Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

E. coli K10 was found to grow anaerobically on molecular hydrogen by reducing nitrate, fumarate, and trimethylamine N-oxide when peptone was added to the culture medium. Molar growth yields based on consumed hydrogen estimated from the amounts of reduction products were all 7.8 g cells/mol, suggesting that 1 mol of ATP was produced in the oxidation of 1 mol of hydrogen. Hydrogenase activity measured in terms of hydrogen evolution was several times higher in cells grown on glucose than in cells grown on hydrogen in the presence of fumarate and trimethylamine N-oxide, while hydrogenase activity measured in terms of hydrogen uptake was unchanged in both cases. The ratio of hydrogenase activities measured in terms of hydrogen uptake and evolution was also high in the extract and centrifugal fractions from cells grown in hydrogen. The soluble fraction and trypsin digest of the precipitate at 100,000 X g were subjected to polyacrylamide disc gel electrophoresis and hydrogenase bands were stained by reduction of benzyl viologen with hydrogen and by oxidation of reduced methyl viologen. The resulting patterns suggest that multiple forms of hydrogenase are present and that the amounts of forms functioning in hydrogen evolution were greatly decresed in cells grown on hydrogen in the presence of acceptors.
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PMID:Hydrogen-dependent growth of Escherichia coli in anaerobic respiration and the presence of hydrogenases with different functions. 36 3

Morphological maturation of the inner root sheath (IRS) and cuticle of the human hair follicle reveals analogies to differentiation processes in other keratinizing epithelia. Detailed biochemical analysis of respective differentiation products, however, has been largely restricted by their low solubility. Herein we provide further evidence for the existence of K1 and K10-derivatives in IRS and hair cuticle based on protein analysis of isolated fractions and immunofluorescence in situ, substantiating our earlier data (Stark, H. J., et al. Differentiation 35, 236-248 (1987)). Extracts from both compartments showed on two-dimensional (2D)-polyacrylamide gels a group of presumptive K1 and K10-turnover products in a wide pI (basic to acidic) and Mr range (56,000-65,000), named IC-I to III and IC-IV, respectively. These components (also found in nail plate) reacted with specific antibodies (to K1 and K10) on Western blots. Weak but distinctive radiolabeling of presumptive precursor spots close to authentic K1 and K10, respectively, and their presence in lower follicle fractions (distant from infundibulum) largely precluded epidermal contamination. Two-dimensional tryptic peptide maps of excised 2D spots from the IC-I to III series revealed high homology to K1, and those from IC-IV components to K10. Immunodetection in frozen sections was improved by trypsin pretreatment and showed distinguished staining for K1 and K10 in IRS ranging from the lower bulbus region up to the "keratinizing zone" of the follicle. Above, the reaction was abruptly abolished which coincides with ultrastructural "melting" of distinct filaments in the intracellular matrix. Thus, our data suggest that differentiation in these follicular compartments (IRS and cuticle) might follow common principles of keratinization.
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PMID:Keratins 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells in the human hair follicle. 170 98

A recent isolate of Mycoplasma fermentans (strain K10, from human leukemic bone marrow) induced a lethal toxicity syndrome in mice. High doses of both viable and inactivated cells were toxic when injected intraperitoneally. Whole lysates and membranes from osmotically shocked cells killed mice, but cytoplasm did not. When membranes were dissolved in detergents and reaggregated by dialysis in the presence of Mg(2+), the lipid-protein complex thus formed was toxic. Lipids extracted from membranes with chloroform-methanol did not kill mice. Protein-rich fractions (obtained by reaggregation plus acetone washes or ammonium sulfate precipitation of dissolved membranes) were also not toxic. No qualitative differences in proteins from three toxic isolates and three nontoxic laboratory strains of M. fermentans were detectable by polyacrylamide gel electrophoresis. The toxic factor contained in reaggregated membranes was heat-stable but sensitive to Pronase, trypsin, and lipase.
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PMID:Toxic membrane fractions from Mycoplasma fermentans. 515 2

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.
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PMID:Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol. 896 93

A parallel-group, double-blind, randomised study was performed to establish the effect of the vitamin D3 analogue KH 1060, in an ointment versus vehicle only, on the epidermal cell characteristics of chronic idiopathic lichen planus; KH 1060 also has marked immunosuppressive activity. A group of 10 patients were treated for 8 weeks with either KH 1060 ointment or vehicle only. In addition to the assessment of clinical scores, keratotome biopsies were taken before and after 8 weeks' treatment. Epidermal cell suspensions were prepared with trypsin and the suspensions incubated with TO-PRO-3 (DNA marker), RKSE 60 (marker for keratin 10-positive cells) and antivimentin (marker for all non-keratinocytes). In nine of the 10 patients, keratotome biopsies were obtained both before and after 8 weeks treatment. The vehicle alone had no significant effect on the clinical severity scores or epidermal cell characteristics. In contrast, the KH 1060 ointment resulted in a statistically significant reduction in the percentage of cells in S- and G2M phase and the percentage of vimentin-positive cells, but it did not affect the percentage of keratin 10-positive cells.
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PMID:The epidermis of chronic idiopathic lichen planus during topical treatment with the vitamin D3 analogue KH 1060. 966 2

