OBJECTIVETo investigate the protective effects of glycyl-glutamine dipeptide supplement on the function of myocardial dynamics in severely burned rats, and to explore its mechanism.

METHODSOne hundred and thirty-six Wistar rats were randomly divided into five groups: i. e, control group (C, n = 8, without burns), burn group (B, n = 32), Gln group (Gln, n = 32), Gly group (Gly, n = 32) and Gly-Gln group (Gly-Gln, n = 32). The rats in the latter four groups were respectively treated with tyrosine (1.5 g x kg(-1) x d(-1)), glutamine (1.0 g x kg(-1) x d(-1)) and tyrosine (0.5 g x kg(-1) x d(-1)), glycine (0.5 g x kg(-1) x d(-1)) and tyrosine (1.0 g x kg(-1) x d(-1)), and Glycyl-glutamine dipeptide (1.5 g x kg(-1) x d(-1)) after receiving a 30% TBSA full-thickness burn on the back. Glutathione (GSH), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cell energy charge (EC) and the index of myocardial dynamics (ASOP, AODP, LVSP, + dp/dtmax) were measured at 12, 24, 48, 72 post-burn hours (PBH).

RESULTSThe content of GSH, ATP, EC and the level of aortic systolic pressure (ASOP), aortic diastolic blood pressure (AODP), left ventricular end diastolic pressure (LVEDP) and maximum rate of intraventricular pressure rise/down (+ dp/dtmax) in B, Gln, Gly, Gly-Gln groups were obviously lower than those in C group (P < 0.01), while the levels of AMP and ADP showed an opposite tendency. Compared with B group, the above indices were ameliorated. The content of GSH (72.7 +/- 1.7) micromol/g in Gly-Gln group at 12 PBH was obviously higher than that in Gln group (67.8 +/- 3.8) micromol/g (P < 0.01). The levels of EC and AOSP were obviously higher in Gly-Gln group than that in Gln group (P < 0.01). The level of GSH, EC, AOSP in Gly-Gln groups were obviously higher than those in Gly group at 48 PBH.

CONCLUSIONGlycyl-glutamine dipeptide, Gly and Gln supplementation after burns can improve the content of GSH and high energy phosphate compound, and suppress the decline of myocardial dynamics function. The effects of Glycyl-glutamine dipeptide is better than single Gly or Gln, indicating that the protective effect on myocardial function after severe burns by Gln and Gly is synergistic.

Animals ; Burns ; drug therapy ; metabolism ; Dipeptides ; pharmacology ; Glutathione ; metabolism ; Glycine ; Myocytes, Cardiac ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

This study was to investigate the effects of DAPT (N-[N-(3,5-difluorophenacetyl-L:-alanyl)]-S-phenylglycine-butyl ester) on cell cycle, apoptosis, differentiation and expansion of hematopoietic stem cells (HSC) of mouse and to elucidate the possible mechanisms. The mRNA expressions of cell cycle-related genes p18, p21, p27, CDK1, CDK2, CDK4, CDK6, and apoptosis-related genes Bcl-2, Bcl-xl, mcl-1, Bax, Bim, p53, Puma were measured by real-time PCR. The cell cycle and apoptosis of Lin(-)c-kit(+)Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells were detected by flow cytometry. The differentiation level of HSC was determined by single cell culture. The expansion of HSC were measured with long-term culture. The results indicated that the mRNA expression of the cell cycle related-genes CDK1, CDK2, CDK4, CDK6, p27 in Lin(-) c-kit(+)Sca-1(+) marked cells increased (P < 0.05), the expression of p18, p21 decreased (P < 0.05), the expression of the apoptosis related-genes Bcl-2, Bcl-xl, Bax, p53, Puma in Lin(-) c-kit(+)Sca-1(+) marked cells increased (P < 0.05), the expression of Bim decreased (P < 0.05), the expression of Mcl-1 had not changed (P > 0.05) after treatment with DAPT 1 µmol/L for 5 d. The changes of cell cycle of Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow had no statistical significance after treatment with DAPT 1 µmol/L for 5 d, CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow at G(0) phase decreased and at G(1) phase increased after treatment with DAPT 1 µmol/L for 5 d (P < 0.05); the apoptotic fractions of Lin(-) c-kit(+) Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow increased after treatment with DAPT 1 µmol/L for 5 d (P < 0.05). The changes of colony number, average number of cells in wells and their differentiation had no statistical significance (P > 0.05) after treatment with DAPT 1 µmol/L for 10 d. Expansion of HSC in bone marrow of mouse decreased after treatment with DAPT 1 µmol/L for 3 d. It is concluded that DAPT not only enhances the exhaustion of CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells of mouse, but also enhances the apoptosis of Lin(-)c-kit(+)Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells of mouse. DAPT also reduces the expansion of HSC. However, the changes of survival and differentiation of single CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in mouse bone marrow cells have no statistical significance.
Animals ; Apoptosis Regulatory Proteins ; metabolism ; Bone Marrow Cells ; metabolism ; Cell Cycle Proteins ; metabolism ; Dipeptides ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Signal Transduction

