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)

We describe a new cellular component of normal mouse thymuses, which is isolated by fractionated trypsin dissociation of minced thymus tissue followed by repeated unit gravity sedimentation. These cells are of unusually large size, with diameters of 30 mum and more. They represent cellular complexes of single large cells filled with high numbers of lymphoid cells. The majority of the engulfed lymphoid cells is not only fully intact, as judged by morphological criteria, but, moreover, includes a high proportion of mitotic figures. Electron microscopic investigations reveal the epithelial character of the large thymic nurse cells (TNC). The peripherally situated cytoplasmic tonofilament streams, and characteristic vacuoles filled with coarse, unidentified material, closely resemble cytoplasmic organelles found in the cortical reticuloepithelial cells described in situ. The internalized lymphocytes are located within caveolae lined by plasma membranes. These TNC caveolae are completely sequestered, and have lost any communication with the extracellular space, as demonstrated by the inability of an electrondense marker, cationized ferritin, to diffuse into the perilymphocytic clefts. The structural interactions between the membranes of the engulfed thymocytes with the surrounding TNC caveolar membranes were investigated both in ultrathin sections and in freeze-etch preparates. Two distinct contact types between both membranes were discerned: (a) complete, close contact along the entire lymphocyte circumference, and (b) more frequently, contact restricted to discrete, localized areas. Judging from their size and distribution, the localized contacts could correspond particle aggregates of freeze-etch preparates, which morphologically resemble certain stages of gap junction. Furthermore, we regularly found square arrays of particles of uniform size, which so far have been thought to be typical for cell membranes actively engaged in ion exchange. Tight junction-like particle arrays, which were present on TNC outer membranes, and probably represented disrupted contacts between adjacent TNC in the intact tissue, could not be found on caveolar or lymphocyte membranes. Finally, one of the most conspicuous specializations of the TNC caveolar membrane were membrane invaginations, which were arranged mainly in groups, and which probably reflect endo- or exocytotoxic events. We investigated the surface antigen phenotype of TNC by indirect immunofluorescence, with monoclonal antibodies against determinants of H-2- complex subregions as well as against lymphocyte differentiation markers. Semiquantification was reached with flow cytofluorimetry, followed by morphological control by fluorescence microscopy. The surface antigen formula of TNC is: Ig(-), Thy-l(-), H-2K(++), I-A (++), I-E/C(+), H-D(++), Ly-1(-), Ly-2(-), Qat-4(-), Qat-5(-), and peanut agglutinin (PNA)(-). Thymic macrophages, which were identified by double fluorescence, with rhodamine- coupled zymosan as a phagocytosis marker, were serologically identical with TNC. Free thymocytes, in contrast, had the following antigen formula: Ig(-), Thy-1(++), H-2K(+/-), I-A(-), I-E/C(-), H-2D(+/-), Ly-1(+/-), Ly-2(+), Qat- 4(-), Qat-5(-), and PNA(+). The unprecedented finding of high numbers of dividing thymocytes sojourning within thymic epithelial cells, and the particular specializations of the TNC caveolar membranes surrounding these engulfed thymocytes is the basis of a hypothesis that postulates that an intraepithelial differentiation cycle is one essential step in, intrathymic T lymphocyte generation.
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PMID:Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: morphological and serological characterization. 696 12

Proliferative activity of murine interleukin-2 (IL-2) dependent T cells (CTLL-2) was detected in the culture supernatant of canine peripheral blood lymphocytes (PBL) stimulated with phytohemagglutinin-P (PHA-P), and was defined as dog IL-2. The highest production of IL-2 was obtained under the conditions in which a PBL population of 2 x 10(6) cells/ml was stimulated with PHA-P at a concentration of 10 micrograms/ml at 38 degrees C for 48 hr. Dog IL-2 activity was significantly inhibited by heating at 65 degrees C, acidification under pH 4, alkalification over pH 10, and trypsin exposure. A peak of dog IL-2 activity was detected in the fraction with a molecular weight of approximately 31,000 by gel filtration. Long-term culture of canine lymphocytes was successful over 10 passages by using dog IL-2 with PHA-P-stimulation every 3 passages. The cultured cells mostly consisted of small- and medium-sized lymphocytes. These cells reacted to anti-dog thymocyte rabbit serum and anti-dog Thy-1 mouse monoclonal antibody. These cells were therefore considered to originate in T-lineage lymphocytes. Cytostasis of PBL from intact dogs reacting to canine transmissible venereal sarcoma cells was increased significantly when PBL was cultured for more than 30 days with homologous IL-2.
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PMID:Characterization of dog interleukin-2 activity. 811 17

The adhesion molecule L-selectin is proteolytically cleaved from the surface of lymphocytes and neutrophils within minutes after stimulation by phorbol ester or calcium ionophores. In contrast to neutrophils, soluble factors have not been shown to induce down-regulation of L-selectin on lymphocytes. We therefore examined whether signals generated by interaction with cell surface receptors could deliver physiological stimuli inducing this regulatory mechanism. While cross-linking of several adhesion molecules (CD2, CD44, alpha 4-integrin, LFA-1) by antibody did not result in a significant reduction of the expression of L-selectin, antibodies against CD45 and Thy-1.2, both involved in the regulation of lymphocyte activation, induced loss of cell surface L-selectin within minutes, even at 4 degrees C, by shedding into the supernatant. Cross-linking of these molecules was shown to be essential, but Fc interactions or adherent cells were not required. A similar response, albeit less effective, was found after cross-linking of CD3. Interestingly, initiation of shedding only occurred in the presence of cell-cell contact, pointing to a second, as yet unknown, signal required. Loss of L-selectin induced by CD45 cross-linking is followed by a rapid re-expression of the molecule upon incubation at 37 degrees C. This reaction is also dependent on specific triggering signals as rapid re-expression was not observed after removal of L-selectin by trypsin. The data indicate that the protein phosphatase CD45 as well as the TCR complex itself in combination with a further, as yet unknown, cell-cell contact-dependent stimulus have a regulatory role in the dynamic control of L-selectin expression in lymphocytes.
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PMID:CD45-mediated signals can trigger shedding of lymphocyte L-selectin. 913 16

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.
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PMID:Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells. 1117 29

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.
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PMID:Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro. 1546 80

Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs) from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total) tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC) including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.
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PMID:Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues. 2423 63


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