Phenylalanine Incorporation Research Articles

Stimulation of skeletal muscle myofibrillar protein synthesis, p70 S6 kinase phosphorylation, and ribosomal protein S6 phosphorylation by inhibition of myostatin in mature mice

Knocking out myostatin activity during development increases the rate of muscle protein synthesis. The present study was done to determine whether postdevelopmental loss of myostatin activity stimulates myofibrillar protein synthesis and the phosphorylation of some of the proteins involved in regulation of protein synthesis rate. Myostatin activity was inhibited for 4 days, in 4- to 5-mo-old male mice, with injections of an anti-myostatin antibody (JA16). The mean myofibrillar synthesis rate increased 19% (P < 0.01) relative to the mean rate in saline-treated mice, as determined by incorporation of deuterium-labeled phenylalanine. JA16 increased phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6 (rpS6) 1.9-fold (P < 0.05). It did not affect phosphorylation of eukaryotic initiation factor 4E-binding protein-1 or Akt. Microarrays and real-time PCR analyses indicated that JA16 administration did not selectively enrich levels of mRNAs encoding myofibrillar proteins, ribosomal proteins, or translation initiation and elongation factors. Rapamycin treatment did not affect the rate of myofibrillar protein synthesis whether or not the mice received JA16 injections, although it eliminated the phosphorylation of S6K and rpS6. We conclude that the normal level of myostatin activity in mature muscle is sufficient to inhibit myofibrillar synthesis rate and phosphorylation of S6K and rpS6. Reversal of the inhibition of myofibrillar synthesis with an anti-myostatin antibody is not dependent on mTOR activation.

Effect of beta-hydroxy-beta-methylbutyrate (HMB) on protein metabolism in whole body and in selected tissues

Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effect. The aim of the study was to examine the role of exogenous HMB on leucine and protein metabolism in whole body and selected tissues. Rats were administered by HMB (0.1 g/kg b.w.) or by saline. The parameters of whole-body protein metabolism were evaluated 24 h later using l-[1- 14C]leucine and l-[3,4,5- 3H]phenylalanine. Changes in proteasome dependent proteolysis and protein synthesis were determined according the “chymotrypsin-like” enzyme activity and labeled leucine and phenylalanine incorporation into the protein. A decrease in leucine clearance and whole-body protein turnover (i.e., a decrease in whole-body proteolysis and protein synthesis) was observed in HMB treated rats. Proteasome-dependent proteolysis decreased significantly in skeletal muscle, changes in heart, liver, jejunum, colon, kidney, and spleen were insignificant. Decrease in protein synthesis was observed in the heart, colon, kidney, and spleen, while an increase was observed in the liver. There were no significant changes in leucine oxidation. We conclude that protein anabolic effect of HMB in skeletal muscle is related to inhibition of proteolysis in proteasome. Alterations in protein synthesis in visceral tissues may affect several important functions and the metabolic status of the whole body.

Noninvasive Imaging of Protein Metabolic Labeling in Single Human Cells Using Stable Isotopes and Raman Microscopy

We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 cm(-1) spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.

Abstract 382: The Bone Morphogenetic Protein Pathway Mediates the Effects of H11 Kinase/Hsp22 on Cardiac Cell Growth and Survival

