Bromoditerpenes from the Red Seaweed Sphaerococcus coronopifolius as Potential Cytotoxic Agents and Proteasome Inhibitors and Related Mechanisms of Action.

Seaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 µM. 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.

Hepatoprotective effect of trypsin/chymotrypsin against olanzapine-induced non-alcoholic steatohepatitis in rats.

Metabolic side effects of atypical antipsychotics are an important cause of deterioration of cognitive function and failure of drug adherence. The antifatty effect trypsin/chymotrypsin (T/C) and their mechanisms of action remain unclear. To investigate possible therapeutic effect of T/C in rat model of chronic olanzapine (OLZ) - induced hepatic steatosis. Twenty rats were divided into two groups: control (C), given distilled water, and O, given 1 mg/kg of OLZ orally daily for 7 weeks. Then, both groups were given T/C 3 enzyme activity unit (EAU)/kg orally as an add-on treatment daily for the next 5 weeks and were named T/C or T/C+O groups. Rat performance in radial arm water maze was tested twice before and after T/C treatment. We measured liver enzymes, alpha-1 antitrypsin, albumin, total protein, direct and total bilirubin, inflammatory cytokines, and lipoprotein serum levels. Liver samples were collected for histopathology and Ki67 expression. The T/C add-on caused significant reduction in OLZ-induced elevation of alanine transaminase (ALT; P < 0.01), aspartate transaminase (AST; P < 0.001), alkaline phosphatase (ALP; P < 0.05), total cholesterol (Tc; P < 0.01), low-density lipoproteins (LDL-c; P < 0.05), steatosis score (P < 0.001), hepatocyte necrosis (P < 0.01), and significantly increased Ki67 expression (P < 0.01). The T/C add-on to OLZ provided protection against hepatic steatosis, elevated enzymes, and disturbed lipid profile and increased Ki67 without disturbing memory function.

Assessing the cognitive and behavioral development of 3-year-old children born from fathers with severe male infertility.

BACKGROUND: Despite the large number of babies born worldwide following intracytoplasmic sperm injection, concerns about the procedure's safety still exist owing to the use of suboptimal spermatozoa. Thus, follow-up of children conceived via intracytoplasmic sperm injection is highly recommended. We propose the use of parent-administered questionnaires to monitor the development of offspring conceived via intracytoplasmic sperm injection. OBJECTIVE: This study aimed to determine whether male infertility treatment affects offspring development. STUDY DESIGN: We compared obstetrical and neonatal outcomes and physical and psychological development of toddlers conceived via in vitro fertilization and intracytoplasmic sperm injection. Once newborns reached 3 years of age, participating patients were sent a set of parent-administered questionnaires, including the Ages and Stages Questionnaires; Prescreening Developmental Questionnaire 2; Peabody Developmental Motor Scales, Second Edition; Social Skills Rating System; Parenting Stress Index, Third Edition; and Child Behavior Checklist for Ages 2-3. Child development was measured by the Ages and Stages Questionnaires; Prescreening Developmental Questionnaire 2; and Peabody Developmental Motor Scales, Second Edition, questionnaires, whereas Social Skills Rating System; Parenting Stress Index, Third Edition; and Child Behavior Checklist for Ages 2-3 questionnaires were used to measure child behavior. The child's developmental or behavioral outcome was considered "abnormal" when he or she scored below average in ≥2 questionnaires from the respective category. We also conducted subanalyses to assess the effects of male genomic integrity, DNA fragmentation, chemical exposure, utilization of surgically retrieved spermatozoa, and extended embryo culture to determine the development of a child conceived via intracytoplasmic sperm injection. RESULTS: A total of 12,306 couples met the inclusion criteria for this study; 1914 of 7433 patients (25.8%) who underwent intracytoplasmic sperm injection and 451 of 4873 patients (9.3%) who underwent in vitro fertilization returned the questionnaires. Our comparison of obstetrical outcomes between the 2 groups did not reveal any significant differences in the mode of delivery distribution, with most mothers having uncomplicated vaginal deliveries. Furthermore, gender distribution, gestational ages, and birthweights were also comparable between children conceived via intracytoplasmic sperm injection and in vitro fertilization. However, children conceived via in vitro fertilization displayed impaired developmental characteristics compared with the intracytoplasmic sperm injection-conceived cohort (adjusted odds ratio, 0.72; 95% confidence interval, 0.5-0.9; P=.0004). There was no difference in child behavior. Furthermore, 3 cases of autism were reported, 1 case from the in vitro fertilization group and 2 from the intracytoplasmic sperm injection group, all conceived from couples with an older male partner. Ages and Stages Questionnaires outcomes were also compared for the offspring conceived via in vitro fertilization and intracytoplasmic sperm injection by gender; however, no significant differences were observed. In addition, 5 separate subanalyses were then conducted exclusively for the intracytoplasmic sperm injection-conceived group. Levels of spermatogenic failure, DNA fragmentation, and chemical exposure did not significantly affect offspring development. Interestingly, although the length of embryo culture did not seem to influence child development, the abnormal behavior rate was significantly higher in children from the day 3 embryo transfer cohort (adjusted odds ratio, 0.4; 95% confidence interval, 0.05-0.34; P=.04). Children conceived via intracytoplasmic sperm injection from ejaculated spermatozoa displayed impaired developmental and behavioral characteristics compared with toddlers conceived from surgically retrieved specimens (adjusted odds ratio, 4.9; 95% confidence interval, 1.2-20.7; P=.05). CONCLUSION: Most children conceived via intracytoplasmic sperm injection and in vitro fertilization are developing well without significant delays. Although the development of a child conceived via intracytoplasmic sperm injection was not affected by most of the variables assessed, those conceived from surgically retrieved spermatozoa were at a considerably lower risk of abnormal developmental and abnormal behavioral characteristics than offspring conceived from ejaculated specimens. However, given the small numbers of respondents available for many subgroups of interest, further studies of outcomes of children born from fathers with severe male factor infertility are warranted.

