednesday, November 10, 2021 at 7:30 AM
Don Haskins Center
2801 N Mesa, El Paso, TX 79968
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Thousands of homeowners up and down the country are locking into super-cheap home loans by remortgaging, new figures have revealed. Figures from UK Finance have revealed that there were 35,600 remortgages in September, around 6,000 more cases than the number of new first-time buyer deals and the number of new homemover deals. In total those remortgage deals were worth a whopping £6.4 billion. It’s a smart move too, with remortgage deals continuing to sit at historically low levels, though with further base rate rises expected they may not be around for long. Here's our guide on how to switch - for more information, see our page on how to save money on your mortgage or if you're buying a house, here's how to make sure your mortgage application is accepted and the best new mortgage deals. If you're buying a house, you may also want to read about getting a mortgage in principle. 1. The pain of the SVR When you take out a mortgage, generally you will initially benefit from a … [Read more...] about How to remortgage: Thousands slashing their mortgage bills and you can too
Best remortgage deals uk
0 Have your say For those have followed every twist and turn of the decade-long equal pay battle in Glasgow, it was common knowledge that any deal would end up costing the city fathers more than a song. Now in what is one of Scotland’s biggest ever remortgaging drives, the country’s largest local authority has agreed to sell off some of its best known music, entertainment and sporting venues to foot the half a billion pound bill. Glasgow City Council yesterday approved plans to finance a £548 million equal pay claim from thousands of women stretching back more than a decade. The council intends to sell off prized assets such as Glasgow Royal Concert Hall and the SEC Armadillo to a wholly owned arms-length company before leasing them back at commercial rates.The firm, City Property Glasgow Investments LLP, is engaging lenders with the aim of refinancing a loan originally taken in 2010.After the funding deal was approved by the council’s … [Read more...] about Glasgow Council to sell key assets before leasing them back in £500m remortgaging deal
But now the FCA wants to change rules to allow lenders to relax checks for mortgage prisoners if they pass a number of conditions, based on whether they are with an active or unactive lender. There are 120,000 customers with firms that are unregulated and a further 20,000 mortgage customers with firms that are no longer lending. For the further 10,000 who are with active lenders the FCA has already agreed a deal which means they can move to a better rate with their existing lender as long as they are up to date with payments, have two years left on their mortgage and up to £10,000 to pay off. If the FCA goes ahead with the rule change for mortgage prisoners it means the remaining 140,000 would be able to apply for a new home loan under an easier affordability test. This test would look at whether their new mortgage costs are more affordable than their current one. In theory it would allow people to move to a cheaper mortgage, although it may not apply to all the 140,000 … [Read more...] about Up to 140,000 mortgage prisoners to get better home loan deal under rule change
Brexit is battering the UK property market, pushing it to its weakest level in more than six years, with almost half of surveyors reporting that sellers and buyers are sitting tight because of political uncertainty. The Royal Institution of Chartered Surveyors (Rics) said its monthly indicators for demand, supply and prices fell to multiyear lows in November. The number of people looking for a new home fell again, with many surveyors attributing this to Brexit uncertainty. The net balance of -21%, down from -15% in October, was the lowest since September 2017. Weaker demand dragged down property prices. The price balance slipped to -11% in November from -10% in October, marking the lowest reading since September 2012. House prices have been falling in London, the south-east and East Anglia, while in the south-west, east Midlands and north-east they have been broadly flat. There were price rises in Northern Ireland, Scotland, West Midlands, Wales, Yorkshire and Humber and the … [Read more...] about UK property market at weakest since 2012 as Brexit takes toll – Rics
THE average two-year fixed mortgage rate has fallen for the first time in a year to 2.49 per cent. This is the first times average rates have dropped since their record low of 2.20 per cent in October 2017. It means now could be the time to remortgage, to lock in low rates. This is especially true for anyone sitting on a standard variable rate mortgage - where the average interest rate has reached 4.89 per cent. Charlotte Nelson, finance expert at comparison service Moneyfacts, said: "[This is] the first time the Moneyfacts UK Mortgage Trends Treasury Report has recorded a reduction to the average mortgage rate since the November 2017 base rate rise. "Many would have assumed that the average rate would have increased in the aftermath of the base rate rise this August, however the opposite seems to be the case. "Providers have started to reignite competition in the market to attract remortgage customers and retain their mortgage books." Homeowners may wish to move fast, as … [Read more...] about Two-year mortgage deals fall for the first time in a year – is now the time to fix your mortgage?