The bone morphogenetic protein (BMP) pathway is a major signaling mechanism during cardiac development but it has no clear function in the post-natal heart. Here, we tested the hypothesis that BMP mediates the physiological effect of the cardiac chaperone H11Kinase/Hsp22 (H11K). Expression of H11K increases during both cardiac ischemia and overload, and its cardiac-specific over-expression in a transgenic (TG) mouse is sufficient to provide major protection against ischemia and to promote cardiac cell growth, which involves the activation of phosphatidylinositol-3-kinase (PI3K) and of its effector Akt. We tested whether H11K-induced activation of PI3K is mediated by BMP. Microarray comparison between hearts from TG and wild type (WT) mice showed an up-regulation of the BMP receptor subunits Alk3 and BMPR-II, as well as of the BMP receptor ligand BMP4, which was confirmed at the protein level (P&lt;0.01 vs WT). Activation of the BMP pathway in TG mice was confirmed by increased phosphorylation of the canonical BMP effectors Smad 1/5/8 (P&lt;0.01 vs WT). The mechanism was further studied in isolated cardiac myocytes. Adeno-mediated over-expression of H11K was accompanied by significant 2–3-fold increase in PI3K activity, phospho-Akt, Smad 1/5/8 phosphorylation and cell growth as measured by [3H]phenylalanine incorporation, and by a 70% reduction in H2O2-mediated apoptosis (all values, P&lt;0.01 vs control). All these changes mediated by H11K in myocytes were abolished upon addition of the BMP antagonist noggin. In pull-down experiments, H11K co-precipitated with both Alk3 and BMPR-II, and increased the association of these two subunits into a functional receptor. Accordingly, Smad 1/5/8 phosphorylation in presence of BMP4 was enhanced by 5-fold upon H11K over-expression, whereas it was decreased by 3-fold upon H11K knockdown (both, P&lt;0.01 vs control), which shows that H11K potentiates the BMP receptor signaling pathway. Therefore, potentiation of the BMP receptor pathway by H11K promotes the activation of the PI3K/Akt pathway and dictates the physiological effects of H11K on cardiac cell growth and survival, which shows a novel role for BMP signaling in post-natal heart. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Lats2 Is a Negative Regulator of Myocyte Size in the Heart

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.

Role of HPF (Hibernation Promoting Factor) in Translational Activity in Escherichia coli

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed 'ribosomal hibernation'. Here, the contributions of HPF to 100S formation and translation were analysed in vitro. HPF bound to, but did not dimerize the 70S ribosome. RMF dimerized and formed immature 90S ribosomes. Binding of both HPF and RMF converted 90S ribosomes to mature 100S ribosomes, which is consistent with the in vivo data. The role of HPF in in vitro translation also was investigated. In an artificial mRNA poly (U)-dependent phenylalanine incorporation assay, HPF bound to ribosomal particles and inhibited translation. In contrast, in a natural MS2 mRNA-dependent leucine incorporation assay, bound HPF was removed and hardly inhibited normal translation. Multiple alignment and phylogenetic analyses indicates that the hibernation system mediated by the HPF homologue, RMF and 100S ribosome formation may be specific to the proteobacteria gamma group. In contrast, most bacteria have at least one HPF homologue, and these homologues can be classified into three types, long HPF, short HPF and YfiA.

The role of AMP-activate protein kinase (AMPK) in the regulation of cardiac hypertrophy

Backgrond: The polyphenol resveratrol has been attributed to some of the cardioprotective effects of red wine consumption, including the ability to inhibit hypertrophy via its antioxidant properties. A recent discovery in hepatocytes showed that resveratrol can activate AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. However, it is currently unknown whether resveratrol can also activate cardiac AMPK and the functional consequence of this. As we have shown that activation of AMPK inhibits protein synthesis associated with cardiac hypertrophy, we hypothesized that an additional mechanism by which resveratrol inhibits hypertrophy is via AMPK activation. &#x0D; Methods: To investigate this, neonatal rat cardiac myocytes were treated with vehicle (Veh) or 50 µM resveratrol (Resv) for 24 hours and 10 µM phenylephrine (PE) to induce hypertrophy. Contributors of the hypertrophic response, such as protein synthesis and specific intracellular signalling pathways, were measured using [3H]-phenylalanine incorporation and immunoblot analysis, respectively. &#x0D; Results: Activation of AMPK was achieved with resveratrol even in the presence of phenylephrine, as shown by increased AMPK phosphorylation (Veh+PE = 0.69±0.06 vs Resv+PE = 1.57±0.29 arbitrary units, p &lt; 0.01). Resveratrol also significantly decreased protein synthesis induced by phenylephrine (Veh+PE = 45702.5±5252.9 vs Resv+PE = 32929.0±4512.9 disintegrations per minute, p &lt; 0.05). This was associated with reduced activities of regulators of protein synthesis, namely p70S6 kinase and eukaryotic elongation factor-2 (eEF2). Specifically, resveratrol countered the effects of phenylephrine by blunting the increase in p70S6 kinase phosphorylation while partially restoring eEF2 phosphorylation. Furthermore, resveratrol dramatically decreased nuclear factor of activated T-cells (NFAT) promoter activity, as shown by reduced luciferase activity driven by a NFAT-responsive promoter (Veh+PE = 171100±7881 vs Resv+PE = 27530±5975 luciferase activity, p &lt; 0.001). &#x0D; Conclusions: As the calcineurin-NFAT pathway is thought to be involved in pathological hypertrophy, this provides further evidence that AMPK activation has therapeutic potential, and shows another mechanism by which resveratrol can prevent hypertrophic growth.