The effect of proteolytic enzymes and pH on GII.4 norovirus, during both interactions and non-interaction with Histo-Blood Group Antigens.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Histo-Blood Groups Antigens (HBGAs) have been described as attachment factors, promoting HuNoV infection. However, their role has not yet been elucidated. This study aims to evaluate the ability of HBGAs to protect HuNoVs against various factors naturally found in the human digestive system. The effects of acid pH and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs were studied, both during interactions and non-interaction with HBGAs. The results showed that GII.4 VLPs and GII.4 HuNoVs behaved differently following the treatments. GII.4 VLPs were disrupted at a pH of less than 2.0 and in the presence of proteolytic enzymes (1,500 units/mL pepsin, 100 mg/mL trypsin, and 100 mg/mL chymotrypsin). VLPs were also partially damaged by lower concentrations of trypsin and chymotrypsin (0.1 mg/mL). Conversely, the capsids of GII.4 HuNoVs were not compromised by such treatments, since their genomes were not accessible to RNase. HBGAs were found to offer GII.4 VLPs no protection against an acid pH or proteolytic enzymes.

Inhibition of chymotrypsin-like activity of the proteasome by ixazomib prevents mitochondrial dysfunction during myocardial ischemia.

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.

The In Vitro Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1.

As more and more protein biotherapeutics enter the drug discovery pipelines, there is an increasing interest in tools for mechanistic drug metabolism investigations of biologics in order to identify and prioritize the most promising candidates. Understanding or even predicting the in vivo clearance of biologics and to support translational pharmacokinetic modeling activities is essential, however there is a lack of effective and validated in vitro cellular tools. Although different mechanisms have to be adressed in the context of biologics disposition, the scope is not comparable to the nowadays widely established tools for early characterization of small molecule disposition. Here, we describe a biotransformation study of the fusion protein tetranectin apolipoprotein A1 by cellular systems. The in vivo biotransformation of tetranectin apolipoprotein A1 has been described previously, and the same major biotransformation product could also be detected in vitro, by a targeted and highly sensitive detection method based on chymotrypsin digest. In addition, the protease responsible for the formation of this biotransformation product could be elucidated to be DPP4. To our knowledge, this is one of the first reports of an in vitro biotransformation study by cells of a therapeutic protein.

α-Chymotrypsin Immobilized on a Low-Density Polyethylene Surface Successfully Weakens Escherichia coli Biofilm Formation.

The protease α-chymotrypsin (α-CT) was covalently immobilized on a low-density polyethylene (LDPE) surface, providing a new non-leaching material (LDPE-α-CT) able to preserve surfaces from biofilm growth over a long working timescale. The immobilized enzyme showed a transesterification activity of 1.24 nmol/h, confirming that the immobilization protocol did not negatively affect α-CT activity. Plate count viability assays, as well as confocal laser scanner microscopy (CLSM) analysis, showed that LDPE-α-CT significantly impacts Escherichia coli biofilm formation by (i) reducing the number of adhered cells (-70.7 ± 5.0%); (ii) significantly affecting biofilm thickness (-81.8 ± 16.7%), roughness (-13.8 ± 2.8%), substratum coverage (-63.1 ± 1.8%), and surface to bio-volume ratio (+7.1 ± 0.2-fold); and (iii) decreasing the matrix polysaccharide bio-volume (80.2 ± 23.2%). Additionally, CLSM images showed a destabilized biofilm with many cells dispersing from it. Notably, biofilm stained for live and dead cells confirmed that the reduction in the biomass was achieved by a mechanism that did not affect bacterial viability, reducing the chances for the evolution of resistant strains.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

Jizanpeptins A-E (1-5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A-E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 µM) compared to chymotrypsin (IC50 = 1.4 to >10 µM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.