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Prosecutors want to review Michael Jackson ‘s financial records, saying they believe serious money troubles drove the entertainer to force his accuser’s family to help him rebut a damaging television documentary.
Assistant District Attorney Gordon Auchincloss requested copies of the documents Friday at a hearing that included several rulings in the singer’s child molestation case, including that “Tonight Show” host Jay Leno may joke about Jackson despite a gag order on prospective witnesses.
Law enforcement officers hug at the Fulton County Courthouse after a judge, a court recorder and a deputy were fatally shot there. (John Bazemore — AP)
Auchincloss told Judge Rodney S. Melville the state only wanted “a concise snapshot” of Jackson’s finances to show the entertainer is “on the precipice of bankruptcy.”
Melville said he would allow only minimal evidence on Jackson’s financial condition. He asked both sides lawyers to confer and reach an agreement by Thursday on what financial records would be released.
“I don’t want to inundate the jury with a lot of figures that don’t tell a story,” the judge said. “I don’t think you can turn the jury into accountants.”
Jackson lawyer Robert Sanger denied Auchincloss’ assertion and said the state’s focus on the singer’s finances today has no relevance to his finances in 2003, the year at issue in the trial.
Despite Sanger’s denial, Auchincloss called Jackson “a spend-a-holic” who has “an insatiable appetite for money.” From 1999 to 2001, he said, Jackson was spending $35 million a year while earning only $11 million to $12 million.
“He is in debt to the tune of $300 million and has liabilities close to $400 million,” the prosecutor said.

domain: law


text:
Median age of the patients was 55 years. The follow-up was complete, with echocardiographic studies and a median period of 32 months.
Results: There were 3 operative and no late deaths. One patient required early reoperation for dehiscence of a patch used to reconstruct the posterior mitral valve annulus. Twenty-four patients were alive at the last follow-up and had a normally
functioning aortic valve prosthesis and no false aneurysms.
Conclusion: Intraoperatively tailored tubular Dacron graft for concomitant reconstruction of the left ventricular SYN-117 research buy outflow tract and replacement of the aortic root is a useful and safe operative technique for patients with destroyed aortoventricular junction.”
“THE SURGICAL MANAGEMENT of hydrocephalus has undergone incredible changes over the past generation of neurosurgeons, including dramatic improvements in imaging, especially computed tomographic scanning and magnetic resonance imaging, and remarkably innovative advances in cerebrospinal fluid valve technology, complex computer models, and endoscopic equipment and techniques. In terms of overall patient outcomes, however, one could conclude that things are a little better, but “”not much.”" This frustrating yet fascinating dichotomy between technological advancements and clinical outcomes makes hydrocephalus, first described by the ancients, as one of the most understated and
see more complex disorders that neurosurgeons treat. The challenge to the next generation of neurosurgeons is to solve this vexing
problem through better understanding of the basic science, improved computer models, additional technological advances, and, most however importantly, a broad-based, concerted multidisciplinary attack on this disorder. This review focuses on the evolution of surgery for hydrocephalus over the last 30 years, the current state of the art of hydrocephalus treatment, and what appear to be the most promising future directions.”
“Objective: The present study examined temporal and spatial patterns of extracellular matrix and smooth muscle cell changes in the ascending aorta with bicuspid and tricuspid aortic valve stenosis.
Methods: Wall specimens were retrieved from both the greater and the lesser curvature (“”convexity”" and “”concavity”") of 14 nonaneurysmal and 12 aneurysmal aortas (aortic ratios 1.2 and 1.5, respectively) and from 3 heart donors (normal). Immunochemistry was performed for detection of apoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling [TUNEL]-positive) and proliferating (Ki-67-positive) smooth muscle cells and for semiquantification of matrix proteins (collagens, fibronectin, tenascin, laminin). Co-immunoprecipitation assessed the extent of Bcl-2-modifying factor binding to Bcl-2, indicating a matrix-derived cytoskeleton-mediated proapoptotic signaling. Polymerase chain reaction allowed for quantification of messenger RNA expression for Bcl-2.

