alpha-chymotrypsin and Disease-Models--Animal

Topics: Animals; Chymotrypsin; Disease Models, Animal; Glaucoma; Lasers; Light; Ocular Hypertension; Radiation Injuries, Experimental; Steroids

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Topics: alpha-Synuclein; Animals; Chymotrypsin; Disease Models, Animal; Mice; Mice, Transgenic; Neurodegenerative Diseases; Parkinson Disease; Proteasome Endopeptidase Complex

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of polyglutamine stretch (polyQ) at the N-terminus of huntingtin (Htt) protein. The abnormally expanded polyQ stretch of mutant Htt makes it prone to aggregate, leading to neuropathology. HAP40 is a 40-kDa huntingtin-associated protein with undefined functions. HAP40 protein has been shown to increase in HD patients and HD mouse model cells. However, recent proteomic analysis provides new evidence that HAP40 protein is decreased in the striatum of HD knockin model mice. In this study, we developed HAP40-specific antibody and showed that both HAP40 mRNA and its encoded protein were reduced in HD striatal neuronal STHDH

Topics: Animals; Autophagy; Cell Line; Chymotrypsin; Corpus Striatum; Disease Models, Animal; Green Fluorescent Proteins; Huntingtin Protein; Huntington Disease; Intracellular Signaling Peptides and Proteins; Mice; Mutant Proteins; p38 Mitogen-Activated Protein Kinases; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Kinase Inhibitors; Protein Subunits; Solubility; Ubiquitin

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.

Topics: Animals; Ceruletide; Chymotrypsin; Disease Models, Animal; Gene Expression Regulation; Glycoproteins; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mutation; Pancreatitis; Prostatic Secretory Proteins; Trypsin; Trypsin Inhibitor, Kazal Pancreatic; Trypsinogen

Topics: Amblyomma; Animals; Anti-Inflammatory Agents; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chymases; Chymotrypsin; Disease Models, Animal; Enzyme Inhibitors; Gene Expression; Glycoproteins; Mice; Rabbits; Rats; Recombinant Proteins; Saccharomycetales; Serpins

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

Topics: Animals; Boron Compounds; Chymotrypsin; Disease Models, Animal; Glutathione; Glycine; Heart; Humans; Membrane Potentials; Mitochondria; Myocardial Ischemia; Oxygen Consumption; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats

The digestive enzyme chymotrypsin protects the pancreas against pancreatitis by reducing harmful trypsin activity. Genetic deficiency in chymotrypsin increases pancreatitis risk in humans and pancreatitis severity in mice. Pancreatic chymotrypsin is produced in multiple isoforms including chymotrypsin B1, B2, C and chymotrypsin-like protease (CTRL). Here we investigated the role of CTRL in cerulein-induced pancreatitis in mice. Biochemical experiments with recombinant mouse enzymes demonstrated that CTRL cleaved trypsinogens and suppressed trypsin activation. We generated a novel CTRL-deficient strain (Ctrl-KO) using CRISPR-Cas9 genome engineering. Homozygous Ctrl-KO mice expressed no detectable CTRL protein in the pancreas. Remarkably, the total chymotrypsinogen content in Ctrl-KO mice was barely reduced indicating that CTRL is a low-abundance isoform. When given cerulein, Ctrl-KO mice exhibited lower intrapancreatic chymotrypsin activation and a trend for higher trypsin activation, compared with C57BL/6N mice. Despite the altered protease activation, severity of cerulein-induced acute pancreatitis was similar in Ctrl-KO and C57BL/6N mice. We conclude that CTRL is a minor chymotrypsin isoform that plays no significant role in cerulein-induced pancreatitis in mice.

Topics: Acute Disease; Animals; Biopsy; Cell Line; Chymotrypsin; Disease Models, Animal; Enzyme Activation; Gene Expression; Humans; Immunohistochemistry; Mice; Mice, Knockout; Pancreas; Pancreatitis; Peroxidase; Serine Endopeptidases; Severity of Illness Index; Trypsin

Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast to trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC due to a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the novel Ctrc+ strain the severity of cerulein-induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis.

Topics: Animals; Ceruletide; Chymotrypsin; Disease Models, Animal; Exons; Genetic Predisposition to Disease; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Pancreatitis; Secretagogues; Severity of Illness Index; Species Specificity

Intrapancreatic activation of the digestive proteases trypsin and chymotrypsin is an early event in the development of pancreatitis. Human genetic studies indicate that chymotrypsin controls trypsin activity via degradation, but there is no evidence of this from animal models. We used CRISPR-Cas9 to disrupt the chymotrypsinogen B1 gene (Ctrb1) in C57BL/6N mice and induced pancreatitis in CTRB1-deficient and C57BL/6N (control) mice by administration of cerulein. CTRB1-deficient mice given cerulein had significant increases in intrapancreatic trypsin activity and developed more severe pancreatitis compared with control mice. CTRB1 therefore protects against secretagogue-induced pancreatitis by reducing trypsin activity. Protease inhibitors developed for treatment of pancreatitis should be designed to target trypsin but not chymotrypsin.

Topics: Animals; Arginine; Ceruletide; Chymotrypsin; Disease Models, Animal; Enzyme Activation; Enzyme Stability; Female; Male; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Proteolysis; Severity of Illness Index; Trypsin

to compare two models of experimental glaucoma by induction of ocular hypertension in rabbits.. Sixteen New Zealand female rabbits, 2-3 kg were used. Model A (n=6): cauterization of episcleral and perilimbar veins of the right eye (RE) with surgical electrocautery. Model B (n=10): Injection of ?-chymotrypsin in posterior chamber of RE. Intraocular pressure (IOP) was measured before and after the induction of ocular hypertension (OHT), once a week at the same time of day for 40 days, with a manual tonometer. The animals were euthanized by CO2 inhalation. In both models the control was the IOP of the left eye (LE). The mean and standard error (SE) values of IOP, expressed in mmHg, were compared statistically by applying Student's t-test with a significance level of p<0.05.. The IOP in LE (control) of model A: was 12.9±1.05 and in model B: 12.9±1.09. There were no significant differences between the models. Model A: The IOP increase in RE was 14.7% (14.8±1.4) with respect to LE. A significant increase in IOP was observed within the first 24 hours: 23.5±1.9 (p<0.05) compared to the control eye. There were no significant differences with subsequent controls. Model B: The increase in IOP in RE was 129.1% (29.6±3.4) with respect to LE. In all cases an increase was observed from Day 1 (p<0.05). The IOP peak in RE was evidenced on Day 25: 35±3.4 (p<0.05). The increase in IOP induced by model B was significantly higher (p<0.01) than in model A. There was loss of ganglion cells of the retina in both models, but the following anatomo-pathological changes were observed only in model B: buphthalmos, subluxation of the lens and increased excavation of the papilla.. This study indicates that model B is the most appropriate method to induce a rapid, controlled increase of IOP in rabbits and, more importantly, that this increase may be sustained over extended periods of time. This model could be useful for evaluating the efficacy of new ocular drug delivery systems and for further studies of the physiopathology of glaucoma.. comparar dos modelos de glaucoma experimental por inducción de hipertensión ocular en conejos y describir cambios anatomo-patológicos.. Se utilizaron 16 conejos New Zealand, hembras, de 2-3 kg. Modelo A (n=6): cauterización de venas epiesclerales y perilimbares en ojo derecho (OD) con cauterio eléctrico quirúrgico. Modelo B (n=10): inyección intracamerular en OD de ?-quimotripsina. Se midió la presión intraocular (PIO) antes y después de la inducción de la hipertensión ocular (HTO), una vez por semana, a la misma hora del día, durante 40 días, con tonómetro manual. Los animales fueron sacrificados por inhalación de CO2. En ambos modelos la PIO del ojo izquierdo (OI).fue tomado como valor control. La media y error estándar (EE) de los valores de la PIO, expresada en mmHg, fueron evaluadas y comparadas estadísticamente aplicando Test T de Student considerando un nivel de significación de p < 0.05.. La PIO en OI (control) del modelo A: fue 12,9±1,05 y en el modelo B: 12,9±1,09. No se observaron diferencias significativas entre ambas. Modelo A: el aumento de la PIO en OD fue 14,7% (14,8±1,4) con respecto a OI. Se observó un incremento significativo de la PIO dentro de las primeras 24 hs: 23,5±1,9 (p<0,05) comparado con el valor del ojo control. No hubo diferencias significativas con los controles posteriores. Modelo B: el aumento de la PIO en OD fue 129,1% (29,6±3,4) con respecto al OI. En todos los casos se observó un incremento desde el día 1 (p<0,05). El pico de PIO en OD se evidenció el día 25: 35±3,4 (p<0,05). El incremento de la PIO inducida en el modelo B fue significativamente mayor (p<0,01) que en el modelo A. En ambos modelos hubo pérdida de células ganglionares de la retina, pero sólo en el modelo B se observaron los siguientes cambios anatomo-patológicos: buftalmus, subluxación del cristalino y aumento de la excavación de la papila. Conclusión: De acuerdo a este estudio, el modelo B aparece como el método más apropiado a los fines de inducir un incremento rápido y controlado de la IOP en conejos y más importante, este incremento sería capaz de mantenerse alto a lo largo de periodos de tiempos extendidos. Este modelo podría ser de gran utilidad para evaluar la eficacia de nuevos sistemas oculares de liberación de fármacos y realizar futuros estudios de la fisiopatología del glaucoma. De acuerdo a este estudio, el modelo B aparece como el método más apropiado a los fines de inducir un incremento rápido y controlado de la IOP en conejos y más importante, este incremento sería capaz de mantenerse alto a lo largo de periodos de tiempos extendidos. Este modelo podría ser de gran utilidad para evaluar la eficacia de nuevos sistemas oculares de liberación de fármacos y realizar futuros estudios de la fisiopatología del glaucoma

