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Biomedical subjects

M D Kramer

Publications and source records attributed to M D Kramer.

At least 145 records · Page 8Linked to original sources
PubMed1988

Human T cell specific proteinase (HuTSP) is encoded by the T cell and natural killer cell specific human Hanukah factor (HuHF) gene.

HuTSP, a serine proteinase which is specifically associated with activated t lymphocytes, was purified to homogeneity and characterized. N-terminal amino acid sequencing revealed identity with a sequence predicted from the human Hanukah factor (HF) gene, which was isolated from a human T cell cDNA library. The dimeric structure of HuTSP, together with its extensive sequence homologies with the murine T cell specific proteinase, MTSP-1, suggests phylogenetic conservation of this serine proteinase family.

Amino Acid Sequence↗
PubMed1988

Determination of frequency of T cells expressing the T cell-specific serine proteinase 1 (TSP-1) reveals two types of L3T4+ T lymphocytes.

TSP-1 is a murine T cell-specific serine proteinase which is exclusively expressed in activated but not in resting T lymphocytes. Among T lymphocyte clones tested so far the enzyme was found to be associated with all Ly-2+ but only with a fraction of L3T4+ lines. Here we have applied a limiting dilution system to determine the frequency of precursor cells of resting L3T4+ and Ly-2+ lymphocytes which can be induced in vitro by antigen/lectin to express TSP-1. T cell subsets were either positively enriched by flow cytofluorometry cell sorting or by negative selection using monoclonal antibodies and complement. Following stimulation of lymphocytes in vitro, individual microwells were tested for growth by visual examination and for the TSP-1 protein/enzyme by analyzing cell lysates using either a specific rabbit anti-TSP-1 antiserum and/or the chromogenic model peptide substrate H-D-Pro-Phe-Arg-p-nitroanilide. Moreover, a large panel of L3T4+ and Ly-2+ T lymphocyte clones generated from primary cultures were similarly investigated. In some cell cultures the presence of TSP-1 was also tested on the mRNA level using a TSP-1-specific oligonucleotide probe. The data show that the majority, if not all, of antigen/lectin-induced-Ly-2+ T cells expressed TSP-1. In contrast, only 12%-27% of the growing lectin or antigen-reactive L3T4+ lymphocytes were positive for the enzyme. Studies performed in parallel with L3T4+ and Ly-2+ lymphocyte populations sensitized in bulk culture showed that under these conditions similar levels of TSP-1-specific mRNA and enzyme activity are detected in both subsets. The finding of primary L3T4+ T lymphocyte clones with distinct patterns of TSP-1 production provides evidence for the existence of two types of L3T4+ effector cells with different functional capacities. The data also suggest a cooperation between distinct L3T4+ lymphocytes for induction of optimal TSP-1 activity in L3T4+ T cells.

Animals↗
PubMed1988

Induction of T cell serine proteinase 1 (TSP-1)-specific mRNA in mouse T lymphocytes.

An oligonucleotide probe corresponding to nucleotides of a cDNA encoding the T cell-associated proteinase 1 (TSP-1) was chosen to study the induction and expression of TSP-1-specific transcripts in mouse T lymphocytes and tissues. We demonstrate that TSP-1 mRNA is only expressed in activated T lymphocytes and is absent from all mouse tissues tested including those containing resting mature T lymphocytes. Expression of the TSP-1 gene was observed in T lymphocytes in vitro in response to either phorbolester (phorbol 12-myristate 13-acetate), Ca2+ ionophore (A23187), lectin or alloantigen. In general, TSP-1 mRNA appeared and peaked later compared to interleukin 2 transcripts. Furthermore, TSP-1 mRNA was inducible in vitro in both Ly-2+ and L3T4+ lymphocyte populations treated with alloantigen and/or lectin. The transcription of the TSP-1 gene was always accompanied by the expression of proteinase activity. High expression of TSP-1 transcripts was also observed in in vivo derived T effector cells specific for lymphocytic choriomeningitis virus. However, TSP-1 mRNA was predominantly associated with virus-specific Ly-2+ T cells and correlated with their proteinase and cytolytic activities. The data suggest that TSP-1 gene transcription is a useful marker to characterize T effector cells in vitro and in vivo.

Amino Acid Sequence↗
PubMed1988

The antiproteolytic compound alpha 2-macroglobulin in human skin.

Monoclonal antibodies recognizing the antiproteolytic compound alpha 2-macroglobulin (MG) were used for immunohistological studies on normal human skin. MG-specific immunoreactivity was found to be localized to the papillary dermis and to be concentrated in the region of the epidermodermal junction. In view of these findings and the possible involvement of proteolytic enzymes and their inhibitors in blister formation, we asked whether MG occurs in the fluid of experimentally induced blisters. MG was identified (by western-blotting) and quantified (by a monoclonal antibody based enzyme immunoassay) in the fluid of experimentally induced suction blisters. Taken together, MG is present in such blister fluid in concentrations 6 times lower than in serum, but still in an antiproteolytic range. These findings allow suggestion of a possible role for the antiproteolytic compound MG in blister formation.