OBJECTIVETo screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells.

METHODSThe function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a (+) and pGBKT7 to construct recombinant expression vectors pET32a (+)-PS1TP2 and pGBKT7-PS1TP2. They were transduced into E. coli Rosetta strain and yeast AH109. The transformed yeast mated with yeast Y187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) for selecting twice and then screening. Then a green fluorescent protein (GFP) expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy.

RESULTSBioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH109, the expression protein showed: (1) the molecular weight of the expressed product was about 41000 Da, and (2) PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1-PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma.

CONCLUSIONA prokaryotic expression vector pET32a(+)-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.

Base Sequence ; Cloning, Molecular ; Dipeptides ; Gene Expression ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Protein Precursors ; metabolism ; Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and micro-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and micro-calpain may be involved in colon epithelial cell death induced by E. histolytica.
Calpain/antagonists & inhibitors/genetics/*metabolism ; *Cell Death ; Cell Line ; Cell Survival/drug effects ; Dipeptides/metabolism ; Entamoeba histolytica/*pathogenicity ; Epithelial Cells/*parasitology ; Gene Knockdown Techniques ; Humans

OBJECTIVETo study the effect of matrix metalloproteinases inhibitor GM6001 in suppressing scar tissue formation in the filtering passage after glaucoma filtration surgery.

METHODSTwenty-four pigmented rabbits (48 eyes) underwent trabeculectomy followed by subconjunctival injection of GM6001 in the right eye (treated eyes) and injection of PBS in the left eye (control) once a day. The intraocular pressure was monitored postoperatively and proliferating cell nuclear antigen (PCNA)- and α-smooth muscle actin (α-SMA)-positive cells in the filtering pathway were detected using immunohistochemistry.

RESULTSOn postoperative days 7, 14, 21, and 28, the intraocular pressure was significantly lower in the treated eyes (GM6001) than in the control eyes (P<0.01). The counts of PCNA- and α-SMA-positive cells were also significantly lowered in the treated than in the control eyes (P<0.01).

CONCLUSIONGM6001 can inhibit excessive proliferation of the fibroblasts in the filtering pathway to suppress scar tissue formation and prolong the existence of the functional filtration bleb in rabbits.

Actins ; metabolism ; Animals ; Cicatrix ; pathology ; prevention & control ; Dipeptides ; pharmacology ; Filtering Surgery ; adverse effects ; Glaucoma ; surgery ; Intraocular Pressure ; Postoperative Complications ; Proliferating Cell Nuclear Antigen ; metabolism ; Rabbits

OBJECTIVETo investigate the potential effects on cognitive function, prognosis, and neuropeptide levels of patients in response to combination therapy with ornithine aspartate plus naloxone for hepatic encephalopathy.

METHODSEighty-four consecutive patients diagnosed with hepatic encephalopathy were randomly divided into two equal groups. The control group (n = 42) received traditional medical treatment, and the research group (n = 42) received the traditional medical treatment as well as the combination therapy with ornithine aspartate plus naloxone. The supplemental treatment was comprised of daily intravenous injection of 10-15 g ornithine aspartate in 250 ml of 5% glucose plus intravenous drip of 3 mg naloxone in 100 ml of 5% glucose, and was given in 7-day cycles for one or two cycles. The cognitive function of patients was assessed by Hasegawa Intelligence Scale (HDS) and Mini-Mental State Examination (MMSE) questionnaires. The effective rate and time duration from coma to consciousness were recorded. Changes in blood ammonia level, markers of liver function, and neuropeptide levels were measured by standard biochemical assays. Intergroup differences were assessed by the Chi-squared test.