Topics: Animals; Chymotrypsin; Disease Models, Animal; Electrocoagulation; Female; Glaucoma; Intraocular Pressure; Rabbits; Reproducibility of Results; Time Factors; Tonometry, Ocular

We sought to verify whether isoflavin-beta (Iso-β), a mixture of isoflavones with antioxidant properties, could prevent thyrotoxicosis-induced loss of muscle mass and the participation of oxidative stress (OS) in the mechanisms of this prevention.. Two experimental periods of thyrotoxicosis induction were used in Wistar rats: 3 and 5 days to assess Iso-β effects before and after thyrotoxicosis-induced muscle wasting. After euthanasia, peritoneal fat and gastrocnemius muscle were collected, weighed, and muscle OS was assessed.. Iso-β prevented the loss of gastrocnemius mass in thyrotoxic rats through the prevention of muscle OS generation during thyrotoxicosis, increasing muscle total antioxidant capacity and decreasing mitochondrial cytochrome c oxidase activity, lipid peroxidation, and protein carbonyl content.. Iso-β decreased oxidative modification of proteins, which is known to exert a major role during proteolysis induction and is present in thyrotoxic myopathy, highlighting the potential action of Iso-β in this complication of the disease. Muscle Nerve 56: 975-981, 2017.

Topics: Animals; Antioxidants; Chymotrypsin; Cyclohexanols; Disease Models, Animal; Drug Administration Schedule; Electron Transport Complex IV; Glycerol; Isoflavones; Male; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Protein Carbonylation; Rats; Rats, Wistar; Superoxide Dismutase; tert-Butylhydroperoxide; Thiobarbituric Acid Reactive Substances; Thyrotoxicosis

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Topics: Animals; Chagas Disease; Chymotrypsin; Disease Models, Animal; Female; Humans; Inhibitory Concentration 50; Kinetoplastida; Leishmaniasis; Mice; Molecular Structure; Molecular Targeted Therapy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrimidines; Species Specificity; Triazoles; Trypanosomiasis, African

Wound dressing materials such as asiaticoside, collagenase, and alpha-chymotrypsin are often used for effective wound healing activity.. In this study, the effects of asiaticoside, collagenase, and alpha-chymotrypsin were studied in rabbit models with open wounds with tissue loss and with full-thickness flank excisions for a period of 21 days.. Three groups of 4 rabbits were examined during trial periods of 7, 14, and 21 days. Four circular wounds measuring 1.5 cm in diameter were made on the dorsal sides of the animals: 2 on the right and 2 the left. Asiaticoside, collagenase, and alpha-chymotrypsin were applied to wounds daily for a period of 7, 14, and 21 days, while 1 gauzed wound served as the control. All biopsy specimens were histopathologically evaluated for recovery. On day 7, microscopic review showed no differences in wound healing between groups.. By day 14, alpha-chymotrypsin showed the quickest reepithelialization (P < 0.05); and by day 21 asiaticoside and collagenase (P < 0.01) showed effective recovery, due to the completion of wound healing for all animals in both groups.. Alpha-chymotrypsin is more effective than the other 2 groups for only 14 days. The effectiveness of asiaticoside and collagenase displayed a more rapid improvement in comparison to alpha-chymotrypsin for healing open wounds with tissue loss for a period of 21 days.

Topics: Animals; Anti-Infective Agents, Local; Bandages; Chymotrypsin; Collagenases; Disease Models, Animal; Rabbits; Triterpenes; Wound Healing; Wound Infection; Wounds and Injuries

Although the proteasome inhibitor known as bortezomib can modulate the inflammatory process through the nuclear factor-kappa B signaling pathway, the immunomodulatory effect of pre-incubated bortezomib has not been fully evaluated for inflammation by infectious agents. Therefore, we evaluated the effect of bortezomib on the expression of inflammatory cytokines and mediators in macrophage cell lines and on survival in a murine peritonitis sepsis model.. Bortezomib was applied 1 hr before lipopolysaccharide (LPS) stimulation in RAW 264.7 cells. The cecal ligation and puncture (CLP) experiments were performed in C57BL/6J mice.. Pre-incubation with bortezomib (25 nM or 50 nM) prior to LPS (50 ng/mL or 100 ng/mL) stimulation significantly recovered the number of viable RAW 264.7 cells compared to those samples without pre-incubation. Bortezomib decreased various inflammatory cytokines as well as nitric oxide production in LPS-stimulated cells. The 7-day survival rate in mice that had received bortezomib at 0.01 mg/kg concentration 1 hr prior to CLP was significantly higher than in the mice that had only received a normal saline solution of 1 mL 1 hr prior to CLP. In addition, the administration of bortezomib at 0.01 mg/kg concentration 1 hr before CLP resulted in a significant decrease in inflammation of the lung parenchyma. Collectively, pretreatment with bortezomib showed an increase in the survival rate and changes in the levels of inflammatory mediators.. These results support the possibility of pretreatment with bortezomib as a new therapeutic target for the treatment of overwhelming inflammation, which is a characteristic of severe sepsis.

Topics: Animals; Boronic Acids; Bortezomib; Cecum; Cell Adhesion Molecules; Cell Line; Cell Proliferation; Cell Survival; Chymotrypsin; Cytokines; Disease Models, Animal; Inflammation Mediators; Ligation; Lipopolysaccharides; Lung; Male; Mice, Inbred C57BL; Nitric Oxide; Proteasome Inhibitors; Punctures; Pyrazines; Sepsis

In this study, we examined the treatment and mechanism of BMMSC on a deep II degree scald of the hamster skin. A deep II degree scald model on the skin of 40 hamsters was duplicated and divided randomly into a stem cell plantation group (group A) and model control group (group B). Skin cells were cultured in vitro until the allogeneic BMMSCs of the 5th generation formed with a cell count of 1 x 10(7)/mL. Local injection plus liquid supernatant smearing was used to plant the cells into the position of the scald in the stem cell plantation group. The control group was given an equivalent amount of normal saline to observe the healing action, and 5 samples were taken in each group after 1, 3, 7, and 14 days for hematoxylin and eosin staining for physiological observation. Polymerase chain reaction was used to detect the amount of chymotrypsin in mast cells. The speed of healing in the stem cell transplantation group was greater than that in the control group; staining results showed that the quality of healing in the transplantation group was better than that in the control group. Chymotrypsin expression was detected in both groups, reaching a peak on day 3. BMMSCs can accelerate wound healing, and chymotrypsin in mast cells may participate in the wound healing process.

