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Biomedical subjects

I Raw

Publications and source records attributed to I Raw.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of an endooligopeptidase A-like protein in PC12 cells: activity modulation by cAMP but not by basic fibroblast growth factor.

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Effect of saponin from Quillaja saponaria (molina) on antibody, tumour necrosis factor and interferon-gamma production.

Saponin has been described to contain adjuvant activity in vaccination protocols, in protection against disease, and on humoral immune response. In this paper we describe the effect of a pure saponin from Quillaja saponaria (molina) on the immune response elicited in mice by two antigens, BSA and Crotalus durissus terrificus (South American rattlesnake) venom. Antibody production as measured by ELISA shows that saponin was able to increase antibody synthesis to both antigens. Moreover, mice immunized with verom plus saponin were completely protected against the lethal effects of the venom. The effect of saponin was also evaluated for cytokine production. Tumour necrosis factor activity about 2.9 times higher than in control mice was detectable in sera from animals immunized with saponin. Interferon-gamma was produced only when BSA and saponin were injected together into the mice.

Adjuvants, Immunologic↗
PubMed1994

Multiparametric analyses of hybridoma growth on glass cylinders in a packed-bed bioreactor system with internal aeration. Serum-supplemented and serum-free media comparison for MAb production.

Monoclonal antibodies are one of the most important products of biotechnology and laboratories and companies all over the world are pursuing their large-scale production. Herein we report a protocol for hybridoma cell cultivation over small glass cylinders inside a 3 liter bioreactor vessel which leads to the production and purification--in order of grams--of one MAb intended for human therapeutic use. This protocol proved to be simple, reproducible and cost effective. Three trials are reported: the first two using conventionally serum-supplemented medium culture and producing 3.15 and 2.1 g of purified MAb in 30 and 21 days respectively, and the third one using serum-free medium culture and producing 6 g of purified MAb in 36 days. We have ascertained the stability of the hybridoma by its cloning directly in serum-free medium. The downstream processing of the serum-free trial was done in a single step, concentrating large volumes of supernatant while simultaneously purifying the antibody.

Animals↗
PubMed1994

A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction.

A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69-kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM3 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HCl, pH 7.4; 50 mM Tris-HCl, pH 7.4/0.75 M MgCl2; 50 mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 micrograms PT, 8.14 micrograms FHA, 6.3 micrograms AC, 3.37 micrograms 69-kDa, 9.54 micrograms FIM2 and 2.23 micrograms FIM3) and from P21 (with 0.175 micrograms PT, 0.28 micrograms FHA, 0.002 micrograms 69-kDa, 0.005 micrograms FIM2 and 0.122 micrograms FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA.

Animals↗
PubMed1994

Kinetic studies on the membrane form of intestinal alkaline phosphatase.

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate.

Alkaline Phosphatase↗
PubMed1993

Preparation of human rabies vaccine in VERO cell culture using a microcarrier system.

1. The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor. Virus titers of about 10(6) LD50/ml were obtained regularly. 2. Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose). The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA. 3. The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2). 4. This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available.

Animals↗
PubMed1993

Purification of superoxide dismutase from placental haemolisate blood: a simple and efficient method.

Superoxide dismutase functions as a scavenger of superoxide radical protecting living organisms. This enzyme has potential use as anti-inflammatory or anti-reperfusion injury drug. Here we present a simple and efficient SOD purification method from human placental blood. Superoxide dismutase from clarified haemolysed placental blood after chloroform and ethanol treatment was purified by DEAE-Sepharose, Phenyl-Sepharose chromatographies and cross flow ultrafiltration. The purified product is 98% pure by SDS-PAGE with 71% yield and specific activity of 2.8 x 10(5) U/mg protein.

Blotting, Western↗
PubMed1992

Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells.

We report here that basic fibroblast growth factor (bFGF)-elicited neurite outgrowth in PC12 cells is potentiated by dibutyryl cyclic adenosine monophosphate (dbcAMP) or forskolin. This property was also described for nerve growth factor (NGF), suggesting that both NGF and bFGF may share common intracellular events leading to neurite outgrowth and synergism with dbcAMP and forskolin. The synergistic effect of dbcAMP and forskolin is specific, since treatment of PC12 cells with bFGF and dibutyryl cyclic guanosine monophosphate (dbcGMP) or phorbol ester did not change the neurite outgrowth response of cells treated with bFGF alone. Furthermore, neurite outgrowth depends on cellular adhesion. Increasing adhesion by plate treatment with poly-d-lysine increases the neurite outgrowth elicited by bFGF alone or bFGF plus dbcAMP. On the other hand, decreasing cellular adhesiveness by plating PC12 cells in semi-solid agarose renders the cells unable to develop neuritic processes. In addition, 3H-methylthymidine incorporation studies showed that bFGF-treated PC12 cells cease growth only when they become fully differentiated after 3-5 days of treatment. In contrast, dbcAMP, which is a poor differentiation factor, is able to block cellular growth after 24 hour treatment. These results suggest that when PC12 cells become differentiated, they stop growing. However, growth inhibition does not necessarily lead to differentiation.

Animals↗
PubMed1992

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.