Treatment of semen samples with α-chymotrypsin alters the expression pattern of sperm functional proteins-a pilot study.

Semen hyperviscosity delays the liquefaction of semen sample and is subjected to limited proteolysis by addition of α-chymotrypsin to reduce the viscosity. α-Chymotrypsin is a proteolytic enzyme involved in degradation of the proteins and polypeptides. Even though α-chymotrypsin improves the handling of hyperviscous samples, its effect on the sperm proteins is not clear. This study was aimed to evaluate the alteration in the expression of sperm functional proteins in samples treated with α-chymotrypsin. Among all the proteins examined in both donor and patient samples, HSPA2 (70 KDa), BAG6 (150 KDa), HIST1H2BA (14 KDa), SPA17 (17 KDa formed after cleavage of C-terminal calmodulin-binding domain), and OXPHOS complexes were undetectable in α-chymotrypsin-treated samples, while the expression of the native SPA17 (20 KDa) was significantly decreased in the α-chymotrypsin-treated samples in comparison with controls. The use of α-chymotrypsin for liquefaction of hyperviscous samples degrades functional proteins of spermatozoa. Intracellular proteins, such as OXPHOS complexes and HIST1H2BA, and sperm surface proteins (HSPA2, BAG6, and SPA17) were degraded in all treated samples. Whether treatment of samples with α-chymotrypsin affects the global proteomic outcome is unclear. More in-depth calibration studies are required to determine the appropriate concentration of α-chymotrypsin for processing hyperviscous semen samples without compromising its protein expression and function. Similarly, the effects of altered protein function on assisted reproductive techniques (ART), such as intrauterine insemination (IUI) and in vitro fertilization (IVF) outcome, are not known and require further research.

Nanodiamond-chymotrypsin and nanodiamond-papain conjugates, their synthesis and activity and visualization of their interaction with cells using optical and electron microscopy.

Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of the dNDs' surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding. Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved the preservation of the enzymes' composition during synthesis. The same assay was used to determine the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain. The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared to be approximately 40% of that of the native enzymes. The optimum pH values and stability under various conditions were determined. The sizes of resulting particles were measured using dynamic light scattering and direct electron microscopic observation. The enzyme conjugates were shown to be prone to aggregation, resulting in micrometer-sized particles. The ζ-potentials were measured and found to be positive for the conjugates. The conjugated enzymes were tested for biological activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules. Incubation with conjugated enzymes results in morphological changes of the cells but does not affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a model of a stepwise conjugate binding to the cell membrane and gradual release of the enzymes.

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor.

Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1-APHC3 from H. crispa, and clusters with the peptides from so named "analgesic cluster" of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10-7 and 7.0 × 10-7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 µM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21-TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.

Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA) binds human erythrocytes independent of Duffy antigen status.

Plasmodium vivax, a major agent of malaria in both temperate and tropical climates, has been thought to be unable to infect humans lacking the Duffy (Fy) blood group antigen because this receptor is critical for erythrocyte invasion. Recent surveys in various endemic regions, however, have reported P. vivax infections in Duffy-negative individuals, suggesting that the parasite may utilize alternative receptor-ligand pairs to complete the erythrocyte invasion. Here, we identified and characterized a novel parasite ligand, Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA), that bound human erythrocytes regardless of Duffy antigen status. PvGAMA was localized at the microneme in the mature schizont-stage parasites. The antibodies against PvGAMA fragments inhibited PvGAMA binding to erythrocytes in a dose-dependent manner. The erythrocyte-specific binding activities of PvGAMA were significantly reduced by chymotrypsin treatment. Thus, PvGAMA may be an adhesion molecule for the invasion of Duffy-positive and -negative human erythrocytes.

Effect of Asiaticoside, Collagenase, and Alpha-chymotrypsin on Wound Healing in Rabbits.

UNLABELLED: Wound dressing materials such as asiaticoside, collagenase, and alpha-chymotrypsin are often used for effective wound healing activity. OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Porcine myofibrillar proteins as potential precursors of bioactive peptides - an in silico study.

Selected porcine myofibrillar proteins have been assessed as potential precursors of bioactive peptides based on in silico analysis. The potential of protein sequences for releasing peptides was evaluated by determining the profile of their potential biological activity and the frequency of occurrence of fragments with a given activity using the BIOPEP database. Digestive enzymes: pepsin, trypsin and chymotrypsin have been used for the in silico proteolysis with the use of the "Enzyme(s) action" tool in BIOPEP. After simulated gastrointestinal digestion the tested sequences of pig myofibrillar proteins are a potential source of a total of 399 peptides with activities such as enzyme inhibition, antioxidative, hypotensive, stimulating or regulating various body functions and antiamnestic activities. Within the intact proteins and after simulated gastrointestinal digestion, dipeptidyl peptidase IV inhibitory peptide sequences were the most frequently observed. The results indicate that pork myofibrillar proteins are a promising source of peptides with biological activity.

Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4.

Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B12 (B12) may be exploited in this area, but an open question is whether B12-peptide conjugates bound to the B12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B12 with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin ß. Prebinding the B12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B12-Ex-4 conjugate relative to Ex-4, when the IF-B12-Ex-4 complex was exposed to 22 µg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 µg/mL of chymotrypsin, and there was no decrease in function at up to 5 µg/mL of meprin ß.

Anti-inflammatory effect of chymotrypsin to autoimmune response against CNS is dose-dependent.

Multiple sclerosis is an inflammatory autoimmune disease of central nervous system (CNS) in which inflammatory cells release pro-inflammatory cytokines, proteases, and other toxic mediators. Proteases are involved in many aspects of inflammatory process. There are many reports regarding the effect of proteases on inflammation. Chymotrypsin is a serine protease with anti-inflammatory effect. We investigated chymotrypsin effect on experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Intra-CSF injection with 0.1 mg/ml, 0.2 mg/ml chymotrypsin, or saline was done on day 7 after EAE induction. Our study demonstrated that 0.1 mg/ml chymotrypsin treatment did not decrease clinical signs, but 0.2 mg/ml chymotrypsin ameliorated clinical signs and manipulated immune response in both brain and spinal cord. Administration of 0.1 mg/ml or 0.2 mg/ml chymotrypsin led to decreased IL-17 along with increased IL-4 and FoxP3 in 0.2 mg/ml chymotrypsin-treated animals. Presumably, chymotrypsin acts in a dose-dependent manner and concentrations of chymotrypsin more than 0.2 mg/ml may have more beneficial effect.

New renin inhibitors--stability and activity determination. Part I.

A series of new six potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-6) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of alpha-chymotrypsin was determined. Compound 5 was unstable, compound 6 was stable, other compounds were partly unstable, compound 2 was stable except kidney homogenate and compound 4 was stable except liver homogenate. Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-6). Compound 2, 4 and 6 showed inhibitory activity (1.4 x 10(-6), 5.2 x 10(-6), 1.5 x 10(-7) M, respectively). Other compounds (1, 3, 5) showed no inhibitory activity up to 10(-5) M.

Zymogen activation confers thermodynamic stability on a key peptide bond and protects human cationic trypsin from degradation.

Human cationic trypsinogen, precursor of the digestive enzyme trypsin, can be rapidly degraded to protect the pancreas when pathological conditions threaten, while trypsin itself is impressively resistant to degradation. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81-Glu82 peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122-Val123 peptide bond by trypsin. Here we demonstrate that the Leu81-Glu82 peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. When cleaved trypsin was incubated with CTRC, the Leu81-Glu82 peptide bond was re-synthesized to establish the same equilibrium. The thermodynamic stability of the scissile peptide bond was not dependent on CTRC or Leu-81, as re-synthesis was also accomplished by other proteases acting on mutated cationic trypsin. The Leu81-Glu82 peptide bond is located within a calcium binding loop, and thermodynamic stability of the bond was strictly dependent on calcium and on the calcium-coordinated residue Glu-85. Trypsinolytic cleavage of the Arg122-Val123 site was also delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation.

Effect of N-ethylmaleimide, chymotrypsin, and H2O2 on the viscoelasticity of human erythrocytes: experimental measurement and theoretical analysis.

The physiological functions of erythrocytes depend critically on their morphology, deformability, and aggregation capability in response to external physical and chemical stimuli. The dynamic deformability can be described in terms of their viscoelasticity. We applied jumping optical tweezers to trap and stretch individual red blood cells (RBCs) to characterize its viscoelasticity in terms of the Young's modulus and viscosity by analyzing the experimental data of dynamic deformation using a 2-parameter Kelvin solid model. The effects of three chemical agents (N -ethylmaleimide, Chymotrypsin, and Hydrogen peroxide) on RBC's mechanical properties were studied by comparing the Young's modulus and viscosity of RBCs with and without these chemical treatments. Although the effects of each of these chemicals on the molecular structures of RBC may not be exclusive, based on the dominant effect of each chemical, we attempted to dissect the main contributions of different constituents of the RBC membrane to its viscosity and elasticity.

Protease-dependent hemagglutinin cleavage contributes to alteration in chicken hemagglutination by the H3N2 influenza A virus.

The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(-) virus]. The Ch(-) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(-) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.