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Biomedical subjects

R B Pepinsky

Publications and source records attributed to R B Pepinsky.

At least 73 records · Page 4Linked to original sources
PubMed1990

An immunoassay to detect human müllerian inhibiting substance in males and females during normal development.

An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.

Adolescent↗
PubMed1990

Studies on the structural properties of lipocortin-1 and the regulation of its synthesis by steroids.

Lipocortin-1 protein synthesis in resting monocytes is under the control of glucocorticoid steroids. This induction occurs at reasonable dexamethasone concentrations, may require concomitant synthesis of transcriptional factors, appears to be cell type specific, and has been observed only in primary tissues in our hands. Variability in the magnitude of the induction suggests that the regulation is complex, involving either additional factors or particular differentiation states. In addition to the induction of intracellular lipocortin-1, steroids cause the appearance of labelled lipocortin-1 on the outer surface of the cells. Whether cell breakage can account for this effect is unclear. Considerable microheterogeneity was found in preparations of recombinant-lipocortin-1. Aspects of N-terminal post-translational processing, N-terminal proteolysis, conformational states and the existence of an air-denatured form lacking alpha-helical structure contributed to this heterogeneity. We believe that these aspects are responsible for the variable biological potency of different preparations. It remains unclear whether this protein actually plays a physiological role in the regulation of the inflammatory response or achieves its effects through membrane binding and subsequent non-physiological perturbation of the cells.

Animals↗
PubMed1989

A dimeric form of lipocortin-1 in human placenta.

We have characterized a 68 kDa lipocortin from human placenta that was identified as a covalently linked homodimer of lipocortin-1 by peptide mapping and sequence analysis. The site of cross-linking was localized within the 3 kDa N-terminal tail region, an exposed domain that contains the phosphorylation sites for protein tyrosine kinase and protein kinase C and is sensitive to proteolysis. Sequence analysis of the corresponding peptide revealed that glutamine-18 was modified, suggesting that the cross-link may be generated by a transglutaminase. By incubating lipocortin-1 with placental membranes and with labelled glycine ethyl ester we observed a Ca2+-dependent labelling of lipocortin-1 within the tail region, supporting this notion. Like lipocortin-1, the dimer inhibits phospholipase Ad2 activity, is a substrate for the epidermal-growth-factor (EGF) receptor/kinase, and display Ca2+-dependent binding to phosphatidylserine-containing vesicles. In preparations from human placenta the dimer is particularly abundant, accounting for approx. 20% of the lipocortin-1.

Amino Acid Sequence↗
PubMed1989

Structure and properties of a human non-pancreatic phospholipase A2.

We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.

Amino Acid Sequence↗
PubMed1989

Lipocortin-1 immunoreactivity in central and peripheral nervous system glial tumors.

We examined the cellular distribution of lipocortin-1 (L-1), a major physiologic substrate for the epidermal growth factor receptor/kinase, in 122 central nervous system (CNS) and peripheral nervous system (PNS) neoplasms using the peroxidase-antiperoxidase technique with a polyclonal antibody specific for L-1. Extensive L-1 immunoreactivity was demonstrated in many CNS tumors; in 11 of 21 glioblastoma multiformes, in five of 12 anaplastic astrocytomas, and in five of 14 astrocytomas. Significant numbers of immunoreactive ependymocytes or astrocytes were also seen in six of 13 ependymomas. In contrast, no immunostaining was detected in the oligodendrocytes in any of ten oligodendrogliomas. PNS tumors, found in two of five malignant nerve sheath tumors, 13 of 15 schwannomas, 13 of 17 neurofibromas, and 14 of 15 traumatic neuromas, also contained considerable L-1 immunoreactivity in Schwann cells or mast cells. These findings raise the possibility that L-1 may participate in the proliferation or subsequent differentiation of neoplastic astrocytes, ependymocytes, and Schwann cells.

Annexins↗
PubMed1989

Human recombinant lipocortin 1 has acute local anti-inflammatory properties in the rat paw edema test.

Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1989

Lipocortin-1 immunoreactivity in the normal human central nervous system and lesions with astrocytosis.

