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Biomedical subjects

M Carraway

Publications and source records attributed to M Carraway.

12 recordsLinked to original sources
PubMed2004

Superoxide dismutase responds to hyperoxia in rat hippocampus.

The brain's anti-oxidant response to highly elevated oxygen (O2) partial pressures is poorly understood. In this study we hypothesized that hyperbaric O2 (HBO2) would stimulate superoxide dismutase (SOD) transcription in the oxidative stress-sensitive rat hippocampus and measured the time course and extent of the changes in hippocampal mRNA for all three SOD isoforms and total SOD enzyme activity. Comparisons were made between exposures to 2 hours of 1 atmosphere pressure normobaric oxygen (NBO); 2 hours of 3 atmospheres HBO2; and room air. Hyperoxia (HBO2 > NBO) was associated with statistically significant increases in transcript levels of the antioxidant enzymes SOD2 (MnSOD) and SOD3 (EC-SOD) at 6 and 18 hours but not SOD1 (Cu, Zn SOD) respectively. Hyperoxia, however, did not affect total hippocampal SOD activity measured at 6 and 24 hours, indicating that the mRNA responses were necessary to maintain the anti-oxidant enzyme activity after oxidative stress.

Animals↗
PubMed2000

Specificity of lipid incorporation is determined by sequences in the N-terminal 37 of apoB.

The N-terminal 17% of apolipoprotein B (apoB-17) is secreted lipid-poor while apoB-41 particles are secreted with a triacylglycerol (TAG)-rich core. Thus, the sequence between apoB-17 and apoB-41 is necessary for the assembly of TAG-rich lipoproteins. To delineate this region, C127 cells were permanently transfected to secrete the N-terminal 29, 32.5, or 37% of apoB. Density gradient centrifugation showed that secreted apoB-29, apoB-32.5, and apoB-37 had peak densities of 1.25, 1.22, and 1.16 g/mL and percent lipid of particle weights of 30, 37, and 49%, respectively. Calculated anhydrous particle diameters were: apoB-29 = 81 A, apoB-32.5 = 88 A, and apoB-37 = 101 A. Immunoprecipitated particles labeled with [(3)H]oleate showed that, as apoB length increased from apoB-29 to apoB-32.5 and apoB-37, the number of TAG (core) molecules per apoB particle increased almost 16-fold from 8 to 32 to 124, while phospholipids and diacylglycerols (surface lipids) increased only slightly from 71 to 87 to 97 molecules, respectively. Thus, sequences in the C-terminus of apoB-29 bind phospholipids and diacylglycerols, sequences between apoB-29 and apoB-32.5 augment TAG binding and sequences between apoB-32.5 and apoB-41 account for the marked incorporation of TAG at a rate of approximately 1 TAG per 2 amino acids. Cryoelectron micrographs of isolated apoB-37 particles revealed mostly spherical particles of approximately 110 A (11.0 nm) with an electron lucent center, consistent with these particles having a TAG core. We suggest that the predicted amphipathic beta-sheets beginning at apoB-29, starts to preferentially recruit core lipids into apoB and propose that the consistent presence of DAG in the secreted particles may have a role in fission of the nascent lipoprotein particles from the endoplasmic reticulum membrane.

Apolipoproteins B↗
PubMed1997

A new restriction fragment length polymorphism from Cryptosporidium parvum identifies genetically heterogeneous parasite populations and genotypic changes following transmission from bovine to human hosts.

Length and restriction site polymorphism within a 2.8-kb threonine-rich open reading frame from Cryptosporidium parvum was identified and used to determine the genotypes of isolates from calves and humans. In agreement with observations of other genetic loci, all calf isolates were identical at this locus. In contrast, human isolates showed two profiles, one found exclusively in humans and one a superposition of both profiles, which were indicative of heterogeneous parasite populations. PCR fingerprints were consistent with a change in the genetic profile of C. parvum isolates following transmission from bovine to human hosts.

Amino Acid Sequence↗
PubMed1996

Water-borne Cryptosporidium: a perspective from the USA.

In the USA, Cryptosporidium parvum has attracted considerable interest due to recent outbreaks of cryptosporidiosis attributed to water supplies contaminated with oocysts. Because of its unprecedented magnitude, the outbreak which occurred in Milwaukee in April 1993 was widely publicized and studied. Information collected during this and other recent outbreaks is being evaluated by regulatory agencies and the water industry with the aim of designing cost-effective measures that will reduce the risk of future water-borne outbreaks, as discussed here by Giovanni Widmer, Margaretha Carraway and Saul Tzipori.

