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Biomedical subjects

C George-Nascimento

Publications and source records attributed to C George-Nascimento.

At least 19 recordsLinked to original sources
PubMed2000

Hepatitis B virus e antigen specific epitopes and limitations of commercial anti-HBe immunoassays.

Current commercial hepatitis B virus (HBV) anti-HBe immunoassays are designed so that anti-HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti-HBe assays, anti-HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586-2595]. Although anti-HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti-HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., alpha interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti-HBe assay must distinguish anti-HBe from anti-HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3-fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti-HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (delta, gamma) which appear to be HBeAg specific have been identified. An anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti-HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes.

Animals↗
PubMed1999

Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection.

The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions. The U. S. Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al., Hepatology 15:19-25, 1992). The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al., Vox Sang. 66:122-129, 1994). The necessity of detecting antibodies to viral envelope proteins (E1 and E2) and to different genotype samples has been demonstrated previously (Chien et al., Lancet 342:933, 1993; Lok et al., Hepatology 18:497-502, 1993). In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome. A nucleic acid sequence consisting of proteins from the viral core, E1, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast. The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies. In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides. This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay. We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase. Because MEFA-6 is fused with human superoxide dismutase (h-SOD), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes. Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.

Antigens, Viral↗
PubMed1998

Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality.

Six mutants of human epidermal growth factor (EGF), which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A approximately = H16A approximately = EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction. On the other hand, the observed enhanced receptor affinities of analogs G12Q, Y13W and H16D highlight surface loci where additional productive receptor-binding contacts can be introduced. Interestingly, at acidic pH analog H16A reveals substantially greater receptor affinity than that of EGF, a property which may offer enhanced therapeutic utility in acidic environments in vivo.

Amino Acid Sequence↗
PubMed1997

Heparin-binding, highly basic regions within the thyroglobulin type-1 repeat of insulin-like growth factor (IGF)-binding proteins (IGFBPs) -3, -5, and -6 inhibit IGFBP-4 degradation.

MC3T3-E1 murine osteoblasts produce insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4)-degrading proteinase activity, which is inhibited by IGFBP-3 and a highly basic, C-terminal domain of IGFBP-3. Of all the other five IGFBPs, IGFBP-5 and -6 share the highest degree of homology with this domain of IGFBP-3; therefore, we investigated whether these two IGFBPs inhibit IGFBP-4 degradation. Both IGFBP-5 and IGFBP-6 inhibit the degradation of 125I-IGFBP-4 by MC3T3-E1-conditioned media, and their inhibitory effects are variably reversed by IGFs. Synthetic peptides containing highly basic, C-terminal regions of IGFBP-5 and IGFBP-6 inhibit 125I-IGFBP-4 degradation, as does an homologous IGFBP-3 peptide, yet each peptide displays a different IC50, with the IGFBP-5 peptide being the most potent and the IGFBP-6 peptide being the least potent. In contrast, a homologous, yet neutral, IGFBP-4 peptide does not inhibit 125I-IGFBP-4 proteolysis, confirming the role of basic residues in the inhibitory process. The IGFBP-3, -5, and -6 peptides, each of which contains the heparin-binding consensus sequence XBBBXXBX, bind heparin, yet the IGFBP-3 and -5 peptides bind heparin with the highest affinities, whereas the IGFBP-6 peptide binds heparin with approximately 10-fold less affinity. Consistent with these regions being involved in proteinase inhibition, heparin completely reverses their inhibitory effects on 125I-IGFBP-4 proteolysis. Together, these data demonstrate that IGFBP-3, -5, and -6 can function as IGF-reversible inhibitors of IGFBP-4 proteolysis, likely through homologous, highly basic, heparin-binding domains contained within the conserved thyroglobulin type-1 motif present in the C-termini of these IGFBPs.

3T3 Cells↗
PubMed1997

Effects of epidermal growth factor administration on repair of acetic acid-induced colonic ulcerations in rats.

The effect of subcutaneous and luminal epidermal growth factor (EGF) administration on acetic acid-induced colonic ulceration was determined in adult rats. Application of acetic acid to the distal colonic lumen caused epithelial denudation, mucosal ulceration and inflammation in the exposed segment. Re-epithelialization was detectable 5 to 7 days later, with near-complete resolution of the lesion by 14 days post-injury. Luminal EGF (1.6 mg/kg bw/day) or subcutaneous EGF (200 micromilligrams/kg bw/day), administered for 4 or 6 days from the time of ulceration failed to enhance re-epithelialization of the acid-exposed segment. However, mucosal and submucosal thickening was attenuated 20-40% by subcutaneous EGF, reflecting a reduction in edema. Luminal EGF had a similar but less substantial effect in the submucosa, but was more effective at attenuating muscularis thickening adjacent to the lesion. In conclusion, administration of exogenous EGF for up to 6 days failed to enhance re-epithelialization of acetic acid-induced colonic ulcerations but did attenuate the associated edematous response.

