Detection and partial characterization of lymphoid cell surface proteases

One of the mechanisms of the skin blistering effect (vesication) of sulfur mustard (bis-(2-chloroethyl)sulfide, HD) is believed to be via the stimulation of specific protease(s) at the dermal-epidermal junction. Cultured normal human epidermal keratinocytes (NHEK) were used as a model to study and characterize protease stimulated by the mustards 2-chloroethyl ethyl sulfide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN(2)) and HD. The results obtained using a chromozym (TRY) peptide substrate protease assay revealed the optimum mustard concentrations and time for protease stimulation to be about 200 microM (CEES), 100 microM (HN(2)) and 100 microM (HD) and 16 h. The mustard-stimulated protease was membrane bound and was inhibited by adding a Ca(2+) chelator (either 2 mM EGTA (ethylene glycol-bis(amino ethyl ether) N,N,N',N' tetraacetic acid) or 50 microM BAPTA AM (1,2-bis(z-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxy methyl ester) alone or in combination), a serine protease inhibitor diisopropyl fluoro-phosphate (DFP, 1 mM), or a protein synthesis inhibitor cycloheximide (35 microM) in the extracellular medium. These results suggest that mustard toxicity may involve the stimulation of trypsin/chymotrypsin-like serine protease, dependent on Ca(2+) and new protein synthesis. Protein purification by gel exclusion and hydrophobic chromatography produced a 70-80 kDa protease, which had an amino acid sequence homologous with a mammalian-type bacterial serine endopeptidase. Based on this information, research is in progress to identify the protease stimulated by HD in NHEK and to determine whether its inhibitors are useful as prospective antivesicant drugs.

Protease and virulence of the extracellular products (ECP) of Vibrio carchariae strain EmI82KL, a causative agent of gastroenteritis in Epinephelus coioides, cultured on different media were studied. The bacteria grown on peptone agar, nutrient agar or brain heart infusion agar produced higher protease activities than that grown on tryptic soy agar (TSA) in terms of protein content. The addition of ethylenediamine di(o-hydroxyphenylacetic acid) or horse serum in TSA enhanced the ECP protease production while the addition of grouper serum apparently reduced the enzyme activity indicating the presence of protease inhibitor(s) in the fish serum. Furthermore, the use of grouper meat or peptone as a single nutrient source remarkably enhanced the production of ECP protease. Adding proteinaceous materials from animal sources (horse serum, grouper meat or peptone) on agar manifested higher ECP protease activity than that from plant source (TSA), indicating the intestine of carnivorous groupers might favour the existence, survival or infection of the bacterium. The protease was a metal-chelator-sensitive serine protease since it was inhibited by 3,4-dichloroisocoumarin and phenylmethanesulfonyl fluoride while about 80% of its activity inhibited by chelating agents (ethylene-diaminetetraacetic acid and ethylene glycol-bis(beta-amino-ethylether) N,N,N',N'-tetraacetic acid). The ECP obtained from each medium was not lethal to the groupers suggesting that the bacterium is low virulent. As grouper is carnivorous, therefore, the role of the protease played in the fish intestine probably is competing for nutrients and/or associated with the cause of edema leading to gastroenteritis.

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-gamma). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-gamma was not affected by the time of treatment. In addition, treatment with rIFN-gamma inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-gamma marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-gamma does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.

It is hypothesized that the balance of cytokines produced by Th1/Th2 subsets of T helper cells plays an important role in the development of autoimmune diseases. Murine collagen-induced arthritis (CIA) is an example of an autoimmune disease in which immunization with cartilage-derived type II collagen induces, firstly, a T cell response to type II collagen and, secondly, the manifestation of a destructive inflammatory response in affected joints. We have investigated the role of Th1/Th2 responses in the development of CIA by monitoring levels of interferon (IFN)-gamma (a Th1 cytokine) and interleukin (IL)-4 and IL-10 (Th2 cytokines), and IL-1 beta and tumor necrosis factor (TNF) (pro-inflammatory cytokines) produced by cultured draining lymph node cells (LNC) from collagen-immunized DBA/1 mice during the induction phase of arthritis and throughout the time of clinical manifestation and subsequent remission of the disease. Although a transient increase in IL-10 was detected 3 days after immunization, Th2 cytokine production was found to be almost completely suppressed 6 days after immunization. In contrast, IFN-gamma was detected in LNC cultures as early as 6 days after immunization and the addition of type II collagen to the culture medium resulted in an approximately 10-fold increase in IFN-gamma production, indicating that a predominantly Th1 response had become established by this time. IFN-gamma production by LNC was found to be further increased at the time of clinical manifestation of arthritis and could be up-regulated by co-culture with type II collagen. IL-10 was not detected in LNC cultures at the onset of arthritis and IL-4, although present, was found to be markedly suppressed in LNC cultures containing type II collagen. These findings indicate that Th1 responses are predominant at the time of onset of arthritis and that the activation of collagen-specific Th1 cells may result in suppression of Th2 activity. IFN-gamma production declined progressively during the progression and subsequent remission of arthritis whereas levels of IL-10 increased and low, though persistent, levels of IL-4 were detected throughout this period. High levels of IL-1 beta and TNF-alpha production were detected at the onset of the disease. The role of Th1 responses in the development of CIA was further emphasized by the observation that immunization of mice with type II collagen in incomplete Freund's adjuvant, which normally fails to induce arthritis, resulted in a predominantly Th2 cytokine profile.

