Biomedical subjects

H L Callahan

Publications and source records attributed to H L Callahan.

6 recordsLinked to original sources

PubMed1992

A member of the aldoketo reductase family confers methotrexate resistance in Leishmania.

Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.

Amino Acid Sequence↗

PubMed1991

Heavy metal resistance: a new role for P-glycoproteins in Leishmania.

P-glycoproteins are responsible for multidrug resistance in tumor cell lines and are thought to have a physiologic role in exporting cellular metabolites. We now report that a P-glycoprotein gene in the H region of the trypanosomatid protozoan Leishmania confers resistance to heavy metals when present in multiple copies. The Leishmania H region is frequently amplified in drug-resistant lines and is associated with metal resistance. Leishmania expression vectors were used to introduce multiple copies of segments of the Leishmania major H region into wild-type L. major promastigotes. Only constructs bearing a segment of L. major DNA containing the P-glycoprotein lmpgpA conferred arsenite resistance. Deletional analysis of the arsenite-resistant construct mapped resistance to the lmpgpA protein coding region. Lines expressing lmpgpA showed resistance to arsenite and trivalent antimonials, but not to pentavalent antimonials, zinc, cadmium, or the typical multidrug-resistant P-glycoprotein substrates vinblastine and puromycin. Transfection of the Leishmania tarentolae P-glycoprotein homologue ltpgpA resulted in a similar resistance profile. Thus, these pgpAs represent a functionally distinct group of P-glycoproteins which exhibit a substrate specificity similar to prokaryotic heavy metal pumps. Additionally, several arguments suggest that pgpAs may play a role in the susceptibility of Leishmania to clinically utilized antimonials.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Dirofilaria immitis superoxide dismutase: purification and characterization.

Superoxide dismutase (SOD) was purified to apparent homogeneity from Dirofilaria immitis, the causative agent of Dog Heartworm disease which is prevalent in the Southeastern United States. The enzyme has a molecular weight of 18,000 under denaturing conditions with an isoelectric point of 5.6. Both values are similar to those found for previously purified helminth SODs. The amino acid analysis shows greater similarity with mammalian SODs than with the published Schistosoma mansoni SOD, probably because the S. mansoni enzyme appears to be an extracellular, not a cytosolic, SOD. Although SOD activity is easily detected in D. immitis homogenates, the hydrogen peroxide scavenging activities of catalase and glutathione peroxidase were below the limits of our assay. This suggests that D. immitis primary defense against oxidants may be SOD. We feel that this line of research may provide valuable insights into a vulnerable area of D. immitis that may be a good target for drug therapy.

Amino Acids↗

PubMed1990

Hydrogen peroxide is the most toxic oxygen species for Onchocerca cervicalis microfilariae.

The toxicity of the active oxygen species hydrogen peroxide, superoxide radical, hydroxyl radical and singlet oxygen to microfilariae (mf) has been studied in vitro, using active oxygen-generating systems and scavengers/inhibitors. Mf viability was monitored by uptake of the radiolabel, [3H]2-deoxy-D-glucose. Hydrogen peroxide and singlet oxygen, but not superoxide radical or hydroxyl radical, are toxic for mf. Hydrogen peroxide was toxic for mf within 2 h at concentrations as low as 5 microM, an amount eosinophils have been shown to release in vitro (Weiss et al. 1986). Catalase and thiourea, but not inactivated catalase, superoxide dismutase (SOD), singlet oxygen scavengers, or hydroxyl radical scavengers, protected mf. Mf have relatively high levels of endogenous SOD but no measurable glutathione peroxidase and low levels of catalase when compared with other parasites (Callahan, Crouch & James, 1988). The low levels of hydrogen peroxide-scavenging enzymes correlate well with mf sensitivity to hydrogen peroxide and the protective effect of exogenous catalase.

Animals↗

PubMed1989

An in vitro radiolabel uptake viability assay for Onchocerca microfilariae.

A radiolabel uptake viability assay for Onchocerca cervicalis using [3H]2-deoxy-D-glucose in Hanks' balanced salt solution, pH 7.5, at 30 C is described and compared to the traditional visual motility assay. A correlation of r = 0.92 between the assays was found, with the radiolabel uptake method apparently a more sensitive indicator of microfilarial viability.

Animals↗

PubMed1986

Cloning the DdeI restriction-modification system using a two-step method.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.

Bacteriophage lambda↗