Addition Of L-leucine Research Articles

English

The production of manganese peroxidase (MnP) by white rot fungus strain L-25 was carried out using potato-processing wastewater and the effects of amino acids in the potato-processing wastewater was investigated. The MnP was efficiently produced from the wastewater by the addition of glucose and the maximum MnP activity linearly increased with an increase in the glucose concentration. The initial pH affected the cell growth and also the production rate of the MnP. The maximum activity and the production rate of the MnP using the potato-processing wastewater-based medium were higher (ca. 2.5- fold) than that of the basal medium. Moreover, amino acids in the wastewater had significant effects on the MnP production. L-Glutamic acid, L-aspartic acid and L-serine induced the MnP secretion, on the other hand, L-phenylalanine, L-tyrosine, L-leucine and L-lysine inhibited it. The addition of L-leucine and L-lysine caused growth inhibition, while, L-phenylalanine and L-tyrosine blocked the MnP biosynthetic pathway. Ammonium ion released from the L-phenylalanine by the L-phenylalanine ammonia-lyase participated in the repression of the MnP biosynthetic pathway of the strain L-25.

Effect of L-Leucine Supplementation on Growth Performance and Carcass Characteristics of Grower-Broiler Chickens Fed Low Protein Diets

Problem statement: Supplementation of broiler diets with cristalline amino acids (i.e. lysine, ethionine and threonine) may support equal broiler growth and improve overall amino acids balance and enable a reduction in CP level of diets. Approach: A trial was conducted to evaluate the effect of supplemental L-leucine in diets containing recommended levels and low crude protein (20 and 18%, respectively) with constant metabolizable energy (3200 kcal kg-1) for broilers from 21-42 day of age. Six experimental diets were formulated with three levels of supplemental L-leucine, 0, 0.5 and 0.67% and two levels of crude protein. A total of 180 1 day-old Cobb broiler chickens were randomly divided into 36 experimental pens, 5 chickens in each pen, with each diet replicated 6 times. The dietary treatments were offered from 21-42 days of age. Feed intake, body weight gain and Feed Conversion Ratio (FCR) were measured on a weekly basis. At the end of the feeding trial the birds were slaughtered and carcass analyses conducted. Results: Feed intake, weight gain and FCR were not affected by increasing levels of L-leucine supplementation. Weight gain was significantly reduced (p<0.05), whereas feed intake and FCR were not significantly affected with decreasing dietary crude protein. A positive response in breast meat yield was achieved by the addition of L-leucine to levels up to 0.5% in the diet but a significant decrease was noted when the level reached 0.67% in diet. Supplementation of L-leucine significantly (p<0.05) decreased the relative weights of the liver and gizzard. However, the addition of L-leucine significantly reduced carcass weights when L-leucine was added at 0.67%. Lowering the dietary protein level also significantly reduced breast yield and carcass weight (p<0.05). However, abdominal fat, gizzard, liver and heart were not affected by protein level. Conclusion/Recommendations: It can be concluded that supplementation of L-leucine at levels up to 0.67% of the diet did not affect performance but deleteratious the carcass weight.

Combined synthesis and in situ coating of nanoparticles in the gas phase

Combined gas phase synthesis and coating of sodium chloride (NaCl) and lactose nanoparticles has been developed using an aerosol flow reactor. Nano-sized core particles were produced by the droplet-to-particle method and coated in situ by the physical vapour deposition of L-leucine vapour. The saturation of L-leucine in the reactor determined the resulting particle size and size distribution. In general, particle size increased with the addition of L-leucine and notable narrowing of the core particle size distribution was observed. In addition, homogeneous nucleation of the vapour, i.e. formation of pure L-leucine particles, was observed depending on the saturation conditions of L-leucine as well as the core particle characteristics. The effects of core particle properties, i.e. size and solid-state characteristics, on the coating process were studied by comparing the results for coated NaCl and lactose particles. During deposition, L-leucine formed a uniform coating on the surface of the core particles. The coating stabilised the nanoparticles and prevented the sintering of particles during storage.

