The study was purposed to investigate the apoptosis of HL-60 cells induced by recombinant common buckwheat trypsin inhibitor (rBTI) and its mechanism. The inhibition rate of rBTI on HL-60 cells was detected by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide); the morphology of HL-60 nuclei was observed by fluorescence microscopy; the apoptosis cells of HL-60 detected by agarose gel electrophoresis and the changes of apoptosis rate was assayed by flow cytometry (FCM), when the HL-60 cells were treated with different concentration of rBTI for 24 hours. The results showed that the growth of HL-60 cells was inhibited evidently after treatment with rBTI in a dose-dependent manner, but there were minimal effects on normal human peripheral blood mononuclear cells (PBMNCs). The nuclei of HL-60 cells showed the characteristics of apoptosis, the analysis by flow cytometry indicated that the apoptosis rate of HL-60 cells was 52% after treatment with rBTI (100 microg/ml), DNA analyzed by agarose gel electrophoresis showed "ladder" pattern. It is concluded that rBTI obviously inhibits growth of HL-60 and induces its apoptosis which provides a foundation for use of recombinant common buckwheat trypsin inhibitor to cure the acute myeloid leukemia.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Fagopyrum ; chemistry ; HL-60 Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Trypsin Inhibitors ; biosynthesis ; genetics ; pharmacology

Animals ; Aquaporins/metabolism* ; Disease Models, Animal ; Glycoproteins/pharmacology* ; Humans ; Lung/metabolism* ; Protective Agents/pharmacology* ; Respiratory Distress Syndrome/physiopathology* ; Sus scrofa ; Trypsin Inhibitors/pharmacology* ; Up-Regulation

OBJECTIVETo study the effects of ulinastatin on coagulation in children who underwent open-heart surgery with cardiopulmonary bypass (CPB).

METHODSFifty children who underwent open-heart surgery for ventricular septal defect were randomly divided into two groups: ulinastatin treatment and control. Before CPB, ulinastatin (1.0×10(4) U/kg) was added to CPB priming fluid only in the ulinastatin treatment group. Activated partial thromboplasin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and international normalized ratio (INR) were measured both before and at 1 hr, 6 hrs and 24 hrs after CPB.

RESULTSThe PT in the ulinastatin group was more prolonged than in the control group at 1 hr after CPB (18.7 ± 0.7 s vs 15.5 ± 0.5 s) and 6 hrs after CPB (17.5 ± 0.6 s vs 15.0 ± 0.6 s). The APTT in the ulinatatin group was also significantly more prolonged than in the control group at 6 hrs after CPB (38.7 ± 3.1 s vs 35.3 ± 3.1 s) and 24 hrs after CPB (34.2 ± 3.0 s vs 31.1 ± 2.6 s).

CONCLUSIONSUlinastatin may prolong PT and APTT after CPB, and thus affects coagulation in children.

Blood Coagulation ; drug effects ; Cardiac Surgical Procedures ; Cardiopulmonary Bypass ; Female ; Glycoproteins ; pharmacology ; Humans ; Infant ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Trypsin Inhibitors ; pharmacology

Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.
Amino Acid Sequence ; Animals ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Spider Venoms ; biosynthesis ; genetics ; pharmacology ; Spiders ; Trypsin Inhibitors ; pharmacology

AIMTo study the protection of casein and protamine against degradation of insulin (INS) by proteolysis enzymes and the effect of these two kinds of protein on the hypoglycemic action of INS solution and enteric-microspheres after administrated orally to rats.

METHODSHPLC was used to determine the remained INS in the solution of alpha-chymotrypsin and trypsin with or without casein or protamine; INS solution and enteric-microspheres were prepared and adiministrated orally to rats together with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC). At the same time, casein or protamine or both of these two kinds of protein were administrated together in order to study their influence on the hypoglycemic effect of INS and microspheres.

RESULTSCasein had a good protection against degradation of INS by alpha-chymotrypsin, but protamine had no protection effect. However, the degradation of INS by trypsin is concerned, the protection effect of protamine on INS was better that of casein. Both of protamine and casein can increase the hypoglycemic effect of INS solution and enteric-microspheres. Co-administrated these two kinds of protein had a better effect. In addition, co-administrated with SNAC, casein and protamine, INS enteric-microspheres had a longer and more potent hypoglycemic effect than that of the solution.

