Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.
 ...
PMID:Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL. 133 12

A role for second messenger-regulated protein kinases in the early post-IL-3 receptor signal transduction pathway was investigated in the mast cell/megakaryocyte line R6-XE.4. The activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was assessed by the ability of the enzyme to phosphorylate histone H1 in the presence of calcium, diacylglycerol, and phosphatidylserine or after proteolytic activation of PKC with trypsin. In high serum-supplemented cells, but not in cells that were preincubated in serum-deficient media for 6 h, subsequent treatment for 15 min with synthetic IL-3 (10 micrograms/ml) caused up to a sixfold increase in the calcium- and lipid-stimulated histone H1 phosphorylating activity of particulate-associated PKC after fractionation on MonoQ. However, there was no corresponding reduction of cytosolic PKC activity. Therefore, IL-3 appeared to modify the activity of preexisting membrane-associated PKC rather than eliciting its recruitment from the cytoplasm in R6-XE.4 cells. This was in contrast to the situation with FDC-P1 cells, where IL-3 induced PKC translocation. IL-3 also stimulated a cytosolic protein kinase that phosphorylated a synthetic peptide patterned after a phosphorylation site in ribosomal protein S6, but this IL did not alter the activity of cAMP-dependent protein kinase.
 ...
PMID:IL-3-induced activation of protein kinases in the mast cell/megakaryocyte R6-XE.4 line. 230 40

A factor that stimulates the incorporation of 75Selenomethionine into the newly formed platelets of recipient mice (thrombopoietin, TPO) has been partially purified from the plasma of thrombocytopenic patients. The activity was precipitated at 60-80% ammonium sulfate saturation and further purified with hydrophobic interaction chromatography. Thrombopoietin was retained by concanavalin-A-Sepharose. Using HPLC size-exclusion chromatography, an approximate molecular weight of 40,000 dalton was calculated. The overall purification factor was about 2,100-fold. TPO was stable in a pH range from 5 to 9 and was heat-sensitive, and the biological activity was destroyed by trypsin treatment and by dithiothreitol. The partially purified molecule did not stimulate the proliferation of megakaryocyte progenitors in vitro and had no effect on the growth of erythroid or granulocyte-macrophage colonies; when administered in-vivo, TPO significantly affected the mean platelet volume and increased the number of small acetylcholinesterase cells in the bone marrow. TPO appears to be specific for the megakaryocytic lineage and active on the postmitotic compartment of megakaryocytes.
 ...
PMID:Partial purification and biochemical characterization of human plasma thrombopoietin. 336 51

The biochemical properties of an in-vitro megakaryocyte growth factor called megakaryocyte potentiator (Mk-POT) were investigated. P388D1 cell conditioned medium (P388D1 CM), was used as the source of Mk-POT. The potentiator activity had an apparent mol. wt of 21 kilodaltons (kd) by gel filtration and was eluted from DEAE-Sepharose pH 8.0 with 0.15 M NaCl. Chromatofocusing revealed three active species with apparent pIs of 4.0, 5.5 and above 6.0. Most Mk-POT activity does not bind to Concanavalin A-Sepharose. Mk-POT activity is sensitive to reduction by dithiothreitol and temperatures above 90 degrees C. Treatment with trypsin, alpha-chymotrypsin and pronase also reduced the Mk-POT activity, but it was not destroyed by RNase A or neuraminidase. It is precipitated in ammonium sulphate solutions of between 60 to 70% saturation, and by 80% ethanol. The Mk-POT activity is stable in solutions of pH 5.0-9.0. The data presented here suggest that megakaryocyte potentiator is either heterogeneous in its properties or more than one molecular species may express the in-vitro Mk-POT activity found in P388D1 CM.
 ...
PMID:Biochemical characterization of an in-vitro murine megakaryocyte growth activity: megakaryocyte potentiator. 348 43

We have previously shown that human megakaryocyte colony-stimulating activity (Meg-CSA) is present in sera from patients with bone marrow megakaryocyte aplasia. In this report, we demonstrate that Meg-CSA is also present in sera from dogs rendered aplastic by 1000 rad total body irradiation. Canine serum Meg-CSA has activity comparable to human when assayed in plasma clot cultures containing human bone marrow mononuclear cells. Because of the uniform high potency and ready availability of aplastic canine sera, it was utilized initially for Meg-CSA purification. Sequential ammonium sulfate precipitation to approximately 80% saturation resulted in recovery of 59%-69% of the serum protein and of 75%-103% of the original serum Meg-CSA. The fraction precipitable between ammonium sulfate saturations of 0% and 44%-50% (fraction I) contained 53%-76% of the initial serum Meg-CSA and 25%-32% of the initial serum protein. This represents an enrichment of Meg-CSA specific activity by over 100%. The Meg-CSA eluted from Sephacryl S-300 in a single peak corresponding to a molecular weight of 175,000. This Meg-CSA peak also contained IgG, but the Meg-CSA did not bind to protein A-agarose. Meg-CSA was 90% inactivated by trypsin digestion for 4 h at 37 degrees C and by exposure to 5mM dithiothreitol for 2 h at room temperature. Exposure to either 6 M guanidine for 1 h at room temperature or 8 M urea for 1 h at 4 degrees C resulted in a 70% loss of Meg-CSA. At culture concentrations capable of stimulating maximal megakaryocyte colony formation, fraction I supported no colony growth by myeloid (CFU-GM) or late erythroid (CFU-E) human hematopoietic progenitor cells. Erythroid burst-promoting activity (BPA) was not detected in fraction I from two of three different aplastic canine sera tested. Therefore, Meg-CSA is functionally distinct from granulocyte-monocyte colony-stimulating factor (GM-CSF), erythropoietin, and BPA. The data indicate that serum Meg-CSA is a 175,000-dalton protein (megakaryocyte colony-stimulating factor, Meg-CSF) in which higher order structure and disulfide bonding are necessary for biologic activity. Partially purified Meg-CSF manifests functional specificity for the CFU-Meg hematopoietic progenitor cell.
 ...
PMID:Human megakaryocyte colony-stimulating factor in sera from aplastic dogs: partial purification, characterization, and determination of hematopoietic cell lineage specificity. 387 46

