Biomedical subjects

A M Barber

Publications and source records attributed to A M Barber.

12 recordsLinked to original sources

PubMed1992

A generalized approach to detection of organophosphate resistance in mosquitoes.

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.

Acetylcholinesterase↗

PubMed1992

pNiXa, a Ni(2+)-binding protein in Xenopus oocytes and embryos, shows identity to Ep45, an estrogen-regulated hepatic serpin.

A Ni(2+)-binding protein (pNiXa, 45 kD, pI 8.5) discovered in Xenopus embryos, was isolated from oocytes. Based on amino acid sequences, pNiXa belongs to the serpin superfamily and shows identity to the cDNA sequence of Ep45, an estrogen-regulated hepatic serpin that contains an (HX)n-motif found in eukaryotic transcription factors. Nondenatured pNiXa, purified by Ni-affinity chromatography, inhibited bovine alpha-chymotrypsin. The presence of pNiXa in embryos when they are susceptible to Ni2+, the high avidity of pNiXa for Ni2+, and the (HX)n-motif point to pNiXa as a molecular target of Ni(2+)-teratogenesis.

Amino Acid Sequence↗

PubMed1990

Expression and characterization of RNase III and Era proteins. Products of the rnc operon of Escherichia coli.

The synthesis rates of ribonuclease III (RNase III) and Era proteins are relatively low, and expression of the era gene is translationally coupled with expression of the rnc gene. Expression of both genes is negatively controlled by RNase III itself. We have constructed plasmids that overproduce RNase III and/or Era proteins under the control of the lambda PL promoter. A plasmid with the rnc gene under PL control expresses RNase III at levels greater than 40% of total cellular protein. Another plasmid with the era gene under PL control and a modified translation-initiation signal produces up to 80% of total cell protein as Era. Each protein has been purified using simple and rapid procedures. Purified RNase III protein specifically processes mRNA transcripts containing known RNase III sites. The purified Era protein binds GDP and GTP and has GTPase activity. Kinetic analysis shows that one molecule of GTP or GDP is bound/Era peptide with a Kd of 5.5 microM for GTP binding and 1.0 microM for GDP binding. The Km of the Era GTPase is 9.0 microM, and the maximum catalyzed rate of GTP hydrolyzed/min/mol of Era protein at 37 degrees C is 9.8 mmol.

Bacterial Proteins↗

PubMed1990

Microplate assay of glutathione s-transferase activity for resistance detection in single-mosquito triturates.

1. Optimum conditions are described for a simple, rapid microplate assay that measures glutathione s-transferase (GST) activity accurately and precisely in small portions of single mosquito homogenates. 2. Up to 10 assay replicates were possible for individual adults and larvae. Concentration of GST activity in the head/thorax region allows blood-fed mosquitoes with abdomens removed to be used in assays. 3. The method allows the use of GST activity as a biochemical character in comparative studies of populations. 4. The microplate assay detects elevated GST activities associated with DDT resistance in Anopheles arabiensis.

Animals↗

PubMed1990

SequenceEditingAligner: a multiple sequence editor and aligner.

Here we present the SequenceEditingAligner system for editing multiple, aligned genetic sequences. This is an interactive multi-window color system that displays more than 3500 nucleotides or amino acids. The system handles nucleic acid or protein sequences with or without secondary structure data. More than 300 sequences, each more than 1500 elements in length, may be analyzed together. With the system scientists can classify elements, align sequences, edit them, find consensus patterns, and simultaneously generate oligomer frequency histograms and other statistics.

Algorithms↗

PubMed1990

CAP binding sites reveal pyrimidine-purine pattern characteristic of DNA bending.

To investigate the intrinsic bending of DNA at sites where proteins bind, we analyzed catabolite gene activator protein (CAP) binding sites and various operators from the viewpoint of DNA bending flexibility. Theoretical conformational analysis. DNase I digestion and x-ray crystallography data indicate that bending of B-DNA is highly anisotropic and sequence-dependent. Certain dimers prefer to bend into the major groove ("major-philic") and others prefer to bend into the minor groove ("minor-philic" dimers). From these data we considered TA, CG, CA:TG and GG:CC as major-philic dimers and AT,AA:TT and GT:AC as minor-philic ones. Analysis of 31 CAP binding sites has identified strong major-philic tendencies 5-7 base pairs (bp) away from the center. In addition, we found minor-philic poly-A tracts extending 4-5 bp away from the proposed major-philic bends. Finally, to analyze the central regions we followed the lead of Shumilov and classified the DNA sites by their spacer lengths [V.Y. Shumilov, Mol. Biol. (Mosk) 21, 168-187 (1987)]. In this way, we identified two subsets of CAP binding sites: one with 6 bp between the TGTGA:TCACA consensus boxes (N6-set) and one with 8 central bp (N8-set). We discovered that the dimer at the center of an N6-set site was usually major-philic, whereas at the center of an N8-set site more often minor-philic. Analysis of phages 434, P22 lambda and trp operators revealed similar results. In conclusion, our data show that CAP binding sites have major-philic and minor-philic dimers at specific positions; the location of these dimers may facilitate wrapping of DNA around CAP. A similar pattern is seen in nucleosomes.

