Application of quartz crystal nanobalance in conjunction with a net analyte signal based method for simultaneous determination of leucine, isoleucine and valine

The aim of the present investigation was to develop a biosensor for the detection of amino acids, Leucine, Isoleucine and Valine based on a quartz crystal nanobalance. leucine (Leu), isoleucine (Ile), and valine (Val) were selectively determined by quartz crystal nanobalance (QCN) sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including Leu, Ile and Val compounds. The selection of the optimal time range involved the calculation of the net analyte sig-nal regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of PRESS value was carried out by applying a moving window strategy. On the base of obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. The results showed that the method was successfully applied for the determina-tion of Leu, Ile and Val.

Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway

Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine＇s action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin （mTOR） complex-1 （mTORC1）. Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine （400 μmol·kg^-1·h^-1） for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 （S6K1） and eukaryotic initiation factor （elF） 4E-binding protein-1 （4E-BP1） phosphorylation in gastrocnemius and masseter muscles （P 〈 0.05）, but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTQRC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of elF2α and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.

Dietary Leucine Requirement for Juvenile Large Yellow Croaker Pseudosciaena crocea (Richardson,1846)

Dietary leucine requirement for juvenile large yellow croaker, Pseudosciaena crocea Richardson 1846 (initial body weight 6.0 g±0.1 g) was determined using dose-response method.Six isonitogenous (crude protein 43%) and isoenergetic (19 kJ g-1) practical diets containing six levels of leucine (Diets 1-6) ranging from 1.23% to 4.80% (dry matter) were made at about 0.7% increment of leucine.Equal amino acid nitrogen was maintained by replacing leucine with glutamic acid.Triplicate groups of 60 individuals were fed to apparent satiation by hand twice daily (05:00 and 17:30).The water temperature was 26-32℃, salinity 26-30 and dissolved oxygen approximately 7 mg L-1 during the experimental period.Final weight (FW) of large yellow croaker initially increased with increasing level of dietary leucine but then decreased at further higher level of leucine.The highest FW was obtained in fish fed diet with 3.30% Leucine (Diet 4).FW of fish fed the diet with 4.80% Leucine (Diet 6) was significantly lower than those fed Diet 4.However, no significant differences were observed between the other dietary treatments.Feed efficiency (FE) and whole body composition were independent of dietary leucine contents (P>0.05).The results indicated that leucine was essential for growth of juvenile large yellow croaker.On the basis of FW, the optimum dietary leucine requirement for juvenile large yellow croaker was estimated to be 2.92% of dry matter (6.79% of dietary protein).

Genome-wide analysis of basic leucine zipper transcription factor families in Arabidopsis thaliana,Oryza sativa and Populus trichocarpa

The basic leucine zipper （bZIP） transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis （Arabidopsis thaliana）, rice （Oryza sativa） and poplar （Populus trichocarpa） constitute a valuable resource for genome-wide analysis and genomic comparative analysis, as they are representatives of the two major evolutionary lineages within the angiosperms： the monocotyledons and the dicotyledons. In this study, bioinformatics analysis identified 74, 89 and 88 bZIP genes respectively in Arabidopsis, rice and poplar. Moreover, a comprehensive overview of this gene family is presented, including the gene structure, phylogeny, chromosome distribution, conserved motifs. As a result, the plant bZIPs were organized into 10 subfamilies on basis of phylogenetic relationship. Gene duplication events during the family evolution history were also investigated. And it was further concluded that chromosomal/segmental duplication might have played a key role in gene expansion of bZIP gene family.