domain: medical


New Orleans gets invitation to bid for 2019, 2020 Super Bowls
City officials and the New Orleans Saints are getting another chance to bring the Super Bowl back to the Big Easy.
News of the announcement comes just one year after the city of New Orleans was denied an opportunity to host the Super Bowl in 2018.
Despite the heartbreak of that decision, Tuesday's announcement is another chance for city officials and the Black and Gold to bring the world's biggest sporting event back to the Crescent City.
New Orleans will be competing with Atlanta, Tampa and Miami to host the Super Bowl in 2019 and 2020. The three cities are considered traditional Super Bowl hosts.
But one person close to the situation told WDSU's Fletcher Mackel, "We think our odds are good if they're inviting us back to the table for a presentation."
New Orleans last hosted the Super Bowl in February 2013. The city has hosted the game a record 10 times overall.
Currently, Atlanta is building a brand new billion-dollar stadium that's partially being funded by the public, which leads many to believe the city will get one of the two games.
Saints owner Tom Benson issued a statement Tuesday evening:
"New Orleans is a Super Bowl favorite city and has been for many years. We are excited to be once again invited and I am confident that our city leaders and the hospitality community will come together and put forth our best bid."


Saturday Night Stereolab – A Spectacle That Rhymes
Stereolab are an alternative music band formed in 1990 in London, England. The band originally comprised songwriting team Tim Gane (guitar/keyboards) and Lætitia Sadier (vocals/keyboards/guitar), both of whom remained at the helm across many lineup changes blah blah read the rest here.
They’re known for experimental krautrock pop lounge music in space, or something. Space definitely figures into their work, what with songs like “Super Falling Star,” “K-Stars,” “Space Moment,” “Sudden Stars,” and “Space Age Bachelor Pad Music” (which, I suppose, is probably as good a description of their music as will ever be).
It’s been said that Stereolab lyrics are often Marxist or socialist, which the band denies. Half the lyrics are in French anyway, so it’s not like I would even know it if they starting singing socialist economic theory in mid-song. the song most notorious for alleged politicalisms is “Ping Pong,” a rather catchy tune –
Original studio version and video here.
Regardless of lyrical messaging, the band has always pushed envelopes out where the Post Office won’t deliver them. Such as “Metronomic Underground,” which I suspect most of you won’t care for but I rather like –
Studio version here.
Singer Laetita Sadier lent her voice to a rather mediocre song by Common for his song “New Wave” in 2002. Her vocals sounded great, but were unfortunately only relegated to the choruses. and yes, she does have a predilection for swearing
My favorite Stereolab song is Miss Modular, heard here live in 2000 –
And the studio version, to close things out with a nicely rhyming spectacle –
Posted on April 2, 2016, in ♫ ♪ ♫, Fun Stuff and tagged 90s, fun stuff, music, radio, Saturday Night Studio. Bookmark the permalink. Leave a comment.
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Blake Snow, Science, FoxNews.com (December 17, 2010)
"Like exploding stars, black holes, dark matter? How about cosmic intrigue, deep space astronomy, or origins of the universe?
Then you're gonna love this.
Researchers at the University of Wisconsin are putting the finishing touches on a giant underground telescope buried beneath the South Pole to help understand said phenomena. Accordingly called the IceCube Neutrino Observatory, completion is expected to occur at 8 p.m. CST, once the last of more than 5,000 optical sensors is buried as much as two miles below the permanent ice cap covering Antarctica...."
Instead of detecting photons, like telescopes we use to observe light, infrared, radio, ultraviolet, and other electromagnetic radiation, this telescope detects neutrinos.
The article's style is breezy, which in the Lemming's opinion helps make a fairly technical subject: fun to read about.
As for why bury the IceCube Neutrino Observatory? Neutrinos pass through just about everything: and are easier to detect when just about everything else is stopped by up to two miles of ice.
- "IceCube Neutrino Observatory: Again"
(December 28, 2010)


