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)

A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.
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PMID:Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry. 826 4

To establish the method for generating a large number of mature human mast cells, we cultured cord blood mononuclear cells (CBMC) in several conditions in the presence of Steel factor (SF). Among several cytokines tested, IL-6 enhanced SF-dependent mast cell growth from purified CD34+ cells for more than 8 wk in culture. When CBMC were cultured instead of CD34+ cells, IL-6 enhanced the mast cell development in the presence but not in the absence of PGE2. PGE2 enhanced the SF- and IL-6-dependent development of mast cells from CBMC probably by blocking granulocyte-macrophage CSF (GM-CSF) secretion from accessory cells, because 1) PGE2, or anti-GM-CSF enhanced the mast cell development induced by SF and IL-6 from CBMC, but not from CD34+ cells; 2) GM-CSF inhibited the enhancing effect of IL-6 on the mast cell development from CD34+ cells; and 3) PGE2 inhibited GM-CSF secretion from CBMC. The mast cells cultured in the presence of SF, IL-6, and PGE2 for >10 wk were 99% pure, and seemed to be functionally mature, because 1) they contained 5.62 micrograms of histamine and 3.46 micrograms of tryptase per 10(6) cells; and 2) when sensitized with human IgE and then challenged with anti-human IgE, the cells released a variety of mediators such as histamine, and an increase in intracellular Ca2+ was found in advance of the activation of membrane movement by using a confocal laser-scanning microscope. Electron-microscopic analysis revealed that some of the cultured mast cells are morphologically mature since they filled with scroll granules and contained crystal granules.
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PMID:Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. 868 36

The epidemiological situation of bacterial meningitis is increasing dramatically. There is no doubt that the lack of proper animal models has hampered the achievement of effective prophylactic and therapeutic means. We report the characterization of the experimental disease caused by Haemophilus influenzae type b (Hib) in mice, taking into account its importance as an etiological agent of such a type of meningitis. The high resistance of C57BL/6, CBA/ J and BALB/cJ mice to Hib infection was proven. LD50 of Hib using trypsin or iron dextran as virulence enhancement factors (VEF), both being similar and more than 1000 times lower than that without any VEF, were determined. Lesions of CNS compatible with meningitis were found in about one third of specimens. Hair bristling, conjunctivitis, diarrhea, depression and prostration were the most characteristic symptoms. The proportion of animals which die is highest on the first day, lower on the second and almost zero after 48 h of infection. Water and food intake was higher in control than in infected animals; nevertheless, there were no differences in body weight increase among the mice after 5 days post-infection. Microorganisms were isolated from CSF and blood after 6 h of infection and positive results remained according to the size of the inoculum. Despite the acuteness of the experimental disease, antibiotic treatment with internationally recommended drugs was shown to be effective. Similar results were achieved when hyperimmune serum vs. Hib was applied.
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PMID:Experimental infection by Haemophilus influenzae type b in inbred mice. 869 54

During allergic disease, leucocytes infiltrate the affected tissues and release their mediators and cytokines. In this way, the local inflammatory process is induced and maintained. Basophilic granulocytes have been demonstrated in lung and sputum of allergic asthmatics, in nasal mucosa and secretion of allergic rhinitis patients, and in skin lesions of atopic dermatitis patients. The number of basophils correlates with the severity of the disease. Analysis of mediator profiles and cellular contents of lavages of nose, skin and lung during allergic late-phase reactions (LPR) have demonstrated histamine, but not tryptase or prostaglandin D2. The histamine-containing cells have been characterized as basophilic granulocytes. This indicates that infiltrating basophils but not mast cells are activated and release their inflammatory contents in the LPR. We are interested in the cellular mechanisms that determine the degranulation of basophils during LPR. Basophil activators, such as allergens and activated complement, are not present at these sites. However, cytokines that prime basophils but do not induce degranulation, such as interleukin-5 (IL-5) and granulocyte/macrophage colony-stimulating factor (GM-CSF), have been detected at sites of LPR. We have now observed that after emptying intracellular Ca2+ stores by means of the Ca2+ adenosine triphosphatase (ATPase) inhibitor, thapsigargin, basophils become extremely sensitive to stimuli that do not affect the Ca2+ stores themselves but that induce degranulation, such as the phorbolester, phorbol myristate acetate (PMA). The most interesting finding was that although both thapsigargin and IL-3, IL-5 or GM-CSF do not induce basophil degranulation by themselves, a 2 min preincubation of basophils with thapsigargin followed by addition of one of these cytokines resulted in extensive histamine release: IL-3 induced 71 +/- 7% histamine release (conc1/2max 6 pM), IL-5 induced 43 +/- 8% histamine release (conc1/2max 41 pM) and GM-CSF induced 57 +/- 10% histamine release (conc1/2max 140 pM). Interestingly, the effect of thapsigargin could be mimicked by platelet-activating factor (PAF) (range 10(-9) to 10(-6) M), although to a lesser extent. Our results indicate that basophil degranulation in tissues during late-phase reactions might be caused by a combination of mediators or cytokines depleting Ca2+ stores, as platelet-activating factor or thapsigargin do, concurrent with activation by interleukin-3, interleukin-5 or granulocyte/macrophage colony-stimulating factor. The response of the basophils towards these cytokines might also be influenced by cell adhesion events, such as binding of basophils via integrins. This is the subject of further study.
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PMID:The role of basophils in allergic disease. 887 Oct 57