Topics: Animals; Bone Marrow Cells; Chymotrypsin; Cricetinae; Diabetes Mellitus, Experimental; Disease Models, Animal; Mast Cells; Mesenchymal Stem Cells; Skin; Soft Tissue Injuries; Stem Cell Transplantation; Wound Healing

Topics: Animals; Chymotrypsin; Disease Models, Animal; Fluorescence; Fluorescent Dyes; Male; Pancreas; Pancreatectomy; Pancreatic Fistula; Pancreatic Juice; Swine

The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608). The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml). In in vivo model, the highest level of parasitemia suppression (≈ 45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

Topics: Actinobacteria; Animals; Antimalarials; Aquatic Organisms; Base Composition; Chymotrypsin; Cluster Analysis; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Inhibitory Concentration 50; Liver; Malaria, Falciparum; Male; Mice; Models, Biological; Models, Molecular; Parasitic Sensitivity Tests; Plasmodium falciparum; Protease Inhibitors; Protein Conformation; RNA, Ribosomal, 16S; Seawater; Spleen; Trypsin

To evaluate the effects of the proteasome inhibitor MG-132 on the expression of nuclear factor (NF)-κB p65, inhibitor (I)-κB, tumour necrosis factor (TNF)-α, and interleukin (IL)-1β in the cartilage and synovial tissues of rats with osteoarthritis (OA), and to investigate the role that the ubiquitin/proteasome system (UPS) plays in the OA process.. A total of 144 adult male Sprague Dawley rats were randomly assigned to four groups: anterior cruciate ligament transaction (ACLT) + MG-132 (ACLT/M), ACLT + dimethylsulfoxide (ACLT/D), sham surgery (Sham), and naïve + MG-132 (naïve/M). Pathological morphology was undertaken. mRNA expression levels of NF-κB p65, I-κB, TNF-α, and IL-1β were determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes were measured using fluorospectrophotometry.. The Mankin scores at all time points in ACLT/M rats were significantly lower than those in ACLT/D rats (p < 0.05). Despite the NF-κB p65 in the synovial tissue at 2 weeks after surgery and IL-1β in the cartilage tissue at 12 weeks after surgery, mRNA expression levels of NF-κB p65, IL-1β, and TNF-α at other time points in ACLT/M were significantly lower than those in ACLT/D (p < 0.05). mRNA levels of I-κB in the cartilage tissue in ACLT/M were significantly higher than those in ACLT/D at 2 weeks after surgery (p < 0.05). mRNA levels of I-κB in the synovial tissue in ACLT/M were higher than those in ACLT/D at all time points, and the difference was significant at 4 weeks after surgery (p < 0.05). MG-132 decreased the activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes in the cartilage and synovial tissues of rats.. The proteasome inhibitor MG-132 delays the progress of OA by alleviating synovial inflammation and protecting the articular cartilage tissue.

Topics: Animals; Anterior Cruciate Ligament; Cartilage, Articular; Chymotrypsin; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endopeptidases; I-kappa B Proteins; Interleukin-1beta; Leupeptins; Male; Osteoarthritis; Physical Conditioning, Animal; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Signal Transduction; Synovial Membrane; Transcription Factor RelA; Tumor Necrosis Factor-alpha

The results of the study of activity of digestive proteases (pepsin, trypsin, chymotrypsin) in homogenates of stomach, pancreas and duodenum in experimental animals have been presented. Rats were exposed to intoxication with carbon tetrachloride (subcutaneous administration of a 50% oil solution of CCl4 in the dose of 0.5 ml per 100 g body weight) for three days and then they were given analysed oils (black nut, walnut and flax oil) intragastrically by gavage at a dose of 0.2 ml per day within 23 days. Pepsin level in gastric mucosa homogenates and chymotrypsin activity in pancreatic homogenates were determined by method of N.P. Pyatnitskiy based on on the ability of enzymes to coagulate dairy-acetate mixture, respectively, at 25 degrees C and 35 degrees C. Trypsin activity in homogenates of pancreatic was determined by method of Erlanger - Shaternikova colorimetrically. It has been established that intoxication with CCl4 decreased the synthesis of proteolytic enzymes of the stomach (by 51%) and pancreas (by 70-78%). Injections of analysed vegetable oils to animals contributed to the normalization of proteolytic enzymes synthesis. The conclusion that there are prospects of using the analysed vegetable oils containing large quantity of polyunsaturated fatty acids (omega-3 and omega-6) for the correction of detected biochemical abnormalities has been done.

Topics: Administration, Oral; Animals; Carbon Tetrachloride Poisoning; Chymotrypsin; Digestive System; Disease Models, Animal; Gastric Mucosa; Male; Pancreas; Pepsin A; Plant Oils; Rats; Trypsin

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity.

Topics: Analysis of Variance; Animals; Body Temperature; Chymotrypsin; Corpus Striatum; Dependovirus; Disease Models, Animal; Dopamine; Dopamine Agents; Male; Methamphetamine; Neurotoxicity Syndromes; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Substantia Nigra; Synaptosomes; Tyrosine 3-Monooxygenase; Ubiquitin-Protein Ligases

Peripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD). Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner.. We evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20) and 40 mg/mL (E40) papain.. The severity of emphysema increased significantly in E20 (60.52 ± 2.8, p < 0.05) and E40 (52.27 ± 4.7; crossed the alveolar intercepts) groups. As compared to the control group, there was a reduction on body (171.6 ± 15.9 g) and muscle weight (251.87 ± 24.87 mg) in the E20 group (157.5 ± 10.3 mg and 230.12 ± 23.52 mg, for body and muscle weight, respectively), which was accentuated in the E40 group (137.4 ± 7.2 g and 197.87 ± 10.49 mg, for body and muscle weight, respectively). Additionally, the thiobarbituric acid reactive substances (TBARS), tert-butyl hydroperoxide-initiated chemiluminescence (CL), carbonylated proteins, and chymotrypsin-like proteolytic activity were elevated in the E40 group as compared to the E20 group (p < 0.05 for all comparisons). The severity of emphysema significantly correlated with the progressive increase in CL (r = -0.95), TBARS (r = -0.98), carbonyl proteins (r = -0.99), and chymotrypsin-like proteolytic activity (r = -0.90). Furthermore, augmentation of proteolytic activity correlated significantly with CL (r = 0.97), TBARS (r = 0.96), and carbonyl proteins (r = 0.91).. Taken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of oxidative stress in a severity-dependent manner.

Topics: Animals; Body Weight; Chymotrypsin; Cricetinae; Disease Models, Animal; Lung; Male; Mesocricetus; Muscle, Skeletal; Muscular Atrophy; Organ Size; Oxidative Stress; Papain; Protein Carbonylation; Pulmonary Emphysema; Severity of Illness Index; tert-Butylhydroperoxide; Thiobarbituric Acid Reactive Substances

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70-90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 μM). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca(2+) wave from 9 to 18 μm/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) wave speed was also observed with a change from physiologic concentrations of carbachol (1 μM) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10-16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca(2+) waves.

Topics: Alcoholism; Animals; Calcium; Carbachol; Chymotrypsin; Dantrolene; Disease Models, Animal; Ethanol; Muscle Relaxants, Central; Pancreas; Peptide Hydrolases; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Trypsin

Carbon monoxide-releasing molecules (CORMs) are a novel group of substances that are capable of modulating physiological functions via the liberation of CO.. This study was undertaken to investigate the effects of CORM-3, a water-soluble CO-releasing agent, on two rabbit models of ocular hypertension.. Ocular hypertension was induced by injecting alpha-chymotrypsin in the rabbit eye. The dose-response effect of CORM-3 on IOP was assessed by topical administration of the drug (0.001, 0.01, 0.1 and 1%). Ocular hypertension was also obtained by weekly subconjunctival injection of betamethasone, and animals were treated topically with CORM-3. A group of animals in both models was treated with the inactive form of the drug (iCORM-3).. CORM-3 induced a dose-dependent reduction in IOP in rabbits treated with alpha-chymotrypsin. A similar reduction in IOP was observed in rabbits with betamethasone-induced ocular hypertension treated with the drug. Treatment with the iCORM-3 had no effect on IOP in both models.. Treatment with CORM-3 is associated with a reduction in IOP in two different rabbit models of ocular hypertension. These results support previous findings on the effect of haem oxygenase-derived CO on IOP and suggest a direct involvement of CO system in the regulation of ocular pressure probably through the modulation of aqueous humour dynamics.

Topics: Animals; Antihypertensive Agents; Chymotrypsin; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Intraocular Pressure; Male; Ocular Hypertension; Organometallic Compounds; Rabbits

Treponema denticola is considered to be an agent strongly associated with periodontal disease. The lack of an animal infection model has hampered the understanding of T. denticola pathogenesis and the host's immune response to infection. In this study, we have established an oral infection model in mice, demonstrating that infection by oral inoculation is feasible. The presence of T. denticola in the oral cavities of the animals was confirmed by PCR. Mice given T. denticola developed a specific immune response to the bacterium. The antibodies generated from the infection were mainly of the immunoglobulin G1 subclass, indicating a Th2-tilted response. The antibodies recognized 11 T. denticola proteins, of which a 62-kDa and a 53-kDa protein were deemed immunodominant. The two proteins were identified, respectively, as dentilisin and the major outer sheath protein by mass spectrometry. Splenocytes cultured from the infected mice no longer produced interleukin-10 and produced markedly reduced levels of gamma interferon relative to those produced by naïve splenocytes upon stimulation with T. denticola. Mandibles of infected mice showed significantly greater bone resorption (P < 0.01) than those of mock-infected controls.

