Leucine

The mechanism of human tumor resistance to the antineoplastic drug bleomycin (BLM) is not known. We now provide evidence implicating metabolic inactivation in the resistance of human Burkitt's (Daudi) lymphoma to BLM. Daudi lymphoma and human head and neck squamous cell carcinoma (A-253) cells grown (s.c.) in nude mice were found to be resistant and sensitive to BLM treatment, respectively. Within l h of s.c. injection of (3H]BLM A2,Daudi xenografts accumulated less BLM and metabolized this drug to a much greater extent than did A-253 xenografts. The BLM-resistant Daudi xenografts metabolized BLM A2 to at least 6 metabolites and only a small proportion of the drug remained as unmetabolized BLM A2. In the BLM-sensitive A-253 xenografts, however, BLM A2 remained the major component. Incubation of BLM \; with Daudi cytosolic fractions resulted in a complex mixture of metabolites similar to that formed by Daudi xenografts in nude mice. This BLM metabolite mixture was biologically inactive in plasmili DNA degradation assays. Treatment of mice bearing Daudi xenografts with an inhibitor of BLM hydrolase, L-fra/w-epoxysuccinyl-leucylamido-(4guanidino)butane (E-64), prior to | 'I I|HI M A2 treatment did not affect the amount of BLM accumulated but inhibited BLM A2 metabolism in the xenografts. Furthermore, although E-64 alone did not inhibit the growth of Daudi xenografts, it potentiated the antitumor activity of BLM. These results indicate that Daudi tumors resist BLM by metabolically inactivating it and that inhibition of BLM metabolism in vivo enhances the antitumor activity of BLM and hence overcomes resistance.

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is ...

There is evidence that changes in branched-chain amino acid (BCAA) levels may correlate with the efficacy of therapeutic interventions for affecting improvement in metabolic control. The objective of this study was to evaluate whether serum concentrations of BCAAs (leucine, isoleucine, valine) could mediate in insulin sensitivity and glucose tolerance after continuous positive airway pressure (CPAP) treatment in patients with obstructive sleep apnea (OSA). A prospective randomized controlled trial of OSA patients with morbid obesity was conducted. Eighty patients were randomized into two groups: 38 received conservative treatment and 42 received CPAP treatment for 12 weeks. Plasma levels of BCAA, glucose tolerance and insulin resistance were evaluated at baseline and after treatment. After treatment, significant decreases of leucine levels were observed in both groups when compared with baseline levels (P < 0.005). With respect to patients with normal glucose tolerance (NGT), pat...

All patients were assessed clinically at Agia Sofia Hospital in Athens. They belonged to three unrelated families from different regions of Greece. Their parents were healthy and nonconsanguineous. All pregnancies and perinatal periods were uneventful. Their ...

... VIROLOGY 71, 134-142 (1976) Cellular Pools of Viral Proteins in 3T3 Cells Chronically Infected with Moloney-Murine Leukemia Virus JES FORCHHAMMER, BJ RN A ... NOMUBA, S., FISCHINGER, PJ, MATTERN, CFT, PEEPLES, PT, BASSIN, RH, and FRIEDMAN, GP (1972). ...

The unimolecular chemistry of [Cu(II)AA(AA – H)]+ complexes, composed of an intact and a deprotonated amino acid (AA) ligand, have been probed in the gas phase by tandem and multistage mass spectrometry in an electrospray ionization quadrupole ion trap mass spectrometer. The amino acids examined include Gly, Ala, Val, Leu, Ile, t-Leu and Phe. Upon collisionally-activated dissociation (CAD), the [Cu(II)AA(AA – H)]+ complexes undergo decarboxylation with simultaneous reduction of Cu(II) to Cu(I); during this process, a radical site is created at the α-carbon of the decarboxylated ligand (H2N1 – •CαH – CβH2 – R; R = side chain substituent). The radical site is able to move along the backbone of the decarboxylated amino acid to form two new radicals (HN1• – CαH2 – CβH2–R and H2N1 – CαH2 – •CβH – R). From the complexes of Gly and t-Leu, only Cα and N1 radicals can be formed. The whole radical ligand can be lost to form [Cu(I)AA]+ from these three isomeric radicals. Alternatively, further...

