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Biomedical subjects

J Flores

Publications and source records attributed to J Flores.

At least 199 records · Page 11Linked to original sources
PubMed1982

Association of cytosol hemoglobin with the membrane in intact erythrocytes.

The problem of demonstrating hemoglobin binding to the erythrocyte membrane in intact cells was approached by observing the quenching of fluorescent membrane probes by hemoglobin as a function of pH. This quenching was studied by measuring fluorescence intensities and decay rates of membrane-bound donors by both right-angle and front-face fluorometry. The donors included 4,4'-bis(isothiocyano)-2,2'-stilbene disulfonate (DIDS) bound to the band 3 protein and 2-, 6-, and 12-(9-anthroyloxy(stearic acid (2-AS, 6-AS, and 12-AS) in the lipid portion of the membrane. The probe fluorescence is quenched progressively as the intracellular pH is decreased from 7.2 to 5.9 and does not depend on the oxygenation state. Since the fluorescence characteristic of the DIDS and x-AS probes in ghosts are independent of pH over this range, this quenching is due to the greater proximity of the donors and hemoglobin. Because hemoglobin binding to the band 3 protein in ghosts has previously been shown under conditions of low ionic strength and hemoglobin concentration, the present results were analyzed by a model in which a hemoglobin molecule is bound or in close proximity to a band 3 protein at pH 6. By using resonance energy transfer theory, we found the distance from DIDS to the bound hemoglobin to be approximately 4 A, which is within the range of distances measured between the DIDS binding site and the band 3 protein cytoplasmic sulfhydryl groups. Furthermore, the pH-dependent fluorescence quenching of 12-AS was stronger than that of 2-AS, showing that a cytoplasmic acceptor is involved and that the average distance between hemoglobin and these probes was greater than that for DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Rescue and serotypic characterization of noncultivable human rotavirus by gene reassortment.

Thirty-three of 50 noncultivable human rotavirus strains from a variety of locations were successfully rescued by gene reassortment. The serotype of each of the 33 strains was investigated by a qualitative cytopathic effect neutralization assay. Nineteen strains resembled the previously characterized human rotavirus serotype Wa, whereas three strains were serologically related to the DS-1 strain. Eleven strains appeared to be serotypically distinct from the Wa and DS-1 strains and thus apparently represent one or more new human rotavirus serotypes.

Cytopathogenic Effect, Viral↗
PubMed1982

Definition of human rotavirus serotypes by plaque reduction assay.

Twenty different human rotavirus reassortants were characterized serologically by a plaque reduction assay as belonging to one of three distinct serotypes. Fourteen were similar if not identical to our prototype Wa strain; two were like the prototype DS-1 strain, and four belonged to a third serotype for which a prototype has not yet been selected. Hyperimmune sera raised against the three serotypes were required to distinguish among them, since postinfection sera had lower titers and were more cross-reactive than hyperimmune sera. These results confirmed the ability of a qualitative cytopathic neutralization test to predict correctly the Wa or DS-1 serotype. A strain of rhesus rotavirus (MMU 18006) was identified as belonging to the newly defined third serotype. Finally, an attempt was made to correlate previously published serotype analysis by neutralization of fluorescent cell-forming units with the results determined by the plaque reduction neutralization assay.

Cross Reactions↗
PubMed1982

Genetic relatedness among human rotaviruses as determined by RNA hybridization.

Viral RNAs from human rotaviruses were compared by gel electrophoresis and by hybridization to probes prepared by in vitro transcription of two well-characterized laboratory strains (Wa and DS-1). Also, the viral RNAs were compared by hybridization to probes prepared from three of the test viruses. Thirteen specimens (diarrheal stools) were obtained from infants and children 5 to 21 months old on a single day at the emergency ward of the Caracas Children's Hospital, and an additional specimen was obtained from the same hospital 6 months before. When the electrophoresed viral RNAs were stained with ethidium bromide and examined by UV light, five different migration patterns (electropherotypes) were distinguished on the basis of differences in mobility of the RNA segments. The hybridization technique that was employed permitted only qualitative comparisons of corresponding genes of different human rotaviruses. Ten of the specimens contained enough virus to yield sufficient RNA for hybridization studies. Eight of the viruses studied by hybridization contained 4 to 11 genes that reacted specifically with the Wa probe to yield double-stranded RNA segments with a mobility similar to that of Wa viral RNA or test virus RNA. The other two viruses contained 11 genes that reacted specifically with the DS-1 hybridization probe to yield double-stranded RNA segments with a mobility similar to DS-1 viral RNA or test virus RNA. A more complex picture emerged when hybridization probes were prepared from three of the test viruses and used to compare the different electropherotypes. Corresponding genes that exhibited similar migration did not necessarily exhibit homology when studied by hybridization. Also, some corresponding genes that exhibited homology did not have the same mobility by gel electrophoresis.