Intranuclear inclusions were found in bone marrow cells of miniature pigs, whose ultrastructure and nature were examined by electron microscope and by cytochemical techniques. The inclusions were found predominantly in the granulocyte series and in a megakaryocyte and plasma cell. The inclusions consisted of closely packed, wavy filaments, 20-40 in number. They varied in size from 0.5-1.0 mum in length and 0.2-0.5 mum in width. The largest inclusion extended across the entire length of the nucleus. Occasionally, an inclusion extending toward the nuclear pore was observed. the intranuclear inclusions showed a tendency to appear frequently in immature granulocytes. The experiment on enzyme digestion for the epoxy sections showed that the inclusions were insoluble in RNase, DNase, pepsin, trypsin and protease solutions. These results seem to suggest that the intranuclear inclusions of blood cells differ from filamentous inclusions which have been noted previously in some neurons.
 ...
PMID:Intranuclear inclusions in the bone marrow cells of miniature pigs. 627 12

Human marrow cells were processed sequentially by density centrifugation and by velocity sedimentation in serum-free Percoll gradients in order to purify megakaryocytes and to determine if these cells are the source of the growth factor derived from platelets. Cell homogenates were made from the resulting fractions and tested for growth-promoting activity(ies) in 3T3 cells and in well characterized human marrow fibroblasts. Growth was evaluated by 3H-TdR incorporation and changes in DNA cell content, as measured by flow microfluorometry. The highest mitogenic activity was derived from homogenates of low density (less than 1.050 g/cu cm), rapidly sedimenting cells. This fraction contained the highest percentage of megakaryocytes. The assessment of growth-promoting activity(ies) derived from various megakaryocyte-enriched marrow cell homogenates containing different proportions of megakaryocytes demonstrated a positive correlation between the number of megakaryocytes and their stimulatory capacity as determined by 3H-TdR uptake. The growth-promoting activities elicited from homogenates of platelets and marrow fractions enriched for megakaryocytes were similar. The dose--response curves for both were parallel, and they were both temperature resistant and trypsin sensitive. These findings implicate megakaryocytes as a source of the growth factor derived from platelets and suggest that megakaryocytes may play a role in the pathogenesis of the marrow fibrosis observed in myeloproliferative disorders by stimulating fibroblast proliferation and collagen secretion.
 ...
PMID:Human megakaryocyte stimulation of proliferation of bone marrow fibroblasts. 747 Jun 27

When interleukin-2 (IL-2) was added to immature, low-ploidy (greater than 80% 2N+4N) megakaryocytes generated in IL-3 and stem cell factor (SCF)-containing liquid cultures of blood mononuclear cells highly enriched in hematopoietic progenitors, a 2- to 6-fold increase in the absolute number of polyploid (more than 8N) megakaryocytes was noted. This effect was found to be indirect and was mediated through natural killer (NK) cells that constitute the major lymphoid cell contaminating day 6 megakaryocyte cell populations. IL-2 had no effect on megakaryocytes generated from CD34(+) cells stimulated with IL-3 and SCF. However, medium conditioned by IL-2-stimulated, but not resting, NK cells (NKCM) contained a trypsin-sensitive factor capable of increasing 2- to 5-fold the number of polyploid megakaryocytes generated in vitro from IL-3 and SCF-stimulated CD34(+) cells. The activity in NKCM was dose dependent and could not be neutralized by an excess of antibodies to IL-6, IL-11, leukemia inhibitory factor (LIF), gp130, stromal cell derived factor-1a (SDF-1a), and thrombopoietin (TPO). Addition of IL-11, but not TPO, to NKCM-containing cultures resulted in further augmentation of polyploidy, with the generation of 50% to 70% polyploid megakaryocytes with a modal ploidy of 16N. This factor is distinct from TPO because it induces endomitosis in IL-3-generated megakaryocytes in vitro, whereas TPO does not, and its activity on megakaryocyte ploidy is not altered by optimal concentrations of TPO. In addition, no message for TPO is detectable in IL-2-stimulated NK cells by reverse transcription-polymerase chain reaction. These findings indicate that IL-2-stimulated NK cells produce a novel peptide, distinct from TPO, IL-6, IL-11, LIF, other gp130-associated interleukins, and SDF1a, that can induce in vitro endomitosis in immature human megakaryocytes in the presence of IL-3 and SCF.
 ...
PMID:Secretion of a unique peptide from interleukin-2-stimulated natural killer cells that induces endomitosis in immature human megakaryocytes. 1175 62

A diagnosis of acute myeloid leukemia was made in a 10-month-old Holstein female calf. The leukemia was macroscopically characterized by great enlargement of the spleen and moderate enlargement of some lymph nodes. Histochemical and immunohistochemical examination disclosed the presence of neoplastic cells either containing metachromatic and tryptase-positive granules or expressing factor VIII-related antigen. The granules, which were positive for naphthol AS-D chloroacetate esterase and did not have particulate contents, were distinct from those of basophilic leukemia cells. This leukemia was thought to be derived from a common myeloid progenitor capable of giving rise to megakaryocyte-erythrocyte progenitors and granulocyte-monocyte progenitors with the ability to differentiate into mast cells.
 ...
PMID:Acute myeloid leukemia with mastocytic and megakaryocytic differentiation in a calf. 2106 15