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We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF) rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ), a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL) rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.
Citation: Chen W, Goff MR, Kuang H, Chen G (2015) Higher Protein Kinase C ζ in Fatty Rat Liver and Its Effect on Insulin Actions in Primary Hepatocytes. PLoS ONE 10(3): e0121890. https://doi.org/10.1371/journal.pone.0121890
Academic Editor: Jonathan Peterson, East Tennessee State University, UNITED STATES
Received: October 20, 2014; Accepted: February 4, 2015; Published: March 30, 2015
Copyright: © 2015 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was financially supported by research grant from Allen Foundation Inc (to GC), start-up fund from the University of Tennessee at Knoxville (to GC), and Scientist Development Grant from American Heart Association (09SDG2140003, to GC). WC received financial support from the China Scholarship Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The normal physiological responses to insulin stimulation in the liver include the increase of glycolysis and lipogenesis, and the reduction of gluconeogenesis [1,2]. In hepatic parenchymal cells, insulin initiates a signaling cascade upon binding to its receptor on the cell membrane, which is followed by the activation of insulin receptor substrates (IRSs) . Tyrosine phosphorylation of IRS1/2 leads to the association and activation of phosphatidylinositol 3-kinase (PI3K). PI3K catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-3,4,5-triphosphate. The latter is a lipid messenger that anchors the downstream effector proteins, e.g. phosphoinositide dependent protein kinase-1, AKT (also known as protein kinase B) and atypical protein kinase C (aPKC), to the cell membrane . For its maximal activation, AKT is phosphorylated by 3-phosphoinositide-dependent kinase 1 (PDK1) at Thr308, and by mammalian target of rapamycin complex 2 (mTORC2) at Ser473 . Replacing either one of them with alanine residue will significantly impair the insulin-induced AKT activity . The activations of AKT and aPKC differentially regulate the transcription of hepatic genes for glucose and lipid metabolism [6–8]. For example, the activation of AKT induces the expression of glucokinase (GCK, gene Gck) to promote glycolysis, and suppresses the expression of cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK, gene Pck1) to reduce gluconeogenesis . On the other hand, the insulin-induced expression of sterol-responsive element binding protein 1c (SREBP-1c, gene Srebp-1c) has been attributed to the activations of AKT [10,11] and aPKC [12,13].
Members of PKC family include classical PKC, novel PKC, and aPKC. Unlike the other two types of PKC, aPKC does not require diacylglycerol or Ca2+ for activation. PKCζ and PKCι/λ (genes Prkcz and Prkci, respectively) are two current members of the aPKC subfamily. PKCζ shares 70% homology in amino acid sequence with PKCι/λ (PKCι in human; PKCλ in mouse) . Both PKCζ and PKCι/λ mediate a variety of physiological events, such as cell polarity establishment, cell motility, immune response and metabolism .
Activation of PKCζ or PKCι/λ has been associated with increased lipogenesis and insulin resistance in multiple tissues and organs [6,14]. The aPKC activation seems to regulate the Srebp-1c transcription and lipogenesis in the mouse liver. For example, in liver-specific PKCλ knockout mice, the basal transcript level of Srebp-1c, but not Gck or Pck1, declined by 50% . In mice with the liver-specific deletion of the regulatory subunit of PI3K, PKCζ/λ activity decreased in association with the reduction of Srebp-1c expression . On the other hand, the insulin-induced activation of aPKC and subsequent Srebp-1c expression were retained in the liver of diabetic mice [15,16]. PKCζ/λ is also activated in the liver of mice fed a high fat diet, which is accompanied by the elevated expression of hepatic Srebp-1c . This effect could be negated by the overexpression of kinase-inactive form of PKCζ . All these observations suggest the role of aPKCs in the regulation of hepatic lipogenesis.
In hepatocytes isolated from individuals with type 2 diabetes, the basal and insulin-induced activation of PKCι were elevated [17,18]. Treatment of these hepatocytes with PKCι inhibitors lowered the expression levels of lipogenic, proinflammatory and gluconeogenic enzymes [17,18]. Moreover, metformin treatment was shown to induce aPKC activities, and increase lipogenic and gluconeogenic enzyme levels in hepatocytes from non-obese human subjects . These data collectively suggest that aPKC isoforms are important players in the pathogenesis of obesity and type 2 diabetes. However, whether aPKC plays a role in the insulin-regulated gene expression in hepatocytes is still unclear.
Zucker fatty (ZF) rat is a genetic model of obesity, hypertriglyceridemia and hepatic insulin resistance . We previously showed that primary hepatocytes from ZF rats fed chow ad libitum exhibited impairment of the insulin-regulated gene expression . Interestingly, the insulin-induced phosphorylation of AKT at Ser473 and Thr308 in primary hepatocytes of ZF rats was not significantly different from that of Zucker lean (ZL) rats . Therefore, we systemically compared the activation states of other components of insulin signal transduction pathway between ZL and ZF hepatocytes. Here, we report the differential expression levels of PKCζ in the liver of ZF and ZL rats, and the effects of PKCζ and PKCι/λ overexpression on the insulin-regulated gene expression in primary rat hepatocytes. We demonstrated that overexpression of PKCζ or PKCι/λ in ZL primary hepatocytes impaired the insulin-regulated gene expression.
Materials and Methods
The reagents for primary hepatocyte isolation and culture including Medium 199, Dulbecco’s Modification of Eagle Medium (DMEM), liver perfusion buffer and liver digest buffer were obtained from Invitrogen (Carlsbad, CA). For RNA extraction, RNA STAT-60 was purchased from TEL-TEST (Friendswood, TX). The reagents for cDNA synthesis and real-time PCR were obtained from Applied Biosystems (Foster city, CA). All real-time PCR primer sets used in this study were synthesized by Sigma-Aldrich (St. Louis, MO). Antibodies against β-actin (#4967), PKCζ (#9368), PKCι (#2998), phospho- PKCζ/λ (p-PKCζ/λ) Thr410/403 (#9378), AKT (#9272), phospho-AKT (p-AKT) Ser473 (#9271), p-AKT Thr308 (#9275), p-AKT Thr450 (#9267), insulin receptor substrate 1 (IRS1, #2382), fatty acid synthase (FAS, #3189), acetyl CoA carboxylase (ACC, #3662), phospho-ACC (p-ACC) Ser79 (#3661) used in this study were purchased from Cell Signaling Technology (Danvers, MA). All other reagents and materials were purchased from Fisher Scientific (Pittsburgh, PA) unless described otherwise.
Animals and diets
Zucker rats were bred and housed under constant temperature and humidity in the animal facility on a 12-hour light-dark cycle. Male ZL (fa/+ or +/+) or ZF (fa/fa) rats at weaning (3 weeks old) were kept on Teklad rodent chow ad libitum (#8640, Harlan Laboratories, Indianapolis, IN) for 8 weeks before liver tissue collection and primary hepatocyte isolation. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Tennessee at Knoxville (Protocols #1256, 1642 and 1863).
Liver tissue collection, total protein preparation and total RNA extraction
The rat was euthanized by primary carbon dioxide asphyxiation, and then secondary cervical dislocation according to regulations. The procedure for liver tissue collection was reported elsewhere [21,22]. In brief, a 10ml syringe with a 21G × 1½" hypodermic needle was used to drain blood from the liver via the inferior vena cava. The liver was excised, sliced, then snap-frozen in liquid nitrogen, and store at -80°C before further analysis. A small portion of the liver tissue was homogenized in 10 volumes of cold whole-cell lysis buffer (1% Triton X-100, 10% glycerol, 1% IGEPAL CA-630, 50mM Hepes, protease inhibitors, pH 8.0), and then centrifuged to remove insoluble matters [21,22]. The protein concentration of the supernatant was determined with PIERCE BCA protein assay kit. Another small portion of the liver tissue was homogenized in 10 volumes of cold STAT-60. Total RNA was extracted according to the manufacturer instructions.
Cultures of primary hepatocytes and 293 HEK cells
The primary hepatocytes were isolated according to published methods . Isolated hepatocytes were seeded on 60-mm collagen type I coated dishes at 2×106 cells per dish and incubated in DMEM (4.5g/L glucose, 8% FBS, 1% penicillin/streptomycin) for 3 hours to allow cell attachment. The attached primary hepatocytes were washed once with PBS and pretreated in medium A (Medium 199 with 100nM dexamethasone, 100nM 3,3’,5-triiodo-L-thyronine, 1nM insulin, and 1% penicillin/streptomycin) for 16–18 hours. HEK 293 cells were seeded and kept in DMEM containing 4.5g/L glucose, 4% FBS, 1% penicillin/streptomycin .
Cloning of the rat Prkci cDNA and subcloning of the rat Prkcz cDNA
Based upon the rat Prkci mRNA sequence (GenBank: EU517502.1), sense 5’-ATC CCC TCA GCC TCC AGC GG-3’ and antisense 5’-ACT GTG ACC GGG CTA ACG GT-3’ primers were designed using Primer-BLAST tools from the National Center Biotechnology Information. For the complete coding sequence of rat Prkci cDNA, PCR was carried out using cDNA derived from total RNA of ZL primary hepatocyte as the template. For subcloning of rat Prkcz, pEYFP-N1 vector containing its complete coding sequence (generous gift from Dr. Ralf Kubitz) was used as the template for the PCR amplification of Prkcz with sense primer 5’-ACC TCG AGA TGC CCA GCA GGA CCG AC-3’ and antisense primer 5’- GTG AAT TCA CAC GGA CTC CTC AGC AGA C-3’. The amplicons containing the complete coding sequences of Prkci and Prkcz cDNA sequences were ligated into pCR2.1 vector through TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol.