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March 22, 2023
The conversation has not stopped, and it will continue. Here I do not mean to suggest any futility; these interventions helped many avoid error and come to see how faith can be intelligible. New discoveries will continue to create new urgencies to think anew about this relationship.
March 8, 2023
No charism is a one and done process, but like the reform of the church, a charism is an ongoing process of listening attentively and lovingly to the Holy Spirit who is, as John of the Cross says, the principal guide of those committed to prayer and to the apostolate.
February 22, 2023
As immigrants, the sisters were not familiar with American culture and history, but that lack of understanding also meant that they were not hindered by baggage associated with the Civil War and negative attitudes about black people.
February 8, 2023
The Eucharistic celebration contains the solution to the violence of technocratic modernity and grounds the many calls in the encyclical to care for our common home in all its facets, from the unborn to non-human creation.
January 25, 2023
All Jesuit colleges had to survive, and the population of their local communities helped to shape their decisions. Western schools like Seattle University were pioneers in admitting women partly because they were located in smaller, less developed cities where the male population was limited.
January 12, 2023
We aspire for an education that seeks to occasion transformations so structural, profound, intimate, and thorough that learners cannot tell what they have learned because it has become one with them.
December 14, 2022
One of the characteristics of the volume is that the contributions represent a wide range of methodologies according to the field of learning of each contributor.
November 30, 2022
There are plenty of topics and questions that are appealing, but we’re particularly interested to see work that explores race, gender, emerging sports, individualized sport, global perspectives, and even transnational comparisons.
October 19, 2022
Perhaps it is obvious, but I believe that we need to know each other’s histories. American Catholics can be pretty tribal—within neighborhoods, parishes, and ethnic communities. But I think that we can benefit from the experiences of others, no matter our backgrounds.
October 6, 2022
The women writers of the Catholic Literary Revival were in their own time well-known and well-read, with no shortage of best-selling authors among their ranks. Most predated and greatly influenced Waugh and Greene. They wrote from a more diverse range of social and political positions than Waugh and Greene, and were often more radical in their use of ninetheenth- and twenthieth-century literary innovations.

domain: library


text:
I have the latest version of NX2. I downloaded the extra picture control for my D700 (Dx1,DX2,DX3,portrait,landscape) and it appears in NX2 when I download my NEF files. So I was happy when I took raw images with my D90, the same picture controls were available when I opened them in NX2 (DX1-3, Portait, landscape). However now that I shoot raw images with the D7000, the picture controls mentioned above do not show up. Is there a reason why it should not show up?
The D90 is not supported either but for some reason whenever I dowloaded my NEF's, I had the option for the other picture controls. No biggie, it was just nice to have it with the D90. The D7000 colors look good right off the camera.

domain: hobby


text:
CD8 Lineage Commitment in the Absence of CD8
Article, Other literature type
Goldrath, Ananda W
Hogquist, Kristin A
Bevan, Michael J
- Publisher: Elsevier BV
Infectious Diseases | Immunology and Allergy | Immunology | Article
The absence of cytotoxic T lymphocyte activity and the failure of MHC class I–restricted T cell receptor (TCR) transgenic thymocytes to mature in CD8α-deficient mice suggest that CD8 may be essential for CD8 lineage commitment. We report that variants of the antigenic peptide that delete TCR transgenic thymocytes from CD8 wild-type but not CD8α-deficient mice can restore positive selection of CD8 lineage cells in the absence of CD8. The positively selected cells down-regulate CD4, up-regulate TCR, respond to the antigenic peptide, and express CD8β mRNA. Interestingly, there was no enhanced selection of CD4+ T cells, implying that the TCR–MHC interaction, even in the absence of CD8, provided instructive signaling for commitment to the CD8 lineage. Our results are discussed in terms of recent models of T cell lineage commitment.

domain: literature