Topics: Alveolar Bone Loss; Amino Acid Sequence; Animals; Antibodies, Bacterial; Bacterial Proteins; Chymotrypsin; Disease Models, Animal; Female; Mice; Mice, Inbred C57BL; Mouth Diseases; Peptide Hydrolases; Porins; Treponema denticola; Treponemal Infections

The variety of animal models used in the study of alcoholic liver disease reflects the formidable task of developing a model that replicates the human disease. We show that oral feeding of fatty acids derived from fish oil and ethanol induces fatty liver, necrosis, inflammation, and fibrosis. Together with the study of oxidative and nitrosative stress markers, cytokines, proteasome function, and protein studies, this model has provided an inexpensive and technically simple method of establishing pathological alcoholic liver injury.

Topics: Administration, Oral; Alanine Transaminase; Alcohol Drinking; Animals; Blotting, Western; Central Nervous System Depressants; Chymotrypsin; Cytochrome P-450 CYP2E1; Dinoprost; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Endotoxins; Ethanol; Fatty Acids; Fatty Liver, Alcoholic; Female; Immunohistochemistry; Liver; Liver Diseases, Alcoholic; Oxidative Stress; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Self Administration; Specimen Handling; Staining and Labeling; Thiobarbituric Acid Reactive Substances

Although it has been suggested that ergot derivatives may play a role in antiglaucoma therapy, little attention has been paid to the ocular hypotensive action of these drugs. Having previously reported that topical natural ergot alkaloids ergocristine alpha-ergocryptine and ergocornine dose-dependently reduce intraocular pressure in ocular normotensive and alpha-chymotrypsin-induced ocular hypertensive rabbits, the aim of the present work was to compare the effect of ergocristine, alpha-ergocryptine and ergocornine on the intraocular pressure and aqueous humor dynamics in ocular normotensive and alpha-chymotrypsin-induced ocular hypertensive rabbits, in order to further explore the ocular actions of these compounds.. Experiments were conducted in albino ocular normotensive and hypertensive rabbits by intracameral injection of alpha-chymotrypsin. Intraocular pressure responses to drug vehicle and seven different doses of topical natural ergot alkaloids were examined, in order to obtain dose-response relationships for comparing the intraocular pressure-lowering effect and potency of these drugs. Tonographies were also performed to ascertain the actions of natural ergot alkaloids on aqueous humor dynamics.. All natural ergot alkaloids tested reduced intraocular pressure in a dose-related fashion. The ocular hypotensive effect was greater in alpha-chymotrypsin-induced ocular hypertensive rabbits for the three compounds tested. All natural ergot alkaloids tested decreased both tonographic outflow facility and, to a greater extent, aqueous humor inflow in ocular normotensive and in alpha-chymotrypsin-induced ocular hypertensive rabbits.. Taken together, our data suggest that these compounds decrease both tonographic outflow facility and, to a greater extent, aqueous humor inflow, which explains their final effect in ocular normotensive and in alpha-chymotrypsin-induced ocular hypertensive rabbits. Reductions in aqueous humor inflow observed after topical application of natural ergot alkaloids in alpha-chymotrypsin-induced ocular hypertensive rabbits can only be explained by a marked inhibition of active secretion of aqueous humor, since processes involved in aqueous humor formation may probably be altered after alpha-chymotrypsin injection.

Topics: Administration, Topical; Animals; Aqueous Humor; Chymotrypsin; Disease Models, Animal; Dose-Response Relationship, Drug; Ergolines; Ergot Alkaloids; Intraocular Pressure; Ocular Hypertension; Rabbits; Tonometry, Ocular

Carbon monoxide (CO) generated from heme may induce vasodilation and exert cyto-protective properties in the eye. This study was undertaken to investigate the effects of hemin, a potent inducer of heme oxygenase-1 (HO-1), on models of ocular hypertension in rabbits.. Ocular hypertension was induced by injecting alpha-chymotrypsin in both eyes under local anesthesia. Only rabbits with an intraocular pressure (IOP) of 25 mmHg or more were used. The dose-response study of the hemin effect on IOP was made by an intravenous injection of the drug (50, 75, and 100 mg/kg) and subsequent IOP monitoring every 6 h. A separate set of animals was pretreated with the HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX, 0.1 mg/kg) 6 h before the vehicle or a 100-mg/kg hemin injection. Ocular hypertension was also obtained by the subconjunctival injection of betamethasone 21-phosphate disodium (4 mg/mL) in both eyes every week for 4 weeks. Only animals with an IOP of 30 mmHg or more were included in the experimental session. A group of these animals was pretreated with ZnPP-IX (0.1 mg/kg) 6 h before the vehicle or a 100-mg/kg hemin injection, and IOP was assessed every 6 hours.. Hemin caused a significant dose-related reduction of IOP in rabbits with alpha-chymotrypsin-induced ocular hypertension. No significant effect was observed in the normotensive eyes of the control animals or on pupil diameter. Pretreatment with the HO-1 inhibitor, ZnPP-IX, abolished the decrease of IOP that was induced by the maximum dose of hemin (100 mg/kg). A similar reduction in IOP was observed in those rabbits with betamethasone-induced ocular hypertension who were treated with 100 mg/kg of hemin. Furthermore, pretreatment with ZnPP-IX prevented the hemin effect on IOP.. The induction of HO-1 by hemin leads to a reduction of IOP in the alpha-chymotrypsin and betamethasone models of ocular hypertension. These results suggest an involvement of CO in the regulation of ocular pressure in rabbits.

Topics: Analysis of Variance; Animals; Betamethasone; Chymotrypsin; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Induction; Heme Oxygenase-1; Hemin; Injections, Intravenous; Intraocular Pressure; Male; Ocular Hypertension; Protoporphyrins; Rabbits; Random Allocation

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.

Topics: alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; Animals; Chronic Disease; Chymotrypsin; Disease Models, Animal; Female; Fetal Development; Inflammation; Leukocyte Elastase; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Proteins; Serpins

A comprehensive investigation of the incidence, distribution, progression and chemical composition of Abeta deposits in the brains of two young (5 years) and seven aged (25-30 years) rhesus monkeys was conducted to determine the similarity of this phenomenon to that in the human. The brains of the young rhesus were devoid of Abeta deposits. In contrast, Abeta deposits were observed within the cerebral cortex of all aged animals. In animals with mild Abeta burden, deposits were observed primarily in association cortical zones. In animals with moderate Abeta burden, many paralimbic cortical zones also contained Abeta deposits. Finally, in an animal with a heavy burden of Abeta, core limbic cortical zones were also involved. The primary sensory and motor cortices were relatively free of Abeta deposits. A higher proportion of plaques contained Abeta40 as compared with Abeta42. Abeta deposits contained a number of other constituents. Cholinesterases were present in nearly 50% of plaques and displayed the exact same biochemical characteristics as those in the human. Nearly 20% of Abeta deposits also contained apolipoprotein E and a smaller proportion contained heparin sulfate proteoglycans and alpha1-anti-chymotrypsin. The latter three chemicals were present in many compact plaques. These results indicate that the distribution, progression and chemical composition of plaques in the aged rhesus monkey display many similarities to those observed in the aged human and Alzheimer's disease. Therefore, despite some differences from the human, the aged rhesus may be a good model for studies of the pathological effects of Abeta in the primate brain.