Generation of Ad-Prkcz and Ad-Prkci recombinant adenoviruses
The inserts containing the complete coding sequences of Prkcz and Prkci were subcloned into pACCMV5 vector to make pACCMV5-Prkcz and pACCMV5-Prkci, respectively. To generate Ad-Prkcz and Ad-Prkci recombinant adenoviruses, pACCMV5 plasmid containing the complete coding sequence of atypical Prkc sequences was co-transfected with JM17 into HEK293 cells using FuGENE®6 Transfection Reagent (Roche) according to manufacturer instructions. After the formation of the viral plaques, the crude lysate was collected and stored at -80°C. The amplification and purification of adenovirus were carried out according to published protocols . The optical density (OD) at 260nm of the adenoviral suspension was determined to estimate the plaque forming units (pfu) of the purified recombinant adenoviruses. We used that 1 OD equals to 1 × 1012 pfu/ml. The purified recombinant adenoviruses were stored at -80°C until being used.
Infection of recombinant adenoviruses and treatments of primary hepatocytes
In experiments using recombinant adenoviruses, purified Ad-Prkcz and Ad-Prkci were added in the medium A (5,000 pfu/cell) to allow the overexpression of PKCζ and PKCι/λ in the primary rat hepatocytes during the pretreatment period, respectively. The pretreated hepatocytes were then washed once with PBS, and treated with medium A containing indicated concentrations of insulin (0nM to 100nM) for 6 hours before total RNA extraction, or for 15 minutes before whole cell lysate preparation.
RNA extraction and real-time PCR analysis
The methods for total RNA extraction and cDNA synthesis were described elsewhere . The gene expression level was determined by real-time PCR with respective primer sets, and normalized to the mRNA level of ribosomal gene 36B4. The nucleotide sequences of primer sets were as follows: Gck, 5′-CGA GAT GCT ATC AAG AGG AGA G-3′ (forward), 5′-TCA CA T TGG CGG TCT TCA TAG-3′ (reverse); Pck1, 5′-AGT CAC CAT CAC TTC CTG GAA GA-3′ (forward), 5′-GGT GCA GAA TCG CGA GTT G-3′ (reverse); Srebp-1c, 5′-GGA GCC ATG GAT TGC ACA TT-3′ (forward), 5′-AGG CCA GGG AAG TCA CTG TCT-3′ (reverse); Pklr, 5′-CGT TTG TGC CAC ACA GAT GCT-3′ (forward), 5′-CA T TGG CCA CAT CGC TTG TCT-3′ (reverse); G6pc, 5′-GAC CTC AGG AAC GCC TTC TAT G-3′ (forward), 5′-ATT GAT GCC CAC AGT CTC TTG A-3′ (reverse); Igfbp-1, 5′-GAT CAC TGA CCT CAA GAA ATG GAA-3′ (forward), 5′-GCG GCA CGT TAA TCT CTC TAA CA-3′ (reverse). The data were presented as either minus Δ cycle threshold (Ct) or the induction fold (ΔΔCt) for which the control treatment group was arbitrarily set as 1.
Immunoblot analysis of proteins
Protein samples (40μg) of the whole cell lysates of primary hepatocytes or liver tissues were resolved in 8% SDS polyacrylamide gel, and then transferred to BIO-RAD Immuno-Blot PVDF membrane (Hercules, CA) as described . Membranes were blocked by 8% non-fat milk, and then probed with specific antibodies (1:1 000 dilution). After gentle wash, membranes were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5,000 dilution). After washing, antigen-bound antibody was detected using ECL Western Blotting Substrate (Thermo Scientific), and subsequently exposure to X-ray films. The films were scanned, and the images were stored for the densitometry analysis using ImageJ software (NIH). Densitometry data for each protein were normalized to β-actin levels in each sample.
Statistical analyses were performed using SPSS 19.0 software. Student t-test was used to compare the means between two groups. One-way ANOVA with LSD post-hoc test was used to compare the means among three or more groups. Data were presented as means ± S.E.M. The number of experiments indicates hepatocyte isolations from different animals. A p value less than 0.05 is considered statistically significant.
PKCζ expression is elevated in the liver of ZF rats
Fig. 1A shows that, despite the well-reported hepatic insulin resistance, the levels of phospho-AKT at Ser473 and Thr450 in the liver of ZF rats were higher than that of ZL rats (please see densitometry analysis in S1 Fig.). The phospho-AKT at Thr308 in the liver tissues could not be detected in the current experimental condition, probably due to non-maximal activation of AKT in ad libitum state, relative less AKT protein amount from hepatocytes in the liver tissue lysate or rapid de-phosphorylation of AKT at Thr308 during the process of euthanasia of the animals. This observation shows that the AKT phosphorylation at Ser473 and Thr450 in ZF rat liver tissue is unimpaired, suggesting the consequence of hyperinsulinemia. In addition, ZF rat liver had higher protein level of FAS, indicating elevation of hepatic lipogenesis.
(A) Immunoblot analysis of the expression levels of the perspective proteins in the liver of ZL (n = 4) and ZF rats (n = 4). (B) The mRNA levels of Prkcz and Prcki in the liver of ZL and ZF rats. ZL and ZF rats at weaning (3 weeks old) were fed chow diet for 8 weeks. Liver samples of rats fed in ad libitum were collected and subjected to analysis of protein and mRNA levels as described in the Materials and Methods. The data were presented mean ± SEM of -ΔCt with animal numbers in parenthesis (* indicates p<0.05 for comparing ZL and ZF using Student’s t-test).
On the other hand, the protein level of PKCζ, but not PKCι/λ, in the liver of ZF rats was higher than that of ZL rats (Fig. 1A). The level of phospho-PKCζ/λ at Thr410/403, which indicated the phosphorylation of both PKCζ and PKC ι/λ, was higher in the liver samples of ZF rats than that of ZL rats (Fig. 1A). The mRNA level of Prkcz, but not that of Prkci, was also elevated in the ZF liver (Fig. 1B). These data collectively demonstrated that the aPKC expression levels changes with the development of hepatic insulin resistance in the ZF liver.
Short-term insulin treatment did not alter protein levels of total and phospho-aPKCs in primary rat hepatocytes
Fig. 2 shows that protein levels of FAS, ACC and phospho-ACC at Ser79 in hepatocytes of ZF rats were higher than that of ZL rats. This result demonstrates that the hepatic lipogenic capability remains elevated in ZF hepatocytes cultured for 20 hours. Insulin treatment dose-dependently induced the levels of phospho-AKT at Ser473 and Thr308 in ZL and ZF primary hepatocytes similarly (Fig. 2). The level of phospho-AKT at Thr450, which was insulin-independent, was slightly higher in ZF hepatocytes than in ZL hepatocytes (Fig. 2). These data demonstrated that the insulin-induced AKT phosphorylation is comparable in both ZL and ZF rat hepatocytes. The impaired insulin-regulated gene expression in ZF hepatocytes may be caused by changes in other components of insulin signaling pathway. On the other hand, the 15-minute insulin treatment did not significantly induce or suppress the protein levels of PKCζ and PKCι/λ and phospho-PKCζ/λ at Thr410/403 in ZL and ZF primary hepatocytes (Fig. 2). There was a slight induction of the total PKCζ when fresh M199 was added. ZF primary hepatocytes had higher level of PKCζ than ZL primary hepatocytes (Fig. 2), which was in line with the finding from the liver tissues (Fig. 1).
The primary hepatocytes from ZL and ZF rats (chow diet, 8 weeks) were pre-treated as described in the Materials and Methods for overnight, and then incubated in fresh medium A with increasing concentrations of insulin (0nM to 100nM) for 15 min. The hepatocyte group remained in pre-treatment medium was indicated by “C”. Immunoblot analysis with antibodies against the indicated proteins was performed. The figure shows the representative result of three parallel experiments obtained from three sets of primary hepatocytes independently isolated from ZL and ZF rats fed chow diet ad libitum.
Overexpressions of PKCζ and PKCι/λ in ZL primary hepatocytes
To investigate whether elevated aPKC protein levels contribute to the hepatic insulin resistance at gene expression level, we constructed recombinant adenoviruses Ad-Prkcz and Ad-Prkci for their overexpressions in ZL primary rat hepatocytes. The expression level of Pckcz or Prkci mRNA was normalized to that of ribosomal gene 36B4, which was stable across all treatment groups (S2 Fig.). In hepatocytes transfected with Ad-β-gal, the relative abundance of Prkcz mRNA (-ΔCt of -7.90) was much lower than that of Prkci mRNA (-ΔCt of -4.20). The overexpression fold of Pckcz or Prkci was calculated using their levels in the Ad-β-gal group as 1. Ad-Prkcz and Ad-Prkci respectively caused overexpressions of Prkcz mRNA (-ΔCt of 3.13, ~2 000 fold, Fig. 3A) and Prkci mRNA (-ΔCt of 3.78, ~250 fold, Fig. 3B, *).
Primary hepatocytes of ZL rats fed chow for 8 weeks were isolated and seeded onto dishes as described in the Materials and Methods. Purified Ad-β-gal, Ad-Prkcz and Ad-Prkci were added in medium A during the overnight pretreatment period to allow the overexpression of β-GAL, PKCζ and PKCι/λ, respectively. The primary hepatocytes were then incubated in fresh medium A with increasing concentrations of insulin (0 nM to 100 nM) for 6 hours before total RNA extraction. Total RNA was extracted, and then subjected to real-time PCR analysis. The data were expressed as fold induction. The gene transcript levels from Ad- β-gal infected primary hepatocytes without insulin treatment were arbitrarily set to 1. (A) The mRNA levels of Prkcz in the ZL primary hepatocytes overexpressing β-GAL, PKCζ and PKCι/λ in response to treatments of increasing concentrations of insulin (mean ± SEM; n = 4; all p<0.05; a>b for comparing insulin treatment groups in hepatocytes overexpressing PKCζ using one-way ANOVA; * for comparing fold inductions among three groups at indicated treatments using one-way ANOVA). (B) The mRNA levels of Prkci in the ZL primary hepatocytes (n = 4) overexpressing β-GAL, PKCζ and PKCι/λ in response to treatments of increasing concentrations of insulin (mean ± SEM; n = 4; all p<0.05; * and # for comparing fold inductions among three groups at indicated treatments using one-way ANOVA).
