AIMTo determine the thermodynamics of binding reaction of arginine oligomer (R8) to bovine submaxillary mucin (BSM) in order to provide the foundation for understanding the influence of mucin on transport of macromolecules through mucosa mediated by arginine oligomer.

METHODSUltracentrifugation sedimentation was employed to investigate the interaction of BSM-R8. The mixtures of R8 with variable concentration and constant volume of BSM were placed on a shaker under oscillation at 25 degrees C to achieve equilibriums of binding reaction, and then centrifuged. The fluorescence intensity of the supernatant was measured by spectrofluorometer. The data were described by two types of binding sites model, the binding parameters of BSM-R8 were obtained by Scatchard plots.

RESULTSAt the low pH values < or = 4.5 and ionic strength > or = 0.2 mol x L(-1), the BSM-R8 interaction was principally electrostatic interaction, the five primary binding sites (n1) predominantly were supplied by sulfate groups, the secondary binding sites apparently depended on pH, in that percent ionization of sialic acid residues (n2) in BSM. At the low ionic strength < or = 0.2 mol x L(-1) and pH 7.0, the BSM-R8 interaction was exceedingly complex, hydrogen bonds, hydrophobic interaction and electrostatic forces were involved in the interaction between R8 and BSM, the binding sites of BSM bound R8 were markedly increased.

CONCLUSIONThere existed evidence that R8 interacted with BSM. The pH and the ionic strength of the binding solution strongly affected the interaction of BSM with R8. The results suggested that the enhancing efficacy of the arginine oligomer for the transport of macromolecules through different site mucosa in body might be variable.

Arginine ; chemistry ; Binding Sites ; Hydrogen-Ion Concentration ; Mucins ; chemistry ; Osmolar Concentration ; Protein Binding ; Thermodynamics

This article analyzed the mechanism of Danggui Sini Decoction(DSD) in improving kidney injury caused by blood stasis syndrome(BSS) in rats. Firstly, 32 female SD rats were randomly divided into the following four groups: a normal group and a BSS group, both receiving an equal amount of distilled water by gavage; a normal+DSD group and a BSS+DSD group, both receiving 5.103 g·kg~(-1) DSD orally for a total of 14 days. Daily cold water bath was given to establish the BSS model, and on the 14th day, BSS rats were subcutaneously injected with 0.8 mg·kg~(-1) adrenaline. Normal rats were subjected to the water bath at 37 ℃ and injected with an equal volume of distilled water. After the experiment, 24-hour urine, serum, and kidney samples were collected for metabolomic analysis, biochemical measurements, and hematoxylin-eosin(HE) staining. The study then employed ~1H-NMR metabolomic technology to reveal the metabolic network regulated by DSD in improving BSS-induced kidney injury and used network pharmacology to preliminarily elucidate the key targets of the effectiveness of DSD. Pathological and biochemical analysis showed that DSD intervention significantly reduced inflammation and abnormal levels of blood creatinine, blood urea nitrogen, and urine protein in the kidneys. Metabolomic analysis indicated that DSD attenuated BSS-induced kidney injury primarily by regulating 10 differential metabolites and three major metabolic pathways(taurine and hypotaurine metabolism, citrate cycle, and acetaldehyde and dicarboxylic acid metabolism). Network pharmacology analysis suggested that the protective effect of DSD against BSS-induced kidney injury might be related to two key genes, ATP citrate lyase(ACLY) and nitric oxide synthase 2(NOS2), and two main metabolic pathways, i.e., arginine biosynthesis, and arginine and proline metabolism. This study, from the perspective of network regulation, provides initial insights and evidence into the mechanism of DSD in improving kidney injury induced by BSS, offering a basis for further investigation into the molecular mechanisms underlying its efficacy.
Rats ; Female ; Animals ; Rats, Sprague-Dawley ; Network Pharmacology ; Drugs, Chinese Herbal/chemistry* ; Metabolomics ; Kidney ; Arginine ; Water

OBJECTIVETo screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1).

METHODSWestern blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1.

RESULTSThe expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites.

CONCLUSIONSThe high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

Arginine ; analogs & derivatives ; analysis ; Cell Line, Tumor ; Chromatography, Liquid ; Glioma ; chemistry ; Humans ; Immunoprecipitation ; Protein-Arginine N-Methyltransferases ; analysis ; physiology ; Repressor Proteins ; analysis ; physiology ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVECorynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.

METHODSQuantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.

RESULTSArginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.

CONCLUSIONThe arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.