Dietary Leucine Requirement of Juvenile Japanese Seabass(Lateolabrax Japonicus)

A 56-day feeding trial was conducted to examine the dietary leucine requirement of juvenile Japanese seabass in sea- water floating net cages （1.5 m × 1.5 m × 2.0 m）. Six isonitrogenous （crude protein 40%） and isoenergetic （gross energy 20 kJ g-1） diets were formulated to contain different concentrations of leucine （0.9%, 1.49%, 2.07%, 2.70%, 3.30% and 3.88% of dry matter）. Crys- talline L-amino acids were supplemented to simulate the whole body amino acid pattern of Japanese seabass except for leucine. Three groups （30 fish individuals each, 8.0g±0.20g in initial weight） were fed to apparent satiation at 5：00 and 17：30 every day. During the experimental period, the water temperature ranged from 26 to 32℃ and salinity from 26 to 30, and the dissolved oxygen was maintained at 7mgL-l. The results showed that weight gain （WG）, nitrogen retention （NR）, feed efficiency （FE） and protein efficiency ratio （PER） were significantly increased when dietary leucine was increased from 0.90% to 2.70% of dry matter, and then declined. WG was the highest when fish were fed D4 containing 2.70% of leucine. No significant differences were observed in body composition among dietary treatments （P 〉 0.05）. Considering the change of WG, the optimum dietary leucine requirement of juve- nile Japanese seabass was either 2.39% of dry matter or 5.68% of dietary protein.

Leucine signaling in the pathogenesis of type 2 diabetes and obesity

Epidemiological evidence points to increased dairy and meat consumption,staples of the Western diet,as major risk factors for the development of type 2 diabetes(T2D).This paper presents a new concept and comprehensive review of leucine-mediated cell signaling explaining the pathogenesis of T2D and obesity by leucine-induced over-stimulation of mammalian target of rapamycin complex 1(mTORC1).mTORC1,a pivotal nutrient-sensitive kinase,promotes growth and cell proliferation in response to glucose,energy,growth factors and amino acids.Dairy proteins and meat stimulate insulin/insulin-like growth factor 1 signaling and provide high amounts of leucine,a primary and independent stimulator for mTORC1 activation.The downstream target of mTORC1,the kinase S6K1,induces insulin resistance by phosphorylation of insulin receptor substrate-1,thereby increasing the metabolic burden of β-cells.Moreover,leucine-mediated mTORC1-S6K1-signaling plays an important role in adipogenesis,thus increasing the risk of obesity-mediated insulin resistance. High consumption of leucine-rich proteins explains exaggerated mTORC1-dependent insulin secretion, increased β-cell growth and β-cell proliferation promoting an early onset of replicative β-cell senescence with subsequent β-cell apoptosis.Disturbances of β-cell mass regulation with increased β-cell proliferation and apoptosis as well as insulin resistance are hallmarks of T2D,which are all associated with hyperactivation of mTORC1.In contrast,the anti-diabetic drug metformin antagonizes leucine-mediated mTORC1 signaling.Plant-derived polyphenols and flavonoids are identified as natural inhibitors of mTORC1 and exert anti-diabetic and anti-obesity effects.Furthermore,bariatric surgery in obesity reduces increased plasma levels of leucine and other branched-chain amino acids.Attenuation of leucine-mediated mTORC1 signaling by defining appropriate upper limits of the daily intake of leucine-rich animal and dairy proteins may offer a great chance for the prevention of T2D and obesity,as well as other epidemic diseases of civilization with increased mTORC1 signaling,especially cancer and neurodegenerative diseases,which are frequently associated with T2D.

Leucine Supplementation in a Chronically Protein-Restricted Diet Enhances Muscle Weight and Postprandial Protein Synthesis of Skeletal Muscle by Promoting the mTOR Pathway in Adult Rats