Protein intake is essential to maximally stimulate muscle protein synthesis, and the amino acid leucine seems to possess a superior effect on muscle protein synthesis compared to other amino acids. Native whey has higher leucine content and thus a potentially greater anabolic effect on muscle than regular whey (WPC-80). This study compared the acute anabolic effects of ingesting 2 × 20 g of native whey protein, WPC-80 or milk protein after a resistance exercise session. 24 young resistance trained men and women took part in this double blind, randomized, partial crossover, controlled study. Participants received either WPC-80 and native whey ( = 10), in a crossover design, or milk ( = 12). Supplements were ingested immediately (20 g) and two hours after (20 g) a bout of heavy-load lower body resistance exercise. Blood samples and muscle biopsies were collected to measure plasma concentrations of amino acids by gas-chromatography mass spectrometry, muscle phosphorylation of p70S6K, 4...

Background: An important aspect of the link between estrogen and breast cancer is whether urinary estrogen levels are representative of the intra-tissue levels of bioavailable estrogens. Methods: This study compares 15 estrogen and estrogen metabolite levels in breast tissue and urine of 9 women with primary breast cancer using a quantitative liquid chromatography-mass spectrometry method. Results: The average levels of estrogens (estrone, 17 beta-estradiol) were significantly higher in breast tissue than in urine. Both the 2 and the 16-hydroxylation pathways were less represented in breast tissue than urine; no components of the 4-hydroxypathway were detected in breast tissue, while 4-hydroxyestrone was measured in urine. However, the 2/16 ratio was similar in urine and breast tissue. Women carrying the variant CYP1B1 genotype (Leu/Val and Val/Val) showed significantly lower overall estrogen metabolite, estrogen, and 16-hydroxylation pathway levels in breast tissue in comparison to women carrying the wild type genotype. No effect of the CYP1B1 polymorphism was observed in urinary metabolites. Conclusions: The urinary 2/16 ratio seems a good approximation of the ratio observed in breast tissue. Metabolic genes may have an important role in the estrogen metabolism locally in tissues where the gene is expressed, a role that is not readily observable when urinary measurements are performed.

Background: The “leucine trigger” hypothesis was originally conceived to explain the post-prandial regulation of muscle protein synthesis (MPS). This hypothesis implicates the magnitude (amplitude and rate) of post-prandial increase in blood leucine concentrations for regulation of the magnitude of MPS response to an ingested protein source. Recent evidence from experimental studies has challenged this theory, with reports of a disconnect between blood leucine concentration profiles and post-prandial rates of MPS in response to protein ingestion.

Aim: The primary aim of this systematic review was to qualitatively evaluate the leucine trigger hypothesis to explain the post-prandial regulation of MPS in response to ingested protein at rest and post-exercise in young and older adults. We hypothesized that experimental support for the leucine trigger hypothesis will depend on age, exercise status (rest vs. post-exercise), and type of ingested protein (i.e., isolated proteins vs. protein-rich whole food sources).

Methods: This qualitative systematic review extracted data from studies that combined measurements of post-prandial blood leucine concentrations and rates of MPS following ingested protein at rest and following exercise in young and older adults. Data relating to blood leucine concentration profiles and post-prandial MPS rates were extracted from all studies, and reported as providing sufficient or insufficient evidence for the leucine trigger hypothesis.

Results: Overall, 16 of the 29 eligible studies provided sufficient evidence to support the leucine trigger hypothesis for explaining divergent post-prandial rates of MPS in response to different ingested protein sources. Of these 16 studies, 13 were conducted in older adults (eight of which conducted measurements post-exercise) and 14 studies included the administration of isolated proteins.

Conclusion: This systematic review underscores the merits of the leucine trigger hypothesis for the explanation of the regulation of MPS. However, our data indicate that the leucine trigger hypothesis confers most application in regulating the post-prandial response of MPS to ingested proteins in older adults. Consistent with our hypothesis, we provide data to support the idea that the leucine trigger hypothesis is more relevant within the context of ingesting isolated protein sources rather than protein-rich whole foods. Future mechanistic studies are warranted to understand the complex series of modulatory factors beyond blood leucine concentration profiles within a food matrix that regulate post-prandial rates of MPS.