Animals↗
PubMed1991

Bioactivity of skeletal muscle proteolysis-inducing factors in the plasma proteins from cancer patients with weight loss.

We determined the circulating level of bioactivity for skeletal muscle proteolysis-inducing factors (PIF) in the blood samples from cancer patients whose body weight loss was greater than 10%. The level of bioactivity was estimated by measurement of tyrosine release from isolated 1at diaphragm muscles incubated with an ultrafiltered fraction of plasma or serum proteins containing molecules from 0 to 25 kDa in molecular weight. Significant levels of bioactivity were detected in 25 of the 50 cancer samples. No activity was found in 18 of the samples from healthy human blood donors. The ability of 13 of the cancer samples to induce muscle proteolysis was significantly inhibited by incubation of muscles in presence of indomethacin (10 microM). The neutralisation of 12 of the cancer samples with the antibodies to recombinant human interleukin-1 (IL-1), alpha and beta forms, partially abrogated the activity in five samples. These results suggest that the accelerated breakdown of proteins induced by the cancer plasma factors is at least in part mediated by IL-1 in cooperation with other active factors not yet defined. Additionally, we have shown that the increased breakdown of proteins induced by PIF in the crude supernatant derived from activated mouse peritoneal macrophages is prevented by the treatment of muscles with either indomethacin or quin-2 (1 microM). These observations provide indirect evidence for a possible causal relationship between the production of PIF and the body-weight loss of cancer patients.

Adolescent↗
PubMed1986

Isolation and characterization of a thrombin-like enzyme from the venom of Crotalus durissus terrificus.

A thrombin-like enzyme was isolated in 6% yield from the venom of Crotalus durissus terrificus by ammonium sulfate precipitation followed by gel filtration on Sephadex G-75 and finally affinity chromatography on Sepharose-1,4-butanediol-diglycyl-p-aminobenzamide eluted with 0.15 M benzamidine. The enzyme behaved like a single component on SDS-PAGE corresponding to a molecular weight of 34 kDa. The specific activity of the enzyme toward bovine fibrinogen was 71 NIH U/mg protein. The pH optimum for the coagulation of human fibrinogen was 8.0. The enzyme hydrolyzes the alpha-chain of fibrinogen, has amidase activity on L-arginine-p-nitroanilide and L-arginine-7-amido-4-methyl-coumarin amino terminal blocked peptides and presents esterolytic activity on N-alpha-tosyl-L-arginine-methylester.

Blood Coagulation↗
PubMed1985

Effect of insulin on the synthesis of rat liver ribosomal and endoplasmic reticulum proteins.

There was reduction in the levels of some liver endoplasmic reticulum proteins from streptozotocin-treated rats compared to those of normal rats as detected by two-dimensional polyacrylamide gel electrophoresis. When insulin was administered 24 h before streptozotocin, the protein patterns were the same as that obtained for normal rats. The comparison of the rate of synthesis of some endoplasmic proteins by streptozotocin-treated rats with that of streptozotocin-treated rats which received insulin 4 h prior to the experiment by the double-labelling technique indicated that insulin increased the rate of synthesis of some liver endoplasmic reticulum proteins. The rates of synthesis of some liver ribosomal proteins from streptozotocin-treated rats were increased and others were decreased when the animals were compared with streptozotocin-treated rats pretreated with insulin 4 h earlier. These results suggest that the generalized decrease of the rate of protein synthesis which has been reported in diabetes is due to a decrease of the rate of synthesis of some ribosomal proteins that may act in chain initiation or have a regulatory function.

Animals↗
PubMed1984

Catecholamines and congenital pain insensitivity.

The congenital pain insensitivity syndrome is accompanied by: a) the presence of a yellow-brown pigment in the basal layer of the epidermis with the histochemical properties of melanin, b) decrease of urinary and blood concentration of dopamine and norepinephrine, c) excretion of melanin and of an abnormal as yet unidentified phenolic metabolite, d) increase in urinary p-hydroxyphenyllactic acid, p-hydroxyphenylacetic acid, indole acetic acid, and e) a high homovanillic acid/vanillylmandelic acid (HVA/VMA) ratio. In this report we show that analgesic patients differ from controls by: a) an increase in norepinephrine excretion after L-tyrosine administration, and b) an increase in norepinephrine, dopamine, DOPA + DOPAC excretion after L-DOPA administration. The administration of L-DOPA eliminates the difference in HVA/VMA ratio between patients and controls. Serum and platelet monoamine oxidase, dopamine beta-hydroxylase and catechol-O-methyltransferase are normal and urinary excretion of biopterins, morphine-like compounds and endorphins is also within the normal range. The comparison of catecholamine metabolism in patients with phenylketonuria, Lesh-Nyham syndrome, congenital sensory neuropathy with anhydrosis and familial dysautonomia on the basis of our data and those in the literature suggests that patients with congenital pain insensitivity display abnormal catecholamine metabolism.

Dopamine↗
PubMed1983

Effect of extra-hepatic Walker sarcoma 256 on the synthesis and degradation of liver cytochromes P-450 and b5.