Formulation, characteristics and aerosolization performance of azithromycin DPI prepared by spray-drying

In this study, a number of dry powder formualtions of azithromycin for inhalation were designed to optimize the composition and further explore the relationship between the composition, the physical properties and the aerosolization performance, hence developing a dry powder inhalation (DPI) of azithromycin (AZI) as an alternative to its counterpart nebulizer. Dry powders of azithromycin with a variety of carrier types and carrier:drug ratios were prepared by spray-drying and characterized afterwards for different physical properties, including particle size and distribution, morphology, flowability, powder density and hygroscopic nature. In-vitro deposition was also evaluated after the aerosolization of powders at 60 L min − 1 via the Aerolizer ® into a twin-stage liquid impinger (TI). It was found that the type and amount of the carrier had significant effects on the aerosolization performance of DPI. The results also showed that the particle size and flowability were two critical physical properties responsible for the aerosolization performance. Specifically, moderate particle size around 5–6 µm produced relatively high respirable fractions (RF). In terms of the flowability, the angle of repose within the range of 43°–52° was in a good linear relationship ( r = 0.9523) with the RF value. In particular, the addition of L-leucine with the carrier:drug ratio of 1:5 showed the highest RF at 37.5%, which indicated that L-leucine is a promising carrier for the dry powder formulation of AZI for inhalation.

Simultaneous synthesis and coating of salbutamol sulphate nanoparticles with l-leucine in the gas phase

Salbutamol sulphate nanoparticles have been simultaneously prepared and coated with l-leucine in the gas phase. Three different ways of coating can be separated based on the operation temperatures used in an aerosol flow reactor. Below the temperature of l-leucine sublimation, formation of the l-leucine layer on the core particle surface takes place via diffusion of l-leucine molecules on the droplet surfaces during droplet drying. At intermediate temperatures, the extent of sublimation of l-leucine depends notably on the concentration, and thus partial evaporation was expected. The l-leucine coating was solely formed via vapor deposition at high reactor temperatures when complete sublimation of l-leucine was obtained. The geometric mean diameter of the core salbutamol particles was approximately 65 nm. In general, particle size increased with the addition of l-leucine. The size distribution remained the same or broadened when the coating layer of the particles was formed via surface diffusion whereas notable narrowing of the distribution was observed when the coating was formed via vapor deposition. Upon desublimation and heterogeneous nucleation on the surfaces of smooth, spherical core particles, l-leucine formed a discontinuous coating with leafy crystals a few nanometers in size.

Solid–liquid equilibria in l-leucine + l-valine + water

Solid–liquid equilibria in the system l-leucine + l-valine + water have been measured at 298 K and ambient pressure. The system was shown to form solid solutions at equilibrium. The data also showed that the equilibrium composition of l-leucine in the liquid phase was only slightly affected by the presence of l-valine; whereas, the equilibrium composition of l-valine increased significantly with the addition of l-leucine. A simple thermodynamic model was used to correlate binary l-leucine + water and l-valine + water solubility data, and then to estimate nonidealities in the solid phase in the ternary system. Surprisingly, the solid solution exhibited significant nonidealities, emphasizing the need for analysis of the solid phase when ternary solubilities are measured.

The Production of Isoamyl Acetate from Amyl Alcohol bySaccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L-leucine and amyl alcohol in fermentations carried out by a high ester-producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro-DL-leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.

Possible involvement of amino acid transporters on S-nitroso-cysteine-induced inhibition of arachidonic acid release in PC12 cells

Previously, we proposed that S-nitroso-cysteine (SNC) was incorporated via the l-type-like amino acid transporters in rat brain slices. In PC12 cells (rat neuronal cell line), SNC inhibited [ 3H]arachidonic acid (AA) release induced by mastoparan (wasp venom peptide). We investigated the involvement of amino acid transporters on SNC-induced inhibition of [ 3H]AA release in PC12 cells. SNC inhibited mastoparan-stimulated [ 3H]AA release in a concentration-dependent manner in normal Na +- and low Na +-containing buffer. The inhibitory effect of 0.6 mM SNC in low Na + buffer decreased by 10 mM l-leucine, l-phenylalanine, l-methionine and l-cysteine. In contrast, l-alanine, l-threonine, l-valine or l-isoleucine showed very limited effects. Addition of l-leucine and l-phenylalanine, but not l-alanine or l-valine, also decreased the inhibitory effect of SNC on ionomycin/Na 3VO 4-stimulated [ 3H]AA release in normal Na + buffer. These findings suggest that SNC is incorporated via the amino acid transporters and inhibits AA release in PC12 cells.

Properties of the Functioning of the Promoter of the Microcin C51 Operon under Different Conditions of Escherichia coli Cell Growth

The level of transcription from the promoter of the microcin C51 operon (P mcc ) depends on the growth phase of Escherichia colicells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase. The functioning of P mcc was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lacoperon. A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the P mcc promoter at the exponential phase of growth; the expression of P mcc –lacduring cell entry into the stationary phase was higher under less favorable medium conditions. The use of composite rich media impaired this feature. The addition of l-leucine (100 μg/ml) to the medium decreased the expression of P mcc –lacin wild-type cells and those carrying the Δlrpmutation. A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from P mcc during cell entry into the stationary growth phase. The capacity of the P mcc promoter and of the wild-type lacZgene promoter upon IPTG induction was virtually the same. A mutation in the ompRgene did not markedly influence transcription started from P mcc .