Leucine tracer has been widely used for examining whole-body protein turnover in humans, but has not been evaluated as an indicator to be used in the indicator amino acid oxidation (IAAO) method. The goal of this study was to determine whether the L-[1-(13C)]leucine isotope is an acceptable indicator by comparing it with an established tracer, L-[1-(13C)]lysine. Healthy men (n = 7; 29.9 +/- 4.8 y old) were fed in random order a diet with 7 graded intakes of phenylalanine without tyrosine. In the first study (n = 5), subjects were administered an excess leucine intake of 65 mg/(kg.d), and in the second study (n = 5), they were given the mean requirement of 45 mg/(kg.d) to determine whether leucine intake affected the pattern of response. Previous IAAO studies using lysine and phenylalanine demonstrated a clear pattern in 13CO2 production, i.e., increasing test amino acid intake resulted in a linear decrease to plateau, with a readily discernable breakpoint indicating the requirement....

Dysregulated anabolic responses to nutrition/exercise may contribute to sarcopenia; however, these characteristics are poorly defined in female populations. We determined the effects of two-feeding regimes in older women (66±2.5y N=8/group): bolus-whey protein (WP-20 g) or novel low-dose leucine-enriched essential amino acids (EAA) (LEAA-3g [40% leucine]). Using (13)C6-Phenylalanine infusions, we quantified muscle (MPS) and albumin (APS) protein synthesis at baseline and both in response to feeding (FED) and feeding-plus-exercise (FED-EX; RE: 6×8 knee-extensions at 75%-1RM). We also quantified plasma insulin/AA concentrations, whole-leg (LBF)/muscle microvascular blood-flow (MBF), and muscle anabolic signaling by phospho-immunoblotting. Plasma insulinemia and EAA-aemia were markedly greater after WP than LEAA (P<0.001). Neither LEAA nor WP modified LBF in response to FED or FED-EX, while MBF increased to a similar extent in both groups, only after FED-EX (P<0.05). In response ...

Adsorption of colicin B to a sensitive strain of Escherichia coli results in rapid cessation of deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Some classes of mutants insensitive to colicin B hyperexcrete a colicin inhibitor into their growth medium. This inhibitor functions by preventing adsorption of colicin B and does not rescue cells to which colicin has already adsorbed. The inhibitor is insensitive to nucleases, proteolytic enzymes, and lysozyme and is not extracted into organic solvents. The inhibitory material has a low molecular weight, which rules out identification as lipopolysaccharide, although purified lipopolysaccharide has some inhibitory activity. Evidence is presented that the inhibitor is enterochelin, an iron chelator which is the cyclic trimer of 2,3-dihydroxybenzoylserine. Enterochelin does not inhibit colicin M, a colicin that is produced by many strains colicinogenic for colicin B.

Branched chain amino acids (BCAA) have been shown to have a general protective effect on the heart in different animal models as well as in humans. However, so far no attempt has been made to specifically elucidate their influence on arrhythmias. Our study was performed to evaluate whether an infusion of either l-leucine or l-valine in a dose of 1 mg kg −1 h −1 10 min before a 7-min period of left anterior descending artery occlusion followed by 15 min of reperfusion, had an effect on arrhythmias measured during the reperfusion phase in the ischemia-and reperfusion-induced arrhythmias model in rats in vivo. The effect of the infusion of these substances on mean arterial blood pressure was monitored throughout the experiment. Both of the tested amino acids exhibited significant antiarrhythmic properties. l-Leucine reduced the duration of ventricular fibrillation (P < 0.05) and l-valine decreased the duration of ventricular fibrillation (P < 0.001) and ventricular tachycardia (P < 0.05). The two amino acids were generally hypotensive. l-Valine lowered blood pressure in all phases of the experiment (P < 0.05) while l-leucine lowered this parameter mainly towards the end of occlusion and reperfusion (P < 0.05). In addition, 30 min infusion of the amino acids in the used dose did not produce any apparent adverse histological changes that were remarkably different from control. In summary, the results of our study suggest that l-leucine and l-valine in the dose that was used attenuates arrhythmias and are hypotensive in their influence. Our findings lend support to the many ongoing investigations into the benefit of the application of l-leucine and l-valine in cardiology like their addition to cardioplegic solutions.

The objective of the present study was to evaluate the antibacterial properties of a plant secondary metabolite - caffeine. Caffeine is present in over 100 plant species. Antibacterial activity of caffeine was examined against the following plant-pathogenic bacteria: Ralstonia solanacearum (Rsol), Clavibacter michiganesis subsp. sepedonicus (Cms), Dickeya solani (Dsol), Pectobacterium atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pseudomonas syringae pv. tomato (Pst), and Xanthomonas campestris subsp. campestris (Xcc). MIC and MBC values ranged from 5 to 20 mM and from 43 to 100 mM, respectively. Caffeine increased the bacterial generation time of all tested species and caused changes in cell morphology. The influence of caffeine on the synthesis of DNA, RNA and proteins was investigated in cultures of plant pathogenic bacteria with labelled precursors: [(3)H]thymidine, [(3)H]uridine or (14)C leucine, respectively. RNA biosynthesis was more affected than D...