CONCLUSIONCasein and protamine can increase the stability of INS in the intestinal fluid by the mechanism of competition and combine with proteolysis enzymes, which will benefit to INS oral administration.

Administration, Oral ; Animals ; Blood Glucose ; metabolism ; Caprylates ; Caseins ; pharmacology ; Chymotrypsin ; antagonists & inhibitors ; Drug Delivery Systems ; Hypoglycemic Agents ; administration & dosage ; pharmacokinetics ; Insulin ; administration & dosage ; pharmacokinetics ; Male ; Microspheres ; Protamines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Solutions ; Trypsin ; pharmacology

OBJECTIVETo investigate the effects of ulinastatin on gut mucosal apoptosis and bacterium translocation in a rat model of sepsis.

METHODSFifty rats were randomly assigned into 4 groups, namely the control (n=5, no operation or drugs), ulinastatin pretreatment (n=15, treated with 25,000 U/kg ulinastatin 2 h before operation), ulinastatin treatment (n=15, treated with 25,000 U/kg ulinastatin 2 h after operation) and sepsis model (n=15, without drug treatment) groups. The rats in the later 3 groups were subjected to cecal ligation and puncture (CLP). At 3, 6 and 12 h after CLP, the rats were sacrificed and the ileum was removed to examine the pathology and apoptosis of the mucosa. The DNA of Bacillus coli in the whole blood was detected using PCR.

RESULTSSepsis caused of epithelial cell loss in the ileal villi, ulceration and blebbing of the lamina propria. Ulinastatin treatment administered before and after the operation both significantly alleviated these morphological anomalies. The sepsis rats showed significantly increased intestinal mucosal apoptotic index as compared with the other 3 groups (P<0.05). Ulinastatin pretreatment, in comparison ulinastatin treatment 12 h after CLP, significantly increased the intestinal mucosal apoptotic index (P<0.05). Bacillus coli DNA was positive in sepsis and postoperative ulinastatin treatment groups but negative in the control and pretreated groups.

CONCLUSIONIncreased intestinal musocal apoptosis and gut bacterial translocation occur in rats following sepsis, and ulinastatin can effectively decrease intestinal mucosal apoptosis and inhibit bacterial translocation.

Animals ; Apoptosis ; drug effects ; Bacterial Translocation ; drug effects ; Female ; Glycoproteins ; pharmacology ; therapeutic use ; Ileum ; drug effects ; microbiology ; pathology ; Intestinal Mucosa ; drug effects ; microbiology ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; Trypsin Inhibitors ; pharmacology ; therapeutic use

OBJECTIVETo investigate the effect of intraluminal administration of ulinastatin (a protease inhibitor) in the intestine on intestinal inflammation in rats with hemorrhagic shock.

METHODSTwenty-eight Wistar rats were randomized into control group (A), intestinal saline perfusion group (B), ulinastatin intestinal perfusion group (C), and intravenous ulinastatin injection group (D) (n=7). The mean arterial blood pressure (MAP) and survival time of the rats were recorded. The changes in human polymorphonuclear cell (PMN) CD11b expression were detected by flow cytometry. The leukocyte count was recorded at different time points after the treatment, and the pathology of the intestinal mucosa was observed comparatively.

RESULTSGroups C and D showed significantly slower reduction of the MAP than groups A and B after hemorrhagic shock (P<0.05). The survival time of the rats was the longest in group C (P<0.05). CD11b expression increased gradually during hemorrhagic shock in all the groups, but the expression level was the lowest in group C (P<0.05). Hemorrhagic shock caused a reduction in leukocyte counts, which remained the highest in group C (P<0.05). Group C also showed the least intestinal pathology among the 4 groups.

CONCLUSIONIntestinal perfusion of ulinastatin can lower the reduction rate of MAP, attenuate plasma activation and intestinal inflammation, and prolong the survival of rats with hemorrhagic shock. These results indicate an important role of protease in intestinal inflammation during hemorrhagic shock.