Arginine ; biosynthesis ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Corynebacterium ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Repressor Proteins ; chemistry ; genetics ; metabolism

The arginyl-tRNA synthetase (ArgRS) catalyzes the esterification reaction between L-arginine and its cognate tRNA(Arg). Previously reported structures of ArgRS shed considerable light on the tRNA recognition mechanism, while the aspect of amino acid binding in ArgRS remains largely unexplored. Here we report the first crystal structure of E. coli ArgRS (eArgRS) complexed with L-arginine, and a series of mutational studies using isothermal titration calorimetry (ITC). Combined with previously reported work on ArgRS, our results elucidated the structural and functional roles of a series of important residues in the active site, which furthered our understanding of this unique enzyme.
Arginine ; chemistry ; Arginine-tRNA Ligase ; chemistry ; Binding Sites ; Catalytic Domain ; Crystallography, X-Ray ; Escherichia coli ; Ligands ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Conformation ; RNA, Transfer ; chemistry ; Structure-Activity Relationship

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.
Arginine ; chemistry ; Aspartic Acid ; chemistry ; Cell Membrane ; physiology ; Conserved Sequence ; Ion Channel Gating ; Ion Channels ; chemistry ; Membrane Potentials ; Protein Structure, Tertiary

BACKGROUND: Bilateral ureteral obstuction (BUO) has been known to decrease the expression of renal aquaporin-2 (AQP2) and Na-K-2Cl cotransporter (NKCC2). The polyuria and urinary concentration defect in postobstructive diuresis (POD) may be explained by these molecular changes. By contrast, chronic infusion of antidiuretic hormone (ADH) has been known to increase the expression of renal AQP2 and NKCC2, but there have been no studies regarding the chronic effect of ADH in molecular level when introducing to POD. We tried to identify the changes of renal expression of AQP2 and NKCC2 in 24 hour BUO rat at POD-7 day and the chronic effect of ADH to the expression of AQP2 and NKCC2 in sham operation rat and in 24 hour BUO rat, at sham operation 7 day and at POD-7 day, respectively. METHODS: Twenty four Spraugue-Dawley rats were divided into four groups. Group I (Control group): sham operation rats(n=6). Group II (BUO group): 24 hour BUO and release of it (n=6). Group III (dDAVP group): dDAVP (1-deamino-8d-arginine vasopressin: V2-receptor-selective agonist) infusion at rate of 20 ng/hour by osmotic minipump subcutaneously for 7 days in sham operation rats (n=6). Group IV (BUO+dDAVP group): dDAVP infusion at rate of 20 ng/hour by osmotic minipump by same method as Group III in 24 hour BUO rats (n=6). All rats were sacrificed at POD-7 day (Group II, Group IV) or sham operation-7 day (Group I, Group III) and renal expression of AQP2 and NKCC2 were analyzed by immunohistochemistry and by Western blot method. Blood and urinary chemistry examinations were done concurrently. RESULTS: BUO group showed increased urine output and decreased urine osmolality (p<0.05) and decreased expressions of AQP2 and NKCC2 compared with Control group {29.1 +/- 4.2% vs. 100 +/- 10.0% (p< 0.05); 40.2 +/- 5.4% vs. 100 +/- 7.9% (p<0.05) respectively}. dDAVP group had decreased urine output from POD-1 day to POD-5 day and increased urine osmolality (p<0.05) POD-1 day to POD-7 day during and increased expressions of AQP2 and NKCC2 compared with Control group {206.5 +/- 19.0% vs. 100 +/- 10.0% (p<0.05); 180.6 +/- 13.3% vs. 100 +/- 7.9% (p<0.05) respectively}. But BUO group showed no difference in urine output and urine osmolality and expressions of AQP2 and NKCC2 compared with BUO+dDAVP group {29.1 +/- 4.2% vs. 42.2 +/- 2.3% (p<0.84); 40.2 +/- 5.4 % vs. 47.9 +/- 4.3% (p<0.91) respectively}. CONCLUSION: BUO and POD show decreased expressions of AQP2 and NKCC2 and the unresponsiveness to chronic ADH infusion may be the pathophysiologic basis of POD such as increased urine output, decreased urine osmolality.
Animals ; Aquaporin 2* ; Blotting, Western ; Chemistry ; Deamino Arginine Vasopressin ; Diuresis ; Immunohistochemistry ; Osmolar Concentration ; Polyuria ; Rats* ; Ureter* ; Ureteral Obstruction* ; Vasopressins

OBJECTIVETo investigate whether leptin receptor Lys109Arg polymorphism influences non-alcoholic fatty liver disease.

METHODSGenomic DNA samples were extracted from blood of subjects who had received a physical examination. Genotyping was performed using oligonucleotide microarray and these fluorescence labeled PCR-amplified fragments were hybridized to allele-specific oligonucleotide probes. The relevant mutation was confirmed by sequencing analysis.

RESULTSA total of 180 subjects (109 males and 71 females) were included in the study, 117 of them had fatty liver disease and the other 63 had no liver problems and served as healthy controls. There were 144 (80%) subjects with GG genotype (Arg109Arg), 33 (18.3%) with GA genotype (Lys109Arg) and 3 (1.7%) with AA genotype (Lys109Lys). The distribution of leptin receptor Lys109Arg polymorphism had no significant difference (P > 0.05) between the fatty liver disease patients (95GG, 21GA and 1AA) and the healthy control subjects (49GG, 12GA and 2AA). The abdominal wall fat was significantly thicker in AA genotype subjects (4.1+/-0.4) cm than that in GA (2.8+/-0.6) cm and GG genotype subjects (2.7+/-0.7) cm (F = 5.197, P = 0.006). The serum cholesterol levels in AA genotype subjects (5.1+/-0.4) mmol/L was significantly lower than that in AG (25.5+/-6.9) mmol/L and GG genotype (27.2+/-8.4) mmol/L subjects (F = 8.164, P = 0.005). There were no significant differences in age, body mass index, hip circumference, waist circumference, blood pressure (BP), percentage of body fat, blood protein, triglyceride, HDL and fasting blood glucose between AA, GG and GA genotype subjects.