Flow cytometry was used to characterize bacterioplankton from two tropical environments in Brazil: the eutrophic Guanabara Bay and the oligotrophic southwest Atlantic Ocean. Bacterial abundance was evaluated by flow cytometry, and cells were stained with SYTO 13, allowing demonstration of differences in nucleic acid content. Bacterial production was also evaluated by means of 3 H-leucine incorporation. Bacterial numbers were different for both sites. In Atlantic Ocean samples, we found a maximum of 5.50 Â 10 5 cells ml À 1 , and low nucleic acid content organisms predominated. In Guanabara Bay, bacterial numbers were one order of magnitude higher than in the ocean, and they varied from outer bay (1.01 Â 10 6 cells ml À 1) to inner bay (6.90 Â 10 6 cells ml À 1). Bacterial activity in ocean samples varied from 4.6 to 126 ng C l À 1 h À 1 , while in the bay, mean values ranged from 1.95 Ag C l À 1 h À 1 (outer bay) to 7.35 Ag C l À 1 h À 1 (inner bay). Values found for both parameters are characteristic of different trophic situations. These results illustrate the utility of cytometric analyses of bacterioplankton populations in characterizing their large spatial and temporal scales of distribution in aquatic ecosystems.

Background: Anabolic resistance is the inability to increase protein synthesis in response to an increase in amino acids following a meal. One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress. The purpose of the present study was to test whether ER stress impairs the response to growth factors and leucine in muscle cells.

Background Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects. Methods We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM). Results...

We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a generich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many c...

We sought to determine if decrements in the mass of fat-free body mass (FFM) and other lean tissue compartments, and related changes in protein metabolism, are appropriate for weight loss in obese older women. Subjects were 14 healthy weight-stable obese (BMI ≥30 ...

A rapid procedure for the recovery of Saccharomyces cerevisiae auxotrophs was developed by exploiting the protection of these mutants from thymineless death when a required metabolite was withheld. The method can be used for thymidine 5&#39;-monophosphate-requiring auxotrophs or wild-type strains blocked in de novo synthesis of thymidylate by folate antagonists.

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT 4 receptor. However the AT 4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT 4 receptor ligands, including Ang IV, Nle 1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leub-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT 4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT 4 ligands also displaced [ 125 I]-Nle 1-Ang IV or [ 125 I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT 4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT 4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Adult male Drosophila subobscu~u were pulse-labelled with aH leucine, and radioactivity incorporated into protein measured for 20 days after the pulse label, by extracting total protein and by autoradiography. The halflife of these proteins which turn over in the adult is approximately 10 days. The rate of synthesis varies greatly between tissues, being greatest in the wall of the mid-gut and almost zero in thoracic flight muscle. Larvae were raised axenically in a medium containing labelled amino acids, and the radioactivity in protein followed for 60 days after emergence by extracting total protein, and for 25 days by autoradiographs. The activity in total protein declined rapidly during the first 4 days after emergence of adults, and then remained constant. Autoradiographs suggest that the initial drop is largely due to the disappearance of the pupal fat body, which contains a large amount of protein at emergence. Ignoring storage protein in the fat body, approximately 20 per cent of the total protein in the adult male turns over, with a half-life of approximately 10 days, and the remaining 80 per cent is not replaced. Among the non-replaced proteins are the structural and mitochondrial proteins of the flight muscles. There is active synthesis of ribosomal proteins.

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavindependent oxidation of proline to ∆ 1-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K i) 30 mM), L-lactate (K i) 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K i) 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 Å. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 Å from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k cat and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.