Low protein intake causes a decrease in protein deposition in most animal tissues. The purpose of this study was to investigate whether leucine supplementation would increase the synthesis rate of protein and muscle weight in adult rats, which chronically consume only 58.8% of their protein requirements. Thirty-six male Sprague-Dawley rats were assigned to one of three dietary treatments including a 20% casein diet （CON）, a 10% casein ＋ 0.44% alanine diet （R）, and a 10% casein ＋ 0.87% leucine diet （RL）. After a 10 d dietary treatment, plasma amino acid levels were measured after feeding, the gastrocnemius muscles and soleus muscles were harvested and weighed, and the fractional synthesis rate （FSR） and mammalian target of rapamycin （mTOR） signaling proteins in skeletal muscle were measured. Regarding the plasma amino acid level, the RL group had the highest concentration of leucine （P 〈 0.05） and the lowest concentration of isoleucine （P 〈 0.05） among the three groups, and the CON group had a lower concentration of valine （P 〈 0.05） than the R and RL groups. Compared with the R and RE groups, the CON group diet significantly increased （P 〈 0.05） feed intake, protein synthesis rate, and the phosphorylation of eukaryutic initiation factor 4E binding protein 1 （4E-BP1）, and decreased the weight of abdominal adipose. Compared with the R group, the RL group significantly increased in gastrocnemius muscle weight, protein synthesis rate, and phosphorylation of both ribosomal protein $6 kinase 1 （56K1） and 4E-BP1. In conclusion, when protein is chronically restricted in adult rat diets, leucine supplementation moderately improves body weight gain and increases muscle protein synthesis through mTOR activation,

Variability in the empirical leucine-to-carbon conversion factors along an environmental gradient

Bacterial production is one of the key parameters to evaluate bacterial role in ocean carbon fluxes.Estimation of bacterial production requires the leucine-to-carbon conversion factors that change widely across environments.However,empirical leucine-to-carbon conversion factors（e CFs） are seldom determined in situ because of time consuming and little is known on regulating factors for the e CFs.During May 2015 to January 2016,fourteen dilution experiments were conducted,from the Zhujiang（Pearl River） Estuary to the coast of the northern South China Sea,to determine spatiotemporal variability in the e CFs and its potential controlling factors along an environmental gradient.The e CFs showed clear spatial variations with the highest（1.27–1.69（kg C）/（mol Leu）） in low salinity waters（salinity〈15）,intermediate（1.03–1.25（kg C）/（mol Leu）） in moderate salinity（salinity of 15–25）,and the lowest（0.48–0.85（kg C）/（mol Leu）） in high salinity waters（salinity〉25）.Substrate availability was responsible for spatial variability in the e CFs.In the pristine coastal waters,low e CFs was related to substrate limitation and leucine incorporated was respired to maximize the survival rather than bacterial production.Hence,the e CFs measurement was needed for estimating bacterial production accurately in various marine environments.

Genetic Analysis of Leucine Content in Indica-Japonica Hybrid Rice (Oryza sativa L.)

Genetic control of leucine content in indica-japonica hybrid rice (Oryza sativa L.) was studied in 35 crosses of F1 and F2 generations, which were derived from crossing 7 male sterile indica rice lines with 5 restorer japonica rice lines along with their parents. Two genetic models and their corresponding statistical methods for quantitative traits of triploid seeds in cereal crops were used for the analysis. The first was the unconditional genetic model, which refers to the analysis of cumulative measurements (from flowering to a specific time) along the developmental stages, while the second was the conditional genetic model, which relates to analysis from one developmental stage to another stage (t - 1→t). The results showed that leucine content of indica-japonica hybrid rice was controlled by the expression of triploid endosperm effect (endosperm additive effect and endosperm dominant effect), cytoplasm effect, diploid maternal plant effect (maternal additive effect and maternal dominant effect) and their environmental interaction effects. Of these effects, endosperm dominant effect and maternal dominant effect were more important at the earlier stages, while endosperm additive effect and maternal additive effect were more important at the later stages of rice grain development under both unconditional and conditional genetic analyses. Due to the high heritabilities, which came from endosperm, maternal and cytoplasm effects for leucine content at different developmental stages, selection for leucine content of indica-japonica hybrid rice would be more efficient at early generations in breeding programs.

