Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×10⁶, aged rats(0.243±0.023)×10⁶). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1β of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.
Animals ; Cytokines ; Microglia ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; Trypsin

In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.
Humans ; Trypsin/chemistry* ; Alkaloids/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

trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections.]]>
Coronavirus ; Diarrhea ; Erythrocytes ; Hemagglutination ; Methods ; Neuraminidase ; Population Characteristics ; Swine ; Trypsin ; Virion

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
Amino Acid Sequence ; Chromatography, Liquid ; Chymotrypsin/metabolism* ; Digestion ; Humans ; Membrane Proteins ; Tandem Mass Spectrometry ; Trypsin ; Vitamin K Epoxide Reductases

In canine acute pancreatitis (AP), inappropriate release and activation of zymogen proteases within the pancreas results in the consumption of serum antiproteases. The aim of this study was to examine whether the serum concentrations of α₂-macroglobulin (A2MG), α₁-antitrypsin (A1AT), and C-reactive protein (CRP) differ between dogs with AP and healthy dogs. Twenty healthy dogs and 20 dogs with AP were included in this study. Concentrations of A2MG, A1AT, and CRP were measured in the sera of healthy dogs and dogs diagnosed with AP. Serum A2MG and A1AT concentrations were significantly lower in dogs with AP than in healthy dogs, whereas the serum CRP concentration was significantly higher. In addition, the concentrations of A2MG and A1AT were significantly higher in AP survivors than in AP non-survivors, while the CRP concentration was significantly lower. However, in both AP survivors and non-survivors, the CRP concentrations showed a negative correlation with A2MG concentrations but not with A1AT. These findings indicate that serum antiproteases and CRP concentrations might be associated with the mortality rate of AP in dogs.
Animals ; C-Reactive Protein ; Dogs ; Humans ; Mortality ; Pancreas ; Pancreatitis ; Peptide Hydrolases ; Protease Inhibitors ; Survivors ; Trypsin

Proteomics is a fast-growing discipline that aims at systematic identification, quantification of proteins and their post-translational modifications in cells. Mass spectrometry-based shotgun proteomics technology is currently one of the mainstream methods for proteomics research. With this method, proteins need to be digested to peptides by site-specific proteases before they can be detected with mass spectrometry. Therefore, site-specific proteases played key roles in this process and so far, a variety of specific proteases have been developed and used in proteomics study. Particularly, the identification, characterization and development of proteases that cleave at the N-termini of corresponding amino acid residues, which are just mirrors to those of typical C-termini proteases, provide novel tools for proteomics analysis. In this review, we summarized the proprieties of LysargiNase, a most recently identified mirror trypsin, and its applications in proteomics research to promote its more widespread usage.
Mass Spectrometry ; Metalloproteases ; chemistry ; metabolism ; Protein Processing, Post-Translational ; Proteomics ; Trypsin ; chemistry

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

A 28-year-old man with a history of alcohol abuse was diagnosed with acute pancreatitis based on clinical symptoms, laboratory findings and computed tomography findings. On the second day of hospitalization, he complained of sudden visual disturbance. The ophthalmologic examination showed Purtscher's-like retinopathy. Two weeks after initial presentation, his vision was significantly improved along with epigastric pain. Retinal arteriolar occlusion by complement-mediated leukoembolization is the proposed pathogenic mechanism of Purtscher's-like retinopathy. Increased activity of proteases such as trypsin, associated with acute pancreatitis might be linked with the production of complement C5a. We report a rare case of Purtscher's-like retinopathy associated with acute pancreatitis.
Adult ; Alcoholism ; Complement C5a ; Hospitalization ; Humans ; Pancreatitis* ; Peptide Hydrolases ; Retinaldehyde ; Trypsin

BACKGROUND: As nonsurgical interventions for vitiligo are not always successful, various surgical modalities have been used in patients with refractory vitiligo. Of these, non-cultured epidermal suspension transplantation (NCES) was recently introduced to treat large recipient sites using cells from small donor tissue. OBJECTIVE: We assessed the effectiveness and safety of NCES as a surgical treatment for patients with refractory vitiligo. METHODS: We retrospectively reviewed 20 cases in 17 patients (11 females; median age 25 years) who underwent NCES from July 2015 through March 2018. Suction blisters (20 mm in diameter) were collected from the patient's inner thigh at a donor-to-recipient area ratio of 1:5. After the addition of 5 mL recombinant trypsin solution to the suction blisters, followed by incubation at 37℃ for 60 min, epidermal cells were manually scraped off the blister surface, and epidermal cell suspension was obtained by centrifugation at 1,500 RPM for 5 min. The suspension was applied to the vitiligo regions after epidermal ablation of those regions. Phototherapy resumed 1 month later. Treatment success was defined as ≥75% repigmentation of the surgical site, and all adverse events were noted. RESULTS: Overall, 85.0% of cases (17/20) exhibited treatment success. Adverse events included hyperpigmentation (20%) and surgical site infection (5%), but the treatment was tolerable in all cases. CONCLUSION: NCES is a reliable surgical option for patients with vitiligo refractory to nonsurgical treatment. Large areas of vitiligo can be treated by NCES, and use of this technique should be encouraged in Korea.
Blister* ; Centrifugation ; Female ; Humans ; Hyperpigmentation ; Korea ; Phototherapy ; Retrospective Studies* ; Suction* ; Surgical Wound Infection ; Thigh ; Tissue Donors ; Transplantation ; Trypsin ; Vitiligo*