Proteolysis during ripening of reduced fat Cheddar cheeses made with different exopolysaccharide (EPS)producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming nonropy and moderately ropy strains of Streptococcus thermophilus were used in making reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making full-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reducedfat cheeses. Cheese made with JFR1 contained higher moisture, moisture in the nonfat substance, and residual coagulant activity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 than in all other cheeses. The HPLC analysis showed a significant increase in hydrophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which contained lower moisture and moisture in the nonfat substance levels and lower chymosin activity than did cheese made with JFR1, accumulated less hydrophobic peptides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar cheese with moisture in the nonfat substance similar to that in its full-fat counterpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar

The branched-chain amino acid (BCAA) leucine acts as both a 'trigger' for the initiation of protein synthesis, and as a substrate for newly synthesized protein. • As a BCAA, leucine can be metabolized within skeletal muscle, leaving open the possibility that leucine metabolites might possess anabolic properties. • One metabolite in particular, β-hydroxy-β-methylbutyrate (HMB), has shown positive effects on lean body mass and strength following exercise, and in disease-related muscle wasting, yet its impact on acute human muscle protein turnover is undefined. • We report here that HMB stimulates muscle protein synthesis to a similar extent to leucine. HMB was also found to decrease muscle protein breakdown. • Our observation that HMB enhances muscle protein anabolism may partly (or wholly) underlie its pre-defined anabolic/anti-catabolic supplemental efficacy in humans.

To determine the effect of glucose availability on glutamine metabolism, glutamine kinetics were assessed under conditions of hyperglycemia resulting from 1) intravenous infusion of 7.5% dextrose in healthy adults and 2) insulin deficiency in young adults with insulin-dependent diabetes mellitus (IDDM). Eight healthy adults and five young adults with IDDM were studied in the postabsorptive state by use of a primed continuous infusion ofd-[U-14C]glucose,l-[5,5,5-2H3]leucine, andl-[3,4-13C]glutamine. Whether resulting from insulin deficiency or dextrose infusion, the rise in plasma glucose was associated with increased glucose turnover (23.5 ± 0.7 vs. 12.9 ± 0.3 μmol · kg−1 · min−1, P &lt; 0.01 and 20.9 ± 2.5 vs. 12.8 ± 0.4 μmol · kg−1 · min−1, P = 0.03, in health and IDDM, respectively). In both cases, high blood glucose failed to alter glutamine appearance rate (Ra) into plasma [298 ± 9 vs. 312 ± 14 μmol · kg−1 · h−1, not significant (NS) and 309 ± 23 vs 296 ± 26 μmol · kg−1 · h−1, ...

The ability to discriminate and respond to chemical signals from the environment is an almost universal prerequisite for survival. Here, we report that the scaffold protein Scribble is essential for odor-guided behavior in Drosophila. Previously, we identified a P-element insert line with generalized sexually dimorphic smell impairment, smi97B. We found that the transposon in this line is located between the predicted promoter region and the transcription initiation site of scrib. A deficiency in this region, Df(3R)Tl-X, and two scrib null alleles fail to complement the smell-impaired phenotype of smi97B. Wild-type behavior is restored by precise excision of the P element, scrib mRNA levels correspond with mutant and wild-type phenotypes, and introduction of a full-length scrib transgene in the smi97B mutant rescues the olfactory deficit. Expression of Scrib is widespread in olfactory organs and the central nervous system. Finally, alternative splicing of scrib generates transcripts...

Quorum sensing is a process of bacterial cell-cell communication that relies on the production, release, and receptor-driven detection of extracellular signal molecules called autoinducers. The quorum-sensing bacterium Vibrio harveyi exclusively detects the autoinducer N-((R)-3hydroxybutanoyl)-L-homoserine lactone (3OH-C4 HSL) via the two-component receptor LuxN. To discover the principles underlying the exquisite selectivity LuxN has for its ligand, we identified LuxN mutants with altered specificity. LuxN uses three mechanisms to verify that the bound molecule is the correct ligand: In the context of the overall ligand-binding site, His210 validates the C 3 modification, Leu166 surveys the chain-length, and a strong steady-state kinase bias imposes an energetic hurdle for inappropriate ligands to elicit signal transduction. Affinities for the LuxN Kinase on and Kinase off states underpin whether a ligand will act as an antagonist or an agonist. Mutations that bias LuxN to the agonized, Kinase off , state are clustered in a region adjacent to the ligand-binding site, suggesting that this region acts as the switch that triggers signal transduction. Together, our analyses illuminate how a histidine sensor kinase differentiates between ligands and exploits those differences to regulate its signaling activity.