Previously it was demonstrated that the levels of large particles (>2 micron) and associated bacterial cell envelope markers increase greatly on occupation in schools; it was hypothesized that the source of both was shed human skin. In the current work to test this hypothesis, room air cleaners were used to collect airborne dust (>50-100 mg) from occupied and unoccupied school rooms which was then subjected to proteomic analysis. Proteins were extracted from the dust and separated using two dimensional gel electrophoresis (2D GE). In situ digestion of protein spots with trypsin released peptides, which were subsequently analyzed by matrix assisted laser desorption/deionization, time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MALDI-TOF-MS-MS). In Coomassie blue stained gels, a single spot generally dominated the 2D gels; this protein was identified by tandem mass spectrometry as K10 epithelial keratin. The results experimentally confirm previous anecdotal reports that human skin is readily shed into air and suggest that increased levels of microbial markers and large particles observed in occupied rooms are also derived from skin.
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PMID:Human K10 epithelial keratin is the most abundant protein in airborne dust of both occupied and unoccupied school rooms. 1817 17

A cell-based wound coverage with keratinocytes and fibroblasts on the basis of a commercially available dermal substitute (Matriderm ((R)), Kollagen/Elastin matrix) was generated, in order to treat wide burn wounds. First the expansion of keratinocytes was optimised and the culturing time was minimised. Raw material was 1-2 cm (2) split skin. Dermis and epidermis were separated by enzymatic treatment with thermolysin. After treatment of both compartments with trypsin and collagenase I, keratinocytes and fibroblasts were isolated and expanded in collagen I coated dishes. After 10 days fibroblasts were seeded on Matriderm ((R)). After cultivation of the fibroblasts-containing matrix for one week keratinocytes were seeded on top. After an additional week of submersed cultivation the matrix was lifted up to the air-liquid interface to initiate epidermal cell differentiation. After 16 days in the air-liquid interphase the matrix was fixed and underwent immunohistochemical and electron microscopic analysis. Histological analysis showed a regularly stratification of the epidermal part. We observed collagen IV, a marker for the basement membrane, between epidermis and dermis. Desmoglein and the differentiation markers involucrine and cytokeratin 10 were found in the suprabasal layers of the epidermis. Electron microscopic analysis showed the basement membrane in the epidermal junction zone as well as cell-cell connections in the form of desmosomes. Late differentiation characteristics, like granular structures and the cornified layer, were found in the stratum granulosum and stratum corneum. Our results demonstrate that a skin equivalent can be generated by using a collagen/elastin matrix, with an expansion rate of 50-100-fold. This skin equivalent may be useful for covering deep wounds.
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PMID:[Development of an engraftable skin equivalent based on matriderm with human keratinocytes and fibroblasts]. 1971 Dec 56

Mycobacterium avium subsp. paratuberculosis is a pathogen which causes a debilitating chronic enteritis in ruminants. Unfortunately, the mechanisms that control M. avium subsp. paratuberculosis persistence during infection are poorly understood and the key steps for developing Johne's disease remain elusive. A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis (SDS-PAGE) followed by LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. avium subsp. paratuberculosis K10 and an cell surface enzymatic shaving method was used to determine the surface-exposed proteins. 309 different proteins were identified, which included 101 proteins previously annotated as hypothetical or conserved hypothetical. 38 proteins were identified as surface-exposed by trypsin treatment. To categorize and analyze these proteomic data on the proteins identified within cell wall of M. avium subsp. paratuberculosis K10, a rational bioinformatic approach was followed. The analyses of the 309 cell wall proteins provided theoretical molecular mass and pI distributions and determined that 18 proteins are shared with the cell surface-exposed proteome. In short, a comprehensive profile of the M. avium subsp. paratuberculosis K10 cell wall subproteome was created. The resulting proteomic profile might become the foundation for the design of new preventive, diagnostic and therapeutic strategies against mycobacterial diseases in general and M. avium subsp. paratuberculosis in particular.
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PMID:Localization of proteins in the cell wall of Mycobacterium avium subsp. paratuberculosis K10 by proteomic analysis. 2037 98

Methods to unequivocally assess and quantify exposure to organophosphate anti-cholinesterase agents are highly valuable, either from a biomonitoring or a forensic perspective. Since for both OP pesticides and various nerve agents the skin is a predominant route of entry, we hypothesized that proteins in the skin might represent an ideal source of unequivocal and persistent biomarkers for exposure to these compounds. In this exploratory study we show that keratin proteins in human skin are relevant binding sites for organophosphates. The thick cornified epithelium of human plantar skin (callus) was exposed to a selection of relevant organophosphorus compounds and keratin proteins were subsequently extracted. After carboxymethylation of cysteine residues, enzymatic digestion of the keratins with pronase and trypsin was performed and the resulting amino acid and peptides were analyzed to assess whether covalent adducts had formed. LC-tandem MS analysis of the pronase digests demonstrated that tyrosine and to a lesser extent serine residues were selectively modified by organophosphate pesticides (both phosphorothioates and the corresponding oxon forms) under physiological conditions. In addition, modification of tyrosine with the nerve agent VX was unequivocally assessed. In order to elucidate specific binding sites, LC-tandem MS analysis of trypsin digests showed two separate tryptic keratin fragments, i.e. LASY*LDK and SLY*GLGGSK, with Y* the modified tyrosine residues, originating from keratin 1/6 and keratin 10, respectively. These preliminary findings, revealing novel binding targets for anti-cholinesterase organophosphates, will form a firm basis for the development of novel (non-invasive) methods for assessment of exposure to organophosphates. Whether this binding will also have biological implications remains an issue for further investigations.
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PMID:Interactions of organophosphates with keratins in the cornified epithelium of human skin. 2252 15

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.
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PMID:Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression. 2345 38


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