Phenylephrine hypertrophy, Ca2+-ATPase (SERCA2), and Ca2+ signaling in neonatal rat cardiac myocytes

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca(2+) transport, and cytoplasmic Ca(2+) signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [(14)C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca(2+)-ATPase protein levels and Ca(2+) transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca(2+) signaling kinetics, measured with a Ca(2+)-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca(2+) signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca(2+) signaling is partially compensated by alternate Ca(2+) transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid,Astronotus ocellatus

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by (3)H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67+/-0.01 mg l(-1) at 28+/-1 degrees C). This depression in RoMR was accompanied by a 50-60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis.

Intestinal ribosomal p70S6Ksignaling is increased in piglet rotavirus enteritis

Recent identification of the mammalian target of rapamycin (mTOR) pathway as an amino acid-sensing mechanism that regulates protein synthesis led us to investigate its role in rotavirus diarrhea. We hypothesized that malnutrition would reduce the jejunal protein synthetic rate and mTOR signaling via its target, ribosomal p70 S6 kinase (p70(S6K)). Newborn piglets were artificially fed from birth and infected with porcine rotavirus on day 5 of life. Study groups included infected (fully fed and 50% protein calorie malnourished) and noninfected fully fed controls. Initially, in "worst-case scenario studies," malnourished infected piglets were killed on days 1, 3, 5, and 11 postinoculation, and jejunal samples were compared with controls to determine the time course of injury and p70(S6K) activation. Using a 2 x 2 factorial design, we subsequently determined if infection and/or malnutrition affected mTOR activation on day 3. Western blot analysis and immunohistochemistry were used to measure total and phosphorylated p70(S6K); [(3)H]phenylalanine incorporation was used to measure protein synthesis; and lactase specific activity and villus-crypt dimensions were used to quantify injury. At the peak of diarrhea, the in vitro jejunal protein synthetic rate increased twofold (compared with the rate in the uninfected pig jejunum), concomitant with increased jejunal p70(S6K) phosphorylation (4-fold) and an increased p70(S6K) level (3-fold, P < 0.05). Malnutrition did not alter the magnitude of p70(S6K) activation. Immunolocalization revealed that infection produced a major induction of cytoplasmic p70(S6K) and nuclear phospho-p70(S6K), mainly in the crypt. A downregulation of semitendinosus muscle p70(S6K) phosphorylation was seen at days 1-3 postinoculation. In conclusion, intestinal activation of p70(S6K) was not inhibited by malnutrition but was strongly activated during an active state of mucosal regeneration.

Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy

AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADalpha) and MO25alpha. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADalpha/MO25alpha expression, LKB1 activity, and AMPKalpha phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADalpha/MO25alpha complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [(3)H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.