Animals ; Arterial Pressure ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation ; enzymology ; metabolism ; Intestines ; enzymology ; metabolism ; Plasma ; metabolism ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; blood ; enzymology ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

OBJECTIVE: To study the effect of urinary trypsin inhibitor (UTI) on STR genotyping with urinary samples. METHODS: Midstream urine samples of 5 male and 5 female volunteers were collected respectively, sub-packaged, added with different concentration of UTI and stored at -80 degrees C. Genomic DNA was extracted from those urinary samples, of which STR profiles were genotyped with IdentifilerTM kit at 8 different time points. Results of genotyping in urinary samples were compared with those of the homogenous blood control samples and the successful rate of genotyping in different group of urinary samples treated with UTI was determined. RESULTS: Fifteen STR loci included in Identifiler system were all detected in control blood samples and urinary samples stored for 1 day. STR locus loss was observed and all 15 STR loci disappeared in female urinary samples untreated with UTI while those storage periods prolonged to 3 and 9 days, respectively. However, all 15 STR loci could be detected in female urinary samples treated with UTI and stored for as long as 9 days. No STR loci could be detected in male urinary samples preserved without UTI for 7 days while 9 STR loci detected preserved with UTI for 9 days. There was no significant difference among the average detection ratios of STR loci in female urinary samples treated with UTI at concentrations of 0.2, 0.4 or 0.6 microg/mL and stored for 30 days, mean of which was as high as 0.8400 +/- 0.0423, statistically higher than that in male urinary samples (0.1600 +/- 0.0423). CONCLUSION Detection rate of STR loci in urinary samples preserved with UTI was increased significantly, which results in prolonging the storage periods of urinary samples for personal identification.
DNA/urine* ; DNA Fingerprinting/methods* ; Female ; Forensic Genetics/methods* ; Genetic Loci/genetics* ; Genotype ; Glycoproteins/pharmacology* ; Humans ; Male ; Microsatellite Repeats ; Polymerase Chain Reaction ; Specimen Handling/methods* ; Time Factors ; Trypsin Inhibitors/pharmacology*

PURPOSE: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). METHODS: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. RESULTS: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. CONCLUSION Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways.
Animals ; Cadherins ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation Mediators ; metabolism ; Injections, Intralesional ; Injections, Intravenous ; Intestinal Diseases ; drug therapy ; etiology ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; Leukocyte Elastase ; metabolism ; Male ; Mucin-2 ; metabolism ; Rats, Wistar ; Sepsis ; complications ; Trypsin ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

OBJECTIVETo study the protective effect of soybean protease inhibitor on LPS-induced lung injury in rats.

METHODFifty male SD rats were randomly divided in five groups, 10 rats in each group as sham-operation group, model control group, positive medicine group, and high, moderate SBTI groups. Except the sham-group, other groups were induced by intratracheal instillation of LPS with a dose of 6 mg x kg(-1). All rats were given drug throughout intraperitoneal injection except the model controlled group, the positive medicine group was given PMSF with a dose of 50 mg x kg(-1), the high dose group of SBTI was given SBTI with a dose of 100 mg x kg(-1), a dose of the moderate group is 50 mg x kg(-1). We examined all rats in seven days. Index exam: cell quantity, activity of neutrophilic granulocyte released elastic protease proteins in BALF, histopathological examination and so on.

RESULTSoybean protease inhibitor can level down the level of total protein, cell quantity, PMN percent, activity of neutrophilic granulocyte in BALF. SBTI level down the content of NF-kappa B in nucleoprotein, while increase the content of I kappa B alpha in plasmoprotein.

CONCLUSIONSBTI is useful in protecting experimental pulmonary injury induced by LPS in rats.

Acute Lung Injury ; chemically induced ; drug therapy ; metabolism ; pathology ; Animals ; Endotoxins ; toxicity ; Granulocytes ; drug effects ; metabolism ; pathology ; I-kappa B Proteins ; metabolism ; Male ; NF-KappaB Inhibitor alpha ; Rats ; Soybeans ; chemistry ; Transcription Factor RelA ; metabolism ; Trypsin Inhibitors ; pharmacology