Extra-hepatic Walker sarcoma 256 produced a marked decrease (approximately 60%) in the levels of cytochrome P-450 and NADP-cytochrome P-450 reductase in rat liver endoplasmic reticulum, and a lesser decrease (approximately 20%) of cytochrome b5 and NADH-cytochrome b5 reductase. Polychlorinated biphenyls induced the synthesis of these cytochromes and reductases to approximately the same extent both in normal and tumor-bearing rats. The double-label technique was used to demonstrate that the synthesis of cytochromes P-450 and b5 was reduced in the liver of tumor-bearing rats. The turnover of P-450 was not affected by the tumor, whereas cytochrome b5 turnover was decreased. It is proposed that Walker sarcoma 256 mainly affects the transcription of cytochromes P-450 and b5 through a toxohormone, and that a regulatory mechanism coordinates the level of each cytochrome and its respective reductase.

Animals↗
PubMed1982

Effect of drugs and hormones on rat liver dimethylaminoazobenzene reductase activity.

1. Rat liver endoplasmic reticulum catalyzes the reduction of 4-dimethylaminoazobenzene (DAB) by NADPH to dimethyl-p-phenylenediamine and p-aminophenol. This azoreductase activity was inhibited by cyanide and cytochrome b5 antibody, but was resistant to carbon monoxide and SKF-525A (beta-diethylaminoethyl-diphenylpropylacetate). 2. DAB azoreductase activity was induced by 20-methylcholanthrene and phenobarbital, and increased in streptozotocin-induced diabetes or fasting. It was repressed by treatment with DAB and its 3'-methyl derivative, but not by several other derivatives with substitutions in the dimethylaminoazobenzene ring. 3. Azoreductase activity, NADPH-cytochrome P-450 reductase, cytochromes P-450 and b5 were measured in liver microsomes prepared from fasted animals and from animals treated with 20-methylcholanthrene, phenobarbital, streptozotocin or 3-aminotriazole plus allyl-isopropylacetamide. No direct correlation could be established between the variations of azoreductase activity and those of cytochromes P-450 and b5, and of NADPH-cytochrome P-450 reductase in these experimental situations. Since these known carriers do not seem to be the limiting factors for the azoreductase activity, the participation of an unknown carrier that can be repressed by dimethylaminoazobenzene is postulated. 4. Dimethylaminoazobenzene treatment did not reduce the rate of synthesis of microsomal proteins but rather increased the turnover rate of proteins with molecular weights of about 17, 30 an 35 kdal. Since streptozotocin increased the synthesis of proteins with molecular weights of 17, 32, and 48 kdal it is suggested that one of these proteins may correspond to the postulated carrier that is the limiting factor in DAB reduction.

Allylisopropylacetamide↗
PubMed1979

Effect of a single dose of inducers and inhibitors on the rate of synthesis of cytochromes and reductases in liver organelles.

Measurement of the effect of drugs on the in vivo rates of synthesis of rabbit liver organelle bound proteins were measured following individual treatments with the inducers phenobarbital, 3-methylcholanthrene and PCB (a mixture of polychlorinated biphenyls) and the inhibitors, cycloheximide, aflatoxin B1, chloramphenicol and actinomycin D. Following their isolation from a homogenate containing the combined livers of 14C-leucine injected experimental animals and 3H-leucine injected control animals, purified fractions of the following proteins were prepared: microsomal cytochrome b5, cytochrome P-450, NADH-cytochrome b5 reductase, NADPH-cytochrome P-450 reductase and proteolipids, outer mitochondrial membrane cytochrome b5, NADH-cytochrome b5 reductase and proteolipids, inner mitochondrial membrane cytochrome c, NADH dehydrogenase and proteolipids, intermitochondrial membrane cytochrome b5 and circulating serum albumin. The effect of a drug was examined by measuring the 14C/3H ratio of leucine incorporation of each fraction; ratios which differed markedly from a control value of 1 represented actual changes in the relative rates of protein synthesis. Increased rates of synthesis of cytochrome P-450 and its reductase, intermitochondrial membrane cytochrome b5 and all three proteolipid fractions resulted from each inducer treatment. Treatments with 3-methylcholanthrene and PCB also increased the rate of synthesis of cytochrome b5 and its reductase in both the microsome and outer mitochondrial membrane. In addition, the PCB treatment increased the rates of synthesis of cytochrome c and NADH-dehydrogenase. The rates of synthesis of cytochromes, reductases and of circulating serum albumin were inhibited following treatments with cycloheximide, aflatoxin B1 and actinomycin D. Actinomycin D appeared to inhibit the release of newly synthesized albumin into the bloodstream while chloramphenicol treatment appeared to inhibit the incorporation of cytochrome c into the mitochondria. After 20 hours of treatment with inhibitors, the inhibitory effect of actinomycin D and cycloheximide were still apparent while the rates of protein synt;esis in chloramphenicol and aflatoxin B1 treated animals increased to levels above the controls. The incorporation of radioactively labeled leucine into the proteolipids of the microsomal, and the outer and inner mitochondrial membranes were inhibited following the treatment with actinomycin D and stimulated following the treatment with cycloheximide.

Aflatoxins↗