Topics: Age Factors; Aging; Amyloid beta-Peptides; Animals; Apolipoproteins E; Brain Chemistry; Brain Diseases; Cholinesterases; Chymotrypsin; Disease Models, Animal; Heparin; Humans; Immunohistochemistry; Plaque, Amyloid; Proteoglycans

The virulence factor Mip (macrophage infectivity potentiator) contributes to the intracellular survival of Legionella pneumophila, the causative agent of Legionnaires' disease. The protein consists of two domains that are connected via a very long alpha-helix (A. Riboldi-Tunnicliffe et al., Nat. Struct. Biol. 8:779-783, 2001). The fold of the C-terminal domain (residues 100 to 213) is closely related to human FK506-binding protein (FKBP12), and like FKBP12, Mip exhibits peptidylprolyl cis/trans isomerase (PPIase) activity. The alpha-helical N-terminal domain is responsible for the formation of very stable Mip homodimers. In order to determine the importance of the homodimeric state of Mip for its biochemical activities and for infectivity of Legionella, a truncated, monomeric Mip variant [Mip((77-213))] was overexpressed in Escherichia coli and characterized biochemically. In vitro isomerase activity assays revealed that the altered protein exhibits full isomerase activity towards peptide substrates. However, the deletion resulted in a dramatic loss in the efficiency of refolding of reduced and carboxy-methylated RNase T(1). By cis complementation of the Mip-negative mutant strain L. pneumophila JR32-2, we constructed the strain L. pneumophila JR32-2.4, which expresses an N-terminally truncated variant of Mip. Infection studies with these strains revealed that the N-terminal part and the dimerization of Mip but not its PPIase activity are necessary for full virulence in Acanthamoeba castellanii. Infection of guinea pigs showed that strains with dimerization-deficient Mip (JR32-2.4) or a very low PPIase activity (JR32-2.2) were significantly attenuated in the animal model. These results suggest a different role of the PPIase activity and the N-terminally mediated dimeric state of Mip in monocellular systems and during the infection of guinea pigs.

Topics: Acanthamoeba; Animals; Bacterial Proteins; Chymotrypsin; Disease Models, Animal; Guinea Pigs; Humans; Immunophilins; Legionella pneumophila; Legionnaires' Disease; Male; Membrane Proteins; Mutation; Peptide Fragments; Peptidylprolyl Isomerase; Recombinant Proteins; Virulence

The role of nitric oxide (NO) in neuronal degeneration of glaucoma is well established, and drugs to inhibit NO production have been introduced in preclinical studies. The present experiments were made to investigate the pharmacological efficacy of a topical formulation of the nonselective nitric oxide synthase (NOS) inhibitor, nitro-L-arginine methyl ester (L-NAME), in an experimental model of glaucoma in rabbits. L-NAME was dissolved in an isotonic, mucoadhesive, viscosized, buffered solution in concentrations of 0.1%, 0.5%, or 1% (w/v). Ocular hypertension (of at least 15 mmHg compared to basal values) was induced by intra-ocular injection of alpha-chymotrypsin. The instillation of L-NAME topical formulations lowered the IOP of hypertensive rabbits in a dose-related manner, with a maximum drop of 12.0 mmHg 60 minutes after administration of the highest concentration. The area under the curve (AUC) of the DeltaIOP (mmHg) versus time (minutes) was 1050.3 +/- 141.7 and 15.1 +/- 2.5 for the 1% L-NAME-treated group and vehicle-treated group, respectively. No change was found in IOP or pupil diameter after instillation of L-NAME eye drops in normotensive rabbits. This study provides the first evidence that topical L-NAME significantly reduces the IOP in a model of ocular hypertension.

Topics: Administration, Topical; Animals; Anterior Chamber; Area Under Curve; Chemistry, Pharmaceutical; Chymotrypsin; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Evaluation, Preclinical; Enzyme Inhibitors; Glaucoma; Injections; Intraocular Pressure; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Ophthalmic Solutions; Rabbits; Solvents

In this study we investigate the changes in intestinal motor responsiveness after mild mesenteric ischemia/reperfusion in anaesthetized rats. Motor responsiveness to pharmacological/electrical stimulation was studied in isolated ileum excised from sham-operated rats or animals which underwent occlusion of superior mesenteric artery (1 h) plus interruption of collateral blood flow and reperfusion for 0, 24, 72 h. Only 24 h reperfusion resulted in a significant suppression in acetylcholine induced contractile response and in indomethacin induced relaxation. In the presence of adrenergic and cholinergic blockade a greater relaxant response to field stimulation (trains 10 s every min, 120 mA, 1 ms and 10 Hz) was unmasked in all groups except 24 h reperfused rats. Such effect was sensitive to N(G)-Nitro-L-arginine methyl ester (NOS unselective inhibitor) and the proteolytic enzyme alpha-chymotrypsin but resistant to aminoguanidine (iNOS selective inhibitor). In conclusion, in this rat model, intestinal mild ischemia/24 h reperfusion induces reversible changes in enteric motility attributable to a decrease in eicosanoids, nitric oxide and neuropeptides availability.

Topics: Acetylcholine; Animals; Chymotrypsin; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Gastrointestinal Motility; Ileum; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Male; Mesenteric Arteries; Mesenteric Vascular Occlusion; Muscle Contraction; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Reperfusion Injury

Erythrocyte invasion by malaria parasites is a complex multistep process involving parasite and erythrocyte receptors. It is a critical stage in the parasite life cycle and, therefore, a logical step in which to intervene to prevent or ameliorate disease. Rodent models of malaria, commonly Plasmodium yoelii, are frequently used for studies of malaria pathogenesis. Little is known, however, about the invasion machinery of rodent malaria parasites. We have found previously that mice congenic for a region of chromosome 1, containing the Duffy antigen/receptor for chemokines (DARC), have different susceptibility to P yoelii infection. Because P vivax, a human parasite, and P knowlesi, a simian parasite, use DARC to enter human erythrocytes, we sought to identify the role of the murine DARC in P yoelii invasion. Using a novel in vivo invasion assay and DARC knock-out mice, we found that DARC knock-out normocytes (mature erythrocytes) had negligible levels of P yoelii invasion compared with wild-type normocytes, demonstrating that DARC is a receptor for invasion of murine erythrocytes. In contrast, DARC knock-out reticulocytes were invaded at a rate similar to that for wild-type reticulocytes. We conclude that there is a DARC- independent pathway for reticulocyte invasion. These findings represent the first identification of a murine malaria receptor on erythrocytes and the first determination that different pathways of invasion exist on normocytes and reticulocytes. Because we show conservation of host-receptor interactions between rodent and human malaria, we can now use this model to identify how immunity can interfere with the invasion process.

Topics: Animals; Antigens, Protozoan; Carrier Proteins; Chymotrypsin; Disease Models, Animal; Duffy Blood-Group System; Erythrocytes; Host-Parasite Interactions; Malaria; Mice; Mice, Knockout; Plasmodium yoelii; Protozoan Proteins; Receptors, Cell Surface; Reticulocytes; Trypsin

To evaluate the ocular hypotensive effect of topical CS-088, an angiotensin AT1 receptor antagonist, and the effect of CS-088 on aqueous humor dynamics.. The effects of CS-088 on intraocular pressure (IOP) were studied in 2 models of rabbit ocular hypertension. Experimental ocular hypertension was induced in albino rabbits by injecting alpha-chymotrypsin into the anterior chamber (alpha-chymotrypsin rabbit). The effects of the single application of CS-088 were examined. Additionally, CS-088 was repeatedly administered over a period of 3 weeks to hereditary ocular hypertensive rabbits (buphthalmic rabbits, JWHR bu/bu) and the IOPs were monitored throughout the experiment. The effects of CS-088 on aqueous humor dynamics were also examined in normal rabbits. In this study, the methods of IOP recovery rate, two-level constant pressure perfusion and fluorescein-dextran perfusion were used respectively to determine the aqueous inflow, outflow facility and uveoscleral outflow (USF).. CS-088 at 1% and 2% significantly lowered the IOP in the alpha-chymotrypsin rabbits with a maximum IOP reduction of 10.1 mmHg. The maximum effect obtained with 2% CS-088 was no greater than that with 1% CS-088. In the buphthalmic rabbits, 2% CS-088 also lowered IOP significantly. Timolol was effective in both models. In the study on aqueous humor dynamics, a slight increase in USF (17%) was seen after a topical application of CS-088 whereas changes in aqueous inflow or outflow facility were not observed.. Topical CS-088 can decrease IOP in rabbits. Despite the USF change, the ocular hypotensive mechanism by CS-088 was not fully determined.

Topics: Administration, Topical; Adrenergic beta-Antagonists; Angiotensin Receptor Antagonists; Animals; Aqueous Humor; Chymotrypsin; Disease Models, Animal; Imidazoles; Intraocular Pressure; Male; Ocular Hypertension; Ophthalmic Solutions; Rabbits; Receptor, Angiotensin, Type 1; Tetrazoles; Timolol

The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.