Deletion of a Conserved, Central Ribosomal Intersubunit RNA Bridge

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

Hypertrophic growth in cardiac myocytes is mediated by Myc through a Cyclin D2-dependent pathway

c-Myc (Myc) is highly expressed in developing embryos where it regulates body size by controlling proliferation but not cell size. However, Myc is also induced in many postmitotic tissues, including adult myocardium, in response to stress where the predominant form of growth is an increase in cell size (hypertrophy) and not number. The function of Myc induction in this setting is unproven. Therefore, to explore Myc's role in hypertrophic growth, we created mice where Myc can be inducibly inactivated, specifically in adult myocardium. Myc-deficient hearts demonstrated attenuated stress-induced hypertrophic growth, secondary to a reduction in cell growth of individual myocytes. To explore the dependence of Myc-induced cell growth on CycD2, we created bigenic mice where Myc can be selectively activated in CycD2-null adult myocardium. Myc-dependent hypertrophic growth and cell cycle reentry is blocked in CycD2-deficient hearts. However, in contrast to Myc-induced DNA synthesis, hypertrophic growth is independent of CycD2-induced Cdk2 activity. These data suggest that Myc is required for a normal hypertrophic response and that its growth-promoting effects are also mediated through a CycD2-dependent pathway.

Effects of environmental hypercapnia on animal physiology: A 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus

Global climate change is associated with a progressive rise in ocean CO 2 concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO 2 stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of 13C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10–15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO 2 concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.

Agonist‐specific activation of the β2‐adrenoceptor/Gs‐protein and β2‐adrenoceptor/Gi‐protein pathway in adult rat ventricular cardiomyocytes

In rat ventricular cardiomyocytes beta2-adrenoceptors (AR) couple to Gs- and Gi-protein, and evidence has accumulated that beta2-AR agonists can differentially activate either Gs- or Gs- and Gi-protein. In this study, in isolated adult rat ventricular cardiomyocytes, we assessed the effects of pertussis toxin (PTX) on beta2-AR agonist (terbutaline (TER), salbutamol (SAL) and fenoterol (FEN)) evoked inhibition of phenylephrine (PE)-induced increase in the rate of protein synthesis (assessed as [3H]phenylalanine incorporation) to find out which beta2-AR agonist activates selectively Gs- or Gs- and Gi-protein. PE (1 microM) increased the rate of protein synthesis from 100% to 130+/-2% (n = 34). FEN, TER and SAL (1 nM-10 microM) inhibited PE-induced increase in the rate of protein synthesis concentration-dependently. FEN inhibited PE effects almost completely (from 132+/-3 to 101+/-1%), whereas TER and SAL caused only partial inhibition (from 131+/-2 to 114+/-2 and 129+/-1 to 111+/-2%, respectively). Pretreatment of cardiomyocytes with PTX (250 ng ml(-1) for 16 h at 37 degrees C) did not affect FEN effects, but converted TER- and SAL-evoked partial inhibition into complete inhibition. Inhibitory effects of the three beta2-AR agonists were markedly attenuated by beta1-AR selective antagonist CGP 20712A (CGP) (300 nM); in contrast, beta2-AR selective antagonist ICI 118,551 (55 nM) inhibited the inhibitory effects of the three beta2-AR agonists only in PTX-pretreated cardiomyocytes,with beta1-AR blocked by CGP. We conclude that, in adult rat ventricular cardiomyocytes, FEN activates selectively the Gs protein-pathway, while TER and SAL activate the Gs- and Gi-protein pathways. Part of the effects of these three beta2-AR agonists appears to be mediated by beta1-AR.