Acetic Acid↗
PubMed1996

Reduced epidermal growth factor receptor expression in hypohidrotic ectodermal dysplasia and Tabby mice.

Patients with hypohidrotic ectodermal dysplasia (HED) and Tabby (Ta) mice lack sweat glands and there is compelling evidence that these phenotypes are caused by mutations in the same highly conserved but unidentified X-linked gene. Previous studies showed that exogenous epidermal growth factor (EGF) reversed the Ta phenotype but the EGF status in HED patients has not been studied at all. Studies reported herein investigated the hypothesis that the EGF signaling pathway is involved in HED/Ta. Fibroblasts from HED patients had a two- to eightfold decrease in binding capacity for (125)I-labeled EGF, a decreased expression of the immunoreactive 170-kD EGF receptor (EGFR) protein, and a corresponding reduction in EGFR mRNA. Reduced expression of the EGFR also was observed in Ta fibroblasts and liver membranes. Other aspects of the EGF signaling pathway, including EGF concentration in urine and plasma, were normal in both HED patients and Ta mice. We propose that a decreased expression of the EGFR plays a causal role in the HED/Ta phenotype.

Animals↗
PubMed1995

Comparison of the distinct effects of epidermal growth factor and betamethasone on the morphogenesis of the gas exchange region and differentiation of alveolar type II cells in lungs of fetal rhesus monkeys.

To compare the effects of epidermal growth factor (EGF) and betamethasone on the morphogenesis of the gas exchange region and the differentiation of the alveolar type II cell during fetal lung development, fetal rhesus monkeys (78% gestation) were treated in utero with EGF (5.33 mg/kg total dose), beta-methasone (2.6 mg/kg total dose) or the carrier, saline (control), every other day for 7 days. EGF-treated monkeys had significantly increased body and adrenal weights. Betamethasone-treated monkeys had significantly decreased body and adrenal weights. Exogenous EGF reduced cytoplasmic glycogen and increased the cytoplasmic organelle and SP-A content within alveolar type II cells. In contrast, exogenous betamethasone did not alter alveolar type II cell cytodifferentiation. Neither EGF nor betamethasone treatment significantly altered the structure of the gas exchange region as shown by a lack of change from controls in alveolar airspace size or in the fraction of the gas exchange region that was potential airspace. We conclude that at clinically relevant doses, EGF greatly accelerates the maturation of alveolar type II cells, whereas betamethasone does not. Exogenous EGF may act directly on alveolar type II cells because these cells contain EGF receptor. Neither EGF nor betamethasone had dramatic effects on the morphogenesis of the gas exchange region.

Amniotic Fluid↗
PubMed1994

Prenatal exposure to epidermal growth factor attenuates respiratory distress syndrome in rhesus infants.

Treatment of nonhuman primate fetuses with epidermal growth factor (EGF) results in histologic and biochemical maturation of their lungs. To determine whether these effects improve lung function postnatally, we studied premature rhesus infants delivered at 78% of gestation after in utero treatment with EGF (n = 5) or placebo (n = 5). Indices of lung function during the 4 d of postnatal care included fractional concentration of inspired oxygen, peak inspiratory pressure, ventilator rate, mean airway pressure, arterial to alveolar oxygen tension ratio, and ventilation index. Statistically significant differences were noted in the time courses of these variables between EGF- and placebo-treated infants. The direction of the differences indicated that the EGF-treated infants had less severe lung disease. Surfactant apoprotein A concentration and lecithin to sphingomyelin ratio were both significantly higher in the amniotic fluid of the EGF-treated group, indicating advanced biochemical maturation in this group of animals. Whereas birth weight was not affected by EGF exposure, adrenal and gut weights, standardized for body weight, were increased significantly. Histologic studies showed advanced cellular maturation with increased parenchymal airspace and decreased parenchymal tissue space in the EGF-treated group compared with the control group. We conclude that prenatal exposure to EGF stimulates biochemical and histologic maturation of the lung and markedly attenuates the clinical severity of respiratory disease in this model of simian respiratory distress syndrome.

Amniotic Fluid↗
PubMed1994

Subcutaneous but not intraluminal epidermal growth factor stimulates colonic growth in normal adult rats.