Interestingly, 0.1-100nM insulin treatments significantly suppressed the expression levels of Prkcz mRNA by 40–60% in hepatocytes transfected with Ad-Prkci (Fig. 3A, black columns). On the other hand, in hepatocytes transfected with Ad-Prkcz, the expression levels of Prkci mRNA in 1-100nM insulin treatment groups were significantly lower than that in hepatocytes with β-GAL overexpression at the corresponding treatments (Fig. 3B, striped columns, #).
Fig. 4 shows that PKCζ, but not PKCι/λ, protein level was overexpressed by ~9.3 folds in hepatocytes transfected with Ad-Prkcz compared to that with Ad-β-gal. Overexpression of PKCζ did not significantly increase phospho-PKCζ/λ Thr410/403 level. Fig. 5 shows that primary rat hepatocytes transfected with Ad-Prkci manifested ~4.0 fold overexpression of PKCι/λ protein and increased phosphorylation of PKCζ/λ at Thr410/403, compared to that with Ad-β-gal. Unfortunately, we tried and failed to develop a method for the determination of activities of PKCζ and PKCƖ/λ using substrates as reported and available antibodies.
Primary hepatocytes of ZL rats fed chow for 8 weeks were isolated and seeded onto dishes as described in the Materials and Methods. Purified Ad-β-gal and Ad-Prkcz were added in ZL primary rat hepatocytes during the overnight pretreatment period to allow the overexpression of β-GAL and PKCζ, respectively. Cells were then incubated in fresh medium A with increasing concentrations of insulin (0 nM to 100 nM) for 15 min. The whole cell lysates were subjected to immunoblot analysis with antibodies against indicated proteins. The figure shows the representative result of three parallel experiments obtained from three sets of animals.
Primary hepatocytes of ZL rats fed chow for 8 weeks were isolated and seeded onto dishes as described in the Materials and Methods. Purified Ad-β-gal and Ad-Prkci were added in ZL primary rat hepatocytes during the overnight pretreatment period to allow the overexpression of β-GAL and PKCι/λ, respectively. Cells were then incubated in fresh medium A with increasing concentrations of insulin (0 nM to 100 nM) for 15 min. The whole cell lysates were subjected to immunoblot analysis with antibodies against indicated proteins. The figure shows the representative result of three parallel experiments obtained from three sets of animals.
Overexpression of PKCζ or PKCι/λ attenuated the insulin signaling cascade in ZL primary hepatocytes
Fig. 4 shows that the levels of insulin-induced phosphorylation of AKT at Ser473 and Thr308, and insulin-independent phosphorylation of AKT at Thr450, were diminished in hepatocytes overexpressing PKCζ, but not β-GAL. There was no change of total AKT. The expression levels of IRS1 were also lowered upon the overexpression of PKCζ. Interestingly, based on the densitometry analysis, there was a slight but significant elevation in the levels of PKCι/λ, FAS, ACC or phospho-ACC at Ser79 in the hepatocytes overexpressing PKCζ compared with Ad-β-gal group (S3 Fig.).
Fig. 5 shows that PKCι/λ overexpression did not significantly change the expression levels of PKCζ and ACC in ZL primary hepatocytes. The levels of FAS and phospho-ACC Ser79 were marginally but significantly elevated (S4 Fig.). PKCι/λ overexpression did not change the total AKT level compared with Ad-β-gal group. Similar to that in PKCζ overexpression groups, the levels of IRS1, insulin-induced phospho-AKT at Ser473 and Thr308, and insulin-independent phosphorylation of AKT at Thr450 were significantly decreased in primary rat hepatocytes overexpressing PKCι/λ in comparison to the Ad-β-gal group. These data collectively demonstrate that overexpression of aPKC attenuates the activation of insulin signaling cascade in primary hepatocytes from ZL rats.
Overexpression of PKCζ or PKCι/ζ impaired the expression levels of insulin-regulated genes in primary hepatocytes from ZL rats
Insulin dose-dependently induced the expression levels of Gck (Fig. 6A) and Srebp-1c (Fig. 6B) transcripts in Ad-β-gal group. Overexpression of PKCζ or PKCι/λ increased the basal levels of Gck and Srebp-1c in ZL primary hepatocytes. However, overexpression of PKCζ or PKCι/λ abolished the insulin-induced Gck and Srebp-1c expressions in ZL hepatocytes.
Primary hepatocytes of ZL rats fed chow for 8 weeks were isolated and seeded onto dishes as described in the Materials and Methods. Purified Ad-β-gal, Ad-Prkcz and Ad-Prkci were added in medium A during the overnight pretreatment period to allow the overexpression of β-GAL, PKCζ and PKCι/λ, respectively. The primary hepatocytes were then incubated in fresh medium A with increasing concentrations of insulin (0nM to 100nM) for 6 hours. Total RNA was extracted, and subjected to real-time PCR analysis. The transcript level of each indicated gene in primary hepatocytes transfected with Ad-β-gal in the absence of insulin treatment was arbitrarily set to 1 for that gene. The mRNA levels of (A) Gck, (B) Srebp-1c, (C) Pck1, (D) Igfbp-1, (E) G6pc and (F) Pklr were expressed as fold inductions (mean ± SEM; n = 4; all p<0.05; a<b, c<d, e>f, g>h, i>j, k>l, m>n, o>p, q>r for comparing the insulin treatment groups of hepatocytes overexpressing Ad-β-gal, Ad-Prkcz or Ad-Prkci using one-way ANOVA; * for comparing fold inductions among Ad-β-gal, Ad-Prkcz and Ad-Prkci groups at the indicated insulin concentrations using one-way ANOVA).
Insulin dose-dependently suppressed the expression levels of Pck1 (Fig. 6C), Igfbp-1 (Fig. 6D) and G6pc (Fig. 6E) transcripts in Ad-β-gal group as anticipated. In the absence of insulin, overexpression of PKCζ or PKCι/λ increased or reduced the basal expression levels of Pck1 (Fig. 6C) or Igfbp-1 (Fig. 6D) transcript, respectively. However, the insulin-suppressed expression of Pck1 was abolished, and insulin-suppressed expressions of Igfbp-1 and G6pc were attenuated in hepatocytes overexpressing PKCζ or PKCι/λ, respectively.
Fig. 6F shows that the mRNA levels of Pklr in hepatocytes overexpressing -GAL, PKCζ and PKCι/λ were not affected by insulin. However, the expressions levels of Pklr in 0-1nM insulin groups were significantly higher in hepatocytes overexpressing PKCζ or PKCι/λ than that overexpressing β-GAL.
Here we show that the expression levels of PKCζ protein and Prkcz mRNA are higher in the liver of ZF rats than ZL rats. This elevation remains intact in primary hepatocytes that have been cultured for more than 20 h. Since the insulin-regulated gene expression is impaired in primary hepatocytes from ZF rats, we hypothesize that the alteration of aPKC expressions may play a role in this impairment. Therefore, PKCζ and PKCι/λ are successfully overexpressed using recombinant adenoviruses in ZL primary hepatocytes. The adenoviral-mediated overexpression of PKCζ or PKCι/λ in primary hepatocytes from ZL rats leads to the impairment of insulin-regulated Gck, Srebp-1c, Pck1, G6pc and Igfbp1 expressions (Fig. 6). Overexpression of either PKCζ or PKCι/λ is sufficient to alter basal and insulin-regulated gene expression in hepatocytes from ZL rats.
The impairment of the insulin-regulated gene expression might be attributed to the reduction of of AKT phosphorylations in cells with overexpression of PKCζ or PKCι/λ., Maximal activation of AKT needs phosphorylations of AKT at both Ser473 and Thr308 as indicated in . Therefore, detection of its phosphorylation using specific antibodies have been a indicator of AKT activation status. It was interesting to note that we could not detect phosphorylation of AKT at Thr308 in the liver tissue lysates of both ZL and ZF rats. Here are the possible reasons. First, the in vivo activation of AKT may be moderate, not maximized, which allows further regulation in response to stimuli. Second, there might be relative less AKT protein amount from hepatocytes in the liver tissue preparations, which requires a larger amount of protein per sample for the detection. Third, the phosphorylation of AKT at Thr308 in the liver may be rapidly de-phoshorylated during the time when the animals were euthanized for the collection of tissue samples. Nevertheless, we demonstrated here that in ZL hepatocytes of Ad-β-gal group, insulin dose-dependently induces the levels of phospho-AKT (Ser473 and Thr308) and IRS1 protein. On the other hand, PKCζ or PKCι/λ overexpression attenuates the insulin-induced phosphorylation of AKT, and decreases the protein level of IRS1, which suggests a diminished signaling cascade initiated from IRS1 upon insulin treatment (Figs. 4 and 5). Without insulin treatment, the expression level of total AKT and p-AKT seem to be not different between the Ad-β-gal control and aPKCs overexpression groups (Figs. 4 and 5). However, higher mRNA levels of Gck, Pck1, Srebp-1c and Pklr were observed in the aPKCs overexpression groups compared to those in the Ad-β-gal control (Fig. 6). This indicates that other mechanisms may contribute to the elevation of the basal expression of those genes in hepatocytes overexpressing PKCζ or PKCι/λ.