The authors have examined the cellular distribution of lipocortin-1 (L-1) in the normal and diseased central nervous system (CNS) using the peroxidase-antiperoxidase (PAP) technique with a polyclonal antibody specific for L-1. L-1 immunoreactivity was evaluated in the frontal cortex, parahippocampal gyrus/lateral ventricle, cerebellum, medulla, and spinal cord from 27 normal human fetuses, neonates, and adults without neurologic disease and in these same regions and representative lesions from 35 patients with diseases producing varying degrees of astrocytosis, including intraparenchymal hemorrhage; embolic, thrombotic, or traumatic infarctions; and Alzheimer's disease (AD). L-1 immunoreactivity was identified in ependymocytes, choroid plexus epithelia, and scattered subependymal astrocytes throughout the ventricular system from 15 weeks gestation through 82 years of age in both normal and diseased CNSs. L-1 immunoreactivity was also detected in reactive astrocytes and many macrophages surrounding each infarction regardless of site or pathogenesis and in scattered reactive astrocytes in people with AD or SDAT. The limited distribution of L-1 in CNS is consistent with the low amounts of L-1 found in brain and suggests that L-1 may participate in the normal function of ependymocytes and the pathophysiology of reactive astrocytosis.

Adult↗
PubMed1989

Autoantibodies to recombinant lipocortin-1 in rheumatoid arthritis and systemic lupus erythematosus.

Corticosteroids may mediate some of their anti-inflammatory effects via induction of a specific 38 kD protein, lipocortin-1. Autoantibodies to lipocortin-1 were measured by enzyme linked immunosorbent assay (ELISA) in 90 healthy subjects and in 63 patients with rheumatoid arthritis (RA), 36 with systemic lupus erythematosus (SLE), 26 with polymyalgia rheumatica, and 13 with chronic airways disease. Sixteen patients with RA receiving prolonged, high steroid doses (prednisolone greater than 7.5 mg/day) had raised IgM antilipocortin-1 levels, while 19 patients with RA untreated with steroids had normal levels. This association was independent of disease activity. In SLE, raised antilipocortin-1 levels were associated with active disease and were independent of steroid treatment. Antilipocortin-1 antibody levels were not raised in patients with polymyalgia rheumatica and chronic airways disease. Thus steroid treatment alone appears insufficient to induce antilipocortin-1 antibodies, unless an underlying autoimmune state is also present. In RA, antilipocortin-1 antibodies may impair anti-inflammatory actions of steroids and render some patients 'steroid resistant'.

Aged↗
PubMed1989

Ontogeny of epidermal growth factor receptor/kinase and of lipocortin-1 in the ovine lung.

We have examined the ontogeny and distribution of the epidermal growth factor receptor/kinase (EGF receptor) and of lipocortin-1, a major cellular substrate of the EGF receptor, in a developmental series of 13 normal ovine fetal lungs (44-145 d of gestation) using the peroxidase anti-peroxidase technique with two extensively characterized polyclonal antibodies recognizing the EFG receptor and one polyclonal antibody recognizing lipocortin-1. Immunoreactive EGF receptor/kinase and lipocortin-1 were detected in conducting airway epithelium by the end of the first trimester of pregnancy before bronchial glands could be identified. This was followed at two-thirds of gestation by immunoreactivity in bronchial glands and large bronchioles adjacent to positive bronchi. By seven-eighths of gestation conducting airway epithelium no longer contained consistently detectable immunostaining for EGF receptor, although lipocortin-1 was identified until term in all levels of conducting airways. In contrast, neither EGF receptor nor lipocortin-1 immunoreactivity was detected in alveolar type I or type II epithelial cells, fibrocytes, chondrocytes, smooth muscle, or endothelial cells at any gestational age. These findings suggest that EGF receptor and lipocortin-1 may participate in early airway development.

Animals↗
PubMed1988

Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment.

Mullerian inhibiting substance (MIS) is a differentiation factor that causes the Mullerian duct to regress during the development of the male reproductive tract. The active form is a disulfide-linked dimer consisting of two identical 70-kDa subunits. Recently, the amino acid sequence for MIS was deduced from its gene sequence and revealed that the carboxyl-terminal region shares homology with transforming growth factor (TGF)-beta. Since TGF-beta is produced as a large latent precursor that requires proteolytic activation for activity, we sought to determine if MIS might undergo a similar processing event. Here we demonstrate that typically 5 to 20% of the protein in MIS preparations is cleaved at a site 109 amino acids from the carboxyl terminus. Concurrent cleavages from both chains of the MIS dimer produces a 25-kDa TGF-beta-like fragment and a high molecular mass complex derived from the amino terminus of the protein. Although the two fragments are noncovalently linked, they remain tightly associated after cleavage, and thus are structurally organized like TGF-beta within its precursor. The same cleavage products also can be generated by limited proteolysis with plasmin, which provides a simple method for converting the entire preparation into the cleaved form. The plasmin-digested MIS is fully active in the organ culture assay.