The Cyclization of High Diversity mRNA Display Libraries

The cyclization of linear peptide sequences shows enormous promise for the development of novel pharmacaphores, owing in part to the loss in conformational entropy upon cyclization. Directed evolution of cyclic peptides in a biological display context has previously been limited to disulfide formation as the sole means to generate peptide macrocycles. The primary disadvantage of this methodology is the inherent instability of the disulfide bond in a reducing intracellular environment. To address this, we have developed a chemical approach to generating covalently cyclized mRNA display libraries based on the cross-linking of primary amines in the linear peptide sequence. MALDI-TOF MS has been employed to verify the specificity of the post-translational cross-linking reaction in a model mRNA- peptide fusion. The incorporation of α-hydroxy phenylalanine via nonsense suppression was used to generate a second model peptide fusion, whose cyclization efficiency could be assayed by hydrolysis of the non-natural ester linkage. Finally, this methodology was used to construct a series of covalently cyclized libraries of varying lengths whose cyclization efficiencies could be directly measured. The largest of these libraries contains >1013 sequences and was found to be ~30% cyclized, effectively the largest cyclic library assembled in a biological display context.

300 GLUCOSAMINE SUPPLEMENTATION DURING IN VITRO MATURATION LEADS TO PERTURBED DEVELOPMENTAL CAPACITY OF BOVINE CUMULUS OOCYTE COMPLEXES

Glucose is a primary energy substrate required for successful in vitro oocyte maturation (IVM). However, most maturation media contain more glucose than that seen in follicular fluid (2.3 mM vs. 5.6 mM in TCM199). Glucosamine (Glc) as an alternative substrate for extracellular matrix during cumulus expansion reduced glucose uptake by bovine cumulus oocyte complexes (COCs, Sutton-McDowall et al. 2004 Reproduction 128, 313–319). As this could enable a reduction in glucose concentrations to physiological levels in IVM medium of COCs, the aim of this study was to investigate the influence of Glc supplementation on oocyte developmental capacity. Bovine COCs were matured in synthetic follicular fluid medium (SFFM, a defined medium based on the composition of follicular fluid, plus 5.6 mM glucose, FSH, hCG and BSA, Sutton-McDowall et al. 2004 Reprod. Fertil. Dev. 16 sup, 204) ± 5 mM Glc. After 24 h, either nuclear maturation (rep = 8, n = 160) or blastocyst development 8 days post-fertilization (rep = 5, n = 400) was determined. Data was arcsine transformed and analyzed by ANOVA, followed by Tukey's test. While the presence of Glc did not affect the completion of nuclear maturation and early cleavage, +Glc led to severely perturbed blastocyst development (−Glc, 32.5 ± 1.9% vs. +Glc, 4.7 ± 3.9%, P &lt; 0.001). Glc supplementation in somatic cells is well-known to down-regulate the phosphatidylinositol-3-kinase (PI3K) signalling pathway, reducing protein synthesis and other cell survival mechanisms. Therefore, oocyte protein synthesis (measured by [2,3,4,5,6-3H] phenylalanine incorporation, rep = 5, n = 200) and embryo development (rep = 6, n = 720) following IVM in SFFM ± Glc ± EGF (a PI3K pathway stimulator) was determined. Glc supplementation led to a 40% decrease in protein synthesis compared to −Glc, while the combination of +Glc + EGF significantly increased protein synthesis by 60%. However, IVM + EGF + Glc did not improve blastocyst rates (main effect: −Glc 41.6 ± 6.6% vs. +Glc, 6.6 ± 1.7%, P &lt; 0.001). Additionally, COCs were also cultured in SFFM ± 50 μM LY294002 (a specific PI3K inhibitor) and nuclear maturation (rep = 5, n = 200) or blastocyst development 8 days post-fertilization (rep = 4, n = 200) was determined. Despite the presence of LY294002 leading to 43% less COCs completing nuclear maturation (P &lt; 0.001), blastocyst development was not affected (mean = 38.8 ± 3.2%). These results demonstrate that Glc supplementation during IVM has no effect on nuclear maturation or early development but is detrimental to oocyte developmental capacity by severely perturbing blastocyst development. However, the diminished developmental capacity appears to be independent of the well-characterized Glc down-regulation of the PI3K signalling pathway. This work was supported by the Australian Research Council (SPIRT, C00107702) and Cook Australia Pty Ltd.

