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C George-Nascimento

Publications and source records attributed to C George-Nascimento.

At least 37 records · Page 2Linked to original sources
PubMed1989

Is dietary epidermal growth factor absorbed by premature human infants?

Urinary epidermal growth factor (EGF) levels from infants receiving breast milk, a rich source of the growth factor, were compared with the levels excreted by infants receiving bovine milk based formulae or total parenteral nutrition that contains very little EGF. Although at 5-10 days after birth there was no significant difference in the urinary EGF output by the three groups of infants, by 13-17 days the urinary EGF output by breastfed infants was higher than by infants fed the two EGF-poor diets. These latter results are consistent with the hypothesis that EGF crosses the gastrointestinal wall to enter the general circulation in the suckling infant.

Adolescent↗
PubMed1989

Lymphocyte proliferative responses to human immunodeficiency virus antigens in vitro.

All HIV seronegative (HIV Ab-) and most HIV seropositive (HIV Ab+) individuals' lymphocytes failed to proliferate in primary cultures in response to purified HIV or to recombinant envelope and core antigens of HIV, even in the presence of recombinant interleukin 2 (rIL-2). Most HIV Ab- and HIV Ab+ individuals' lymphocytes, however, could proliferate or be induced by rIL-2 to proliferate in response to lysates of Escherichia coli or Saccharomyces cerevisiae. These findings indicate selective defects in lymphocyte proliferative responses to HIV antigens before the development of AIDS in which lymphocytes are unable to proliferate in response to any antigens. These defects in cell-mediated immune responses to HIV antigens are likely to play an important role in the pathobiology of HIV infections. Although intact HIV or glycosylated gp120 envelope protein of HIV are involved in these defects, a non-glycosylated recombinant form of the HIV gp120 envelope (ENV2-3) and p25 core proteins did not inhibit antigen- or mitogen-driven lymphocyte proliferation.

AIDS-Related Complex↗
PubMed1988

Characterization of recombinant human epidermal growth factor produced in yeast.

Four different forms of human epidermal growth factor (h-EGF) are found in the culture medium of a recombinant strain of Saccharomyces cerevisiae. These forms were characterized after purification using reverse-phase high-performance liquid chromatography. The most abundant form of secreted recombinant h-EGF has leucine at the carboxyl terminus and is identical with gamma-urogastrone. A second species is identical with the most abundant form except that it lacks the carboxyl-terminal leucine. This form appears to be the product of a carboxypeptidase found in the growth medium. The other two forms of recombinant h-EGF are the respective oxidation products of the above where the single methionine residue has been converted to methionine sulfoxide. These four forms of recombinant h-EGF are fully active; they bind to the EGF receptor of A431 cells as well as stimulate mitotic activity of human foreskin fibroblasts with equal specific activity. The location of the disulfide bonds in the predominant form of recombinant h-EGF was determined following digestion with thermolysin. The amino acid compositions of the resulting peptides showed that the placement of disulfide bonds in recombinant h-EGF is identical with that in murine EGF.

Amino Acid Sequence↗
PubMed1988

Luminal hydrolysis of recombinant human epidermal growth factor in the rat gastrointestinal tract: segmental and developmental differences.

Epidermal growth factor (EGF), present in high concentrations in the milk of various species, is biologically active following oral administration to young animals. Although in vivo studies show gastrointestinal processing of dietary EGF during early postnatal development, the relative importance of luminal and mucosal digestion in such processing is undefined. To characterize the luminal metabolism of dietary EGF in the developing gastrointestinal tract, we incubated human recombinant 125I-EGF in vitro at 37 degrees with luminal fluid from the stomach and various segments of the small intestine of 12 day old suckling and 31 day old weanling rats and analyzed the resulting reaction products. The rate of EGF hydrolysis as determined by generation of acid soluble material was greater in weanling small intestine than in suckling, with maximal hydrolytic capacity observed in the mid-jejunum and ileum. Minimal hydrolysis was observed with stomach fluid from both age groups, and EGF retained its ability to elute as a single species on Sephadex G-25 columns and to bind to monospecific affinity columns and placental membrane receptors. Incubation with suckling small intestinal fluid produced little change in the chromatographic profile on Sephadex G-25, but a reduction in antibody and receptor binding was observed. In contrast, incubation with weanling small intestinal fluid yielded both a more pronounced loss of EGF-like material on G-25 columns and a greater reduction in receptor and antibody binding. We conclude that little luminal EGF degradation occurs in the rat stomach during the suckling and weanling periods, but that in the lumen of the small intestine breakdown increases during postnatal development.

Animals↗
PubMed1988

Proteolysis of mitochondrial-coded and nuclear-coded proteins found in yeast mitochondria.