The intake of approximately 70 g of alcohol impairs liver protein metabolism in healthy humans. To establish the threshold at which alcohol impairs hepatic protein metabolism in humans we compared the effects of 500 mL of water (control study), 300 (28.4 g ethanol) or 750 mL (71 g ethanol) of table wine on hepatic protein metabolism in three groups of healthy nonalcoholic volunteers. Hepatic protein metabolism was estimated (L-[1-14C]leucine infusion) by measuring the fractional secretory rates of albumin and fibrinogen during the overnight postabsorptive state (basal) and the subsequent administration of water or two different amounts of wine (300 or 750 mL) given with a liquid glucose-lipid-amino acid meal. During the meal, water did not affect fibrinogen fractional secretory rate and increased albumin fractional secretory rate by approximately 50% (P &lt; 0.01). The 300 mL of wine increased albumin secretory rate by only approximately 20% (P &lt; 0.01 vs. basal, P &lt; 0.04 vs. w...

Background: The ELAC2 gene has been proposed to be a prostate cancer susceptibility gene and is being referred to as HPC2, in part because three case–control studies suggested that two common polymorphisms (Ser217Leu and Ala541Thr) are associated with risk. However, four subsequent larger studies have not confirmed this association. In five of the seven total studies, subject selection was influenced by prostate-specific antigen (PSA) levels. We examined the association and possible effect of subject selection in a larger study and a meta-analysis. Methods: In a population-based study in Australia, 825 case patients and 732 control subjects were genotyped for the Ser217Leu and Ala541Thr polymorphisms of ELAC2. Odds ratios (ORs) for prostate cancer were estimated by unconditional logistic and polytomous regression. A meta-analysis was conducted combining our data with those from seven published studies. The association of genotype with the logarithm of plasma PSA levels in control su...

L-type amino acid transporter 1 Cholangiocarcinoma 4F2hc miR-7 Currently, there is no effective treatment for cholangiocarcinoma (CCA), which is the most prevalent in the northeastern part of Thailand. A new molecular target for the treatment of CCA is, therefore, urgently needed. Although L-type amino acid transporter 1 (LAT1) is highly expressed in CCA cells, its role in malignant phenotypes of CCA cells remains unclear. This study aimed to investigate the impact of LAT1 on proliferation, migration, and invasion of KKU-M213 cells, the CCA cells derived from Thai patients with intrahepatic cholangiocarcinoma. Results showed that KKU-M213 cells expressed all LAT isoforms (LAT1, LAT2, LAT3 and LAT4). The expressions of LAT1 and its associated protein 4F2hc were highest whereas those of LAT2 and LAT4 were extremely low. Treatment with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced L-leucine uptake concomitant with an inhibition of cell motility and, to a lesser extent, on cell proliferation. It also induced a time dependent upregulation of LAT1 and 4F2hc expressions. Similarly, cell migration and invasion, but not proliferation, were reduced in LAT1 knockdown KKU-M213 cells. In addition, silencing of LAT1 inhibited the expressions of 4F2hc mRNA and protein whereas the expression of microRNA-7, the 4F2hc down-regulator, was increased. Furthermore, the phosphorylation levels of ERK1/2 and p70S6K were reduced after LAT1 knockdown. Collectively, these results suggest that suppression of cell invasion and migration in LAT1 knockdown KKU-M213 cells may be partly mediated through the inhibition of the 4F2hc-signaling pathway by the up-regulation of microRNA-7. Based on this finding, LAT1 may be a potential therapeutic target for treating CCA.

This study examined the impact of leucine (Leu) derived from complete meals on stimulation of skeletal muscle protein synthesis (MPS). Expt. 1 examined time course changes in translation initiation and MPS after a meal. Male rats (;300 g) were trained for 5 d to eat 3 meals/d providing 20, 50, and 30% of energy from whey protein, carbohydrates, and fats, respectively. Plasma and skeletal muscle were collected at time 0 (baseline) after 12 h of food deprivation and then at 45, 90, 135, 180, and 300 min after a 4-g meal. Plasma Leu increased at 45 min and remained elevated through 180 min. MPS peaked at 45-90 min and returned to baseline by 180 min. Plasma Leu correlated with phosphorylation of ribosomal protein p70 S6 kinase (r ¼ 0.723; P , 0.05), eukaryotic initiation factor 4E binding protein-1 (r ¼ 0.773; P , 0.05), and MPS (r ¼ 0.608; P , 0.05) over time. Expt. 2 examined 3 levels of protein intake (10, 20, and 30% of energy) from 2 sources (wheat and whey) with different Leu contents (;6.8 and ;10.9%, respectively) on stimulation of initiation and MPS. Rats were trained to eat 3 meals/d providing 14, 56, and 30% of energy from protein, carbohydrates, and fats. On d 6, MPS was evaluated at 90 min after rats consumed 1 of the 6 test meals. Whey protein stimulated initiation and MPS more than wheat and the differential response related to greater plasma Leu responses in the whey groups. These studies demonstrate that peak activation but not duration of MPS is proportional to the Leu content of a meal.