Child, Preschool↗
PubMed1982

In vitro transcription of two human rotaviruses.

The RNA polymerase activities of a cultivatable (Wa) and a noncultivatable (DS-1) strain of human rotavirus were studied. Under optimal conditions, transcription of all of their RNA segments occurred, as evidenced by the hybridization of labeled transcripts to genomic RNA. Cross-hybridization between the two viruses showed that none of their 11 genes were completely homologous. The transcription products could be translated in vitro, yielding proteins with an electrophoretic pattern resembling that obtained with proteins labeled in vivo during infection with the Wa virus.

Cell-Free System↗
PubMed1981

Fluorescence polarization study of human erythrocyte membranes with 1-phenyl-3-(2-naphthyl)-2-pyrazoline as orientational probe.

The emission and polarization spectra of 1-phenyl-3-(2-naphthyl)-2-pyrazoline (PNP) in various environments were studied. Compared to the widely used orientational membrane probe 1,6-diphenylhexatriene (DPH), PNP is five times less photolabile and since its fluorescence emission maximum is at longer wavelengths (lambda max approximately 445 nm), it is more suitable for use with intact erythrocytes. The limiting fluorescence anisotropy of PNP is 0.385. In erythrocyte ghosts, the steady-state emission anisotropy of PNP is a decreasing formation of wavelength and its temperature dependence parallels that of DPH, dropping from 0.298 at 2 degrees C to 0.185 at 38 degrees C when averaged between 420 and 470 nm.

Diphenylhexatriene↗
PubMed1981

Antigenic characterization of human and animal rotaviruses by immune adherence hemagglutination assay (IAHA): evidence for distinctness of IAHA and neutralization antigens.

An immune adherence hemagglutination assay (IAHA) and a modified enzyme-linked immunosorbent assay for antigenic characterization of human rotaviruses were developed. The designations of type 1 and type 2 were identical to those established previously by specific complement fixation, enzyme-linked immunosorbent assay, and immune electron microscopy. By IAHA (and modified enzyme-linked immunosorbent assay) certain animal rotaviruses were found to be closely related to human rotavirus type 1. The pattern of IAHA reactivity and the cell culture neutralization serotype were found to be distinct properties. The separation of neutralization and IAHA reactivity was apparent when animal rotaviruses which were distinguishable from each other by neutralization assays were found to share IAHA specificity. Further evidence for the dissociation of the neutralization and IAHA specificities was found in studies of human and bovine rotaviruses which underwent genetic reassortment during coinfection. Thus, it appeared that the IAHA and neutralization antigens were coded for by different genes. In view of these findings, we suggest that the term serotype be reversed to identify the antigen that reacts with neutralizing antibodies as is customary for other viruses and that the term subgroup (instead of serotype) be used for the specificity detected by specific complement fixation, enzyme-linked immunosorbent assay, and now IAHA.

Animals↗
PubMed1981

Distinctive ribonucleic acid patterns of human rotavirus subgroups 1 and 2.

The ribonucleic acid migration patterns of 7 subgroup 1 and 16 subgroup 2 human rotaviruses recovered from four geographic areas were compared. The subgroup 1 ribonucleic acid patterns had strikingly slower-moving segments 10 and 11, suggesting a correlation between the ribonucleic acid pattern and the subgroup specificity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

A rapid fluorometric method for determining bilirubin levels and binding in the blood of neonates: comparisons with a diazo method and with 2-(4'-hydroxybenzene)azobenzoic acid dye binding.