Antibodies, Monoclonal↗
PubMed1988

Release of biologically active fragments from human plasma-fibronectin by murine T cell-specific proteinase 1 (TSP-1).

We have studied the proteolytic activity of TSP-1, a tissue-specific serine proteinase expressed by activated murine T cells, on human plasma fibronectin and have investigated by affinity chromatography the biological activity of the polypeptides released after limited proteolysis. A Mr approximately 2.9 x 10(4) peptide bound to denatured collagen (gelatine) but not to heparin, a Mr approximately 3.0 x 10(4) fragment contained heparin-binding but not collagen-binding sites and a third Mr approximately 3.5 x 10(4) peptide did not express either of the two activities. All larger fragments - an array of five to six polypeptides with relative molecular masses between 15 x 10(4) and 19 x 10(4) - were bound to heparin-Sepharose but not to gelatine-Sepharose and could be eluted with high salt concentration. These data confirm recent results suggesting multiple, proteinase-resistant domains with discrete biological functions within fibronectins. The release of small, biologically active fibronectin fragments by TSP-1, an enzyme specifically released by activated T cells upon contact with antigen, suggests a role for this enzyme in the numerous cellular functions elicited by T lymphocytes in vivo.

Animals↗
PubMed1988

Coronary artery bypass in septuagenarians. Analysis of mortality and morbidity.

Elective coronary artery bypass surgery can be performed with an expected operative mortality of 1-3%. However, the effects of age on morbidity and mortality in patients undergoing this procedure remain controversial. To analyze morbidity and mortality in septuagenarians undergoing isolated coronary artery bypass surgery, we compared the results in 685 septuagenarians with those in 3,142 patients under the age of 70 years, all of whom underwent this procedure from January 1981 to December 1986. A larger percentage of elderly patients had triple-vessel disease (89% vs. 71%), left main coronary artery obstruction (34% vs. 16%), and ejection fractions less than 45% (68% vs. 41%). A larger percentage of septuagenarians had perioperative myocardial infarction (8% vs. 2%), required prolonged ventilatory support (10% vs. 3%), and had major neurological complications (4% vs. 1%). Mortality rates were significantly higher in elderly patients (7% vs. 2%) but did not correlate with the severity of coronary artery disease, the anginal pattern, or the diminishment of ventricular function. Major causes of mortality were pulmonary failure, renal failure, or both, sepsis, and neurological complications. These data suggest that elderly patients have an increased risk of cardiac and noncardiac morbidity and mortality after coronary artery bypass surgery. Higher mortality rates in this age group appear attributable to noncardiac organ failure. Late follow-up studies failed to show any significant difference among patients based on age alone.

Aged↗
PubMed1987

A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus-associated reverse transcriptase.

TSP-1, a murine T cell specific proteinase, is expressed in cytolytic T lymphocytes and secreted upon their interaction with antigen bearing target cells. In searching for possible extracellular substrates of the enzyme in the physiological environment of cytolytic effector cells, we have investigated the proteolytic activity of TSP-1 on retroviral proteins. It is shown that reverse transcriptase derived from the retrovirus Moloney murine leukemia virus is inactivated by TSP-1 via limited proteolysis. The data suggest the possibility that cytolytic T lymphocytes are able to interfere with retroviral replication by secreting a serine proteinase which degrades viral proteins.

Animals↗
PubMed1987

The T cell-specific serine proteinase TSP-1 is associated with cytoplasmic granules of cytolytic T lymphocytes.