Base Sequence↗

PubMed1990

HIV-1 reverse transcriptase: structure predictions for the polymerase domain.

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.

Amino Acid Sequence↗

PubMed1988

Plasmodium fragile: detection of a ring-infected erythrocyte surface antigen (RESA).

A ring-infected erythrocyte surface antigen (RESA) has been detected by modified immunofluorescence assay in erythrocytes infected with the simian malaria parasite, Plasmodium fragile. This RESA, of Mr 95,000, shares many characteristics with the RESA initially found in the human malaria parasite P. falciparum. Both antigens are found in the membrane of erythrocytes infected with young asexual parasite stages, in merozoite-enriched preparations, and in parasite culture supernatant. Since the RESA of P. falciparum has been shown to confer protective immunity and since P. fragile infection of rhesus monkeys mimics P. falciparum infection in humans, the finding of a RESA in P. fragile underlines the importance of this species as an animal model for antimalarial vaccines.

Animals↗

PubMed1987

Detection of antibodies in human sera to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum using the synthetic peptide (NANP)3 in an enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibody in human sera to a synthetic peptide, Asn-Ala-Asn-Pro (NANP)3, derived from the repeating amino acid sequence found in the surface circumsporozoite protein of Plasmodium falciparum sporozoites. One hundred four sera from U.S. residents were used to determine a cut-off value for reactivity. Test sera were considered reactive when the absorbance was greater than that at the 95th percentile of the control sera. Sera from 112 Kenyans living in an area of holoendemic malaria transmission were tested. Of the total number of sera, 65% had detectable antibody to (NANP)3. The percentage of reactive sera increased from 41% in sera from children under 4 years of age to 85% in sera from adults 20 to 39 years of age. The high exposure to malaria parasites of the Kenyans was reflected in indirect fluorescent antibody assay titers to blood stage P. falciparum parasites. All of the Kenyan sera had antibody present at titers greater than 1:256.

Adolescent↗

PubMed1986

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.

Amodiaquine↗

PubMed1985

Evaluation of sporontocidal compounds using Plasmodium falciparum gametocytes produced in vitro.

A test system that uses infective gametocytes from in vitro cultures was developed for evaluating the sporontocidal activity of antimalarial compounds. In evaluating the system, pyrimethamine and cycloguanil (dihydrofolate reductase inhibitors) and primaquine (8-aminoquinoline) were tested against pyrimethamine-sensitive and pyrimethamine-resistant strains of Plasmodium falciparum. The drugs were administered to Anopheles either in a blood meal containing infective gametocytes or in a noninfective meal 2-4 days later. The mosquitoes were dissected 9-10 days after they received the infective blood meal, and the sporontocidal effect of the drugs was evaluated by the number of oocysts found in the gut. Both cycloguanil and pyrimethamine had marked sporontocidal activity. The susceptibility pattern of the strains to the sporontocidal effect of pyrimethamine and cycloguanil was similar to the susceptibility of their asexual blood stages in vitro to the schizontocidal effect of the compounds. The sporontocidal effect was observed only when the compounds were administered at the same time as the infective blood meal, but not when they were given 2-4 days later. No sporontocidal activity was observed with primaquine. This system permits more reliable quantitative observations than have been possible with previous methods.

Anopheles↗

PubMedDate not supplied

Finger-loops, oncogenes, and metals. Claude Passmore Brown memorial lecture.

Certain DNA-binding proteins that regulate gene expression contain single or multiple copies of short polypeptide sequences, approximately 30 residues long, consisting of combinations of four Cys or His residues at defined spacing, so that Zn++ is complexed in tetrahedral coordination with the respective thiol-sulfur and/or imidazole-nitrogen atoms. The Zn++ ion evidently serves as a strut that stabilizes folding of the domain into a 'finger-loop', which is capable of site-specific binding to double-stranded DNA. This article reviews the evidence (a) that finger-loop domains have been highly conserved during evolution, (b) that they furnish one of the fundamental mechanisms for regulating gene expression, and (c) that a metal ion (e.g., Zn++) is required for binding of finger-loops to DNA and for their biological functions. The authors' search of amino acid sequences of 38 transforming proteins identified possible finger-loop domains in the myc, fms, fps, raf-1, rfp, src, syn, yes, erbA, int-1, and TGF-alpha gene-products. The search incidentally revealed possible finger-loop domains in human insulin receptor, which may provide a mechanistic explanation for recent observations that insulin, after binding to its cell surface receptor, is translocated to hepatocyte nuclei and becomes bound to chromatin. Zn++-coordination sites in finger-loop domains are proposed as potential targets for metal toxicity; substitution of Ni++, Co++, or Cd++ for Zn++ in finger-loops of transforming proteins is suggested as an hypothetical mechanism for metal carcinogenesis.

Amino Acid Sequence↗