Biomedical subjects

E Reich

Publications and source records attributed to E Reich.

At least 109 records · Page 6Linked to original sources

PubMed1985

Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase-type plasminogen activator.

Controlled extracellular proteolysis is catalyzed in part by the secretion of plasminogen activators. As a step in the study of the expression of these enzymes in mouse tissues, we have isolated five cDNAs encoding the mouse urokinase-type plasminogen activator from a cDNA library prepared with size-selected mRNA from MSV-transformed 3T3 cells. The longest cDNA insert contains the entire coding region of mouse urokinase, 58 base pairs of the 5' non-coding region, and the entire 3' non-coding region, which is 942 base pairs long. The deduced protein sequence, which starts with a signal peptide of 20 amino acids, shows extensive homology to that of human and porcine urokinase. However, in contrast to these enzymes, mouse urokinase contains no N-glycosylation site. Bacteria harbouring one of the recombinant plasmids synthesize and secrete into their periplasm a protease indistinguishable from mouse urokinase.

Amino Acid Sequence↗

PubMed1985

Isolation and characterization of a Mr = 38,000 protein from differentiating smooth muscle cells.

In culture, vascular smooth muscle cells grow and form a confluent monolayer of cells. Under appropriate conditions, regions of the monolayer can be induced to draw away from the substrate and form multicellular nodules. The ultrastructure of the cells in the nodules appears to be similar to that of differentiated smooth muscle cells. The process of nodulation is associated with the synthesis of a unique protein whose molecular weight is estimated from gradient gel electrophoresis to be 38,000 (38-kDa Protein). The protein is secreted into the culture medium and can be detected either by metabolic labeling or by staining with Coomassie Blue. Partial purification of 38-kDa Protein was achieved using affinity chromatography. The protein is adsorbed to heparin-agarose, but not to gelatin-agarose. The concentration of 38-kDa Protein in nodular conditioned medium is estimated at 1.9 micrograms/ml and less than 0.01 microgram/ml in conditioned medium made from monolayer cells. The presence of 5% fetal bovine serum in the labeling medium does not affect 38-kDa Protein synthesis. Cross-reactivity with fibronectin was evaluated using polyvalent antibodies to 38-kDa Protein. The 38-kDa Protein is not antigenically related to fibronectin. Furthermore, we establish that the protein is not qualitatively influenced by the presence of ascorbate (50 micrograms/ml), beta-aminoproprionitrile fumarate (50 micrograms/ml) heparin (10 ng/ml), or fibronectin (20 micrograms/ml) in the culture medium. We find that the added components neither suppress 38-kDa Protein synthesis in nodular cultures nor enhance 38-kDa Protein synthesis in monolayer cultures. The 38-kDa Protein is not detected in either monolayer or nodular cell layers and appears to be a secreted protein. Its appearance in nodular conditioned medium during nodulation suggests a relationship with that process.

Animals↗

PubMed1985

[Polyvinyl iodine in ophthalmic surgery].

The importance of minimizing the number of bacteria on the conjunctive before intraocular surgery has been undisputed for many years now. Bacteriological considerations led to antiseptic measures being preferred to antibiotics. Until recently there was no antiseptic agent available which was tolerated by both the cornea and the conjunctiva. The good results obtained in the treatment of bacterial and viral keratoconjunctivitis with PVP iodine led to this study of the preoperative preparation of 41 eyes prior to cataract extraction.

Cataract Extraction↗

PubMed1985

Gene expression and cAMP.

By comparing the 5'-flanking region of the porcine gene for the urokinase form of plasminogen activator with those of other cAMP-regulated genes, we identify a 29-nucleotide sequence that is tentatively proposed as the cAMP-regulatory unit. Homologous sequences are present (i) in the cAMP-regulated rat tyrosine aminotransferase, prolactin, and phosphoenolpyruvate carboxykinase genes and (ii) 5' to the transcription initiation sites of cAMP-regulated Escherichia coli genes. From this we conclude that the expression of cAMP-responsive genes in higher eukaryotes may be controlled, as in E. coli, by proteins that form complexes with cAMP and then show sequence-specific DNA-binding properties. The complex formed by cAMP and the regulatory subunit of the type II mammalian protein kinase might be one candidate for this function. Based on several homologies we suggest that this subunit may have retained both the DNA-binding specificity and transcription-regulating properties in addition to the nucleotide-binding domains of the bacterial cAMP-binding protein. If this were so, dissociation of protein kinase by cAMP would activate two processes: (i) protein phosphorylation by the catalytic subunit and (ii) transcription regulation by the regulatory subunit.

Animals↗

PubMed1985

Coordinate inhibition of plasminogen activator and tumor growth by hydrocortisone in mouse mammary carcinoma.