Amino Acids↗
PubMed1988

Insulin and epidermal growth factor stimulate phosphorylation of a 170-kDa protein in intact hepatocytes immunologically related to lipocortin 1.

Lipocortin 1 is a steroid-induced, calcium-regulated membrane binding protein (Mr = 39,000) which is a substrate for the epidermal growth factor receptor kinase in intact cells. Using a polyclonal antibody to human recombinant lipocortin 1, we have identified a 170-kDa phosphoprotein in freshly isolated rat hepatocytes which shares antigenic determinants with lipocortin 1. The protein was recognized by four different anti-lipocortin 1 antisera, and antibody binding was inhibited by a 100-fold molar excess of human recombinant lipocortin 1 over antibody. Based on Coomassie Blue staining, the 170-kDa lipocortin-related protein is abundant (approximately 100 ng/10(6) cells) in rat liver, while lipocortin 1 itself is found in very low amounts. Epidermal growth factor and insulin stimulated phosphorylation of this 170-kDa protein in intact rat hepatocytes. The increase in phosphorylation was more pronounced in hepatocytes from dexamethasone-treated animals. The phosphorylation occurred exclusively on serine residues and was maximal 30-60 min after hormone addition. The 170-kDa protein was localized in the cytoplasm in the absence of calcium, while increasing calcium concentration led to partial association with the membrane compartment in rat liver. This 170-kDa protein represents a new member of the class of proteins whose serine phosphorylation is regulated by insulin and EGF and may belong to the family of lipocortin-related molecules.

Amino Acids↗
PubMed1988

Lipocortins 1 and 2 as substrates for the insulin receptor kinase in rat liver.

Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.

Adenosine Triphosphate↗
PubMed1988

Five distinct calcium and phospholipid binding proteins share homology with lipocortin I.

We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.

Amino Acid Sequence↗
PubMed1988

Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C.

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.

Amino Acid Sequence↗
PubMed1988

Stimulation of pancreatic islet beta-cell replication by oncogenes.

Although the growth potential of the pancreatic islet beta cells is limited, glucose, cAMP, and certain polypeptide growth factors have been reported by other workers to exert modest stimulatory effects on beta-cell replication. To further assess means through which beta-cell growth can be stimulated, selected oncogene constructs linked to a rat insulin promoter were introduced by means of electroporation into free islet cells prepared from fetal rats and adult hyperglycemic obese (ob/ob) mice. The uptake and expression of the added oncogenes were sufficiently efficient to exert effects on beta-cell physiology in short-term experiments (less than or equal to 4 days). Stimulation of islet cell [3H]thymidine incorporation was observed after transfection with src alone or the combination of myc and ras. The effect observed in the fetal islet cells with src was more pronounced than any effect previously reported. Transfection with the src oncogene resulted in phosphorylation of lipocortin I and was paralleled by an increased immunofluorescence against src-like immunoreactivity in a majority of the electroporated cells. It is concluded that electroporation can induce sufficiently efficient expression of added oncogene constructs to study their effects on cells that are not readily transformable into continuously growing cell lines. Furthermore, the results suggest that beta-cell replication might be manipulated extrinsically by inserting appropriate growth-promoting genes into these cells.

Animals↗
PubMed1987

Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2.

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.

Amino Acid Sequence↗
PubMed1986

Purification and partial sequence analysis of a 37-kDa protein that inhibits phospholipase A2 activity from rat peritoneal exudates.

We have purified from rat peritoneal exudates a 37-kDa protein that inhibits phospholipase A2 activity. It is the predominant phospholipase inhibitor protein in these preparations and also is detected in a wide variety of cell lines. Levels of expression range from 0 to 0.5% of total protein. In the peritoneal preparations, the inhibitor is partially proteolyzed into a series of lower mass forms, including species at 30, 24, and 15 kDa. These fragments all are immunoreactive with an antibody raised against the 37-kDa protein. The rat protein also is immunoreactive with an antibody developed against a 6-kDa phospholipase inhibitor protein from snake venom. The primary structure of more than half of the rat inhibitor has been deduced by protein microsequence analysis. These sequences are closely related to sequences from its human analogue, which we recently cloned and expressed (Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Properties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins collectively referred to as lipocortin.

Amino Acid Sequence↗