Epidermal growth factor (EGF) was administered by chronic subcutaneous or intracolonic infusion into normal adult rats to determine the effect on colonic growth. Subcutaneous infusion of 200 micrograms EGF/kg/day for 7 days increased the cross-sectional mass and protein content of the muscularis and mucosal layers of the proximal colon, with the distal colon showing less response. In the mucosa, subcutaneous EGF induced proportional increments in the number of cells per crypt, and in the number of cells positively labelled for PCNA, while maintaining a normal crypt growth fraction. In contrast, an 8-fold higher dose of EGF administered intraluminally had no effect on colonic mucosal or muscularis growth. This lack of bioactivity was unlikely to reflect rapid luminal degradation as radiolabelled EGF remained stable in the colonic lumen for at least 4 h. The results demonstrate that the normal adult colon is responsive to subcutaneously delivered EGF, particularly the proximal colon, whereas EGF may not be active on the normal colon when presented from the luminal direction.

Animals↗
PubMed1992

Incomplete process of recombinant human platelet-derived growth factor produced in yeast and its effect on the biological activity.

Partially purified recombinant human Platelet-derived Growth Factor BB homodimer isolated from yeast culture media contains variable amounts of unprocessed PDGF-BB. This unprocessed PDGF-BB is found as a result of incomplete cleavage of the precursor to form the mature protein. Although the signal peptide is efficiently removed, a fraction of the PDGF secreted has an extended sequence corresponding to the truncated yeast alpha-factor leader. The data suggest that it is the amino acid chain from the truncated a-factor leader and not the sugar moiety attached to it that is responsible for the higher mitogenic activity found in this unprocessed molecule compared to highly purified PDGF-BB.

Amino Acid Sequence↗
PubMed1992

Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.

Amino Acid Sequence↗
PubMed1991

Effect of epidermal growth factor on the fetal development of the tracheobronchial secretory apparatus in rhesus monkey.

Fetal rhesus monkeys were treated with recombinant human epidermal growth factor (EGF) to determine if EGF can induce maturation of the tracheobronchial secretory apparatus. At 75% of gestation, EGF was administered simultaneously into both the amniotic fluid and fetal abdominal cavity at an average dose of 66 micrograms/kg body weight, by each route over a 7-d period. At the end of the treatment period, the fetuses were delivered and either euthanized immediately or after maintenance on ventilatory support for 6 h. The lungs were removed, and the trachea and one lobe of the right lung was fixed and embedded for light microscopy. The left lung was lavaged with saline, and the collected fluid was used to quantify released secretory product. Secretory product was also measured in amniotic fluid as well as in situ on histologic sections of tracheal epithelium. When the tracheas of EGF-treated monkeys were examined, the epithelium was found to be taller and to contain a greater proportion of secretory cells and a smaller proportion of intermediate cells than the control group. However, there was no significant change in the total number of cells per millimeter of basal lamina or in the proportion of the epithelial population made up of basal or ciliated cells. There was more secretory product stored in the epithelium and submucosal glands and increased quantities of respiratory secretions in both lung lavage and amniotic fluid of EGF-treated monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Molecular cloning of a human basic fibroblast growth factor receptor cDNA and expression of a biologically active extracellular domain in a baculovirus system.

A cDNA clone encoding a human fibroblast growth factor (FGF) receptor was isolated from a hepatoma cell line cDNA library. The cDNA encodes a three immunoglobulinlike-domain FGF receptor that is similar to a human placental FGF receptor cDNA but lacks two amino acids. The variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain FGF-receptors, can be explained by an alternate splicing mechanism. We have used a baculovirus expression system to produce high levels of a soluble, extracellular domain form of the FGF receptor (EC-FGF receptor). Spodoptera frugiperda (Sf9) insect cells infected with recombinant EC-FGF receptor viruses synthesized and secreted an EC-FGF receptor of apparent Mr = 58,000. The EC-FGF receptor purified from conditioned media of infected Sf9 cells by lentil lectin affinity chromatography was shown to bind basic FGF with high affinity (Kd = 1-5 nM), to inhibit the binding of radioiodinated basic FGF to its high affinity receptor and to inhibit endothelial cell proliferation. Furthermore, binding of basic FGF to the EC-FGF receptor was shown to be significantly enhanced by heparin. The availability of biologically active FGF receptors will allow an analysis of their interaction with members of the FGF family of proteins and viruses of the herpes family that have been shown to use the FGF receptor system for cell entry.