This study describes the localization of the previously purified T cell-specific serine proteinase, termed TSP-1 (M. M. Simon et al., EMBO J. 1986. 5: 3267), within cytoplasmic granules of cytolytic T cell lines (CTLL). Subcellular fractionation of disintegrated CTLL (ruptured by nitrogen cavitation) was accomplished by Percoll density gradient centrifugation of cell lysates (postnuclear supernatant). Individual fractions were tested for proteinase activity on chromogenic peptide substrates and for the presence of TSP-1 by Western blot analysis. In addition, each fraction was assayed for cytolytic activity against sheep red blood cells (SRBC), for protein and for additional marker enzymes to assess the enrichment for cellular organells. All serine enzyme-type molecules including TSP-1 expressed by CTLL were identified by labeling cell lysates or gradient fractions with the serine proteinase-specific affinity ligand tritiated diisopropyl fluorophosphate [( 3H]DFP) in the presence or in the absence of class-specific or enzyme-specific proteinase inhibitors and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data demonstrate that the Percoll gradient fraction, which was shown by morphological examination in the electron microscope to be highly enriched for cytoplasmic granules, also contained greater than 80% of proteinase activity in addition to the granule-associated structures cytolysin and arylsulfatase. The identity of the granule-associated proteinase in two independent cell lines, CTLL HY3-Ag3 and CTLL 1.D.9, with the serine proteinase TSP-1 is indicated by its specificity for the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide, its sensitivity to class-specific as well as TSP-1-specific enzyme inhibitors and by its reactivity with a polyvalent TSP-1-specific rabbit antiserum. Both CTLL contain a [3H]DFP-labeled protein that migrates with a molecular mass of 60 kDa under nonreducing conditions and with 30 kDa under reducing conditions and which can be inactivated by the TSP-1-specific inhibitor H-D-Pro-Phe-Arg-chloromethylketone. CTLL HY3-Ag3 (a long-term culture CTLL with natural killer-like activity) but not CTLL 1.D.9 (an antigen-specific short-term cultured CTLL) express in addition a further [3H]DFP-binding protein which migrates with 27 kDa under nonreducing or reducing conditions. No substrate specificity was found for this molecule. The possible function of the granule-associated serine proteinase TSP-1 is discussed.

Amides↗
PubMed1987

A novel serine proteinase (HuTSP) isolated from a cloned human CD8+ cytolytic T cell line is expressed and secreted by activated CD4+ and CD8+ lymphocytes.

A novel proteinase, termed human T cell-associated serine proteinase (HuTSP), has been highly purified from a human CD8+ T lymphocyte clone. By using a panel of chromogenic model peptide substrates the enzyme was found to specifically recognize L-arginine and to cleave Tos-Gly-Pro-Arg-nitroanilide with high efficiency at a pH optimum of 10.5-11. Exposure to class-specific proteinase inhibitors revealed that HuTSP is a serine endopeptidase. The enzyme has a mol. mass of approximately 50 kDa (non-reduced) and of approximately 25-30 kDa (reduced) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting HuTSP to be a disulfide-linked dimer. The enzyme is shown to be inducible by lectin in both CD4+ and CD8+ lymphocytes. Moreover, HuTSP was detected in a number of independent CD4+ and CD8+ T cell clones and was found to be released from effector cells upon ligand binding to the CD3-T cell receptor complex.

Clone Cells↗
PubMed1987

A monoclonal antibody recognizing plasminogen and plasmin--altered reactivity in psoriatic lesions.

To study the organization of the plasminogen activator/plasminogen-plasmin (PA/PG-P) system in human epidermis we raised monoclonal antibodies with specificity for human plasminogen and plasmin (PG-P). Monoclonal antibody P2, which appeared most suitable for immunohistology, is a mouse monoclonal antibody of IgG1 subtype, specific for the precursor enzyme plasminogen and for the high molecular weight chain of the active enzyme, plasmin. Immunofluorescence analysis of normal human epidermis revealed that reactivity with P2 was confined to the basal cell layer. In psoriatic lesions, however, this regional organization was not found. Immunoreactivity in this case was scattered throughout all the hyperproliferating cell layers, which is taken as evidence for an altered distribution of PG-P in psoriatic lesions. In psoriasis other components of the PA/PG-P system have previously been found to be altered. In this context our findings add to the hypothesis that this system may be involved in the pathology or the pathogenesis of psoriatic lesions.

Antibodies, Monoclonal↗
PubMed1987

Cloned cytolytic T-effector cells and their malignant variants produce an extracellular matrix degrading trypsin-like serine proteinase.

This report describes the distribution of a trypsin-like proteinase in defined homogeneous cytolytic T-cell lines (CTLL) and their in vitro and in vivo derived malignant T-lymphoma variants. By means of chromogenic peptide substrates, we found the enzyme to attack preferentially at the carboxy terminus of arginine, in particular when non-polar amino acids were present in the amino terminal neighbouring position. The enzyme was identified by means of various inhibitors as a serine type proteinase having a pH optimum around 8 X 5. Affinity chromatography in connection with molecular sieving resulted in a 200-fold purification and indicated a molecular weight (MW) of about 50,000 for the proteinase. The enzyme was found to be highly expressed in antigen-specific CTLL as well as in their tumorigenic variants. Both intact lymphocytes of all CTLL tested and Triton X-100 lysates or enriched proteinase preparations thereof were able to degrade a high molecular weight protein (casein) and to release high molecular weight split products from the sulphated proteoglycans in subendothelial extracellular matrix. The results are discussed with respect to the invasiveness of normal and malignant T lymphocytes and the proteinase is suggested to be crucially involved in the process of cellular migration in vivo.

Animals↗
PubMed1986

Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes.