A simple, rapid fluorometric method for determining the albumin-bound bilirubin concentration, total blood bilirubin concentration, and the bilirubin reserve-binding capacity of albumin was clinically evaluated using blood specimens from 79 neonates. This study showed that these bilirubin determinations, made by means of the Bell Laboratories hematofluorometer, correlated well with plasma bilirubin levels obtained by a diazotization (Jendrassik-Grof) method. Hematofluorometer reserve-binding capacities correlated very well with 2-(4'-hydroxybenzene)azobenzoic acid (HABA) dye reserve-binding capacities for specimens of artificially jaundiced adult blood. For specimens of neonatal blood the HABA dye reserve capacity was, on the average, higher than the hematofluorometer reserve-binding capacity, particularly for specimens from low-birth-weight babies (less than 2,000 gm). Comparison of HABA reserve capacity and hematofluorometer reserve capacity for high-birth-weight babies (greater than 2,000 gm) gave data very similar to those for adult blood specimens. The specific bilirubin-binding capacity of albumin was found to be greater for infants whose birth weight exceeded 2,000 gm than for the lower birth weight group. The total blood bilirubin concentration obtained by the hematofluorometer is shown to be significantly higher than the concentration of bilirubin bound to albumin, an indication of other important compartments of bilirubin in blood.

Adult↗
PubMed1978

Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.

Adenylyl Cyclases↗
PubMed1977

Application of the zinc protoporphyrin (ZP) test as a monitor of occupational exposure to lead.

Zinc protoporphyrin, the predominant abnormal metabolite that appears in whole blood as a result of chronic lead absorption, can be assayed fluorometrically in diluted whole blood with great sensitivity. An extremely simple and rapid test for the effects of lead absorption is described that is based on this assay. Results of a field trial show a characteristic dose-response relationship between zinc protoporphyrin and lead, and indicate that this test should be seriously considered as a monitor for the effects of chronic lead adsorption.

Adult↗
PubMed1977

Comparison of zinc protoporphyrin and free erythrocyte protoporphyrin in whole blood.

The recent change in emphasis from blood lead to erythrocyte protoporphyrin as the primary diagnostic test for screening for childhood lead poisoning has made it desirable to develop the quantitative relationship between the two protoporphyrins, free erythrocyte protoporphyrin (FEP) and zinc protoporphyrin (ZP), that are now widely used for screening purposes. ZP was found to be equal to 1.4 EP, thus permitting the measurement of either parameter, and its conversion to the other.

Child, Preschool↗
PubMed1977

Filter paper-disc method for the determination of zinc protoporphyrin in blood.

Zinc protoporphyrin, an abnormal metabolite resulting from undue lead absorption, can be quantitatively determined in blood that has been spotted and dried on filter paper. This procedure, which yields values that correlate well (r = 0.96) with those obtained by direct determination in whole blood, offers advantages in convenience of collection, storage, and transportation that recommend it for field use. The light sensitivity of the zinc protoporphyrin, however, mandates that the specimens be protected from excessive light exposure until analyzed.

Filtration↗
PubMed1976

The activation of adenylate cyclase by cholera toxin: possible interaction with the nucleotide regulatory site.

The application of cholera toxin to intact cells causes a stimulation of adenylate cyclase activity. The effect is characterized by a lag period followed by a progressive rise in enzyme activity over several hours. Only a few minutes' exposure to the toxin is required to produce effects lasting over several days. Stimulation of adenylate cyclase by cholera toxin in broken cell preparations requires the presence of nicotinamide-adenine dinucleotide (NAD) and an unidentified component of the cytosol. Guanyl nucleotides and certain non-hydrolysable analogues of guanosine triphosphate also stimulate adenylate cyclase. Stimulation by the analogues results in a highly activated enzyme which has characterisitcs similar to those of adenylate cyclase after stimulation by cholera toxin. Thus the stimulation is irreversible, the enzyme may be "solubilized" by non-ionic detergents in the activated state, and responses to certain hormones are enhanced. Therefore the possibility exists that cholera toxin acts on the guanyl nucleotide regulatory protein of the adenylate cyclase complex. In exploring this possibility it was found pretreatment with cholera toxin not only blocked the stimulatory effect of subsequently added guanylylimidodi-phosphate (GppNHp) but that the latter reduced the stimulation by toxin. Similarly, pretreatment with GppNHp blocked the effect of cholera toxin. The similarities in the effects of cholera toxin and GppNHp, together with the mutual interference of their activities, suggests that cholera toxin acts at the same regulatory site at which guanyl nucleotides exert their effects on adenylate cyclase.

Adenylyl Cyclases↗