The rate of degradation of radioactive labeled mitochondrial proteins synthesized both in vitro and in vivo by isolated yeast mitochondria and growing yeast cells respectively, has been studied. It was found that the in vitro-synthesized mitochondrial proteins are rapidly degraded by an energy-dependent proteolytic system. Under the same experimental conditions the in vivo-synthesized mitochondrial proteins are slowly degraded to a limited extent by a protease which is slightly inhibited by ATP. During this period, the mitochondria are coupled and metabolically active. It is proposed that mitochondria possess an energy-dependent proteolytic system that recognizes as substrates either "abnormal" proteins or unassembled protein subunits encoded in the mitochondrial genome. An apparently different system, which is independent of energy, seems to be responsible for the slow and limited degradation of "normal" mitochondrial proteins.

Adenosine Triphosphatases↗
PubMed1987

Antigenicity and immunogenicity of domains of the human immunodeficiency virus (HIV) envelope polypeptide expressed in the yeast Saccharomyces cerevisiae.

Expression vectors were constructed for the production of various domains of the envelope gene product of the SF-2 isolate of human immunodeficiency virus (HIV) in the yeast Saccharomyces cerevisiae. Serum specimens from HIV seropositive blood donors reacted in immunoblot assays with recombinant polypeptides from both the gp120 and gp41 coding regions of env. Polypeptides from both domains were purified and injected into experimental animals. Antibodies raised in rabbits to env-2, a recombinant polypeptide representing the majority of the protein moiety of gp120, reacted with fully glycosylated native gp120 of HIV-SF2 virions. In addition, these env-2 antisera showed reactivity with viral gp120 of divergent HIV isolates. A 121 amino acid polypeptide (env-5), representing the region of gp41 stretching between the two hydrophobic domains of the protein, elicited antibodies in rabbits that reacted with glycosylated, native gp41. Thus, selected domains of the HIV env gene expressed in genetically engineered yeast, are recognized by sera from HIV infected humans, elicit antibodies that react with native HIV glycoproteins and provide a source of envelope antigens for evaluation as potential subunit vaccines for HIV.

Animals↗
PubMed1986

Enhancement of epidermal regeneration by biosynthetic epidermal growth factor.

Epidermal regeneration depends on mitosis and migration of keratinocytes. Epidermal growth factor is known to stimulate growth of keratinocytes in vitro, thus it might be expected to promote wound healing. The results of this study show that topical application of biosynthetic human epidermal growth factor accelerates epidermal regeneration in split-thickness wounds and partial-thickness burns. The significant enhancement of epidermal regeneration suggests the potential for clinical use of epidermal growth factor for accelerating healing of burns, wounds from trauma, diabetic ulcers, skin graft donor sites, and others.

Animals↗
PubMed1986

Differential antibody responses of individuals infected with AIDS-associated retroviruses surveyed using the viral core antigen p25gag expressed in bacteria.

Infection with the retrovirus that is the etiological agent of acquired immune deficiency syndrome (AIDS) is characterized by the development of antiviral antibodies. To generate reagents for studying immune responses to individual viral proteins, we have produced viral antigens in microorganisms by recombinant DNA techniques. Large amounts of the major core protein (p25gag) of an isolate of the AIDS retrovirus (AIDS-associated retrovirus; ARV-2) have been directly expressed in Escherichia coli. Recombinant p25gag (R-p25gag) has been purified and used in an enzyme-linked immunosorbent assay (ELISA) for antibodies to p25gag. Serum samples obtained from 100 individuals with AIDS, AIDS-related complex (ARC), or potential exposure to the virus through sexual contact with AIDS or ARC patients (contacts) were tested first in an ELISA with disrupted whole virus to determine which of the subjects had mounted an antibody response to the virus (virus seropositive) and then in the p25gag ELISA to determine if they had antibodies to this particular viral antigen. We observed a decrease in the proportion of virus seropositive individuals with antibodies to p25gag among patients groups in which the disease was more advanced; contacts were often positive (71%), ARC patients less frequently positive (48%), and AIDS patients only rarely positive (16%). Our results suggest that monitoring p25gag seropositivity of infected individuals may be useful for predicting either the prognosis or the stage of the disease.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals.

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.

Acquired Immunodeficiency Syndrome↗
PubMed1986

A genetically engineered, mutant human alpha-1-proteinase inhibitor is more resistant than the normal inhibitor to oxidative inactivation by chemicals, enzymes, cells, and cigarette smoke.