Aldosterone directly stimulates cardiac myocyte hypertrophy

Background Clinical and experimental studies suggest that aldosterone modulates myocardial hypertrophy. From in vivo studies, it is not possible to distinguish between direct actions on myocyte growth and effects of mechanical load. In this study we tested the hypothesis that aldosterone induces myocyte hypertrophy in low-density, serum-free cultures of neonatal rat ventricular myocytes. Methods and results Hypertrophy was quantified by [ 14C]-phenylalanine incorporation and confocal microscopic assessment of myocyte surface area. Aldosterone caused a 27% increase in protein incorporation (EC 50 = 40 nmol/L) and a 29% increase in myocyte surface area compared with the vehicle control. This response was associated with increased mRNA levels of atrial natriuretic factor, α- and β-myosin heavy chain measured by RNase protection assay, and it was suppressed by the mineralocorticoid receptor blocker spironolactone. Analysis of early signaling events showed that aldosterone stimulation acutely translocated protein kinase C (PKC)-α to the membrane fraction and increased the levels of phosphorylated ERK1/2 and JNK. PD 98059, an inhibitor of the ERK activator MEK (mitogen-activated protein kinase kinase) and bisindolylmaleimide I, an inhibitor of PKC activation, each blocked aldosterone-stimulated hypertrophy. Conclusion Aldosterone directly stimulates hypertrophy in neonatal rat ventricular myocytes. The growth response is dependent on the mineralocorticoid receptor and is associated with activation of ERK, JNK, and PKC-α.

The ribosome-associated inhibitor A reduces translation errors

Recently we have reported about a novel stress response protein (pY or RaiA) associated with Escherichia coli ribosomes that inhibits translation at the aminoacyl-tRNA binding stage. Here we show that leucine misincorporation during in vitro poly(U) translation is inhibited by this protein much stronger than the incorporation of phenylalanine. The miscoding counteraction by RaiA is especially strong at the concentrations of magnesium ions close to those observed in vivo and diminishes at higher magnesium concentrations. The results obtained suggest that the anti-miscoding activity of RaiA could be the main function of the protein, rather than the inhibition of translation. The role of the protein in adaptation of cells to environmental stress is discussed.

Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus

In the renal hypertrophy that occurs in diabetes mellitus, decreased proteolysis may lead to protein accumulation, but it is unclear which proteins are affected. Because the lysosomal proteolytic pathway of chaperone-mediated autophagy is suppressed by growth factors in cultured cells, we investigated whether the abundance of substrates of this pathway increase in diabetic hypertrophy. Rats with streptozotocin (STZ)-induced diabetes were pair-fed with vehicle-injected control rats. Proteolysis was measured as lysine release in renal cortical suspensions and protein synthesis as phenylalanine incorporation. Target proteins of chaperone-mediated autophagy were measured in cortical lysates and nuclear extracts by immunoblot analysis. Proteins that regulate chaperone-mediated autophagy [the lysosomal-associated membrane protein 2a (LAMP2a) or the heat shock cognate protein of 73 kD (hsc-73)] were measured in lysosomes isolated by density gradient centrifugation. Proteolysis decreased by 41% in diabetic rats; protein synthesis increased at 3 days, but returned to baseline by 7 days. The abundance of proteins containing that chaperone-mediated autophagy KFERQ signal motif increased 38% and individual KFERQ containing proteins [e.g., M2 pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pax2] were more abundant. LAMP2a and hsc73 decreased by 25% and 81%, respectively, in cortical lysosomes from diabetic vs. control rats. The decline in proteolysis in acute diabetes mellitus is associated with an increase in proteins degraded by chaperone-mediated autophagy and a decrease in proteins which regulate this pathway. This study provides the first evidence that reduced chaperone-mediated autophagy contributes to accumulation of specific proteins in diabetic-induced renal hypertrophy.