Effects of Valine and Leucine on Some Antioxidant Enzymes in Hypercholesterolemic Rats

Objective: Reactive oxygen species (ROS) are involved in the endothelial-mediated disorders within atherosclerosis. Considering that an oxidant/antioxidant imbalance might be a key factor in the damaging ROS-mediated effects, the present study intends to determine the influence of a high-fat diet, associated with essential amino acids—valine and leucine, upon the experimental animals, through evaluation of plasmatic level of some antioxidant enzymes. Material and Methods: The study was conducted on 32 male Wistar rats, which were fed with cholesterol, valine and leucine, for 60 days. The animals were divided into four groups, according to the received diet: the first group—standard diet;the second group—cholesterol (C);the third group—cholesterol and valine (C + V);the fourth group—cholesterol and leucine (C + L). Evaluations of the oxidative status, through plasma levels of the antioxidant enzymes: superoxide dismutase (SOD) and glutathione peroxidise (GPx), were made for the four mentioned groups of animals, at the beginning of the study (R0), after one (R1) and two months (R2). Results: The average values of SOD and GPx in group of animals fed exclusively with cholesterol (C) were significantly higher compared to the third group where cholesterol was supplemented with valine (C + V) or fourth group fed with cholesterol and leucine (C + L) (p < 0.001), after one month as well at the end of the experiment (two months). There were no significant differences in the levels of SOD and GPx between group III and group IV (p < 0.05) at the end of the experiment. Conclusion: Our results showed that valine and leucine decreased the serum levels of SOD and GPx and therefore they were useful antioxidants, which could improve the endothelial dysfunctions associated with atherosclerosis. Moreover, analysis of the oxidative status in the context of atherosclerotic mediated endothelial damage suggests that deviation from normal to alter endothelial status may be conditioned by an oxidants/antioxidants imbalance.

Homeobox leucine zipper proteins and cotton improvement

Transcription factors play key roles in plant development and stress responses through their interaction with cis-elements and/or other transcription factors. Homeodomain associated leucine zipper proteins (HD-Zip) constitute a family of transcription factors that are characterized by the presence of a DNA-binding domain closely linked with leucine zipper motif functioning in dimer formation. This type of association is unique to plants and considered as an excellent candidate to activate developmental responses to altering environmental conditions. Cotton is the most important fiber plant with a lot of local and commercial uses in the world. HD-Zip proteins not only have key roles in different stages of vascular and inter-fascicular fiber differentiation of cotton but also are suggested to have an important role against abiotic stress that is one of the key factors limiting cotton productivity. Plants have developed various strategies to manage stress conditions through a combination of metabolic, physiological and morphological adaptations. These adaptive changes rely largely on alterations in gene expression. Therefore, transcriptional regulators play a crucial role in stress tolerance. Being a transcription factor HD-Zip might be a useful target for genetic engineering to generate multiple stress tolerance in susceptible plants. In the following chapter, we discussed how the HD-Zip proteins would play a useful role for cotton development both in fiber production and stress adaptation.

Behaviors of Leucine and Isoleucine in Ion Mobility- Quadrupole Time of Flight Mass Spectrometry

In this study, ion mobility separation coupled with tandem mass spectrometry （IM-tandem MS） was utilized to investigate the ionization behaviors of two amino acids including leucine and isoleucine. Under the electrospray ionization （ESI） mode, two protonation sites in each molecular sturcture caused two forms of protomer. One arose from the amino being protonated （amino-protomer） and the other from the carboxyl being protonated （car- boxyl-protomer）. In the two-dimensional （drift time, m/z） spectrum, the protomers had the same mass, but the dis- tinguishable drift times and fragmentation patterns. For the characterization purpose, the theoretical collision cross section （CCS） values of the protomers were calculated and proven to be consistent with the experimental. Moreover, the quantified relationship between the amino acids and their protomers was evaluated. It showed that the abun- dance of the carboxyl-protomer was proportional to the concentration of the amino acid, whereas that of the amino- protomer did not have the same trend. Under the atmospheric pressure chemical ionization （APCI） mode, only the carboxyl-protomer was observed. In addition, the amino-protomer and the cluster ions observed under ESI were absent completely. The results demonstrate that the ionization mode impacts heavily on the ionization behaviors of leucine and isoleucine not only on the form of therir protomers but also On the quantified relationship.

Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment

The Arabidopsis （Arabidopsis thaliana L.） genome encodes for four distinct classes of homeodomain leucinezipper （HD-ZIP） transcription factors （HD-ZIPI to HD-ZIPIV）, which are all organized in multi-gene families. HD-ZIP transcription factors act as sequence-specific DNA-binding proteins that are able to control the expression level of target genes. While HD-ZIPI and HD-ZIPII proteins are mainly associated with environmental responses, HD-ZIPIII and HD- ZIPIV are primarily known to act as patterning factors. Recent studies have challenged this view. It appears that several of the different HD-ZlP families interact genetically to align both morphogenesis and environmental responses, most likely by modulating phytohormone-signaling networks.

Effects of dietary leucine supplementation on the gene expression of mammalian target of rapamycin signaling pathway and intestinal development of broilers

This experiment was to investigate the effects of dietary leucine supplementation on the gene expression of mammalian target of rapamycin(mTOR) signaling pathway and intestinal development of broilers. A total of 384 one-day-old broilers were randomly assigned into 4 treatments with 6 replicates(16 broilers per replicate). Broilers in these treatment groups were offered the following diets with 1.37,1.77,2.17 and2.57% of leucine. These diet treatments were named 1.37 TM, 1.77 TM, 2.17 TM, and 2.57 TM. The experiment lasted 21 days and all birds had free access to feed and water. Results indicated that there was no significant difference in body weight, average daily gain and average feed intake among all treatments(P > 0.05). The broiler duodenal villus height in 2.57 TM was the lowest, but the highest occurred in1.37 TM on d 7 and 14(P < 0.05). The villus height in the jejunum and ileum increased along with leucine level from 1.37 to 2.17%. The villus height of jejunum was significantly higher in 2.17 TM than in 1.37 TM on d 7 and 14, and the ratio of villus height to crypt depth(V:C) in the duodenum, jejunum and ileum increased significantly(P < 0.05) on d 21. The gene expression level of mTOR in the duodenum decreased with increasing leucine level and was higher in 1.37 TM than in 2.57 TM on d 7 and 14(P < 0.05). On d 14 and 21 of the trial, the expression of S6 K1 in the duodenum was higher in 1.37 TM than in 2.57 TM(P < 0.05), and the expression of mTOR, S6 K1 in the jejunum and ileum increased with increasing leucine level form 1.37 to 2.17%, whereas a significant difference occurred between 1.37 TM and 2.17 TM(P< 0.05).In conclusion, the addition of leucine fails to enhance the growth performance of broilers. However,leucine can improve intestinal development by enhancing villus height and V:C ratio in the jejunum and ileum. Moreover, the expression of mTOR, S6 K1 increased as the level of dietary leucine was elevated from 1.37 to 2.17%.

Leucine modulates the IPEC-J2 cell proteome associated with cell proliferation, metabolism and phagocytosis