Mechanism of the stimulation of Ca 2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Comparison of the T helper cell response induced by respiratory syncytial virus and its fusion protein in BALB/c mice

Molecular States of Fungal Nuclease Composed of Heterogeneous Subunits as Estimated from the Effects of Urea and Chelating Agents

A correlation between the ratio of the cell surface protease activity to phosphatase activity and the complexity of the pattern of cell surface exposed polypeptides of Leishmania promastigotes was demonstrated for various strains grown under similar conditions. The ratio of the cell surface protease activity to acid phosphatase activity was high for L. major and L.b. panamensis and it correlates with the expression of a single polypeptide of 63 KDa on their cell surface. Intermediate and lower ratios of these enzymatic activities relate with more complex radio-iodinated patterns: two main bands in L.b. guyanensis (70 and 58 KDa) and L.b. braziliensis (72 and 60 KDa) and three main bands 65, 50, 27 KDa in all L.m. mexicana strains tested. Evidence is presented that the acid phosphatase located on the L.m. mexicana cell surface is not an artifact due to a secondary absorption of the secreted acid phosphatase from the culture medium. These results confirm the Leishmania antigen cell surface heterogeneity. The implications on the biology of Leishmania and the clinical manifestation of leishmaniasis are discussed.

Comparative study of Agkistrodon acutus venoms from china and taiwan

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

Evaluation of the Effect of EDTAC, CDTA, and EGTA on Radicular Dentin Microhardness

Further characterization of the anticoagulant proteinase, cerastase F-4 from cerastes cerastes (Egyptian sand viper) venom

Calcium, magnesium, and zinc are divalent metal ions essential for good health. Other divalent metals, such as cadmium, lead, and arsenic, are toxic. Turning to common metal-chelating agents, researchers have found that they can use nuclear magnetic resonance spectroscopy and magnetic resonance imaging to quantitatively sense and distinguish among all these metals in blood serum (Anal. Chem. 2016, DOI: 10.1021/acs.analchem.6b03546). Gil G. Westmeyer of the Technical University of Munich and colleagues labeled the metal-coordinating carboxyl groups of ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) with 13C. When either of the compounds binds the metals, 13C NMR spectra show well-separated chemical shifts that allow the researchers to distinguish between the metals. For example, they were able to quantitate millimolar concentrations of Ca2+ in human serum in the presence of competing concentrations of Mg2+. The researchers were further abl...

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rβ1 and IL-12Rβ2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rβ2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rβ2 expression can be prevented by treatment with IFN-γ, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rβ1 and β2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-γ secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-γ secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-γ production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-γ secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.

The role of complement in host resistance to infection with Trypanosoma lewisi was studied in normal, C4-deficient, and C3-depleted rats. Complement levels were measured in normal rats throughout the course of infection. A drastic reduction of total complement and C4 hemolytic activities occurred, and C3 levels measured immunochemically were decreased. Although total complement and C4 levels were regularly reduced to less than 10% of preinfection levels regardless of parasite numbers, the degree of C3 consumption correlated with the parasitemia. C3 levels varied from 100% of preinfection in rats with light infections to 35% in animals with heavy parasitemias. Recovery to normal levels followed trypanosome elimination from the peripheral blood. The infection had no significant effect on C6 hemolytic activity. Parasitemias and C3 levels in C4-deficient rats did not differ from those of normocomplementemic controls. Depletion of C3 and late-acting components by cobra venom factor during the reproductive or adult stages of infection did not alter the parasitemias. In addition, T. lewisi and immune serum caused complement activation in vitro, which could be inhibited with ethylene glycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid or ethylenediaminetetraacetic acid. It is concluded that T. lewisi infection in rats result in activation of the classical complement pathway with extensive consumption of the early-acting components, as well as a low degree of activation of the alternative pathway. However, complement does not appear to play a major role in the control and termination of the infection.