RESULTSThe HDS and MMSE scores of the research group were significantly higher than those of the control group after therapy. The effective rate, time duration from coma to consciousness, blood ammonia, the liver function markers alanine aminotransferase, gamma-glutamyl-transpeptidase and total bilirubin, and the neuropeptides arginine vasopressin and beta-endorphin were remarkably improved after treatment in the research group, as compared with that in the control group.

CONCLUSIONSupplementing the traditional treatment for hepatic encephalopathy with ornithine aspartate plus naloxone combination therapy provides better therapeutic outcome than traditional treatment alone.

Adult ; Dipeptides ; therapeutic use ; Female ; Hepatic Encephalopathy ; drug therapy ; metabolism ; psychology ; Humans ; Male ; Middle Aged ; Naloxone ; therapeutic use ; Neuropeptides ; metabolism ; Prognosis

The rapid increase in the prevalence of metabolic syndrome, which is associated with a state of elevated systemic oxidative stress and inflammation, is expected to cause future increases in the prevalence of diabetes and cardiovascular diseases. Oxidation of polyunsaturated fatty acids and sugars produces reactive carbonyl species, which, due to their electrophilic nature, react with the nucleophilic sites of certain amino acids. This leads to formation of protein adducts such as advanced glycoxidation/lipoxidation end products (AGEs/ALEs), resulting in cellular dysfunction. Therefore, an effective reactive carbonyl species and AGEs/ALEs sequestering agent may be able to prevent such cellular dysfunction. There is accumulating evidence that histidine containing dipeptides such as carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-methyl-L-histidine) detoxify cytotoxic reactive carbonyls by forming unreactive adducts and are able to reverse glycated protein. In this review, 1) reaction mechanism of oxidative stress and certain chronic diseases, 2) interrelation between oxidative stress and inflammation, 3) effective reactive carbonyl species and AGEs/ALEs sequestering actions of histidine-dipeptides and their metabolism, 4) effects of carnosinase encoding gene on the effectiveness of histidine-dipeptides, and 5) protective effects of histidine-dipeptides against progression of metabolic syndrome are discussed. Overall, this review highlights the potential beneficial effects of histidine-dipeptides against metabolic syndrome. Randomized controlled human studies may provide essential information regarding whether histidine-dipeptides attenuate metabolic syndrome in humans.
Amino Acids ; Anserine ; Carbohydrates ; Cardiovascular Diseases ; Carnosine ; Chronic Disease ; Dipeptides ; Fatty Acids, Unsaturated ; Histidine ; Humans ; Inflammation ; Metabolism ; Oxidative Stress ; Prevalence ; Sequestering Agents

OBJECTIVETo study the effect of L-alanyl-L-glutamine (Ala-Gln) on the levels of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) in the intestinal tissues of low-birth-weight (LBW) newborn rats with hypoxia/reoxygenation-induced intestinal injury.

METHODSPregnant rats were fed with or without smoking. The rats born by those fed without smoking were included in group A; for the rats born by those fed with smoking, normal-birth-weight rats were included in group B, and LBW rats were randomly divided into control group (group C), hypoxia/reoxygenation (H/R) group (group D), and Ala-Gln group (group E). Each group consisted of 24 newborn rats. The rats in groups D and E received H/R treatment twice a day for three consecutive days to establish an intestinal injury model; the rats in group E were intraperitoneally injected with Ala-Gln (10 ml/kg) before daily H/R treatment, while those in groups C and D were given an equal dose of normal saline by intraperitoneal injections. On days 4, 7, and 10 after birth, 8 rats were sacrificed in each group to collect intestinal tissues. The IGF-1 levels in intestinal tissues were measured using ELISA, and IGF-1R levels were measured by immunohistochemistry.