The effects of recombinant human granulocyte CSF (rhG-CSF) and recombinant human granulocyte-macrophage CSF (rhGM-CSF) on the recombinant human stem cell factor (rhSCF)-dependent development of human mast cells from fetal liver progenitors were examined. Mast cells were identified by immunohistochemical staining for tryptase and by flow cytometric analysis of surface Kit expression. Only rhGM-CSF affected mast cell development. When rhGM-CSF (1, 10, or 100 ng/ml) and rhSCF (50 ng/ml) were added to cell cultures from day 0, both the percentage and absolute numbers of mast cells were diminished after 4 wk compared with cultures exposed to rhSCF alone. Half of the maximal response was achieved at a dose of rhGM-CSF between 0.1 and 1 ng/ml. The Kit+ cells developing in the presence of rhGM-CSF and rhSCF exhibited an intensity of surface Kit expression comparable to that of cells exposed to rhSCF alone. Also, if the initial exposure to rhGM-CSF was delayed for 1 to 3 wk, attenuation of mast cell development waned. These findings are consistent with uncommitted progenitor cells being diverted to nonmast cell lineages by rhGM-CSF, while cells committed to a mast cell lineage, albeit immature, appear to be resistant to the lineage directives of rhGM-CSF. Exposure of fetal liver cells to rhGM-CSF for 1 to 3 days before addition of rhSCF further diminishes the number of mast cells that develop compared with the simultaneous addition of these growth factors on day 0. Whether administration of rhGM-CSF to humans before or together with rhSCF diminishes the mast cell hyperplasia that occurs with rhSCF alone remains to be determined.
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PMID:Recombinant human granulocyte-macrophage colony-stimulating factor (CSF), but not recombinant human granulocyte CSF, down-regulates the recombinant human stem cell factor-dependent differentiation of human fetal liver-derived mast cells. 921 2

HTLV-1 (human T-cell leukemia virus type I) associated myelopathy (HAM) is a demyelinating disease. We showed that the CSF of patients and heated CSF of normal subjects induce a segmentary demyelination in rat nerves, and potentiate trypsin in vitro. Here we further characterize the neuropathy induced by the CSF of patients. Peroneal nerves injected 5-8 days before with native or heated CSF of patients, besides extensive demyelination, presented proliferation of myelinating and nonmyelinating Schwann cells, axonal sprouting, fine fibres with a few turns of myelin, disarray of nonmedullated bundles, desmosome-like junctions, and coated pits and vesicles in Schwann cells and axons. The normal CSF was innocuous to the nerve in its native form, but after heating, it induced a neuropathy in all, similar to that elicited by the CSF of patients. Our findings indicate that the CSF of HAM patients contains a thermostable pathogen for nerves of the rat; a thermostable pathogen also occurs in the normal CSF although its activity is checked by endogenous thermolabile factors. We suggest that the pathogen present in the CSF of HAM patients participates in the disease.
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PMID:Cerebrospinal fluid of HTLV-1 associated myelopathy patients induces axonal sproutings and Schwann cell proliferation in the rat sciatic nerve. 970 Jun 98

Mast cells are found frequently in close proximity to blood vessels, and endothelial cells are likely to be exposed to high concentrations of their granule mediators. We have investigated the proinflammatory actions of the major mast cell product tryptase on HUVEC. Addition of purified tryptase was found to stimulate thymidine incorporation, but induced little alteration in cell numbers, suggesting it is not a growth factor for HUVEC. Expression of ICAM-1, VCAM-1, and E-selectin was not altered following incubation with tryptase, but the potent granulocyte chemoattractant IL-8 was released in a dose-dependent fashion in response to physiologically relevant concentrations, with maximal levels in supernatants after 24 h. The actions of tryptase on HUVEC were inhibited by heat inactivation of the enzyme, or by preincubating with the protease inhibitors leupeptin or benzamidine, suggesting a requirement for an intact catalytic site. Reverse-transcription PCR analysis indicated up-regulation of mRNA for IL-8 as well as for IL-1 beta in response to tryptase or TNF-alpha. However, tryptase was a more selective stimulus than TNF-alpha and did not induce increased expression of mRNA for granulocyte-macrophage CSF or stimulate the release of this cytokine. Leukocyte accumulation in response to tryptase may be mediated in part through the selective secretion of IL-8 from endothelial cells.
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PMID:The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells. 971 64