The nucleotide sequence of chicken invariant chain (Ii) was determined, and the amino acid sequence similarity with human Ii is 61%. Certain regions important for the biological function of human Ii are highly conserved between chicken and mammals. The cytoplasmic tail of chicken Ii fused to the plasma membrane reporter molecule neuraminidase relocated the protein to endosomes. Moreover, like the mammalian orthologs, the cytoplasmic tail was found to contain two independent leucine-based endosomal sorting signals. Chicken Ii was found to interact with human Ii and crosslinking studies also indicate that chicken Ii assembles as a trimer. The chicken Ii can furthermore bind the human MHC class II (HLA-DR1). Many of the functional properties between the chicken Ii and its mammalian orthologs are thus maintained in spite of their sequence differences.

developed a new method to determine the fractional synthesis rate (FSR) and breakdown rate (FBR) of muscle protein. This method involves a pulse tracer injection and measurement of enrichment in the arterial blood and muscle at three time points. The calculations of FSR and FBR are based on the precursor-product principle. To test this method, we gave a pulse injection of L-[ring-13 C6]phenylalanine of 4-6 mg/kg in five rabbits. The measured FBR value (0.233 Ϯ 0.060%/h) was almost identical (P ϭ 0.35) to that (0.217 Ϯ 0.078%/h) estimated from a leg arteriovenous balance model (Biolo G, Chinkes D, Zhang X-J, and Wolfe RR. J Parenter Enteral Nutr 16: 305-315, 1992). The measured FSR value tended to be lower than that estimated from the leg model (0.125 Ϯ 0.036 vs. 0.185 Ϯ 0.086%/h; P ϭ 0.14), possibly because the new method measures only muscle FSR, whereas the leg balance model also includes skin and bone contributions. The pulse tracer injection did not affect muscle protein kinetics as measured by leucine and phenylalanine kinetics in the leg. In another five rabbits, we demonstrated that sampling could be reduced to either one or two muscle biopsies when multiple pulse injections were used. This method can be completed in 1 h with one muscle biopsy and has technical advantages over currently used methods.

Previous investigations have shown the efficacy of right-(RCPL) and left-(LCPL) circularly polarized light in promoting the asymmetric photolysis of racemic organic substrates and producing measurable enantiomeric excesses (e.e.s) when photolysis is incomplete. Synchrotron radiation, polychromatic and having out-of-plane components which are elliptically and ultimately circularly polarized, has been suggested as a universal source of RCPL and LCPL on a cosmic scale. The more prevalent right-(REPL) and left-(LEPL) elliptically polarized components have never been investigated for similar capabilities. The present study, using a 212.8 nm laser beam to mimic the synchtrotron radiation, explores the potential of REPL and LEPL in this context and finds a qualitative trend indicating that each induces asymmetric photolysis in the same sense as RCPL and LCPL, but to a lesser degree.

We recently showed that parenteral administration of amino acids to premature infants immediately after birth is safe and results in a positive nitrogen balance and increased whole-body protein synthesis. However, we did not determine organ-specific effects; albumin, produced by the liver, is an important protein, but its concentration is often low in premature neonates during the first few days after birth. The objective of the study was to test the hypothesis that the fractional and absolute albumin synthesis rates would increase with the administration of amino acids after birth, even at low nonprotein energy intake. Premature infants (&lt;1500 g birth weight), who were on ventilation, received from birth onward either glucose only (control group, n = 7) or glucose and 2.4 g amino acid kg(-1) d(-1) (intervention group, n = 8). On postnatal day 2, all infants received a primed continuous infusion of [1-(13)C]leucine, and mass spectrometry techniques were used to determine the inco...