Topics: Animals; Apoenzymes; Body Weight; Chlormethiazole; Chlorzoxazone; Chymotrypsin; Cysteine Endopeptidases; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP2E1 Inhibitors; Disease Models, Animal; Ethanol; Hydroxylation; Immunohistochemistry; Liver; Liver Diseases, Alcoholic; Male; Multienzyme Complexes; Organ Size; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Trypsin

Increased intraperitoneal pressure is associated with physiological changes including alterations of intraocular pressure (IOP). We have previously shown that IOP is not adversely affected by increased intraperitoneal pressure up to 15 mm Hg in women with no preexisting eye disease. The aim of this study was to measure IOP changes associated with increased intraperitoneal pressure (up to 15 mm Hg) of 2 h duration in 12 rabbits with alpha-chymotrypsin-induced glaucoma. A reliable model of glaucoma was created by injecting alpha-chymotrypsin into the posterior chamber of the right eye in 12 rabbits. Thereafter, 5 of the 12 rabbits with glaucomatous eyes were treated with topical timolol. The left eye was used as a control. During pentobarbital general anesthesia, increased intraperitoneal pressure up to 15 mm Hg was created by intraperitoneal CO2 insufflation. Body temperature and expired CO2 were kept constant throughout the study. IOP measurements were made using an electronic pneumotonometer. IOP, mean arterial pressure, heart rate, and central venous pressure were recorded in head-up and head-down positions before, during, and after increased intraperitoneal pressure. The IOP of both eyes, in both treated and untreated rabbits, increased significantly from baseline only when increased intraperitoneal pressure associated with the head-down position resulted in a significant increase in central venous pressure. However, the IOP increase remained within the diurnal range. The major finding of this study is that, in a reliable model of glaucoma, CO2 pneumoperitoneum was associated with an increase in IOP when a head-down position was combined with pneumoperitoneum.. In rabbits with alpha-chymotrypsin-induced glaucoma, increased intraperitoneal pressure (up to 15 mm Hg) resulted in a significant intraocular pressure increase when pneumoperitoneum was associated with the head-down position. However, the intraocular pressure increase remained within the diurnal range.

Topics: Adjuvants, Anesthesia; Administration, Topical; Anesthesia, General; Animals; Antihypertensive Agents; Blood Pressure; Body Temperature; Carbon Dioxide; Central Venous Pressure; Chymotrypsin; Disease Models, Animal; Glaucoma; Head-Down Tilt; Heart Rate; Insufflation; Intraocular Pressure; Male; Ocular Hypertension; Pentobarbital; Pneumoperitoneum, Artificial; Posture; Pressure; Rabbits; Reproducibility of Results; Time Factors; Timolol; Tonometry, Ocular

Arylpiperazine derivatives were synthesized and investigated in this study. Two animal models, including an intraocular pressure (IOP) recovery method and an alpha-chymotrypsin-induced glaucoma model, were used to determine the ocular pharmacological effects of the arylpiperazine derivatives. In the IOP recovery method, New Zealand rabbits with normal IOP were instilled with 50 microliters of 0.5% eye drops, then 10% sodium chloride solution was infused through the ear marginal vein. The relative percent of IOPs were calculated, then delta IOPt% was obtained from the difference of IOPt% between the treated and controlled eye. In the alpha-chymotrypsin-induced glaucoma model, the induced glaucoma rabbits were topically instilled with 0.5% arylpiperazines onto the eyes, and then the IOP changes were calculated to evaluate the effect of eye drops. Our results showed that in the IOP recovery method, BG31 and YCT2-2 demonstrated a very significant effect for reducing IOP; delta IOPt% were -27.6 and -25.5 for BG31 and YCT2-2, respectively. Two other compounds, C219 and C220 also lowered IOP, but the effects were less significant. In alpha-chymotrypsin-induced glaucoma, the maximum effect of YCT2-2 on the IOP was found at 5 hrs. The delta IOP and delta delta IOP were -12.5 +/- 1.7 and -5.8 +/- 1.1 mmHg (p < 0.01), respectively. For BG-31 and C220, there existed a trend to increase IOP with time. In the study, we found that YCT2-2 with higher solubility in the acidic condition was correlated to the significant IOP lowering effect.

Topics: Administration, Topical; Animals; Chymotrypsin; Disease Models, Animal; Female; Glaucoma; Intraocular Pressure; Male; Ophthalmic Solutions; Piperazines; Rabbits; Tonometry, Ocular

Experimental animal models of thrombosis have been established in several species to examine factors responsible for thrombotic disorders in man. One technical facet of all thrombosis models is the need to quantitate cell deposition on thrombogenic surfaces, and this is routinely accomplished with radioisotopic labeling of specific components. Data reported here demonstrate that formalin-fixed thrombi can be hydrolyzed with chymotrypsin allowing recovery and quantitation of platelets and erythrocytes incorporated within the clot. Recovery of platelets from in vitro generated, model thrombi averaged 99 +/- 10% (mean +/- 1 SD; n = 7; range 88-116%) of calculated content; recovery of erythrocytes was 94.1 +/- 1.1% (n = 6) as measured by recovery of cellular hemoglobin after chymotrypsin hydrolysis of clots. Chymotrypsin was also shown to release platelets and erythrocytes from string-bound thrombi generated in vivo with an arterio-venous shunt model in beagle dogs. Platelet recovery from these string clots after chymotrypsin hydrolysis was independently verified with a quantitative Western blot assay of platelet antigens. These data demonstrate that experimental thrombi can be hydrolyzed with chymotrypsin, thereby not only eliminating the need for radioisotopes, but also permitting flow cytometric analysis of cells comprising the thrombus.

Topics: Animals; Blotting, Western; Chymotrypsin; Disease Models, Animal; Dogs; Erythrocyte Count; Flow Cytometry; Hydrolysis; In Vitro Techniques; Platelet Count; Thrombosis

The aim of this study was to set-up and validate the use of a radio-telemetry system in order to record IOP in chronic ocular hypertensive animals. The transmitter of a miniaturized radio-telemetry system was implanted in rabbits, and its catheter was tunnelled subcutaneously to the superior conjunctival sac and inserted into the midvitreous. Implantation was performed in chronic ocular hypertensive rabbits induced by an injection of alpha-chymotrypsin into the posterior chamber of the eye. The effects of 0.5% timolol maleate, 2% dorzolamide hydrochloride and 1% epinephrine were assessed and compared after topical administration in this model. Implanted radio-telemetric system into the vitreous allowed IOP measurement for more than 6 months. In this study, circadian IOP kinetic profiles were monitored in all animals over 24 h for 3 weeks. Timolol maleate was found significantly potent in reducing IOP, while changes depended on the nyctemeral period. Dorzolamide hydrochloride induced a very large IOP reduction and was found to be also well effective at night. We evidenced a biphasic time-dependent effect after topical epinephrine, with a long lasting IOP increase occurring after the administration. This change was found to be related to side effects resulting from a poor ocular tolerance of this drug in the rabbit, leading to either a complete eye closure or a higher blinking rate. By using our method, we confirmed the pressure pulses and undershoots occurring during blinking. Radio-telemetry in chronic glaucoma rabbits appears as a refined method to assess anti-glaucoma drug activity, 24 hours a day, for long-term periods in unrestrained animals, while also providing information on the ocular side effects of eye drops.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Carbonic Anhydrase Inhibitors; Chymotrypsin; Circadian Rhythm; Disease Models, Animal; Epinephrine; Female; Glaucoma; Intraocular Pressure; Ophthalmic Solutions; Rabbits; Sulfonamides; Telemetry; Thiophenes; Timolol

It has recently been shown that the infusion of oleic acid into the rat pancreaticobiliary duct causes a reproducible and long-lasting atrophy of the exocrine pancreas. The effects of this pancreatic atrophy on non-invasive pancreatic function tests have not been fully characterized. This study was undertaken to determine which pancreatic function test was most useful in determining pancreatic insufficiency in this model. Pancreatic insufficiency (PI) was induced in male Wistar rats by oleic acid infusion and three pancreatic function tests were compared in these animals and saline controls. The coefficient of fat absorption on a 5 or 45% fat diet and bentiromide testing could not differentiate animals with or without PI, but fecal chymotrypsin levels were excellent discriminators. All animals with PI had fecal chymotrypsin levels below 67 U/g feces whereas all saline controls were above this level. We conclude that, in this model of PI, the fecal chymotrypsin concentration is the best non-invasive test to determine pancreatic insufficiency.