The neural cell adhesion molecule (N-CAM) is a cell recognition molecule involved in cellular migration, synaptic plasticity, and CNS development. A 105- to 115-kDa isoform of N-CAM (cleaved N-CAM or cN-CAM) is increased in schizophrenia in hippocampus, prefrontal cortex, and CSF. We purified and partially characterized cN-CAM, a putative novel isoform, and confirmed that the first 9 amino acids were identical to exon 1 of N-CAM, without the signal sequence. Analysis of trypsin-digested cN-CAM fragments by matrix-assisted laser desorption ionization on a time-of-flight mass spectrometer (MALDI-TOF) yielded peptides that could be identified as being derived from the first 548 amino acid residues of the expected N-CAM amino acid sequence. Immunological identification with four specific N-CAM antisera directed toward cytoplasmic, secreted, variable alternative spliced exon, or GPI epitopes failed to indicate other known splice variants. Neuraminidase treatment of cN-CAM produced a minor alteration resulting in a faster migrating immunoreactive band, indicating partial glycosylation of cN-CAM. Membranous particles from cytosolic brain extract containing cN-CAM were obtained by ultracentrifugation; however, CSF contained few such particles. cN-CAM and synaptophysin were colocalized on these particles. Both cN-CAM and N-CAM 180 were present in synaptosomal preparations of human brain. Following incubation of synaptosomes or brain tissue without protease inhibitors, N-CAM 180 was degraded and cN-CAM was increased. A cN-CAM-like band was present in human fetal neuronal cultures, but not in fetal astrocyte cultures. Thus, cN-CAM represents a protease- and neuraminidase-susceptible fragment possibly derived by proteolytic cleavage of N-CAM 180. An enlargement in ventricular volume in a group of adult patients with schizophrenia over a 2-year interval was found to be correlated with CSF cN-CAM levels as measured at the time of the initial MRI scan (r = 0.53, P = 0.01). cN-CAM is associated with ventricular enlargement; thus, the release of N-CAM fragments may be part of the pathogenic mechanism of schizophrenia in vulnerable brain regions such as the hippocampus and prefrontal cortex. Alternatively, the increases in cN-CAM in schizophrenia may be a reflection of a more general abnormality in the regulation of proteolysis or of extracellular matrix stability.
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PMID:Characterization of human cleaved N-CAM and association with schizophrenia. 1168 38

Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant human granulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokine orientation on its binding to receptors from human placenta. Competition enzyme linked immunoabsorbent assays (ELISA) revealed that mAb 8C2 would be directed to a linear epitope, whereas mAb 6E3 would delimit a more assembled epitope. Gel-filtration high performance liquid chromatography (HPLC) of the immune complexes formed by incubating [(125)I]hG-CSF with each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. In addition, mAb 6E3 completely inhibited [(125)I]hG-CSF binding to human placental microsomes. Although [(125)I]mAb 6E3 was unable to bind to preformed hG-CSF-receptor complexes, [(125)I]mAb 8C2 did recognize hG-CSF previously bound to receptors, suggesting that epitope 8C2 would remain accessible in the hG-CSF-receptor complex. To identify the cytokine region defined by mAbs, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C, trypsin and alpha chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation. Results showed that mAb 6E3 would be directed to a conformation-dependent epitope located close to the hG-CSF binding domain and included into the sequence 1-122/123, whereas mAb 8C2 recognized the region 41-58, which represents a linear epitope left exposed after cytokine binding to receptors from human placenta.
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PMID:Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors. 1168 84

We report the in vitro and in vivo effects of granulocyte macrophage colony stimulating factor (GM-CSF), a known inhibitor of in vitro mast cell differentiation, in a patient with benign, adult-onset systemic mastocytosis. In vitro effects of GM-CSF on bone marrow cultures before the start of treatment showed a marked inhibition of mast cell marker expression [tryptase, Kit, and high-affinity IgE receptor (FcepsilonRIalpha)] at both protein and mRNA levels. Therefore, the patient was treated with daily injections of GM-CSF for 10 weeks. After an initial improvement, increasing worsening of clinical symptoms was noted, and the patient refused further treatment. Lesional skin biopsies showed an increase of toluidine blue-positive mast cells, compared with uninvolved skin, with further significant increase after treatment. Similar results were obtained on staining for mast cell-specific tryptase and Kit, as well as for CD1a and FcepsilonRIalpha. These findings show that GM-CSF inhibits human bone marrow mast cell differentiation in vitro, and also in mastocytosis. However, GM-CSF apparently enhances recruitment of mast cell as well as dendritic cell precursors into the tissue during systemic treatment. These findings and the observed adverse clinical effects in the present patient make it unlikely that GM-CSF monotherapy will be beneficial for the treatment of mastocytosis.
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PMID:Effect of granulocyte macrophage colony-stimulating factor in a patient with benign systemic mastocytosis. 1170 99


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