-Bestatin is a potent inhibitor of ~inopeptidase B, an enzyme which is found in abundance in the membrane of monocytes and macrophages. Binding of Bcstatin to cells in the histiocytic linage up~~ulates colony stimuIating activity (both in viiro and in Go). which subsequently increases hematopoietic and hematologic values. We report that the treatment of mice with Bcstatin upregulates the frequency and absolute numbers of colony forming unit-granulocyte-macrophage (CFU-GM), as well as the entry of CFU-GM into S phase (a measure of progenitor cell activity). As a result, there is an increase in bone mar~w cellularity in cycl~hosphamidc myelosuppre~ed mice and an increase in the absolute neu~ophil count in normal and myelosuppressed mice. The therapeutic application of this hematopoietic modulator has been demonstrated in combination cyclophosphamide and Bestatin protocols. While Bestatin has significant therapeutic activity for minimal metastatic disease, therapy models in which the hosts have greater metastatic tumor burdens requires combination ~hemoimmunothempy for a significant therapeutic effect. Because myelosuppression as a them~utic indication for Bestatin has only recently been recognized, few clinical studies have been undertaken with appropriate surrogates of hematopoietic activity. However, the preliminary clinical evidence of hematopoietic activity by this non-toxic dipeptide. as reviewed here, suggests that this may be an appropriate drug for the treatment of myelosuppressed patients. Thus, Bestatin as an Orally active biological response modifier (BRM) has significant therapeutic activity for metastatie disease via multiple mechanisms including hematopoietie stimulation and macrophage activating properties. Bestatin I hemopaietir stimulation I marrophage activation R&urn6-Proprietis h&matopoiWiques et thbapeutiques de la Bestatine chez les souris normales et my&odGprimbs. La Bestatinc cst un inhibitettr puissant de L'aminopeptidase B, enzyme quc I'on trouve en abondance darts la membrane dcs monoc_vtes et ties musrophages. La fixation de la Bestatine sur les cellules de la l&t& histiocytaire r&de positivemcnt l'~~t~v~t~ de .stitttttlat~on des ~o~onie.~ farrssi bit% in vitro &in vivo), cette acti~~it~ au~mentant d son tour les populations cri&daires tl~~J~fQl~O~~~~~i!eS et i~~nratofo~iques. Lcs autetrrs rapportent ici 9~ ~adminjstratiun de Bestatirte ri des souris permet d'obtcnir unc au.qmentation de la fr&quence et du nontbre absolu des unit& formatrices de colonies de granuiocytes-t~laerop~t~t.~es (~FU-~M~ de m8me que i'entr& des CFU-GM en pltase S {tm indicateur de l'activitci des cellule~ l~ro~~t~itrircs~. En cotts~qttence de ces ph~nont~nes, on observe une ait~ntentation de la ceiiula~.t~ dans la ntoelIe usseuse cite: des souris ayant subi une myhlosuppression par le cyclophospltantidc. On observe une augmentation du nontbre absolu des ncutrophiles aussi birtt chez lcs souris normales que the-_ les mye'lod6prinrt!es. L'int&Pt thkapeutique de ce modulateur de l'hknaropoii;se a iti d&tom& ri I'aide de prorocoles associant cyclophosphamide et Bestatine. La Bestatine montre une activitP t~t~rapet~tiqtte si~~tif~~oti~~e au cows dc ntaladies trPs peu nt~tastas~es. En revanche, dans ies modkies ou I'h&e est cnvahi de m&astascs tumorales plus importantes. i'association d'une citintio-intmunot~t~rapie est nkessaire pour obtenir un @et tb&apeutique significutif. La myklodt!pression n'ayant itt: reconnue que rtkemment comme indiratiun thtfrapeutique de la Bestatine, pru d'&udes cliniques ont t% entreprises avec des substituts approprids de l'activite' h&matopo~ktique. ~~attntoins. /es l~rel~t~ers itr~itntents eliniques pass& it+ en revue en faveur d'une actisitc! ~t~matopo~~tique de re dipeptide d&ourvu de tosicitf, strg+wzt yrc'il pourrait s'agir d'un nt~di~at~leni adupth pour le rraitement de mafades nty~lod~~rio;~s. Ainsi ia Bestatitte en tant que modifcateur de la rkponse biologique (BMR) actif per OS, poss6de une activitP thf+apeutiW@ signifiicativr par f'internt&diaire de micanismes multiples parmi lesquels ia stimulation de SItintatopoi'Pse et des propricilis ~acti~~ation dtt t~t~t~ro~~~t~~e.