Effects of ascorbic acid on hepatic nuclear binding of L-tryptophan

This study investigated the in vitro effects of ascorbic acid on nuclear L-tryptophan receptor binding of rat liver. Ascorbic acid at 10 −6M to 10 −4M inhibited the in vitro 3H-tryptophan binding to hepatic nuclei and nuclear envelope receptors. Addition of dithiothreitol, a protective agent for sulfhydryl groups, did not affect the inhibitory binding. Addition of L-leucine did reduce the inhibitory binding. Addition of NaCl (0.125 × 10 −4M) did not influence the inhibitory binding effect of ascorbic acid but decreased the inhibitory effect of the NaCl on nuclear tryptophan binding. In vivo administration of ascorbic acid before or along with L-tryptophan decreased the L-tryptophan-induced stimulation of hepatic protein synthesis.

Influence of l-leucine on l-tryptophan binding to rat hepatic nuclei

This study was concerned with the effect of l-leucine on l-tryptophan's actions (nuclear receptor binding and protein synthesis) on rat liver. Earlier studies dealing with 3H-tryptophan binding to rat hepatic nuclei in vitro revealed that some amino acids, such as l-alanine, l-phenylalanine, and l-tyrosine, but not others, such as l-leucine, competed for such binding. This study evaluated the influence of l-leucine together with unlabeled l-tryptophan on 3H-tryptophan binding to hepatic nuclei in vitro. Whereas l-leucine alone had little effect, l-leucine together with unlabeled l-tryptophan (excess, 10 −4M) caused significantly less inhibition of 3H-tryptophan binding to hepatic nuclei than did unlabeled l-tryptophan alone (9% vs. 68% inhibition, respectively). l-leucine (10 −10 to 10 −4M), when added together with unlabeled l-tryptophan (10 −7 to 10 −4M), abrogated the inhibition of binding because of the unlabeled l-tryptophan alone. Other in vitro 3H-tryptophan binding effects were: 1) l-isoleucine and l-valine revealed binding effects similar to that with l-leucine; 2) the dipeptide, TRP · LEU, acted similarly (but somewhat less) to that of l-leucine; and 3) l-alanine and sodium selenite, each of which was inhibitory to 3H-tryptophan binding, showed less inhibition when l-leucine was also added. In vivo effects were investigated after tube-feeding water, l-leucine or/and l-tryptophan to rats 10 min before killing and revealed the following results: 1) using isolated hepatic nuclei for in vitro 3H-tryptophan binding revealed less specific binding to nuclei of rats tube-fed l-tryptophan alone than to nuclei of those tube-fed l-tryptophan plus l-leucine, suggesting an in vivo inhibitory effect of l-leucine; and 2) using hepatic microsomes for in vitro 14C-leucine incorporation into protein revealed greater stimulation by l-tryptophan alone (+61%) than by l-tryptophan plus l-leucine (+8%) over control (water alone). Thus, the addition of l-leucine appears to have an inhibitory effect on actions attributable to l-tryptophan alone.

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of α-isopropylmalate synthase

Mutants of the ‘miso’ yeast, Zygosaccharomyces rouxii, that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro- dl-leucine-resistant mutants, an analogue of l-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of α-isopropylmalate synthase, one of the enzymes used for l-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of l-leucine, a feedback inhibitor.

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of alpha-isopropylmalate synthase.

Mutants of the 'miso' yeast, Zygosaccharomyces rouxii, that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro-DL-leucine-resistant mutants, an analogue of L-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of alpha-isopropylamalate synthase, one of the enzymes used for L-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of L-leucine, a feedback inhibitor.

Marine Vibrio species produce the volatile organic compound acetone

While screening aerobic, heterotrophic marine bacteria for production of volatile organic compounds, we found that a group of isolates produced substantial amounts of acetone. Acetone production was confirmed by gas chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The major acetone producers were identified as nonclinical Vibrio species. Acetone production was maximal in the stationary phase of growth and was stimulated by addition of l-leucine but not the other common amino acids, suggesting that leucine degradation leads to acetone formation. Acetone production by marine vibrios may contribute to the dissolved organic carbon associated with phytoplankton, and some of the acetone produced may be volatilized to the atmosphere.

Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Manganese(II)-bicarbonate-mediated catalytic activity for hydrogen peroxide dismutation and amino acid oxidation: detection of free radical intermediates.

To examine the structural identities of reactive free radicals and the mechanism of the oxidative modification of proteins, we used EPR and spin-trapping methods to investigate the oxidation of amino acids by H2O2 as well as the decomposition of H2O2 itself catalyzed by Mn(II) ions. Superoxide and hydroxyl radicals (O2-. and OH.) were trapped by a spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), in a reaction mixture containing Mn(II) and H2O2 in bicarbonate/CO2 buffer. When Hepes was used in place of bicarbonate buffer, superoxide radical was not observed, indicating the importance of bicarbonate buffer. With addition of L-leucine to a similar reaction mixture, a leucine-derived radical that replaced the DMPO-superoxide adduct was detected in the absence and presence of DMPO. Using various isotope-enriched L-leucines, we successfully identified this radical as a hydronitroxide, -OOC(R)CHNHO.. The data are consistent with the formation of a transient "caged" OH. in the inner coordination sphere of Mn(II). This caged OH. is likely to undergo an intramolecular hydrogen-atom abstraction from the Mn-bound H2O2 or amino acid. Two reaction schemes are proposed to account for the experimental results shown here and in the preceding papers.

Insulin release from a cloned hamster B-cell line (HIT-T15) the effects of glucose, amino acids, sulphonylureas and colchicine

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.

Opp-lac Operon fusions and transcriptional regulation of the Escherichia coli trp-linked oligopeptide permease.

The transcriptional regulation of the Escherichia coli trp-linked opp operon that encodes the oligopeptide permease was investigated by using lambda plac Mu51-generated lac operon fusions. Synthesis of beta-galactosidase by strains harboring oppA-lac, oppB-lac, and oppD-lac fusions occurred at a basal level when the fusion-containing strains were grown in minimal medium. The addition of L-leucine or L-alanine to exponentially growing, aerobic cultures or shifting the aerobic fusion-containing strains to anaerobic growth medium increased the synthesis of beta-galactosidase from all opp-lac fusions. When transcription of the opp operon was induced by L-leucine, the differential rate of beta-galactosidase synthesis from each opp-lac fusion increased 8- to 10-fold; this increased rate of lacZ expression from the opp-lac fusions resulted in a 5- to 6-fold increase in total beta-galactosidase activity after maximum expression was achieved. Importantly, when F'123 derivatives harboring independently isolated E. coli opp-lac operon fusions were introduced into E. coli and Salmonella typhimurium, the data clearly demonstrated that the E. coli opp operon was expressed identically and responded to the same transcriptional regulatory signals in both E. coli and S. typhimurium. A comparison of beta-galactosidase synthesis by E. coli strains harboring an opp-lac operon fusion and either an oppE+ locus or an oppE mutation demonstrated that the reduction in peptide transport produced by the oppE mutation does not result from a decrease in the level of opp operon transcription.

Physiological concentrations of L-leucine control the release of prolactin from cultured rat pituitary cells.

In an attempt to further define the optimum experimental conditions for the in vitro study of PRL endocrinology, the effect of extracellular leucine concentration on PRL secretion was assessed. Rat pituitary cells, maintained in short term monolayer culture, were washed free of serum and placed in Minimum Essential Medium (MEM; 0.39 mM leucine) supplemented with nonessential amino acids and 0.1% gelatin. After the establishment of a constant rate of basal PRL release, the MEM was replaced with leucine-free MEM (supplemented as above), and the rate of PRL release was followed. PRL release from the cells in the leucine-free medium fell within 15 min to a rate 30% of that seen in the controls. Return to the control rate was achieved by the addition of L-leucine only. The D-isomer, isoleucine, valine, proline, leucyl-leucine, and leucine-agarose were ineffective. In the short term, the rate of PRL synthesis was found to be independent of extracellular leucine and the inhibition of release seen upon removal of leucine, and the subsequent recovery upon replacement of leucine was also observed when this experiment was conducted in the presence of 50 micrograms/ml cycloheximide. The L-leucine recovery was found to be dose dependent, with an increase in leucine concentration from 20 to 40 mg/liter causing a tripling of the PRL release rate. The addition of 10(-7) M TRH showed that leucine can be limiting in the response to this stimulant and allowed a response to as little as 10 mg/liter leucine to be discerned. From these experiments, it is concluded that physiological concentrations of leucine specifically and profoundly affect the release of PRL by a mechanism independent of protein synthesis. This leucine requirement for release has not been previously reported.