CONCLUSIONLeptin receptor Lys109Arg polymorphism may be involved in the regulation of distribution of abdominal wall fat thickness and cholesterol metabolism. Whether leptin receptor Lys109Arg polymorphism is in any way related to fatty liver disease is still not known.

Adult ; Arginine ; chemistry ; genetics ; Fatty Liver ; etiology ; genetics ; Female ; Genotype ; Humans ; Lysine ; chemistry ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Receptors, Cell Surface ; genetics ; Receptors, Leptin

OBJECTIVETo investigate the influence of arginine hydrochloride and arginine acetate on the immune function and acid-base balance in rabbits with severe burns.

METHODSOne hundred and ten flap-eared rabbits were used in the study, in which 8 served as normal control, while the rest were inflicted with 30% TBSA full thickness burn. All the rabbits were divided into 10 groups, i.e. normal control (C, n = 14), burn control (B, n = 14, with intravenous infusion of Ringer's solution), 0.3 g/kg arginine hydrochloride (AH, n = 12), 0.3 g/kg arginine acetate (AA, n = 10), 0.6 g/kg AH (n = 10), and 0.6g/kg AA (n = 10) groups, 1.2 g/kg AH (n = 10), 1.2 g/kg AA (n = 10), 2.4 g/kg AA (n = 14) and 2.4 g/kg AH (n = 12) groups. AA and AH in different doses were fed to rabbits in corresponding groups 2 times a day for 7 days. The changes in the immune function, acid-base balance, chloride ion metabolism, and mortality were determined.

RESULTSDisorder in immune system was found after severe burns, with enhanced immune function at the beginning and weakening afterwards. The lymphocytic transformation rate, the CD4/CD8 ratio, the phagocytosis rate and the chemotactic index of white blood cells on 7 post burn day (PBD) were obviously lower in B group compared with C group (P < 0.05 or 0.01). These indices were obviously higher in 1.2, 2.4 g/kg AA and AH groups than those in B group on 7 PBD (P < 0.05 or 0.01). There was no difference in improvement of immune functions between 0.3, 0.6g/kg AH, AA group and B group. The values of blood pH, base excess (BE), buffer base (BB), HCO(3)(-) level in AH group were significantly lower than those in C group on 7 PBD (P < 0.05 or 0.01), while there were no obvious changes in AA group, they were obviously higher than those in AH group (P < 0.05 or 0.01). The contents of chloride ion in but 2.4 g/kg AH group during 5 to 7 PBD were obviously higher than those in C group and 2.4 g/kg AA group (P < 0.05 or 0.01), while no difference was found between 2.4 g/kg AA and C groups. The mortality in B group was obviously higher than that in 0.3, 0.6, 1.2 g/kg AH and AA groups (P < 0.05 or 0.01), but significantly lower than that in 2.4 g/kg AA and AH groups (P < 0.05).

CONCLUSIONDisorders in immune functions were observed in severely burned rabbits. Administration of arginine acetate as well as arginine hydrochloride could enhance the immune function, but arginine acetate seemed to be safer than arginine hydrochloride. Excessive dosage should be avoided to prevent a rise of the mortality.

Acetates ; chemistry ; therapeutic use ; Acid-Base Equilibrium ; Animals ; Arginine ; chemistry ; therapeutic use ; Burns ; immunology ; physiopathology ; therapy ; Enteral Nutrition ; Female ; Male ; Rabbits ; Wound Healing

In the current study, we synthesized a 16-residue-long peptide VR with the aim of inspecting the feasibility to design beta-hairpin-like antimicrobial peptide. The peptide was designed by alternating arrangement of arginine and valine and linking two stranded antiparallel beta-sheet with a short loop segment (DPG) and a disulfide bridge. Antimicrobial and hemolytic activities were investigated. Melittin was chosen as a control peptide. We also tested bactericidal kinetics and salt sensitivity. Results show that VR had similar antibacterial activity compared with melittin. However, VR displayed much less hemolytic activity than melittin. These results suggest that VR had higher cell selectivity than melittin. The antibacterial activity of VR was not inhibited in the presence of 25 and 50 mmol/L NaCl. VR still possessed antibacterial activity in the presence of 100 mmol/L NaCl. Collectively, the de novo peptide VR displayed high antimicrobial activity, low hemolytic activity, and salt resistant, indicating that VR was a promising candidate for novel antimicrobial applications.
Anti-Infective Agents ; chemistry ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; chemistry ; pharmacology ; Arginine ; chemistry ; Bacteria ; drug effects ; Drug Design ; Escherichia coli ; drug effects ; Hemolysis ; drug effects ; Microbial Sensitivity Tests ; Staphylococcus aureus ; drug effects ; Valine ; chemistry