Topics: Absorption; Animals; Chronic Disease; Chymotrypsin; Chymotrypsinogen; Dietary Fats; Disease Models, Animal; DNA; Exocrine Pancreatic Insufficiency; Feces; Lipase; Male; Oleic Acid; Oleic Acids; Pancreas; Pancreatic Function Tests; Rats; Rats, Wistar

Topics: Animals; Chymotrypsin; Disease Models, Animal; Drug Therapy, Combination; Humans; Penicillin G Procaine; Rabbits; Serum Bactericidal Test; Syphilis; Time Factors; Treponema pallidum

A newly synthesized topical carbonic anhydrase inhibitor, 6-hydroxyethoxy-2-benzothiazole sulfonamide (6-HS), was administered systemically and topically to alpha-chymotrypsin-induced glaucoma rabbits to evaluate its ocular hypotensive effect. A significant IOP lowering effect was observed after topical application of 50 microL of 3% 6-HS gel, but a dose of 50 microL of 3% 6-HS suspension failed to reduce IOP. The maximal magnitude of reduced IOP after topical gel instillation was 24.4%, very close to the result obtained following intravenous injection of 6 mg/kg of 6-HS (23.3%). However, the blood levels of 6-HS after topical instillation with 3% 6-HS gel was much lower than that following 6 mg/kg of 6-HS injected intravenously (less than 5%). Since a lower dose of 6-HS (1 mg/Kg) administered intravenously did not cause a significant drop in IOP, it is reasonable to deduct that the ocular hypotensive effect of 6-HS applied topically can then be attributed to the inhibition of intraocular carbonic anhydrase activity. It was also noted that a larger dose of intravenous administration of 6-HS (20 mg/Kg) had a more profound IOP and blood pressure reducing effect with moderate metabolic acidosis.

Topics: Animals; Benzothiazoles; Carbonic Anhydrase Inhibitors; Chymotrypsin; Disease Models, Animal; Ethoxzolamide; Female; Glaucoma; Intraocular Pressure; Male; Rabbits

We have developed a model system for studying proliferative retinopathy in vitro using bovine retinal explants cultured in collagen gels. Cellular outgrowth from retinal explants occurred after 7 days as single cells from peripapillary explants or as cell sheets and tubular outgrowths from peripheral retinal explants. Single cell outgrowths were shown immunohistochemically to be endothelial cells or macrophages; sheetlike and tubular outgrowths also constituted cords of endothelial cells with stromal macrophages, but in addition glial cells were closely associated with the endothelial cells. Cellular outgrowths were preceded by extensive macrophage activation within the ischemic retinal explant, suggesting that macrophage-derived angiogenic factors may be important in inducing retinal endothelial cell proliferation. In addition, glial cell-derived factors may have a role in the development of vessellike tubular structures from endothelial cell outgrowths.

Topics: Animals; Cattle; Cell Division; Cell Movement; Chymotrypsin; Collagen; Culture Techniques; Disease Models, Animal; Endothelium; Factor VIII; Gels; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; Microscopy, Phase-Contrast; Retinal Diseases; Trypsin; Vimentin

Oral pancreatic enzyme replacement therapy generally benefits patients with severe pancreatic deficiency. However, the fate of oral pancreatic supplements in the digestive lumen and their possible effects on circulating gut hormones are only partially known. The purpose of this article is to validate an experimental model that produces total pancreatic insufficiency in pigs, and to study the fate of orally administered Eurobiol, a whole pancreas lyophilized preparation, and its effects on circulating plasma levels of five digestive hormones. Pancreatic insufficiency was created by pancreatic duct ligation, and the duodenal, jejunal and ileal contents were sampled through cannulas before a normal meal and 0.5-24 h later. Blood samples were taken at the same times, and plasma neurotensin, pancreatic polypeptide, secretin, cholecystokinin (CCK), and gastrin were measured. In pigs with pancreatic insufficiency, Eurobiol, given during the meal, induced a significant increase in all enzyme activities in the duodenum and the jejunum, and in the levels of amylase, trypsin, and chymotrypsin in the ileum, relative to placebo. In the duodenum, the peak concentrations of enzyme activities were 19, 11, 17, and 29% (p less than 0.001) of the postprandial peak activities measured in control pigs with an intact pancreas for lipase, amylase, trypsin, and chymotrypsin, respectively. In the jejunum, the same activities were, respectively, 30, 11, 25, and 36% (p less than 0.01-0.001) of normal peaks. In pigs with pancreatic insufficiency, basal and integrated meal-stimulated neurotensin levels were increased; basal, peak, and integrated meal-stimulated pancreatic polypeptide and secretin levels were increased, whereas gastrin and CCK were not different from controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Amylases; Animals; Cholecystokinin; Chymotrypsin; Disease Models, Animal; Exocrine Pancreatic Insufficiency; Gastrins; Intestines; Lipase; Male; Neurotensin; Pancreas; Pancreatic Extracts; Pancreatic Polypeptide; Secretin; Swine; Trypsin

The phenomenon of glioma killing by lymphokine activated killer cells (LAK) was studied. We demonstrate that LAK cells generated by culturing the lymphokine interleukin-2 (IL-2) with peripheral blood lymphocytes from brain tumour patients destroys autologous glioma. The rat 9L glioma model was used to show that LAK killing was tumour-selective as glioma but not syngeneic normal brain tissue was destroyed. The susceptibility of both human and 9L rat glioma to LAK cell killing was markedly diminished by pretreating glioma cells with trypsin or chymotrypsin, but was unaffected by pretreatment with neuraminidase, glycosidases, sodium periodate or hydrocortisone. These results suggest that the cell surface determinant on glioma cells responsible for its tumour selective lysis by LAK is a protein sensitive to trypsin and chymotrypsin. The tumour-selective killing of glioma by LAK in vitro prompted the initiation of a Phase I study in which ten patients with malignant glioma have been treated with direct intracerebral injection of IL-2 or LAK without evidence of systemic or brain toxicity.

Topics: Adult; Aged; Animals; Borohydrides; Brain Neoplasms; Cell Line; Chymotrypsin; Cytotoxicity, Immunologic; Disease Models, Animal; Female; Glioma; Humans; Interleukin-2; Killer Cells, Natural; Lymphocyte Activation; Male; Middle Aged; Periodic Acid; Rats; Rats, Inbred F344; Trypsin

The nonelastolytic proteases trypsin and chymotrypsin were administered to hamsters 24 hours after intratracheal injection of elastase. Severity of the disease, extent of degradation and resynthesis, new cross-link formation, and the levels of the enzyme lysyl oxidase, which mediates the cross-link formation, were compared with the same parameters measured in hamsters with experimental emphysema induced by elastase alone. Increases in mean linear intercept indicated that a more severe form of the disease was produced. Although elastin degradation after 1 week was similar in both groups, resynthesis of the elastin destroyed by the elastolytic insult was significantly impaired in the animals injected sequentially with elastase and trypsin or chymotrypsin. Formation of new elastin as monitored by 14C-lysine incorporation into the elastin specific cross-links desmosine and isodesmosine was reduced approximately 40%, although there was no significant difference in the levels of lysyl oxidase activity. It is suggested that the most likely mechanism compatible with the recorded observations involves destruction of the microfibrillar component of the elastic fiber by trypsin or chymotrypsin, resulting in the absence of the requisite template for resynthesis of the pulmonary elastin.

Topics: Animals; Chymotrypsin; Cricetinae; Desmosine; Disease Models, Animal; Elastin; Female; Isodesmosine; Lung; Lysine; Mesocricetus; Pancreatic Elastase; Protein-Lysine 6-Oxidase; Pulmonary Emphysema; Trypsin

Two animal models have been employed to examine the role of pancreatic polypeptide, a potent and selective inhibitor of pancreatic exocrine secretion, in the treatment of acute pancreatitis. In one model pancreatitis was induced by feeding young female Swiss Webster mice an ethionine-supplemented, choline-deficient diet for 48 hr. Animals (N = 30 per group) were injected subcutaneously every 8 hr for 7 days with pancreatic polypeptide (0, 2, 20, and 200 micrograms/kg/day). Treatment with 20 and 200 micrograms/kg/day pancreatic polypeptide significantly (P less than 0.05) reduced mortality from a control rate of 70% to 42% and 33%, respectively. Treated animals also exhibited significant (P less than 0.05) decreases in pancreatic content of activated chymotrypsin and an improvement in pancreatic histology. Pancreatic polypeptide was effective whether treatment was started before or at the same time the test diet was introduced. In contrast, pancreatic polypeptide failed to protect dogs with acute pancreatitis induced by retrograde injection of the pancreas with bile, which may reflect the rapid and mechanical nature of pancreatic damage in this animal model.