A body of evidence has suggested that hormones which modulate plasminogen activator production by cultured tumor explants in vitro may have a qualitatively comparable effect on the growth of the same tumors in vivo. As a test of this correlation and to explore its potential for predicting the in vivo response of tumors to hormones, we have studied here the effect of hydrocortisone on the growth of primary and first generation transplants of mouse mammary tumor virus-determined mammary tumors in BALB/c X DBA/8 F1 (hereafter called CD8F1) mice; hydrocortisone had been found previously to inhibit plasminogen activator production by explants of these tumors. The results were: (a) hydrocortisone reversibly blocked the growth of palpable primary tumors; growth resumed at control rates following withdrawal of exogenous hormone; (b) hydrocortisone inhibited the growth of first-generation tumor transplants when administered either before or after the appearance of palpable tumors; (c) pretreatment with hydrocortisone both delayed the appearance of primary tumors and greatly reduced tumor incidence in susceptible mice; a substantial part of the decrease in tumor incidence was apparently irreversible; (d) hydrocortisone reduction of tumor growth was accompanied by inhibition of tumor plasminogen activator content, and these effects displayed a similar dose dependence (enzyme content of tumor lysates was measured by the 125I-fibrin plate assay); the enzyme present in control and hormone-treated tumors was predominantly of the urokinase type. These findings suggest that plasminogen activator production and mammary tumor growth in CD8F1 mice are coordinately regulated and thus encourage the view that plasminogen activator might be useful as an in vitro marker for predicting the in vivo response of tumors to hormones.

Animals↗

PubMed1984

cDNA and gene nucleotide sequence of porcine plasminogen activator.

We have isolated cDNA and genomic clones coding for porcine plasminogen activator (urokinase, uPA). The cDNA is 2375 nucleotides long: it consists of a 5'-non-coding region (104 nucleotides), an open reading frame of 1329 nucleotides, and 3'-non-coding region of 942 nucleotides apart from the poly A tail. The genomic segment corresponding to the transcribed sequence is 5.85 kb long; it is composed of 11 exons and 10 introns. The 5'-flanking genomic region contains a number of sequences of potential regulatory significance, including possible hormone receptor binding sites and a sequence which we tentatively propose may be involved in activation of transcription by cAMP. The full sequence of both cDNA and genomic clones, the latter including 1.3 kb of flanking region, is presented and discussed, and the deduced amino acid sequence compared with that of human uPA.

Amino Acid Sequence↗

PubMed1984

Antibodies to interleukin 2. Effects on immune responses in vitro and in vivo.

Antibodies to highly purified mouse interleukin 2 (IL-2) were raised in rabbits; a 1:500 dilution of antiserum completely blocked the in vitro mitogenic effect of 10(-9) M IL-2. The antisera functioned effectively to immunoprecipitate biosynthetically labeled IL-2 and the purified immunoglobulins were useful in the construction of affinity columns for the adsorption and one-step immunopurification of IL-2. The antibodies were apparently specific for IL-2 among the lymphokines, they did not block the biological effects of IL-1, IL-3, gamma-IFN, B cell stimulating factor(s), and cytotoxic T cell differentiation factor(s). When anti-IL-2 was added to the in vitro reactions, it blocked mixed leukocyte reactions (MLR) and associated lymphocyte proliferation, the in vitro generation of cytotoxic T cells, and antibody formation as assessed by erythrocyte-specific plaque-forming cells (PFC). When injected into mice, anti-IL-2 antibodies also reduced the formation of cytotoxic lymphocytes in response to allogeneic cells, suggesting that endogenous IL-2 participates in such reactions in vivo. Taken together, the results indicate that these IL-2 antibodies will be useful adjuncts in the analysis of immune response both in vivo and in vitro.

Animals↗

PubMed1984

Purification and characterization of a plasminogen activator secreted by a pig kidney cell line.

The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.

Amino Acids↗

PubMed1984

Cytotoxic T cells both produce and respond to interleukin 2.

Interleukin 2 (IL-2) is a T cell-derived lymphokine that serves as a cofactor for the in vitro response of T lymphocytes to antigen and plays an important role in regulating the growth and/or differentiation of these cells (1, 2). It has been postulated (2, 3) that IL-2 is produced by a discrete regulatory T cell subset, with its effects being exerted on a second, functionally distinct subpopulation of T cells. Cytotoxic T cells have been included in the IL-2-responsive subset (3). Several models of immune regulation have further assumed that the T lymphocyte pool is divided into a complex array of genetically preprogrammed T cell subtypes, each performing a specific regulatory or effector function (4, 5). However, recent results from several laboratories (6-8) have failed to support such a strict functional subdivision of the T cell pool. The availability of highly purified mouse IL-2 (1) prompted us to reevaluate the distinction, if any, between IL-2-producing and IL-2- responsive T cells. For this purpose, we resorted to a cell-cloning procedure using activated T lymphocytes that were maintained only for short periods in culture. T cell clones were tested for cytotoxic activity, responsiveness to IL-2, and for the capacity to produce IL-2 after appropriate stimulation. We found no evidence for the existence of a major functional subdivision involving these parameters among alloantigen-activated T cells: the majority of clones analyzed could perform all three functions.