GCK catalyzes the phosphorylation of glucose into glucose-6-phosphate, which is the rate-limiting step of glycolysis in the liver . The induction of the hepatic Gck expression depends on the activation of insulin signaling pathway in the liver . PKCζ (Fig. 4) or PKCι/λ (Fig. 5) overexpression diminished phosphorylation of AKT at Ser473 along with the decrease of IRS1 protein expression. It has been reported that aPKCs probably act as negative feedback regulators of insulin signaling pathway by promoting serine phosphorylation and tyrosine de-phosphorylation of IRS proteins both in vitro and in cell lines [25–27]. Thus, the reduction of the activation of insulin signaling pathway due to PKCζ or PKCι/λ overexpression may impair the insulin-regulated Gck expression. Additionally, the insulin-induced Gck expression has been suggested to be mediated by transcription factors, such as hepatocyte nuclear factor 4, forkhead box protein O1, SREBP-1c, liver x receptor α, and peroxisome proliferator-activated receptor γ [28–31]. Since the knowledge about the non-kinase activities and endogenous substrates of PKCζ or PKCι/λ is limited, it is possible that PKCζ or PKCι/λ directly regulate Gck expression through controlling the activation of certain transcription factors at its promoter, which increases the Gck basal mRNA level.
PEPCK catalyzes the rate limiting enzymatic reaction in gluconeogenesis, by which oxaloacetate is converted into phosphoenolpyruvate . In the liver, the insulin-suppressed Pck1 expression has been thought to be purportedly mediated through three pathways: (1) the phosphorylation and inactivation of forkhead box proteins by insulin [33,34]; (2) antagonizing the glucagon-induced PPARγ co-activator 1 expression ; (3) the phosphorylation of CREB-binding protein via aPKCs [36,37]. Interestingly, insulin-induced activation of aPKCs was shown to suppress Pck1 expression via this phosphorylation of SREBP-binding protein . However, our results show the induction of Pck1 expression upon overexpression of PKCζ or PKCƖ/λ in the absence of insulin. The activities of aPKC isoforms are elevated in the liver of individuals with type 2 diabetes . Treatment of hepatocytes from these individuals with aPKC inhibitors decreases the basal transcript levels of gluconeogenic genes . Hepatic inhibitors of PKCƖ have been show to correct the metabolic abnormalities in rodents . Here, aPKCs overexpression not only increases the basal transcript of Pck1 mRNA, but also abrogates the insulin-suppressed Pck1 expression. Additionally, aPKCs overexpression attenuates the insulin-suppressed G6pc expression. Therefore, our data suggest that aPKCs are prominent positive regulators of hepatic gluconeogenesis.
SREBP-1c is a master regulator of the hepatic expression of lipogenic genes . The insulin-induced Srebp-1c expression requires the activation of PI3K, and the mTORC2-mediated phosphorylation of AKT at Ser473 . Based on the use of pharmacological inhibitors, the activation of mTORC1 downstream of AKT is identified as an indispensible step in the induction of Srebp-1c . On the other hand, the activation of PI3K subsequently activates aPKC in the liver, which promotes Srebp-1c expression and lipogenesis [12,13]. Despite the diminished activation of AKT and the abolished insulin-induced Srebp-1c expression, PKCζ or PKCι/λ overexpression increases the basal transcript level of Srebp-1c in the primary hepatocytes from ZL rats (Fig. 6C). Interestingly, PKCζ or PKCι/λ overexpression only marginally induced the protein levels of FAS and ACC (Figs. 4 and 5). One possibility is that the elevation levels of transcripts might not have been translated in the presence of overexpression of PKCζ or PKCι/λ. Alternatively, diminished insulin signaling pathway results in decreased activation of p70 S6-kinase, which is required for the SREBP-1c processing to generate nuclear SREBP-1c . Moreover, there might be a lag time between the elevation of Srebp-1c mRNA and the increase of SREBP-1c protein, and the down-stream targets. Nevertheless, the protein levels of ACC and are maintained at a high level in the hepatocytes and liver samples of ZF rats (Figs. 1 and 2), demonstrating the metabolic changes associated with these insulin-resistant animals. Further investigations are needed to delineate the sequential events associated with the elevation of the hepatic lipogenesis upon insulin stimulation and activation of AKT and aPKC isoforms.
It is interesting to note that the Prkcz mRNA and PKCζ protein, but not that of Prkci mRNA and PKCι/λ protein levels are elevated in the liver of ZF rats. Insulin at 1nM and up significantly induces the expression levels of Prkci mRNA, a process that is blunted in the presence of PKCζ overexpression. On the other hand, insulin does not affect the expression of Prkcz mRNA. Additionally, PKCι/λ overexpression alone does not affect Prkcz mRNA expression. However, PKCι/λ overexpression introduced the insulin-mediated suppression of Prkcz mRNA expression. These data indicate that there are crosstalks between PKCζ and PKCι/λ pathways, which may mutually regulate the expression of each other at mRNA levels in response to hormonal or nutritional stimuli. The alteration of this mutual regulation mechanism probably is associated with the development of the impairment of insulin signaling cascade. Further studies are warranted.
It is worth noting that, in ZL primary hepatocytes, the overexpression of aPKCs and the attenuated insulin signaling cascade lead to abolishment of insulin-regulated expression of Gck, Pck1 and Srebp-1c (Fig. 6). In contrast, ZF rats showed unimpaired phosphorylation of AKT at Ser473 and Thr450 despite increased PKCζ levels (Figs. 1 and 2). This difference may explain that the insulin-regulated expression of Gck and Pck1 is impaired rather than abolished in ZF primary hepatocytes . Additionally, in high-fat diet fed mice, increased basal AKT phosphorylation was observed in the liver with indication for the development of insulin resistance . Additionally, elevated aPKC activity was shown to attenuate AKT-dependent FOXO1 phosphorylation in the liver of diet-induced obesity mice, despite concurrent elevated hepatic AKT activity . These data collectively suggest that activation statuses of both AKT and aPKC are critical in the regulation of hepatic insulin sensitivity.
In summary, we have shown that the expression level of PKCζ is elevated in insulin-resistant animals. Overexpression of PKCζ or PKCƖ/λ in ZL primary hepatocytes reduces the activation of AKT probably through the reduction of IRS1 expression, demonstrating the attenuation of the activation of insulin signal pathway. This alteration of the expression levels of aPKC leads to the impairment of the insulin-regulated gene expression in primary hepatocytes. Our data demonstrate the critical roles of aPKCs in the regulation of hepatic insulin-sensitivity at gene expression level, and provide insights into the development of the insulin resistance and type 2 diabetes.
S1 Fig. Densitometry analysis of target proteins in the livers of ZL and ZF rats.
Densitometry analysis was performed using ImageJ software (NIH). The data for each protein were normalized to β-actin levels in each sample. One-way ANOVA with LSD post-hoc test was used to compare the means. Error bars represent S.E.M. * indicates a p value less than 0.05.
S2 Fig. Ct number of the ribosomal gene 36B4 in ZL hepatocytes transfected with Ad-β-gal, Ad-Prkcz and Ad-Prkci, respectively.
Primary hepatocytes of ZL rats fed chow for 8 weeks were isolated and seeded onto dishes as described in the Materials and Methods. Purified Ad-β-gal, Ad-Prkcz and Ad-Prkci were added in medium A during the overnight pretreatment period to allow the overexpression of β-GAL, PKCζ and PKCι/λ, respectively. The primary hepatocytes were then incubated in fresh medium A with increasing concentrations of insulin (0 nM to 100 nM) for 6 hours before total RNA extraction. Total RNA was extracted, and then subjected to real-time PCR analysis with primer pairs for 36B4. The data were the raw Ct number of 36B4 for 0 nM insulin treatment groups (mean ± SEM; n = 4).
S3 Fig. Densitometry analysis of target proteins in ZL hepatocytes transfected with Ad-β-gal or Ad-Prkcz.
Densitometry analysis was performed using ImageJ software (NIH). The data for each protein were normalized to β-actin levels in each sample. Two-way ANOVA was used to determine the contribution of insulin treatment and adenovirus overexpression to total variance. Error bars represent S.E.M. * indicates that adenovirus overexpression accounts for a significant percentage of total variance with a p value less than 0.05. a<b, c<d<e, which indicate insulin treatments account for a significant percentage of total variance with a p value less than 0.05.
S4 Fig. Densitometry analysis of target proteins in ZL hepatocytes transfected with Ad-β-gal or Ad-Prkci.
Densitometry analysis was performed using ImageJ software (NIH). The data for each protein were normalized to β-actin levels in each sample. Two-way ANOVA was used to determine the contribution of insulin treatment and adenovirus overexpression to total variance. Error bars represent S.E.M. * indicates that adenovirus overexpression accounts for a significant percentage of total variance with a p value less than 0.05. a<b, a’<b’, c<d, e<f, which indicate insulin treatments account for a significant percentage of total variance with a p value less than 0.05.
We thank Dr. Ralf Kubitz for the kind gift of pEYFP-N1 vector containing the complete coding sequence of rat Prkcz.
Conceived and designed the experiments: WC GC. Performed the experiments: WC MRG HK. Analyzed the data: WC MRG GC. Contributed reagents/materials/analysis tools: GC. Wrote the paper: WC GC.
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domain: literature


text:
With hopes of bringing great, affordable steak dinners to everyone who wanted one, Sizzler originally opened in California in the late 1950s. Now, this restaurant offers affordable dinners, period, with a buffet of a wide array of offerings for all to enjoy.
You never now what to expect from the entree portion of the Sizzler Buffet but you know it will be good. Options include seafood, barbecue, chicken, steaks, and various International styles. You can always count on a fabulous soup and salad bar, a few select appetizers, and a spread of desserts that will have your mouth watering.
Sizzler Dorado is a steak house where they proudly serve their take on American cuisine. Anticipate and dress for a dining experience that is very casual. When's the best time to come? They're open for both dinner and lunch, so make time to try it. You could discover flavors you've been missing all your life.
Sizzler Dorado is found toward the center of Dorado.