Amino Acid Sequence↗
PubMed1990

Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor.

Long-term storage of recombinant human epidermal growth factor (EGF), an important promoter of cell division, results in its conversion to a new species that elutes later than native EGF on a reverse-phase column. This new species, called EGF-X, has only 20% of the biological activity of native EGF. Peptide mapping indicated that the primary structure of EGF-X differs from that of native EGF solely within the first 13 residues. N-Terminal sequencing of EGF-X revealed that about 30% of the polypeptides have been cleaved at the Asp-3/Ser-4 bond. In addition, the yields after the His residue at position 10 were extremely low, indicating that a chemical modification occurs at residue 11 that is incompatible with Edman degradation. We hypothesized that aspartic acid 11 had been converted to an isoaspartyl residue, and this was confirmed with L-isoaspartyl/D-aspartyl methyltransferase, an enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues but does not recognize normal L-aspartyl residues. EGF-X, but not EGF, was found to be a substrate of this enzyme, and proteolytic digestion of EGF-X with thermolysin localized the site of methylation to a nine-residue peptide containing position 11. We did not observe formation of the isoaspartyl derivative in EGF that had been denatured by reduction of its disulfide bonds. In addition, replacement of the aspartyl residue at position 11 with glutamic acid resulted in a fully active EGF derivative that does not form detectable amounts of EGF-X. We propose that conversion of this aspartyl residue to isoaspartate is a significant nonenzymatic degradation reaction affecting this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis.

High-performance tandem mass spectrometry has been employed to structurally characterize intact O-linked glycopeptides and establish the complexity and extent of glycosylation for recombinant human platelet-derived growth factor B chain (rhPDGF-B) expressed in yeast. In addition, liquid secondary ion mass spectrometry (LSIMS) and Edman degradation have been employed to verify the protein sequence. LSIMS of high-performance liquid chromatographically fractionated proteolytic digests confirmed the complete amino acid sequence predicted by the human PDGF-B gene structure. Potential glycopeptides (as indicated by a mass shift of 162 or 324 Da from the mass of a predicted cleavage product) were sequenced using tandem mass spectrometry and Edman degradation. Ultraviolet matrix laser desorption mass spectrometry of rhPDGF-B dimer was used to determine the molecular weight distribution for the intact recombinant glycoprotein. In addition to the presence of unmodified peptides, corresponding peptides bearing monomannosyl moieties were found on serine 26 and threonines 20, 63, 88, 90 and 101. Further, dimannosyl moieties were found on threonines 6 and 63. These data reveal the presence of O-linked glycosylation at sites which do not fortify the concept of a consensus sequence involving proline residues, but which strengthen the concept of secondary and tertiary structure requirements. The advantages of high-energy collisionally induced dissociation analysis of O-linked glycopeptides over conventional base elimination and borohydride reduction and other mass spectrometric techniques are presented for the first time.

Amino Acid Sequence↗
PubMed1990

Importance of hypervariable regions of HIV-1 gp120 in the generation of virus neutralizing antibodies.

Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120. Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited. The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals. However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies. Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

Animals↗
PubMed1989

Minimal hydrolysis of epidermal growth factor by gastric fluid of preterm infants.

Epidermal growth factor (EGF), present in high concentrations in milk, may play a role in growth of the gastrointestinal tract. Resistance to proteolytic degradation in the stomach is necessary if ingested EGF is to function within the gastrointestinal tract. Although EGF stability to low pH and proteases predicts gastric survival, the extent of digestion in the stomach remains to be defined. Consequently, we measured gastric degradation of 125I-human recombinant EGF in preterm infants with an in vitro method in which EGF was incubated at 37 degrees C with stomach fluid at pH 1.8, 3.2, and 5.8 followed by analysis of degradation products. As maximal acid proteolytic activity is present one hour after feeding in preterm infants, fluid was obtained at that time from 18 infants with a mean gestational age at birth of 30.4 (3.0) (SD) weeks and a postnatal age of 26.3 (12.7) days at sampling. Incubations for up to 60 minutes revealed minimal loss of trichloroacetic acid precipitable radioactivity, in contrast to the substantial hydrolysis of iodinated casein which occurred under the same conditions. Chromatography of reaction mixtures on Sephadex G-25 showed a single major peak of radioactivity which coeluted with EGF. Epidermal growth factor also retained greater than 75% of its ability to bind to anti-EGF affinity columns and placental membrane receptors after incubation with gastric fluid. These data support the concept of substantial gastric survival of ingested EGF in a potentially biologically active form in preterm infants.

Epidermal Growth Factor↗