Animals↗

PubMed1984

[Radiochemical purity studies on 10 radiopharmaceuticals].

Ten radiopharmaceuticals in frequent clinical use were examined for their radiochemical purity. Thin-layer chromatography, paper chromatography and electrophoresis were used. The activity of the separated components was measured in a gamma sample changer. The assumed chemical identity of the radiochemical impurities was evaluated by comparing the results with data described in the literature. For all radiopharmaceuticals tested, the radiochemical purity was found to be not lower than the value claimed by the manufacturer and to be in accordance with the requirements of the European Pharmacopoeia as far as those pharmaceuticals are listed therein.

Pharmaceutical Preparations↗

PubMed1983

A proenzyme from chicken plasma similar to human plasma prekallikrein.

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.

Amino Acids↗

PubMed1983

Diamphotoxin. The arrow poison of the !Kung Bushmen.

We have purified the arrow poison extracted from Diamphidia nigro-ornata pupae by the !Kung Bushmen of Southern Africa, and named it diamphotoxin. The toxin is a single chain polypeptide of Mr = 60,000 with an isoelectric point of pH 9.5. It blocks neuromuscular function and is cardiotoxic and hemolytic, and the minimum lethal dose for mice is 25 pg (less than 0.5 fmol). The toxin binds tightly to cells, permitting the resolution of two distinct phases. Erythrocytes exposed to toxin in the absence of divalent cations show no apparent lesion (phase I). After washing and addition of 1 mM Ca2+, there occurs a rapid efflux of K+ followed by the loss of hemoglobin (phase II). The pH optimum for phase I is pH 6.7 and for phase II pH 8.6. The action of the toxin is noncatalytic, requiring a solution concentration of approximately 65 toxin molecules/cell for hemolysis of sheep erythrocytes under standard conditions. Ca2+ ions induce a conformational change in the free, purified toxin molecule. We propose that this change also occurs in membrane-bound toxin. Hemolysis would result from the formation of channels permitting the diffusion of small cations.

Amino Acids↗

PubMed1983

Complex-formation and inhibition of urokinase by blood plasma proteins.

We have studied the formation of covalent complexes between 125I-urokinase (125I-UK) and proteins in human plasma. Although 125I-UK reacts with many proteinase inhibitors in purified systems, the predominant complexes formed in plasma are with antithrombin III (ATIII) and alpha 2-macroglobulin (alpha 2M). 125I-UK interacts with purified alpha 2M or alpha 2M in plasma to form a characteristic pattern of multiple complexes whose Mr values by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis are in the range of 380 000-720 000, under non-reducing conditions, and 180 000-430 000 after reduction. We also examined the inhibition of UK amidolytic activity by plasma and by purified ATIII. In the presence of saturating concentrations of ATIII and heparin, an apparent first-order rate constant of 6.8 X 10(-1) s-1 was calculated for the inhibition of urokinase. In contrast, the rate constant for the formation of covalent ATIII-UK complexes was lower, suggesting the inhibition of UK proceeds first via the formation of transient non-covalent intermediates that are then transformed more slowly into covalent end products. The observed rate constants for enzyme inhibition or complex-formation with plasma or purified inhibitors are insufficient to account for the reported clearance rate of injected UK in vivo.

Antithrombin III↗

PubMed1983

Antibodies to plasminogen activator inhibit human tumor metastasis.

The human carcinoma HEp3 grows on the chorioallantoic membrane and metastasizes to the chicken embryo with kinetics that are quantitatively predictable. We have used this experimental system to test whether plasminogen activator produced by the tumor is required for metastasis. Rabbit antibodies were raised against human urinary urokinase; these cross-reacted with and blocked the catalytic activity of HEp3-PA but did not inhibit chicken PA. When administered intravenously to embryos that had received an inoculum of HEp3 cells, the anti-urokinase antibodies did not inhibit tumor growth at the site of primary inoculation on the chorioallantoic membrane, but they either prevented or strongly inhibited metastasis to the embryo lung. Antibody treatment delayed the onset of pulmonary metastasis, indicating that plasminogen activator is required during early stages of the process.

Animals↗

PubMed1983

Hormonal regulation of plasminogen activator mRNA production in porcine kidney cells.