RESULTSThere were no significant differences in IGF-1 and IGF-1R levels between groups A and B at all time points. The levels of IGF-1 and IGF-1R in group C kept increasing, were higher than those in other groups on day 7 (P<0.05), and reached a normal level on day 10, without significant differences compared with those in groups A and B. Group D had significantly lower IGF-1 and IGF-1R levels than group C at all time points (P<0.05). The levels of IGF-1 and IGF-1R in group E were lower than those in group C on days 4 and 7 (P<0.05), but they increased to approximately the levels in group C and were significantly higher than those in group D on day 10.

CONCLUSIONSIntrauterine and postnatal hypoxia may induce intestinal injury in LBW newborn rats, and parenteral administration of high-dose Ala-Gln can reduce hypoxia-induced intestinal injury. Therefore, Ala-Gln has a protective effect against hypoxia-induced intestinal injury.

Animals ; Birth Weight ; Dipeptides ; pharmacology ; Female ; Hypoxia ; metabolism ; Insulin-Like Growth Factor I ; analysis ; Intestines ; chemistry ; Male ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Receptor, IGF Type 1 ; analysis

The aim of this study was to explore the effect of DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) on proliferation in vitro of human multiple myeloma cell line RPMI8226 and its underlying mechanism. The proliferation of RPMI8226 cells was detected by CCK-8 method; flow cytometry was employed to assay the cell apoptosis rate;the expressions of Notch1 and Hes1 proteins were detected by Western blot. The results indicated that the proliferation of human RPMI8226 cells significantly decreased after treatment with DAPT 0.5 - 5.0 µmol/L for 24 - 72 h (P < 0.05) in a concentration- and time-dependent manner. DAPT significantly induced apoptosis of RPMI8226 cells (P < 0.05). The expressions of Notch1 and Hes1 proteins were gradually downregulated with the increase of DAPT concentration. It is concluded that the DAPT can inhibit the proliferation of RPMI8226 cells, which may be related with the down-regulation of the protein expression of Notchl and Hes1.
Apoptosis ; drug effects ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dipeptides ; pharmacology ; Homeodomain Proteins ; metabolism ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptor, Notch1 ; metabolism ; Transcription Factor HES-1

OBJECTIVETo study the expression of lung Krüppel-like transcription factor (KLF2/LKLF) in lung tissues of rats with chronic obstructive pulmonary disease (COPD) and the relationship between KLF2 and NF-E2-related factor 2 (Nrf2), and make further explore the effects of KLF2 on the expression of gamma-glutamylcysteine synthetase (gamma-GCS).

METHODSTwenty-two male SD rats were randomly divided into a COPD group (n = 10) and a normal control group (n = 11). The rat model of COPD established by cigarette smoking and intratracheal instillation of lipopolysaccharide (LPS), and lung tissues were obtained. The expressions of KLF2, Nrf2, gamma-GCS mRNA and protein in lung tissues were measured by immunohistochemistry (IHC), Western blot, in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR). To explore the relationship between KLF2 and Nrf2 protein,we utilize the method of co-immunoprecipitation (CO-IP).

RESULTSIHC and Western blot showed that protein expressions of KLF2, Nrf2, gamma-GCS were higher in the lung tissues from rats with COPD than those in the control groups (all P < 0.05). The levels of KLF2, gamma-GCS mRNA were markedly increased in the COPD group (all P < 0.01) while Nrf2 mRNA expression in COPD group had no significant difference with that in control group ( P > 0.05). CO-IP result showed that KLF2 were obviously present in immunoprecipitates of Nrf2 (P < 0.01) . Linear correlation analysis showed that the level of KLF2 protein was positively correlated with the level of Nrf2 protein (P < 0.05), and KLF2, Nrf2 proteins were positively correlated with gamma-GCS mRNA and protein (all P < 0.05).

CONCLUSIONThe expression of KLF2 is significantly up-regulated in COPD, which maybe up-regulate gamma-GCS mRNA expression by increasing Nrf2 expression and nuclear translocation against oxidative stress.

Animals ; Dipeptides ; metabolism ; Kruppel-Like Transcription Factors ; metabolism ; Lung ; pathology ; physiopathology ; Male ; NF-E2-Related Factor 2 ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; pathology ; physiopathology ; Rats ; Rats, Sprague-Dawley