There are certainly some strong points to this area, like Dorado Del Mar Golf Club and Dorado Beach. The latter of the two is the most convenient spot to visit the beach, and nestles itself within a reasonable distance of the restaurant.
If you're looking to find another restaurant in the region with some similar menu items and/or comparable prices, there is no shortage of nearby places to consider, including Blue Seahorse Bar & Grill.
Then again, if you're looking for something different, this region of Puerto Rico features an impressive selection of restaurants, where you will encounter many different cuisine styles and flavors. For instance, you might also consider trying Creole fare at Metropol Dorado, which is a short distance away west-southwest.
Although prices will vary slightly depending on if you go for the buffet or a special item, you can expect to pay less than $10(USD) per person, including drinks and dessert.
Location: Carretera 693 Int 658 Km 1.5 , Dorado, Puerto Rico
Phone: (787) 665-7061
Help us improve! We welcome your corrections and suggestions.

domain: geography


text:
February's almost over, and that means one thing: March. And what does March mean? March 1st is one of four times a year the DCI reserves to make changes to the banned and restricted lists. And in case you missed last week talking about a whole new environment or Aaron Forsythe's article comparing Standard and Extended, there are big changes around the corner.
A: Yes, Cohort can attack if he has Haste (via Warchief) and was played that turn. The only thing the Cohort cares about is if somebody was played-- who doesn't worry him.
Q: Q: I have heard the argument that the Glasskite ability is only based on targeting, and not spell specific. Your thoughts? --Judi E.
A: Glasskites only counter the first spell or ability played. It doesn't matter how many times a given spell can or did target it.
The Glasskite ability is "Whenever this creature becomes the target of a spell or ability for the first time in a turn, counter that spell or ability.” This can be dissected into "Whenever
Having dissected the wording, let's ask some questions. The questions to ask are:
Who? This Creature
What? Becomes the target of a spell or ability for the first time
When? This turn
If you play Shock targeting such a creature, the trigger goes on the stack. If you respond with a Terror, the ability recognizes that its creature has already become the target for the first time and does not trigger for the Terror.
A: The Gatekeeper and Veins both would trigger. The Gatekeeper would be destroyed as a state-based effect. You'd lose 2 life from the Veins and take 2 damage from the Gatekeeper.
A: Awe Strike reads in part "The next time target creature would deal damage…" Since Awe Strike doesn't include something like "to you", it will prevent all of the Heartless damage, and you'll gain that much life (half of your life rounded down plus have of your opponent's life rounded down will be added to your life).
Q: If I have Shirei, Shizo's Caretaker in play and a creature with power 1 goes to my graveyard, then I play Otherworldly Journey on Shirei, can I stack the "at end of turn" effects in such a way to bring back the little guy? --Daniel V.
A: No, once Shirei leaves play the game loses track of who he was. The Shirei that comes into play after his Journey is not considered the same as the one that left.
A: No, putting a copy of a Spirit creature into play isn't the same thing as playing a Spirit spell.
*Extra*: Teardrop Kami and Kiki-Jiki would combo with Auriok Champion just fine though. The Auriok triggers of creatures entering play rather than being played (ie: played as a spell). You won't have huge amounts of creatures at once, but you could generate a huge amount of life from the Auriok triggers.
A: Well, do you think that Guiltfeeder's "fear" makes it easier to sneak him past blockers and thus make your Ninja more likely to spring forth? Do you think that once the Guiltfeeder is unblocked its ability will trigger causing life loss in your opponent, and that you also have an unblocked creature that would be available for Ninjutsuing someone like Ink-Eyes, Servant of Oni into play early enough to also be able to hit your opponent for five damage and trigger Ink-Eyes' ability? Do you think Ink-Eyes reanimating creatures from your opponent's graveyard makes Guiltfeeder less effective next time? If so, then they do.
Q: If my opponent plays Blessed Reversal, and I use Ninjutsu to return my two unblocked creatures, will he gain 3 life for each of the creatures I returned to my hand? --Offer Z.
A: No, he won't gain life for those creatures. However, he will gain life for the Ninja that show up. There are multiple results possible…
You could Ninjutsu one unblocked creature for another and have the same number of creatures attacking. The life gain will be the same.
You could Ninjutsu one unblocked creature and respond by revealing the same Ninja to Ninjutsu another creature (you'll have to spend the Ninjutsu cost twice). The Ninja will be put into play by the last ability, and the first ability will "do nothing" since the Ninja is already in play, but you have one fewer creature, so your opponent won't gain as much.
*Extra*: If your opponent wanted to make sure you don't pull Ninja tricks, he or she could play Blessed Reversal before declaring blockers. Since blockers have yet to be declared, you won't be able to use Ninjutsu yet.
Q: When Yukora, the Prisoner is unblocked, and I return him to play via Ninjutsu, would his ability make me sacrifice the Ninja put into play, or can I arrange the triggered abilities on the stack so the Ninja lives? --Chad
A: You don’t get a choice about arranging Yukora's triggered ability and the activated Ninjutsu ability-– the Ninja will always live.
You announce Ninjutsu, putting the ability onto the stack and returning Yukora to your hand as part of the cost to play the ability. This triggers Yukora’s ability, which goes on the stack on top of the Ninjutsu ability. The triggers resolves before the Ninja enters play.
Q: I was thinking: do multiple Overblaze effects stack? --Scalemx
A: "Stack" isn't the best word. Yes, they multiply. So one makes damage x2. Two would make damage x2 and x2 (x4). The big jump starts after the second one-- a third would make the damage x2 x2 x2… for a total of x8!
A: Yes, if you repeatedly activate Genju of the Fields, it will get the “Whenever this creature deals damage, you gain that much life” text multiple times. A Plains that has that text twice and deals two damage will make you gain four life. (Three times will be six life.)
Q: How does Dokai, Weaver of Life's Elementals token creatures work? Are they */*s where * is equal to your lands all the time or just the number when you made the token? --Pat T.
A: Each token will be X/X according to how many lands you controlled as it was being made. You could have an 11/11 token, play two lands, and then make another token. The second one would be 13/13 while the first is still 11/11.
A: No, Extraplanar Lens adds extra mana "of any type that land produced". Since only was produced, you'll only get extra.
*Extra*: If you had a land like the Odyssey ones that produce two colors at once you can get either color. Tapping a Mossfire Valley for will let the Lens produce either or
A: Yes, Uyo copies spells and copies of spells. Uyo isn't picky about the how it got on the stack.
Q: Does having a Samurai of the Pale Curtain in play prevent regeneration because the creature is not sent to the graveyard? --Stefan A.
A: No, Samurai of the Pale Curtain does not stop regeneration. Regeneration replaces destruction, and since the creature never was destroyed, it would never be sent to the graveyard.
Pre-Kamigawa Block Questions
Q: Is there a rule that says Gaea's Cradle taps for one green mana even if you have no creatures in play? --Sam
A: No, there isn't. To figure out how much you get multiply it by the number of creatures you control. Mathematically, multiplying anything by 0 results in 0.
A: Your normal draw is replaced by the Words effect, and you'll get to deal two damage to a target creature or player. An activated Words of War means the draw that would kill you never happens.
Q: My opponent has played a Forest with an enchantment on it that says whenever this land becomes tapped, put a 1/1 squirrel token into play (sorry forgot name). He also has a card that states: tap an untapped creature: untap target land (forgot name too). He taps the forest and taps the squirrels and creates about 150 of them. Would he die because of mana burn from the Forest? --Joe
A: No, he's not going to burn at all. You're thinking of Squirrel Nest and Earthcraft, a combo that is popularly referred to as "Squirrelcraft". The key is that the squirrels aren't made by tapping the Forest for mana; they're made by tapping the land for the Nest rather than tapping it for mana (you can't do both).
A: The Death Speakers is going to die.
Protection from black does the following for Death Speakers:
Death Speakers can't be Damaged by black sources (the damage is prevented)
Death Speakers can't be Enchanted or Equipped by black things
Death Speakers can't be Blocked by black creatures
Death Speakers can't be Targeted by black spells or abilities
An easy way to remember this is to remember the word DEBT.
Since No Mercy is not trying to Damage, Enchant, Block, or Target the Death Speakers, No Mercy's ability will work fine.
You can play Ninja tricks against No Mercy though.
What you could do is stack the damage from a creature, let that damage resolve, and in response to the No Mercy trigger (since the creature is still unblocked), use Ninjutsu to return the creature. The fresh Ninja won't be who the No Mercy is looking for, and the creature to be destroyed will be safely in your hand.
A: It doesn't. The Orrery effect only applies to spells of the given types, it does not change the time at which they would normally be playable, and that's what the Staff cares about.
A: No. Fear is now a keyword, and even though it means that a creature "can't be blocked except by black or artifact creatures", the word "black" no longer appears on the card. Older cards were errataed to receive the keyword ability instead of their printed text. This includes older cards like Shadowmage Infiltrator and, well, Fear.
Q: What exactly is interrupt?
A: In Magic terms, an interrupt was a spell that could target other spells (or abilities). 6th Edition cleared up many rules nightmares like interrupts and the "Interrupt Window". Errata was issued to adjust cards for 6th Edition, and interrupts are now instants.
*Extra*: Oddly enough, Red Elemental Blast was misprinted in Alpha with the type "Instant". This was fixed in Red Elemental Blast to "Interrupt", but with 6th Edition, it regained the misprinted but now correct type-- instant.
Q: I have found some old cards with "phasing" or "phase out", but I don't know the meaning of it. Please, can you help me? --Paul
A: Phasing [CR 502.15] is an ability from the Mirage block that made permanents stay in play only half the time.
You start your turn with a phasing critter…
Before you actually untap, that creature and anything on it (equipment, artifacts, counters) gets set aside (in the "Phased-Out" zone). Then you untap, and your turn proceeds as normal.