Plasminogen activator (PA) production in LLC-PK1 pig kidney cell culture is induced to high levels by calcitonin and vasopressin, both of which stimulate adenylate cyclase, or by other compounds that also raise intracellular cAMP levels. Enzyme induction is transiently sensitive to inhibition by actinomycin D, suggesting that increased concentrations of cAMP mediate the inducing effects of the hormones by enhancing the transcription of PA-mRNA sequences. We tested this hypothesis by measuring PA-mRNA sequences in the Xenopus oocyte translation system which showed a 15-20-fold enhanced PA-synthesizing capacity when supplied with poly(A)+RNA from induced cells, above that obtained from uninduced cell RNA. Changes in PA-mRNA levels measured by Northern hybridization using cloned PA-specific cDNA gave results that agreed well with those obtained from translation assays. Pretreatment with high concentrations of cycloheximide did not block calcitonin-induced PA-mRNA synthesis, indicating that PA gene activation was a primary transcriptional result of calcitonin stimulation and did not require new protein synthesis.

Animals↗

PubMed1983

Changes in malignant phenotype of a human carcinoma conditioned by growth environment.

The human epidermoid carcinoma HEp3 exhibits highly malignant growth in chicken embryos that disappears progressively in cell culture. When studied by clonal analysis, loss of tumorigenicity was apparent within 20 generations and essentially complete after 40 generations in culture; affected all clones; and occurred under conditions which excluded the selection of variants based on superior growth rate in culture. Once ostensibly lost, the malignant phenotype reappeared after prolonged exposure of nontumorigenic cells to in vivo conditions. This similarly affected all clones assayed over a wide range of inoculum sizes. There was no evidence that HEp3 populations were heterogeneous with respect to tumorigenicity, or that the results were due to preferential selection of genetically determined subpopulations. It is suggested that the malignant phenotype of HEp3 is expressed in response to conditions in the physiological environment.

Air↗

PubMed1983

Plasminogen activators of the pituitary gland: enzyme characterization and hormonal modulation.

We studied plasminogen activator (PA) of the rat pituitary gland in organ and cell monolayer culture. Both anterior and intermediate lobes contain, synthesize and secrete a mixture consisting of the two known types of PA: urokinase and so-called tissue PA. Both enzymes were formed essentially by all PA secreting cells, and PA was identified specifically in mammotrophs, corticotrophs, and luteinizing hormone containing gonadotrophs. Pituitary PA production was modulated on exposure to a variety of biological effectors: anterior lobe PA secretion was stimulated by agents that raised intracellular cAMP concentration; his process depended on de novo enzyme synthesis. Enzyme production was repressed by androgens and glucocorticoids. When anterior lobe cultures were maintained in plasminogen-free media, the extracellular, secreted forms of ACTH consisted almost exclusively of the high molecular weight forms (31,000 and 23,000); the smaller forms (13,000 and 4,500) were also found in the extracellular medium of cultures supplemented with plasminogen. In contrast, the size distribution of intracellular ACTH species was unaffected by the presence of plasminogen. These results resemble those previously obtained with pancreatic islets and are consistent with the possibility that plasmin, generated by PA secretion, participates in prohormone processing. PA synthesis in intermediate lobe explants was stimulated by exposure to dibutyryl cAMP, and repressed by hydrocortisone. In accordance with the dopaminergic control of intermediate lobe function in some vertebrates, apomorphine strongly repressed PA synthesis in intermediate, but not anterior lobe cultures.

Adrenocorticotropic Hormone↗

PubMed1983

Modulation of plasminogen activator in rodent mammary tumors by hormones and other effectors.

The production of plasminogen activator (PA) and its regulation by hormones and other effectors were studied in organ cultures of primary rat and mouse mammary tumors. PA was quantitated using the radioiodinated fibrin plate method. The level of PA in tumor tissue was 10- to 100-fold higher than that in normal rat or mouse mammary glands; the rates of PA secretion were 10- to 1000-fold higher in the tumor cultures. PA production was stimulated by prolactin and pituitary extracts in N-nitrosomethylurea- and 7,12-dimethylbenz(a)anthracene-induced rat tumors but not in mammary tumor virus-induced mouse tumors; hydrocortisone inhibited PA production in all three tumor categories. Sex hormones and agents such as cholera toxin and retinoic acid effectively modulated enzyme production by some tumors. Three major points of interest emerge from our findings: (a) the pattern of tumor PA response to hormones differs qualitatively and quantitatively from that previously determined for the normal mammary; (b) the profile of responses of tumor PA and tumor growth to hormones shows numerous correlations suggesting that these two parameters may be coordinately regulated; (c) pituitary extracts contain an apparently novel factor that stimulates rat mammary tumor PA synthesis.

Animals↗