Leucine can affect intestinal protein expressions, and improve mucosal immune function. However, little study has been conducted to determine the change of protein component by leucine treatment in intestine epithelial cells. The present study was to cover the key proteins and cell pathways that could be regulated by leucine treatment in porcine intestinal epithelial cell line(IPEC-J2) cells with the approach of proteome analysis. A total number of 3,211 proteins were identified in our approach by searching the database of Uniprot sus scrofa. Among identified proteins, there were 101 proteins expressed differently between control group and leucine group. Compared with the control group, there were 50 up-regulated proteins and 51 down-regulated proteins in leucine group. In these proteins, leucine treatment decreased the expression of some proteins including pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase,E3 ubiquitin ligase, cathepsin D, caspase 3 and caspase 6, and increased the levels of some proteins, such as some eukaryotic translation initiation factors, ubiquitin carboxyl-terminal hydrolase, DNA-related RNA polymerase II, urokinase plasminogen activator, cyclin-dependent kinase inhibitor 2 b, Mut L homolog 1,5-methylcytosine binding domain 4, polymerase d, a-tubulin, syntaxin 18, Ras homolog D, actin related protein 2/3 complex and cofilin. Via the analysis of Gene Ontology and pathways, these proteins in IPECJ2 cells were related with some physiological functions, such as protein metabolism, glycolysis, cell proliferation, apoptosis and phagocytosis. Thus, these results suggest that leucine affects gut barrier function possibly via regulating cell proliferation and apoptosis, metabolism and phagocytosis.

Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway

The aim of this study is to investigate the effects of leucine（Leu） and histidine（His） on the expression of both the mammalian target of rapamycin（mTOR） signaling pathway-related proteins and caseins in immortalized bovine mammary epithelial cells（CMEC-H）, using a single supplement through Western blotting. The Earle＇s balanced salt solution（EBSS） was set as the control group and other treatment groups, based on the EBSS, were added with different concentrations of Leu or His, respectively. The results showed that, compared with the control group, the expression of caseins and the phosphorylation of mTOR（Ser^2481）, Raptor（Ser^792）, e IF4E（Ser^209）, and e EF2（Thr^56） increased with the Leu concentrations ranging from 0.45 to 10.80 mmol/L（P〈0.01）. The P-4EBP1（Thr^37） at 10.80 mmol/L Leu, and P-RPS6（Ser^235/236） at 5.40 to 10.80 mmol/L Leu all decreased. Similarly, the His supplementation from 0.15 to 9.60 mmol/L increased the expression of αs2-casein, β-casein, κ-casein, P-mTOR（Ser^2481）, P-Raptor（Ser^792）, P-S6K1（Thr^389）, P-4EBP1（Thr^37）, P-e IF4E（Ser^209）, and P-e EF2（Thr^56）（P〈0.01） in CMEC-H, whereas the αs1-casein expression was only reduced at 9.60 mmol/L His, G protein β subunit-like protein（GβL） at 0.15 and 9.60 mmol/L His, and P-RPS6 at 4.80 to 9.60 mmol/L His. Our linear regression model assay suggested that the αs1-casein expression was positively correlated with P-mTOR（P〈0.01）, P-S6K1（P〈0.01）, and P-e EF2（P〈0.01） for the addition of Leu, while the expressions of β-casein（P〈0.01） and κ-casein（P〈0.01） were positively correlated with P-e EF2 for the addition of His. In conclusion, the milk protein synthesis was up-regulated through activation of the mTOR pathway with the addition of Leu and His in CMEC-H.

Crystal structure of a plant leucine rich repeat protein with two island domains

Leucine rich repeat(LRR)domain,characterized by a repetitive sequence pattern rich in leucine residues,is a universal protein-protein interaction motif present in all life forms.LRR repeats interrupted by sequences of 30 70 residues(termed island domain,ID)have been found in some plant LRR receptor-like kinases(RLKs)and animal Toll-like receptors(TLR7-9).Recent studies provide insight into how a single ID is structurally integrated into an LRR protein.However,structural information on an LRR protein with two IDs is lacking.The receptor-like protein kinase 2(RPK2)is an LRR-RLK and has important roles in controlling plant growth and development by perception and transduction of hormone signal.Here we present the crystal structure of the extracellular LRR domain of RPK2(RPK2-LRR)containing two IDs from Arabidopsis.The structure reveals that both of the IDs are helical and located at the central region of the single RPK2-LRR solenoid.One of them binds to the inner surface of the solenoid,whereas the other one mainly interacts with the lateral side.Unexpectedly,a long loop immediately following the N-terminal capping domain of RPK2-LRR is presented toward and sandwiched between the two IDs,further stabilizing their embedding to the LRR solenoid.A potential ligand binding site formed by the two IDs and the solenoid is located at the C-terminal side of RPK2-LRR.The structural information of RPK2-LRR broadens our understanding toward the large family of LRR proteins and provides insight into RPK2-mediated signaling.