Topics: Animals; Bile; Chymotrypsin; Disease Models, Animal; Dogs; Ethionine; Female; Mice; Pancreatic Polypeptide; Pancreatitis; Trypsin

The antiglaucomatous effects of Glauplex 2 and pilocarpine nitrate on alpha-chymotrypsine-induced experimental glaucoma were studied in 8 rabbits. Changes in intraocular pressure were measured over a period of 12 hours after a single instillation of Glauplex 2 or two instillations of 2.6 p. cent pilocarpine nitrate at t = 0 and t = 6 hours. The antihypertensive effect of a single instillation of Glauplex 2 was shown to be approximately equivalent to that of two instillations of 2.6 p. cent pilocarpine nitrate.

Topics: Animals; Chymotrypsin; Delayed-Action Preparations; Disease Models, Animal; Glaucoma; Ophthalmic Solutions; Pilocarpine; Rabbits; Tonometry, Ocular

Alterations in the pancreatic secretion of fluid and of enzymes in response to either pilocarpine (15 mg/kg) or an octapeptide of cholecystokinin (0.1 microgram/kg) have been found in rats that received daily injections of reserpine (0.5 mg/kg) for 7 days. During a 3-hr secretory period, significant reductions in the volume of pancreatic juice and in the total output of protein, amylase, and trypsin were observed in these animals. In the first hour of the secretory response, however, protease output was increased in the treated animals, particularly that of chymotrypsin, which was also increased in the longer secretory period following pilocarpine, but not cholecystokinin, stimulation. Zymogen granules isolated from the pancreas of the treated rats by differential centrifugation in a 0.3 M sucrose buffer had increased specific activities of the proteases when compared to those of untreated controls. Ultrastructurally, zymogen granules isolated from the pancreas of the treated rats showed changes in density, with bizonal and trizonal configurations being frequently observed, and had less distinct limiting membranes. In some, the membrane appeared broken at intervals, and there was granular material, presumably derived from the granule contents, lining the surface of the granule. It is concluded that pretreatment with reserpine inhibits fluid secretion and alters enzyme secretion in the rat exocrine pancreas. The latter effect is related to a nonparallel storage of amylase and proteases in the secretory granules induced by the drug treatment, probably through an action on protein synthesis or intracellular transport. An accumulation of proteases may lead to activation of these enzymes and to granule lysis. Inasmuch as the reserpine-treated rat has been proposed as an experimental model for cystic fibrosis, these findings are relevant in terms of possibly pathogenetic mechanisms in this disease.

Topics: Amylases; Animals; Cholecystokinin; Chymotrypsin; Cystic Fibrosis; Disease Models, Animal; Male; Pancreas; Pilocarpine; Rats; Reserpine; Trypsin

The injection of alpha-chymotrypsin into the posterior chamber of the eye is known to produce an experimental ocular hypertension of long duration in animals. The present study reports the pathological changes which occur in the eye during the first nine months after the ocular injection of alpha-chymotrypsin in rabbits. Six weeks after treatment most of the eyes showed a buphthalmia and an intraocular pressure elevation which varied greatly from animal to animal. The anterior chamber angle of the treated eyes showed a progressive enlargement. Several days after the enzyme injection a transient increase in thickness of the cornea and Descemet membrane was noted. Cupping of the optic disc, characterized by a total disappearance of the optic nerve head fibers and an excavation beginning at margins of the retina appeared after four months and in most cases were present seven months after the treatment. More or less prominent retinal degeneration was also evidenced three months after enzyme injection. The results indicate alpha-chymotrypsin-induced occular hypertension in the rabbit leads after several months to pathological change in the eye analogous to that observed in human glaucoma.

Topics: Animals; Anterior Chamber; Chymotrypsin; Cornea; Descemet Membrane; Disease Models, Animal; Eye; Glaucoma; Intraocular Pressure; Male; Optic Disk; Rabbits; Retina; Tonometry, Ocular

The effect of timolol, propranolol, epinephrine, and isoproterenol on intraocular pressure (IOP) (measured by tonometry) were compared after topical administration in conscious rabbits. Epinephrine and isoproterenol decreased IOP in normotensive rabbits, whereas propranolol had no effect. Timolol produced only a slight and inconsistent lowering of IOP in normotensive rabbits. All four agents reduced IOP elevated by an oral water load; the adrenergic agonists were substantially more active than the two beta-adrenergic blocking agents. In alpha-chymotrypsin-induced ocular hypertension, epinephrine, isoproterenol, and timolol were essentially equally effective, whereas propranolol exhibited only weak activity. In this latter model, timolol did not lose its effectiveness after multiple instillations (three/day) over an 8-day period. The concentration of timolol in the acqueous humor after topical application of effective hypotensive doses was relatively high as compared to that found in plasma. In addition, topical doses of timolol required to lower IOP were considerably greater than those needed to reduce or block the ocular hypotensive activity of isoproterenol. The mode of action and therapeutic implications of beta-adrenergic blocking agents in glaucoma are discussed.

Topics: Adrenergic beta-Antagonists; Animals; Aqueous Humor; Chymotrypsin; Disease Models, Animal; Epinephrine; Intraocular Pressure; Isoproterenol; Male; Propanolamines; Propranolol; Rabbits; Sympathomimetics; Timolol; Water

Topics: Animals; Cats; Cattle; Chickens; Chymotrypsin; Disease Models, Animal; Dogs; Glaucoma; Haplorhini; Humans; Hydrophthalmos; Light; Primates; Rabbits; Rats; Swine

The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 32SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 35SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-anti-trypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.

Topics: alpha 1-Antitrypsin; Animals; Cartilage, Articular; Chymotrypsin; Cytoplasmic Granules; Disease Models, Animal; Enzyme Inhibitors; Glycosaminoglycans; Humans; Immune Sera; Joint Diseases; Kinetics; Leukocytes; Oligopeptides; Pancreatic Elastase; Peptide Hydrolases; Phenylalanine; Proteoglycans; Rabbits; Sulfur Radioisotopes

Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.

Topics: Animals; Arthritis; Cartilage, Articular; Cattle; Chemical Phenomena; Chemistry; Chondroitin Sulfates; Chromatography, Gel; Chymotrypsin; Cytoplasmic Granules; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Glycosaminoglycans; Humans; Immunodiffusion; Joint Diseases; Leukocytes; Nasal Septum; Pancreatic Elastase; Proteoglycans; Sodium Dodecyl Sulfate; Viscosity

Pancreatitis was induced by injection of autologous bile into the main pancreatic duct of dogs. An initial fall in blood pressure was accompanied by appearance of large quantities of active trypsin, chymotrypsin, and elastase in pancreatic exudate with full saturation of protease inhibitors. The enzymes soon appeared in ascitic fluid and lymph, but only in the form of complexes with alpha1-antitrypsin, and alpha2-macroglobulin. No such complexes were detected in venous blood indicating short half-life in the circulation. These studies confirm the release of pancreatic enzymes during bile-induced pancreatitis, and quantify an important protective role for plasma protease inhibitors in this situation.

Topics: alpha 1-Antitrypsin; alpha-Macroglobulins; Animals; Ascitic Fluid; Bile; Chymotrypsin; Disease Models, Animal; Dogs; Exudates and Transudates; Female; Half-Life; Male; Pancreatic Elastase; Pancreatitis; Peptide Hydrolases; Trypsin

Topics: Animals; Anti-Inflammatory Agents; Blood Coagulation; Chymotrypsin; Disease Models, Animal; Esterases; Fibrinolysin; Injections, Intravenous; Injections, Subcutaneous; Jugular Veins; Ligation; Methods; Phlebography; Pyrazoles; Rabbits; Streptokinase; Thrombin; Thrombophlebitis; Thrombosis; Vena Cava, Inferior

Topics: Animals; Chymotrypsin; Dietary Fats; Dietary Proteins; Disease Models, Animal; Feces; Female; Intestinal Absorption; Jejunum; Ligation; Lipase; Lipid Metabolism; Lipids; Male; Pancreatic Diseases; Pancreatic Ducts; Pancreatin; Proteins; Swine; Trypsin

Topics: Adhesiveness; Animals; Biomechanical Phenomena; Chymotrypsin; Collagen; Contact Lenses; Cornea; Disease Models, Animal; Eye Diseases; Humans; Keratitis; Microscopy, Electron, Scanning; Rabbits; Surface Properties; Time Factors

Topics: Animals; Anti-Inflammatory Agents; Aprotinin; Chymotrypsin; Corticosterone; Depression, Chemical; Dimethyl Sulfoxide; Disease Models, Animal; Edema; Heparin; Heparinoids; Histamine H1 Antagonists; Humans; Male; Methysergide; Ointments; Phenylbutazone; Rats; Stimulation, Chemical; Tibial Fractures; Time Factors; Wounds and Injuries