Journal Article↗
PubMed1996

Identification of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat.

Oocysts of the protozoan parasite Cryptosporidium parvum are found in most surface waters and can contaminate municipal water supplies, as demonstrated by recent outbreaks of cryptosporidiosis. A method capable of fingerprinting C. parvum isolates from the environment would facilitate the study of epidemiology and transmission cycles and aid in the implementation of preventive measures to reduce water contamination by oocytes. In this study, we report polymorphism in C. parvum isolates on the basis of analysis of random amplified polymorphic DNA and nucleotide sequences in a region of the 18S rRNA and the internal transcribed spacer 1. Isolate-specific primers for these two regions were designed, and PCR tests capable of discriminating between isolates were developed. In both PCR assays, the five C. parvum isolates analyzed segregated into two subgroups. One group consisted of isolates that originated directly from human patients, and the other group had various host origins and had been propagated in laboratory animals. These results demonstrate the feasibility of distinguishing C. parvum isolates by sequence-specific PCR tests.

Animals↗
PubMed1993

Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli.

The fate of heteroduplex molecules containing 5-, 7-, 9-, 192-, 410-, and 514-base loops after transformation of wild-type and various mutant strains of Escherichia coli has been examined. No evidence for repair was obtained for the wild type or for strains with mutations in the following genes: mutS, recA, recBC sbcBC, recD, recF, recJ, recN, recO, recR, recBC sbcBC recF uvrA, recG ruvC, ruvB, lexA3, lexA51, uvrA, nfo xth nth, polA(Ts), or pcnB. These results rule out the involvement of the SOS system and most known recombination and repair pathways. Repair of heteroduplex molecules containing 410- and 514-base loops was observed when a 1-base deletion-insertion mismatch was present nearby. The repair of both the mismatch and the loops was directed by the state of dam methylation of the DNA chains and was dependent on the product of the mutS gene. A high efficiency of repair (95%) was found even when the mismatch and the loops were 1,448 nucleotides apart. We conclude that multibase loops in DNA can be removed only as a consequence of corepair by dam-directed mismatch repair.

Bacterial Proteins↗
PubMed1990

Mutations produced by DNA polymerase III holoenzyme of Escherichia coli after in vitro synthesis in the absence of single-strand binding protein.

Single-stranded plasmid DNA, containing the mnt gene, was replicated in vitro with DNA polymerase III holoenzyme. Escherichia coli mutH bacteria, defective in mismatch repair, were transformed with the products of in vitro synthesis. Mutations in mnt were readily identified and 33 out of 65 isolates were single base changes including transition, transversion and frameshift mutations. The remaining 32 isolates were deletions of apparently random length and substitutions (deletion/insertions). The intergenic deletions as well as the transition and frameshift mutations were identical to those previously isolated from mismatch repair-defective cells in vivo.

Base Sequence↗
PubMed1987

Spontaneous mutations occur near dam recognition sites in a dam- Escherichia coli host.

The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.

Chromosome Deletion↗
PubMed1986

Analysis of forward mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in the bacteriophage P22 mnt repressor gene.

We describe the isolation and genetic characterization of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations in the phage P22 mnt repressor gene cloned in plasmid pBR322. Mutations in the mnt repressor gene or its operator on this plasmid, pPY98, confer a tetracycline resistance phenotype, whereas the wild-type plasmid confers tetracycline sensitivity. Cells carrying pPY98 were briefly exposed to MNNG to give 20 to 40% survival and a 50- to 100-fold increase in tetracycline-resistant cells. DNA sequence analysis showed that 29 of 30 MNNG-induced mutations were GC-to-AT transitions and one was an AT-to-GC transition. About 80% of the mutations are in three hotspots. This mutation spectrum is consistent with the proposed mechanism of mutagenic action of MNNG, which involves mispairing of an alkylated base, O6-methylguanine. The mnt gene may be a useful target for determining mutagenic specificity at the nucleotide level because forward mutations are easily isolated, the target size is small, and the DNA sequence changes of mutations can be determined rapidly.

Base Sequence↗