Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.     

Proteome of the phytopathogen <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>: a global expression profile

Background  

Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).     

Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.     

Synthesis and analysis of selenomethionine metabolites

This paper describes the enzymatic synthesis of selenomethionine metabolites of the transmethylation and polyamine synthesis pathways: adenosylselenomethionine, adenosylselenohomocysteine, decarboxylated adenosylselenomethionine, and methylselenoadenosine. These compounds and the corresponding methionine metabolites were simultaneously separated by a single HPLC run. The sensitivity of the HPLC method is about 20 pmol per compound. The method may be used for direct analysis of the metabolite levels in tissues or cells treated with selenomethionine and it provides an assay method for the pulse-chase type of analysis of relative flows for both selenium- and sulfur-containing compounds in transmethylation and polyamine pathways.     

Hydrolysis of histones by proteinases.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.     

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.     

Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival

Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine). Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.     

Effects of deer,mice and dwarf bamboo on the emergence,survival and growth of <Emphasis Type="Italic">Abies homolepis</Emphasis> (Piceaceae) seedlings

In the subalpine mixed forest of Mt. Ôdaigahara, mid-western Japan, the understory is dominated by dwarf bamboo (Sasa nipponica), which is the major forage of overly populous sika deer (Cervus nippon). In the present study, we monitored the survival and growth of Abies homolepis seedlings over 5years to determine how they responded to the experimental removal of dwarf bamboo and to the exclusion of sika deer and mice (Apodemus argenteus and A.speciosus). Deer and dwarf bamboo reduced the survival of seedlings but had different effects on growth. The stems of seedlings were shorter in the presence of deer, indicating that taller seedlings were apt to be browsed by deer, whereas the diameters of seedlings were smaller in the presence of dwarf bamboo, mainly owing to its shading effect. The presence of mice decreased the number of seedlings germinating in a particular site, but had no effect on seedling survival after germination. There was no significant indirect effect whereby the survival of seedlings was predicted to be facilitated by the decreased biomass of bamboo because of grazing by deer. We supposed that this might be because the direct negative effect of deer was so large as to conceal the positive indirect effect.     

Effect of handling of plasma samples on histamine radio-enzyme assay

1. Freeze-thawing of plasma samples increased the histamine level at physiological histamine concentrations as analyzed by radio-enzyme assay, but not by high performance liquid chromatography. 2. Heating of plasma samples decreased the histamine levels. 3. Enzymatic or non-enzymatic formation of histamine during handling of plasma samples was not detected. 4. Some albumin preparations contain histamine.