Alpha-1-proteinase inhibitors (alpha 1PI) containing methionine (Met-358) or valine (Met----Val-358) at the reactive center were synthesized in and purified to homogeneity from recombinant yeast. The pure proteins were exposed to 1 of 4 different oxidizing systems: N-chlorosuccinimide (chemical oxidation), myeloperoxidase plus peroxide and halide (enzymatic oxidation), activated neutrophils (cellular oxidation), or gas-phase cigarette smoke. The effect of these treatments on the leukocyte elastase inhibitory function of both proteins was then assessed. After brief exposures, substantial inactivation of the normal inhibitor occurred, whereas the mutant inhibitor remained fully active. More prolonged exposures led to complete inactivation of the normal protein and partial inactivation of the mutant inhibitor. These results suggest that the reactive center methionyl residue in alpha 1PI is more rapidly affected by oxidants than are other oxidizable residues in the inhibitor; however, Met-358 is not the only residue in alpha 1PI whose modification can lead to the inactivation of the elastase inhibitory capacity of this protein.

Binding Sites↗
PubMed1986

Properties of synthetic-gene recombinant human epidermal growth factor: comparison with the natural growth factor from human urine and milk.

The receptor-binding, immunological and biological properties of synthetic-gene human epidermal growth factor (EGF), produced by recombinant techniques in yeast have been compared with those of mouse submaxillary gland EGF and partially purified EGF from human urine and milk. The three forms of human EGF produced parallel concentration curves in radio-receptor assays using AG2804 Simian virus 40-transformed human lung fibroblasts and iodinated mouse or recombinant human EGF. Equivalent receptor-binding activities of urine and milk EGF were equipotent with recombinant human EGF in an homologous radioimmunoassay using recombinant human EGF antiserum with 125I-labelled recombinant human EGF, while none of the preparations was effective in competing for binding of 125I-labelled mouse EGF to mouse EGF antiserum. Urine, milk and recombinant human EGF stimulated protein synthesis and inhibited protein degradation in cultured AG2804 fibroblasts with identical potency. On a weight basis, mouse EGF was equipotent with recombinant human EGF in competitive binding to cell receptors and in effects on protein synthesis or protein degradation in AG2804 fibroblasts. It is concluded that recombinant human EGF is indistinguishable from the natural growth factor from urine or milk and shares similar biological properties with mouse EGF.

Animals↗
PubMed1985

Effect of ethionine on the in vitro synthesis and degradation of mitochondrial translation products in yeast.

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.

Adenosine Triphosphate↗
PubMed1981

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII.

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.

Amino Acids↗
PubMed1978

The use of orthacryl two-dimensional polyacrylamide gel electrophoresis to identify and compare the subunit polypeptides of bovine heart and yeast cytochrome c oxidases.

A two-dimensional electrophoretic method which takes advantage of the "migration anomalies" experienced by some polypeptides on gels of different porosities has been successfully used to resolve the seven subunit polypeptides of yeast cytochrome c oxidase and the nine polypeptides associated with bovine cytochrome c oxidase. The two-dimensional maps provided by this method reveal clear differences between these two cytochrome c oxidases.

Animals↗
PubMed1976

Effect of lipids on the reconstitution of D-lactate oxidase in Escherichia coli membrane vesicles.

An unsaturated fatty acid auxotroph lacking D-lactate dehydrogenase activity has been isolated from Escherichia coli ML 308-225 dld-3. While NADH oxidase activity in membrane vesicles prepared from the mutant cells grown in a variety of unsaturated fatty acids is comparable to that of previously isolated fatty acid auxotrophs, D-lactate oxidase activity is absent. However, D-lactate oxidase ativity can be restored when vesicles are incubated with a purified preparation of D-lactate dehydrogenase obtained from wild type cells. The effect of altering the fatty acid composition of the membrane on the reconstitution of D-lactate oxidase activity was examined. Binding of purified D-lactate dehydrogenase was not affected by either the lipid composition of the membrane vesicles or the temperature during reconstitution. However, the reconstitution of D-lactate oxidase activity was strongly influenced by the fatty acid composition of the membrane lipids. The temperature dependence of the reconstituted activity was analyzed. Temperature transitions were not observed with membrane vesicles supplemented with oleic or linolenic acid but palmitelaidic acid-enriched vesicles exhibited a transition temperature at 30 degrees. Attempts to reconstitute the elaidic-supplemented vesicles at temperatures below 30 degrees failed to yield active D-lactate oxidase and the vesicles aggregated. At 42 degrees, aggregation did not occur and D-lactate oxidase activity was obtained to a level of 10% of that found with membranes from the parent strain (ML 308-225). These results suggest that although binding of D-lactate dehydrogenase is independent of the physical state of the membrane, reconstitution of the D-lactate oxidase activity in membrane vesicles is dependent upon the fatty acid composition of the phospholipid, hence the physical state of the membrane.

Cell Membrane↗