On the next turn, you're about to untap. But wait! You have a phased out critter. Phase the critter back into play (including anything that was attached to it). Now you get to actually untap all your permanents-- including the freshly returned creature.
Other points to remember about Phasing:
Phasing in lets that permanent function as though it has Haste
Phasing in does NOT trigger "comes into play" triggers
Phasing out does trigger "leaves play" triggers
Phasing out removes all damage on the permanent
Permanents that phase in are considered the same permanent that left play (the game normally forgets about a permanent if it leaves play)
Q: During a recent game we encountered a problem deciding who won our match. Both of us have Platinum Angel but no way to remove the other one's Angel. So is it a draw or what? --Adam & Steve
A: Games that cannot be completed are a draw. It doesn't matter how I-should-be-dead you are or your opponent is when you both have Platinum Angels in play.
That's all for this week. Next weekend is Grand Prix Seattle. If you're in the Pacific Northwest and looking forward to throwing down with hundreds of your fellow Magic players, Seattle Center will be the place to be. The Extended action is sure to be red hot with the format as open as it is. I'll be seeing you there, and then it's off to Pro Tour Atlanta a week later.

domain: game


My Ryan Stiles interview is in this week's News and Review.
You can read it by clicking here. Unfortunately I won't be able to go see him as I'm reading at the premier party for the new Morbid Curiosity Magazine that night in SF. The current issue has my Colon story.
Too bad. Stiles is one funny, funny man. I'm sure the shows gonna be a blast.


text:
Doria is a Western style Japanese dish which usually consists of white sauce and rice. Doria looked like a European dish, however it was invented by a Swiss chef in Japan.
Apparently, the dish “Doria” was named after the Genoese statesman “Andrea Doria” from the noble class family in Genoa, Italy.
I will introduce the unique Meat Sauce Eggplant Doria instead of using white sauce to make it at home easily. You would enjoy eating this dish with your family and/or friends!
Ingredients (3 - 4 plates):
- Rice for 3 - 4 people
- 1/2 eggplant
- Ground beef (1/2 lbs, 0.23kgs)
- 1 small can of sliced mushrooms (113g, 4oz)
- 1/2 bin tomato pasta sauce
- 1 clove garlic
- 1 teaspoon olive oil
- 5 or more small leaves parsley
- 1 cube *Japanese consomme
- 1 teaspoon salt & pepper
- 6 sliced cheese (I used sliced cheddar cheese)
- Spray butter or spray olive oil
*Japanese consomme is made of vegetable extract, beef extract, chicken extract, yeast extract, soy sauce, salt, sugar and so on. It is similar to vegetable, chicken and beef stock.
-> See pictures A, B & C.
Here is how to make it at home. Cooking time is about 40 minutes - 50 minutes.
- Cook rice. Meanwhile, cut garlic into small pieces. Cut an eggplant into bite size chunks.
- Preheat olive oil in a frying pan with medium heat. Add garlic and stir for 1 minute. -> See picture D.
3. Add ground beef in the frying pan with medium heat and stir for 3 - 5 minutes or until ground beef is fully cooked. -> See picture E.
4. Add the eggplant and stir for about 3 - 5 minutes. -> See picture F.
5. Add mushrooms and stir until the eggplant and mushrooms are fully cooked. -> See picture G.
6. Add tomato pasta sauce and stir for 1 minute. Mix occasionally. -> See picture H.
7. Add Japanese consomme and salt & pepper, and stir for 3-5 minutes. Remove from heat. -> See pictures I & J.
8. Spray oil or butter on a small - medium size glass tray as shown in the picture. -> See picture K.
9. Put the cooked rice on the glass tray as shown in the picture. -> See picture L.
10. Add meat sauce from step 7. on top of the rice as shown in the picture. -> See picture M.
11. Add sliced cheese on top of the meat sauce. -> See picture N.
12. Heat oven up to 400 °F (205 °C) without preheating. Put the meat sauce doria into the oven for about 15 - 20 minutes. (Heating time may vary and depending on the oven which you use.) Remove from the heat.
13. Add parsley leaves on top.

domain: food


On 23 January 1988 a young and inexperienced three-piece rock band entered a studio in Seattle, Washington to record a demo they hoped would arouse the interest of a local label. Running through ten songs that they had developed on the city’s live circuit the tape was recorded in one afternoon with few overdubs for barely $150. That evening, the band took to the stage at the Community World Theater in Tacoma and performed the same songs in front of an enthusiastic audience, appearing under the name Ted, Ed and Fred.
The tape that they had recorded earlier that day would reach the ears of only a handful of listeners, yet it would be the man responsible for overseeing the session who would ultimately bring the group to the attention of rising label Sub Pop. ‘That demo was seventies riff rock with a slightly weird post-punk angularity,’ producer Jack Endino told journalist Everett True years later. ‘The thing that separated it from anything else was the singing. His voice had a lot of character and he had a weird ear for melody, he wouldn’t be following the guitar riffs like typical idiot riff-rock.’ A few months after the recording of the demo the band changed their name to Nirvana.
1988 was an important milestone for the Seattle music scene, as the newly-formed Sub Pop signed several promising acts onto its roster. Soundgarden had released their first EP Screaming Life a few months earlier and were now commencing work on its follow-up while Mudhoney would issue their own mini-album Superfuzz Bigmuff later that year. Both groups, along with Nirvana, would become key figures in the city’s so-called grunge scene, a style of alternative rock that would reinvent the genre and ultimately bring an end to the popular-yet-critically reviled ‘hair metal’ that was dominating Los Angeles. Many of the bands that would emerge from Seattle during this time were heavily influenced by such post-punk rock acts as Sonic Youth and Dinosaur Jr., as well as the Pixies, whose song structure and pop sensibility would inspire Nirvana’s more commercial work.
Long before becoming the reluctant voice of a generation, frontman Kurt Cobain grew up in a dysfunctional family, with his parents divorcing when he was a child and the youngster being forced to move home at regular intervals. He was given a guitar when he was just eight, a present from his Aunt Mari, whose home Cobain would record his first demo at several years later. ‘As soon as I got my guitar I just became so obsessed with it,’ he later explained to biographer Michael Azerrad.
While his own music would be heavily inspired by punk, his earlier influences would come from a variety of different artists such as AC/DC, the Beatles, Aerosmith and even ABBA. But it would not be until 1985, when Cobain was eighteen, that he would finally form his own group, which he christened Fecal Matter. This short-lived band would feature several members of popular local act the Melvins, while Cobain and two of his fellow musicians would record a home demo entitled Illiteracy Will Prevail.
Despite the collapse of Fecal Matter Cobain had enjoyed performing in a band and writing songs with other artists, but it would be with another local musician, Krist Novoselic, that he would finally find the right chemistry. ‘This is around the time I met this person named Kurt Cobain,’ said Novoselic in his autobiography Of Grunge & Government: Let’s Fix This Broken Democracy!. ‘If I am to speak about independence I need to mention one of the most independent people I’ve ever met. Kurt was a completely creative person – a true artist.’ Along with drummer Aaron Burckhard, Cobain and Novoselic formed their own group and after long discussions finally settled on Skid Row. Ironically, there was another group who had formed on the other side of America, in the New Jersey town of Toms River, that had also taken the same name, eventually releasing their eponymous debut in 1989 to considerable success.
Even as they began to perform together as Skid Row Cobain and his new bandmates also collaborated with other local musicians around Seattle including members of the Melvins, resulting in such side projects as the Sellouts, Stiff Woodies and Brown Cow, yet writing and playing original material was always his main objective. Skid Row made various appearances in the area, both at house parties and small clubs around Aberdeen, Raymond and Seattle. During a party at a house in Raymond the band were so drunk that they managed to alienate most of their audience.
In an interview Options years later Cobain recalled the evening; ‘We were really drunk so we started making spectacles of ourselves. Playing off the bad vibes we were giving to the rednecks, you know – jumping off tables and pretending we were rock stars…Then we started playing Sex Bomb for about an hour and our girlfriends were hanging on us and grabbing our legs and doing a mock lesbian scene and that really started freaking out the rednecks.’
Soon it became clear that their relationship with Burckhard was coming to an end and so Cobain and Novoselic began searching for a replacement, something that would haunt the band until the arrival of Dave Grohl in 1990. With Burckhard out of the picture Cobain ran an ad in a local magazine called the Rocket, in which he declared, ‘SERIOUS DRUMMER WANTED. Underground attitude, Black Flag, Melvins, Zeppelin, Scratch Acid, Ethel Merman. Versatile as heck.’
Meanwhile, they had become close friends with the Melvins and, in particular, Dale Crover, their well-respected drummer following the departure of Mike Dillard. In January 1988 Cobain contacted a studio in Seattle called Reciprocal Recording and spoke to the producer Jack Endino about booking a recording session. Endino had already gained modest acclaim in the area due to his own group Skin Yard, yet it was his work as a producer that had earned him a reputation around town. It would be the involvement of Crover that had impressed Endino the most and so he agreed to produce the session himself.
Although the band were still relatively inexperienced the threesome had energy, a raw and unpolished sound and a professional drummer overcompensating for his bandmates’ shortcomings. The songs were basic and the lyrics unsophisticated, yet Endino was impressed by what he heard. Among the ten tracks that were recorded at Reciprocal two would be included on the original issue of the band’s debut album, while most of the others would surfaced on the 1992 compilation Incesticide. One of the tracks recorded during this session, Pen Cap Chew, would remain unfinished due to running out of tape during the recording. With the band unwilling to spend a further $40 for another reel they agreed that Endino should fade the song out before the end. The final cost for the session would come to a total of $152.44 and from that they had ten songs that they could try to tempt labels with.