Leucine increases mucin 2 and occludin production in LS174T cells partially via PI3K-Akt-mTOR pathway

Mucin 2 and occludin play a crucial role in preserving the intestinal mucosal integrity. However, the role for leucine mediating intestinal mucin 2 and occludin expression has little been investigated. The current study was conducted to test the hypothesis that leucine treatment could increase mucin 2 and occludin levels in LS174 T cells. The LS174 T cells were incubated in the Dulbecco's Modified Eagle Medium(DMEM)supplementing 0, 0.5 and 5 mmol/L L-leucine for the various durations. Two hours after the leucine treatment, the inhibitor of mammalian target of rapamycin(mTOR) and protein kinase B(Akt) phosphorylation in LS174 T cells were significantly increased(P < 0.05), and the mucin 2 and occludin levels were also significantly enhanced(P < 0.05). However, the pretreatment of 10 nmol/L rapamycin, which was an mTOR inhibitor, or 1 μmol/L wortmanin, which was an inhibitor of phosphatidylinositol 3-kinase(PI3 K), completely inhibited leucine-induced mTOR or Akt phosphorylation(P < 0.05), and significantly reduced leucine-stimulated mucin 2 and occludin levels(P < 0.05). These results suggest that leucine treatment promotes the mucin 2 and occludin levels in LS174 T cells partially through the PI3 K-Akt-mTOR signaling pathway.

Investigation of Peptoid Chiral Stationary Phases Terminated with N＇-Substituted PhenyI-L-proline/leucine Amide

Eight peptoid chiral stationary phases （CSPs） terminated with N＇substituted phenyl-L-proline or L-leucine amide were prepared and evaluated under normal phase mode. With 59 racemic analytes, we compared the enantio- meric separations on CSPs terminated with p-methylphenyl, p-chlorophenyl and unsubstituted phenyl. For short peptoid selectors containing only one S-N-（1-phenylethyl） glycine （Nspe） unit, the terminal p-methyl substituent did not affect chiral recognition abilities significantly. In L-proline amide terminated CSPs, p-chloro substituent resulted in obviously inferior selectivity while in L-leucine amide terminated CSPs, it worked much better. Longer peptoid selectors containing two more Nspe units generally performed much better than the shorter ones, due to the great contributions of peptoid chain to chiral recognition. Meanwhile, the effects of the terminal substituent on selectivity were found changed on these CSPs. For CSPs terminated with L-leucine amide, the terminal p-chloro substituent in longer selector no longer produced the best recognition ability; the CSP with unsubstituted phenyl instead performed best. Comparison of these peptoid CSPs varied in terminal substituents and chain length was conducted to gain a better understanding of the chiral recognition mechanism of this type CSP and promote the development of more useful CSPs.

Contributions of a Position Amino Acid Residues to the Conformational Stability of GCN4 Leucine Zipper

The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and stability of parallel coiled coils. The changes of the circular dichroism spectra show that the displace- ment of the a position polar asparagine and the increase of asparagine in the GCN4 leucine zipper can reduce the α-helix content of the coiled coil structure. The mutants are less stable than the natural peptide in guanidine hydrochloride. The results show that the interaction between the polar asparagine contributes to the conformational stability of the coiled coil. Both the conformation and the number of polar residues in the coiled coil also affect the α-helix content and its resistance to the denaturant. The conclusions provide evidence describing the folding process of proteins including coiled coils in vivo.