We describe the purification of a T cell specific serine proteinase derived from a cloned murine cytolytic T lymphocyte line. Analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mol. wt of approximately 60 kd under non-reducing conditions and of approximately 30 kd under reducing conditions. The proteinase cleaves the model peptide substrate H-D-Pro-Phe-Arg-NA, at the 4 nitroanilide (NA) group with high efficiency. Much lower or no activity of the enzyme is found against synthetic peptide substrates carrying other amino acid (AA) sequences at position P2, P3 adjacent to L-arginine or against substrates in which AA other than L-arginine are bound to the NA group. Optimal pH for the cleavage of H-D-Pro-Phe-Arg-NA is in the range of 8.0-8.5. The enzyme is strongly inhibited by inhibitors of serine proteinases, diisopropylfluorophosphate, phenylmethane-sulfonyl fluoride, m-aminobenzamidine, aprotinin, and leupeptin but not by inhibitors of either thiol-, metallo- or carboxyl-proteinases. We propose the designation TSP-1 (T-cell derived serine proteinase 1) for this enzyme. TSP-1 has the capacity to stimulate B lymphocytes for proliferation in the absence of antigen.

Animals↗
PubMed1986

Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T cell line and its expression by functionally distinct T cells.

We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed.

Animals↗
PubMed1986

A specific serine proteinase is inducible in Lyt-2+,L3T4- and Lyt-2-,L3T4+ T cells in vitro but is mainly associated with Lyt-2+,L3T4- effector cells in vivo.

Recently, we and others reported on the expression of a serine proteinase in long-term cultured murine T lymphocyte cell lines. In an attempt to explore the distribution and possible regulation of this enzyme in T lymphocyte subsets, we performed the presented detailed study. We found that the proteinase is not expressed by thymocytes and resting T cells but can be induced by lectin or antigen in combination with lymphokine sources in vitro in macrophage-depleted unselected T cells as well as in both T cell subsets (Lyt-2+,L3T4- and Lyt-2-,L3T4+) separated by flow cytofluorometry. Furthermore, it appears that cell-associated proteinase activity is increasing with prolonged culture period of sensitized T lymphocytes and that it is higher in antigen-activated as compared to lectin-activated T cells. When tested for substrate specificity the T cell-associated proteinase was shown to preferentially cleave model peptide substrates carrying L-arginine at position P1 in combination with nonpolar amino acids at position P2 and P3. As concluded from its sensitivity to proteinase inhibitors the enzyme can be classified as a serine proteinase and by molecular sieving at high ionic strength it was shown to have a mol. mass of approximately 50-60 kDa. Analysis of in vivo activated T cells revealed that this particular proteinase was expressed in flow cytofluorometry sorted lymphocytic choriomeningitis virus-specific Lyt-2+,L3T4- cytolytic T lymphocytes but not in Lyt-2-,L3T4+ T cells presensitized with either Listeria monocytogenes or I-A alloantigens. The data demonstrate that the two T cell subsets (Lyt-2+,L3T4-; Lyt-2-,L3T4+) have distinct in vitro induction requirements for the expression of proteinase and that after activation of T cells in vivo the enzyme is preferentially associated with Lyt-2+,L3T4- effector cells.

Animals↗
PubMed1986

Giant leiomyoma of esophagus.

This case report of a 37-year-old man with giant leiomyoma details some of the problems encountered in diagnosing and treating this rare form of the disease. Initial studies at another institution were interpreted as demonstrating achalasia, and thoracotomy was later undertaken because of a mistaken diagnosis of a pericardiac mass, which led to a biopsy and a resulting esophagopleural fistula. Subsequent esophagectomy and drainage of empyema space were employed to remove the tumor, which had ulcerated and bled, as well as to drain the empyema cavity. The tumor measured 20.5 cm long and weighed 540 gm. Gastrointestinal continuity was reestablished by colon interposition, and the patient has been well for the succeeding 6 years.

Adult↗
PubMed1986

A fast-acting cytotoxin derived from Con A-activated porcine leukocytes. I. Biochemical characteristics and target cell specificity.

We here describe a new cytotoxin which was detected in serum-free culture supernatants of mitogen stimulated porcine leukocytes. The factor was precipitated at 35-45% (NH4)2 SO4 concentration, sensitive to heat, low pH, and trypsin, thus indicating its protein nature. Column chromatography on hydrophilic or hydrophobic matrices revealed amphiphilic properties. The smallest unit which was sufficient to induce complete target cell lysis had a molecular weight of 33K. This porcine cytotoxin (PCT) could be distinguished functionally from lymphotoxin (LT) and tumor necrosis factor (TNF). It is a fast acting mediator which exerts strong cytostatic and cytotoxic anti-tumor activity using a panel of T lymphoma target cells.

Animals↗