The sound that was starting to emerge from Seattle at this time was the antithesis to the sex, drugs and rock ‘n’ roll attitude of such pop rocks bands as Poison and Bon Jovi, who had begun to dominate the metal scene during the mid-1980s with their infectiously catchy upbeat songs and power ballads. The Seattle scene had already started to produce some strong local talent, with members of the influential Green River breaking off into new groups that included Mudhoney and Mother Love Bone, the latter eventually evolving into Pearl Jam following the untimely death of their singer Andrew Wood in 1990. Endino had worked with several of these bands and had an ear for potential and following the recording of Cobain and his new group he knew that he had heard something special. Endino made copies of the tape – what fans often refer to as the Dale Demo – and passed them out among his friends, one of which was Jonathan Poneman.
To be honest, I don’t even really know what to make of this tape
Along with his business partner, Bruce Pavitt, local radio DJ and music promoter Poneman launched the independent record label Sub Pop in Seattle in 1987, naming the company after a fanzine that Poneman had self-published. Their first discovery was Soundgarden, whose first EP Screaming Life the label had financed and released to modest acclaim. While Pavitt was somewhat reluctant to take a chance on Endino’s new discovery Poneman was excited by the demo and tried to convince his partner. As he later recalled in an interview with Carrie Borzillo, ‘He told me, ‘There’s this kid who came in who looks like a car mechanic. He came in with Dale Crover. He lives in Olympia and his name is Kurt.’ And he says, ‘To be honest, I don’t even really know what to make of this tape. It’s awesome but they just bashed it out and it’s really unlike anything I’ve really heard. The guy’s got an amazing voice.”
By this point, having toyed with the a variety of names – including Throat Oyster and Windowpane – the band finally settled on Nirvana, despite a British act having already performed under the same name during the late 1960s. Poneman was finally able to convince Pavitt to take a chance on the young band, but with Crover turning his attention back to the Melvins, Nirvana were once again searching for a new drummer. Following a handful of gigs with a young-yet-bad tempered drummer called Dave Foster, Cobain and Novoselic were introduced to Chad Channing, a resident of Bainbridge Island and former member of local speed metal act Stone Crow, who had seen the band perform at Evergreen State College in Olympia and was invited to replace Foster.
Nirvana would return to Reciprocal on 11 June, almost five months after the recording of the demo, to work with Endino once again, this time to record a single that was to be released by Sub Pop. This would be the first of three sessions – the remaining two taking place on 30 June and two weeks later on 16 July – which would result in a handful of leftover tracks, including such titles as Floyd the Barber, Mr. Moustache and early live favourite Spank Thru. But the main focus would be on the two tracks that were to be included on the single.
While not the band’s first choice Sub Pop had insisted that they record a cover of Love Buzz, a track originally released by Dutch rockers Shocking Blue in 1969. It had found its way into several of Nirvana’s set lists, yet Cobain was understandably frustrated that the A-side would not be one of their own songs. On the reverse side, however, was an original compositions called Big Cheese, a track that had not been recorded during their earlier demo session.
Sub Pop released the Love Buzz/Big Cheese single in November 1988, the same month that saw the arrivals of such diverse offerings as GN’R Lies by Guns N’ Roses and Delicate Sound of Thunder by Pink Floyd. Despite its limited release the single generated a minor buzz among music critics, not only in America but also in Britain and soon the band found that their profile had been raised. ‘After the Love Buzz seven-inch came out they started getting a few more people to their shows,’ Endino was quoted as saying in the book Grunge is Dead: The Oral History of Seattle Rock Music. ‘And Kurt was still trying to figure it out – he would play guitar and his singing just didn’t have the power. Ultimately, I think he concluded that he needed to play simpler songs so that he could sing better.’ Already much of what they intended to record for their debut album had become staples of their live sets and soon it was decided that they were to return once again to Reciprocal to commence work on a full-length release.
A fourth musician was signed to the label as a member of Nirvana; Jason Everman. A close friend of Channing, Everman’s time with the band would be brief and his musical contributions limited yet he would play an important part in the history of Nirvana for one simple reason: he offered to pay the studio fees to record the album, then under the working title Too Many Humans. With Endino once again at the producer’s desk the recording sessions for the album would come to a total of $606.16 and while Everman would not play a single note he would be credited as guitarist on the inlay sleeve, as an acknowledgment to his non-musical contribution to the band. On 24 December Nirvana made their way to Reciprocal for the first of several sessions that would ultimately result in their first album which, by the time of its release, would be retitled Bleach.
While only Cobain and Novoselic had been long-serving members of the band they were well-rehearsed by the time they arrived at the studio to record the songs that they had worked hard to develop during their live shows. As Channing recalled in Grunge is Dead, ‘Recording Bleach happened fast. We were in there for I think a week. Recording at Reciprocal – a really cool scene. I always remember Jack sitting there and he had a circle of snacks around him – chocolate bars. We did a lot of weird experimenting with snare sounds. We actually took two snare drums, took the bottom head off one of them and the top head off another, combined the two drums together. So there ended up being this gap between the two snares but I used double-sided screws to link them together. Another thing – Blew was a song that initially was played in D. So when we went to record that song it had turned out that we were already in D and that everyone turned down, or at least Krist did, to C – it was even lower.’
From the very beginning Cobain had become the principal songwriter, with most tracks often based around his guitar riffs, yet while he is now regarded one of the greatest lyricists of his generation he gave little thought to the words at the time of recording their debut album. ‘With Bleach I didn’t give a flying fuck what the lyrics were about,’ he confessed in an interview with Spin in October 1993 while promoting the release of their third and final studio album In Utero. ‘Eighty percent were written the night before recording. It was like, ‘I’m pissed off. Don’t know what about. Let’s just scream negative lyrics and as long as they’re not sexist and don’t get too embarrassing it’ll be okay.’ I don’t hold any of those lyrics dear to me.’
Both the song arrangement and lyrics to many of the tracks recorded during these sessions were relatively basic, in part due to the band’s inexperience but also as they had little time to waste in the studio, often cutting a song with only one or two takes. Nirvana would return to Reciprocal several times over the next month, the first couple of sessions taking place in between Christmas and New Year and then again in mid-January, with the sessions coming to an end on 24 January 1989.
‘Bleach was made in three days,’ Endino would later explain. ‘It was divided up into various little bits here and there, but I mean the total was thirty hours. In those days it was sort of more of a lark. You can only afford three days? Okay, let’s see if we can make a record in three days! And we would do it.’ While at the time Channing was the group’s official drummer two songs would be included on the final track list that featured Crover – Floyd the Barber and Paper Cuts – while a third, Downer, would later be included as a bonus on the CD release.
Bleach was released in the United States in June 1989, two months before it made its way to Europe and three months before Soundgarden’s major label debut Louder Than Love. While it would take the release of Nirvana’s sophomore album Nevermind to bring Bleach to the attention of a wide audience the reviews were mostly positive, praising both the raw guitar and production. Yet it would be Cobain, once again talking to Spin in early 1992, who would be the most critical towards the album. ‘I couldn’t even tell you shit like when Bleach was released. I couldn’t even name the songs on the album.’
Put Bleach up against any Mudhoney release around this time and see who wins
The record would later be criticised in the book Chunklet Presents the Overrated Book: The Only Book You’ll Ever Need, in which the authors stated, ‘It’s time to out everyone who claims that Bleach was Nirvana’s best album. Let there be no arguments: the songwriting, arrangements, creativity and (yes) production is much more impressive on later releases. This is simply a good document of a competent Pacific Northwest rock band circa late 80s. Put Bleach up against any Mudhoney release around this time and see who wins.’
To coincide with the release of Bleach Nirvana appeared at Seattle’s Moore Theatre on 9 June at an event called Lamefest ’89, sharing the bill with local groups Mudhoney and TAD, before taking to the road for a short tour of the United States later that month, commencing on 22 June at San Francisco’s the Covered Wagon, despite barely earning enough money from each show for fuel to drive to the next venue. Novoselic, speaking in Of Grunge & Government said, ‘We toured constantly, driving all over North America, playing from British Columbia, Canada to Baja California, Mexico, Montreal to Florida, Texas and Nebraska, gigging at dozens of little hole in-the-wall clubs. I really enjoyed seeing our vast land, playing shows one town and one night at a time. Small clubs were where people on the outside of the mainstream converged.’
Despite Nirvana having recently released their first album and now touring to promote their music, internal conflicts were putting added pressure on the band. And once again the issue was with the drummer, particularly as far as Cobain was concerned. While the events tend to vary depending on who is telling the story, the common opinion was that Channing was somewhat mistreated and alienated from his bandmates. ‘Sometimes I just felt sorry for Chad,’ Pavitt told Michael Azerad in the biography Come As You Are: The Story of Nirvana. ‘You could tell that…I didn’t feel that they treated him with a lot of respect.’ Elsewhere, Endino concurred, ‘I can’t speculate on why they fired Chad but I think it was personal, that they didn’t get along. Because I think his drumming had come along quite a ways by the time they got rid of him. He had a style he was adding. He was getting there.’ Regarding their decision to fire Channing, Cobain simply told Azerad, ‘He really had bad timing and he wasn’t a very powerful drummer.’
While Cobain may have been openly critical of several former band members, even going as far as telling one interviewer that prior to adding Dave Grohl to the group for the recording of Nevermind ‘all the other drummers we had pretty much sucked,’ Channing himself has remained somewhat tight-lipped and diplomatic about the experience. ‘I have no regrets because I always thought that things just sort of fall in place for a reason. For example, I was that perfect puzzle piece for the band at the time and then they needed another piece to do other things and stuff,’ he explained in a 2018 interview with KaaosTV. ‘Our differences were strictly on a musical level. We always stayed friends. In fact, I remember the first time I saw them with Dave at this place called the OK Hotel. It was the first time I’d seen the guys in probably a year or so and it was really good to see them. It was like, ‘Hey, how’s it going?’ It was very friendly.’
By the time that Channing departed from Nirvana in 1990 the band were already on the verge of landing a major record deal, but without a full-time drummer this seemed highly unlikely. After briefly considering Dan Peters of Mudhoney, who would agree to provide drums on the band’s next single Sliver, Cobain and Novoselic attended a show in San Francisco where a hardcore punk band called Scream were performing. Impressed with the drummer, an energetic and charismatic young man called Dave Grohl, they became determined to entice him into Nirvana.
‘Someone told me who they were and I was thinking, ‘What, that’s Nirvana? Are you kidding?’ Because on the cover of Bleach they looked like psycho lumberjacks,’ Grohl was quoting as saying in Paul Brannigan’s biography This is a Call. ‘But I loved Bleach, I thought it was great. It had everything that I really loved about music. It had the Beatles influence on About a Girl and then songs like Paper Cuts and Sifting were heavy as balls. And Negative Creep was amazing. And girls liked Nirvana.’ Grohl would take to the stage for the first time as an official member of Nirvana on 12 October 1990 at the North Shore Surf Club in Olympia, Washington.
As Rolling Stone would recall twenty-seven years later, ‘In a clip filmed during that performance the band is seen playing to a sweaty, engaged crowd and it showcases their new drummer, shirtless and hair flying as he propels the songs’ frenetic, thunderous beats. Grohl’s live Nirvana premiere included a rendition of Shocking Blue’s Love Buzz, which Nirvana had recorded as a single in 1988. The trio also performed Nirvana’s Scoff and Sliver as well as a cover of Devo’s Turn Around.’ By this point the new line-up of Nirvana had already entered the studio to commence work on what would become their major label debut, having courted the interest of DGC Records. This time taken under the wing of Butch Vig, a producer whose résumé included Gish, the recently-released first album from the Smashing Pumpkins, Cobain, Novoselic and Grohl would divide their time between studios in Wisconsin and California, yet despite the good fortune that had befallen them Cobain proved to be unpredictable. Kurt was awesome to work with but he was incredibly moody,’ recalled Vig in an interview with NME decades later. ‘That was the thing, was to figure out when he was going to be focused or when he was going to disappear into himself.’ Regardless, Grohl’s first release with his new band was the single Smells Like Teen Sprit, which was released through Geffen on 10 September 1991 and would eventually become an anthem for the Generation X teens of the ’90s. Their second studio album Nevermind followed two weeks later. The world was never quite the same again…


text:
- Detection and quantification of circulating immature platelets: agreement between flow cytometric and automated detection. Journal of thrombosis and thrombolysis. 2016 Academic Article GET IT
Renal Denervation: Past, Present, and Future.
Reviews in cardiovascular medicine.
Times cited: 1
- Suicide left ventricle due to conduction disturbance following transcatheter aortic valve replacement and reversal with restoration of sinus rhythm: Is there life after death?. The Journal of invasive cardiology. 2015 Academic Article
- Association of immature platelets with adverse cardiovascular outcomes. Journal of the American College of Cardiology. 2014 Academic Article GET IT
- Museum of TMH Multimodality Imaging Center: Evaluation of mechanical aortic valve endocarditis and fistula formation by cardiac computed tomography. Methodist DeBakey cardiovascular journal. 2014 Academic Article GET IT
Immature platelet fraction (IPF) determined with an automated method predicts clopidogrel hyporesponsiveness.
Journal of thrombosis and thrombolysis.
Times cited: 20


text:
Sunrise Cove - There’s a small island in Sunrise Cove, about 50’ x 50’. It really packs a lot in. There are three separate beaches and a big mountain there, maybe 6 feet tall, with a grassy mushroom top. It looks like something out of Munchkin land. I think it’s big enough to spread a table cloth out for a picnic. You can row our rubber boat there. If you take your time rowing, I think you can cover the distance in about three minutes. There is an aerial picture of Sunrise Cove (below). You can see this little island and its triangular beaches. Just a fun place. This is all part of what you will see as we ride around. If we are doing a mid-day cruise for lunch, that's one of the stopping places.
Swimmin’ Hole – there’s a very smooth, soft sandy beach in Sunset Cove that's almost warm on a sunny day - Depending on what the tide is doing, we can bring our yacht really close to the beach. We can use the dinghy for closer support and general frolicking. If anybody gets the hankerin’ to get wet, you can jump right off the swim step into a foot and a half of mild water. By the way, at the back of the yacht, we have a warm, fresh water outside shower for rinsing off.
Give us a call to schedule your adventure!
Capt. Robin 360-927-8216
I’m not going to describe sunsets in detail to anybody here, but picture 20 miles of glassy water facing west while dining and enjoying the view. If its smooth water you will get two sunsets for the price of one. One in the sky and one on the water You cannot capture this on your big screen at home. All this can be experienced without your colleagues back at the office knowing your where-abouts. If you have the need to communicate, we can usually get phone reception about 75% of the time. I love cell phones (no one knows where you are).
Join us for an afternoon tour or evening sunset cruise and dinner off the North Shore of Orcas Island specializing in the scenic beauty of Sucia Islands State Park. We board for dinner late afternoon and return late sunset to dark.
Leaving from North Shore Orcas Island we offer two journeys:
We leave the marina around 5:00 or 6:00PM for our ride through Sucia Islands State Park. It's a nice tour through all of this sceanery before we settle in for dinner. If it’s calm enough we can stop out in the kelp beds for our main dinner break. There’s nothing quite like stopping out in the middle when it’s calm. I do mean stopped, no engines idling, no generators running, no noise. You may hear heavy breathing outside. This will be sea lions, seals, otters, maybe some porpoises cruising by. They will all look at us to see what’s going on. I like to stop in the kelp beds, lots of critters there.
Keep in mind that this is a very personal cruise. There are typically only 6 of us on board- myself, our chef, and you honeymooners. The flight deck, or up on the sundeck, is the perfect place to watch this all unfold. The flight deck has its own couch and table, stools, etc., it's not like an aircraft cockpit there's plenty of elbow room. The sundeck above has comfortable couches for everybody.
THIS IS YOUR PERSONAL CRUISE. We never mix parties. If you didn't bring them, you
won't meet them. (Except our chef and the Skipper) If there's only two of you, then
that's our party.
Brandt's Landing, Orcas Island
Floating into the Islands
Sandstone formation at Sucia Island.
Views around Sucia Island
Lounge to your heart's content
After dinner sunset wonderment
Beautiful sandstone formation
Arial shot of Sunrise Cove
Dinner at Sunset Cove - We'll settle in for awhile in a place I call Sunset Cove, perfectly calm waters to dine by. Take a look below at the arial view of Sunset Cove (it's really called Shallow Bay, but I like to re-name stuff). The photo shows calm waters and even if a little breeze shows up it won't affect us because we're so sheltered. You can take the skiff over to the beach, if you want, while dinner is cooking. When you gaze into the shallow waters and see a few crabs scurryng around, you will then realize why we have taken you to this truly sacred place.
Mid-day Putt - cruise around with a snack or have lunch. The greater portion of the day shows no sunset. Up to six folks or a small group can take a sightseeing ride around the islands. Lots to see. Food doesn't have to be involved at all, or you can bring your own snacks.


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Tetrapodenia Gleason, Bull. Torrey Bot. Club 53: 289. 1926.—Type: T. glandifera Gleason [=Burdachia sphaerocarpa Adr. Juss.].
Trees or shrubs; stipules intra- and epipetiolar, completely connate, persistent; petiole eglandular; lamina bearing several–many glands in abaxial surface. Inflorescence terminal, single or 2 or 3 together, each usually divided near base into 3 (–5) axes, each axis a raceme of short 1–6-flowered cincinni; bracts and bracteoles persistent, the lowest bracteole and alternate subsequent bracteoles bearing 1 large eccentric abaxial gland; pedicel circinate in bud. Sepals leaving outermost petal exposed in enlarging bud, all biglandular; corolla bilaterally symmetrical, the posterior petal strongly differentiated from the lateral 4, with a thick erect claw and large marginal glands at least on proximal half of limb and often continuing over the apex; petals pink or white, glabrous; limb of outermost petal concave; lateral petals with the margin entire; androecium radially symmetrical, glabrous; stamens 10, all fertile; filaments opposite sepals slightly longer than those opposite petals, connate at base; anthers all ± alike or some larger than others, the locules parallel and rounded at apex and often exceeded by the thick, fleshy connective; pollen radially symmetrical, 3-colporate; receptacle glabrous on both sides of stamens; gynoecium at least apparently radially symmetrical; ovary with the 3 carpels completely connate, 1 ± anterior and 2 ± posterior, 3-locular but 1 of the posterior locules empty and smaller; styles 3, slender and subulate with the stigmas very small, slightly internal and decurrent. Fruit dry, indehiscent, a fibrous or aerenchymatous nut without a bony endocarp, 9–28 mm long, 8–19 mm in diameter, symmetrically spheroidal or conoidal, containing only 1 seed. Chromosome number unknown.
Two or three species in lowland forests near rivers or in low, periodically flooded places, in Guyana and Amazonian Venezuela, Colombia, Brazil, and Peru. The dry indehiscent fruits are well adapted for dispersal by water. [map]
Burdachia is closely related to another genus of Amazonian trees bearing fibrous or aerenchymatous fruits, Glandonia, but it is readily distinguished by its persistent intrapetiolarly connate stipules, glabrous filaments, and anthers with rounded locules and thick, fleshy, apically enlarged connectives. In flower Burdachia can be confused with Byrsonima, but that genus never has glands on the leaves or bracteoles.
Etymology: This genus was named for Karl Friedrich Burdach (1776–1847), professor of medicine at Leipzig, Dorpat, and Königsberg (Wittstein, 1856).

domain: biology