Biomedical subjects
J M Kelly
Publications and source records attributed to J M Kelly.
Evidence of genetic recombination in Leishmania.
In the genus Leishmania there has been no convincing demonstration of genetic exchange, and it has been proposed that reproduction is clonal. However, preliminary characterization of two strains of Leishmania isolated from wild animals in a zoonotic focus of cutaneous leishmaniasis in the Eastern Province of Saudi Arabia, has suggested that they may represent hybrids of Leishmania major and Leishmania arabica. Evidence presented here strongly supports this hypothesis. Isoenzyme analysis and molecular karyotyping of cloned organisms indicated that the putative hybrids are distinct from other species of Leishmania, and possess characteristics of both L. major and L. arabica. Experiments using highly specific probes demonstrated that kinetoplast minicircle DNA from the putative hybrid contained L. major-specific, but not L. arabica-specific sequences. DNA fingerprinting data obtained using 6 genomic DNA probes were consistent in all cases with a L. major/L. arabica recombinant genotype, and implied both diploidy and allelic segregation. These observations suggest that sexual reproduction may generate genetic diversity within natural Leishmania populations.
A sensitive repetitive DNA probe that is specific to the Leishmania donovani complex and its use as an epidemiological and diagnostic reagent.
Leishmania donovani (HU3 strain) metacyclic promastigotes generated in vitro were used to construct a cDNA library in the bacteriophage vector lambda gt10. A cDNA clone (Lmet 2), was isolated by differential screening with metacyclic-derived or log-phase promastigote-derived cDNA. The clone insert was comprised predominantly of four copies of an imperfect 60-bp repeat motif, which was represented in the genome by multiple tandem repeats distributed among at least six chromosomes. The corresponding mRNA transcript was a developmentally regulated 12-kb doublet. The Lmet 2 sequence was entirely specific to L. donovani donovani, L. donovani (East Africa), L. donovani infantum and L. donovani chagasi, even when genomic Southern blots and slot blots of other Leishmania species were washed at low stringencies. Twenty-two strains of L. donovani were clearly detected by radiolabelled Lmet 2 cDNA probe, with signals of approximately equal intensity, irrespective of geographical origin, which encompassed widely dispersed endemic regions. The probe could detect DNA from fewer than 100 organisms and identified small numbers of promastigotes in infected sand flies. Amastigotes were also detected in impression smears of organs from infected hamsters. The Lmet 2 probe is likely to be a valuable reagent for clinical diagnosis and epidemiological investigations.
Syntheses of tolrestat analogues containing additional substituents in the ring and their evaluation as aldose reductase inhibitors. Identification of potent, orally active 2-fluoro derivatives.
A series of aldose reductase inhibitors were prepared which were analogues of the potent, orally active inhibitor tolrestat (1). These compounds (5, 7, 9, and 10) have an extra substituent on one of the unoccupied positions on the naphthalene ring of 1. Primary amide prodrugs of several members from the series 5 and 7, namely 6 and 8, respectively, were also prepared. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. In general, compounds in series 5, 7, 9, and 10 were potent inhibitors of bovine lens aldose reductase. 2-Halo-substituted analogues from the series 5, 7, and 9 exhibited high activity in the nerve of the 4-day-galactose-fed rat, and in several instances, the primary amide prodrug 8 enhanced the in vivo potency of the respective carboxylic acid 7. Two 2-fluoro-derivatives, 8a and 9a, had especially high activity in vivo and were chosen for additional studies. These compounds were found to be approximately equipotent to tolrestat in the sciatic nerve of the galactose-fed rat and the STZ rat, as judged by their ED50's in these assays. Although primary amide analogue 8a did not have intrinsic inhibitory activity toward aldose reductase, it was metabolized to an active form in vivo and also in vitro within the sciatic nerve.
Characterization of monoclonal antibodies to a rat liver vasopressin receptor.
1. Balb/c mice were immunized against a vasopressin binding protein purified from rat liver. The hybrids produced from two cell fusions were screened against this receptor. Three hybrids were selected, cloned and expanded in serum-free media. The monoclonal antibodies (MoAb) secreted by these three hybrids were of the subclass IgM and were able to immunoprecipitate [125I]-labelled purified receptor. 2. All three MoAb bound to the purified solubilized receptor, crude liver and kidney membranes in a concentration-dependent manner. However, the binding of MoAb to the membranes did not inhibit the binding of [125I]-[d(CH2)5,Sar7]AVP, a selective V1 receptor radioligand, to the liver membrane-bound receptor. 3. These results suggest that the three MoAb recognize epitopes on the V1 receptor which are not denatured by solubilization, but are common to both rat liver and kidney membranes.
Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans.
The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented. The gene contains no introns. The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein. The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein. A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition. Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation. This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.
Nitrous oxide and internal tamponade during vitrectomy.
We analysed the nitrous oxide composition of the intraocular gas bubble following vitrectomy and fluid-air exchange in 12 patients. Samples were taken under standardised conditions at 20 minutes after completion of the fluid-air exchange. Analysis was conducted by a Medishield MS2 mass spectrometer. The percentage composition of nitrous oxide in the samples varied between 4 and 21% (mean 9%). This influx of nitrous oxide was associated with an increase of intraocular pressure. Pressure rises of greater than 20 mm Hg were not seen owing to venting of gas through the sclerostomies. An inverse relationship was noted between the extent of retinal detachment preoperatively and the amount of nitrous oxide entering the eye. A possible explanation for this relationship is proposed. The importance of nitrous oxide movement is stressed.
Cellular energy metabolism and regulation.
Consistent with the increased demand for nutrients imposed by lactation and growth, those tissues directly involved in the digestion, absorption, and processing of the required additional nutrients show response to these states. During lactation, the rumen, upper intestine, and liver increase in size, and more energy is spent on Na+,K+ transport and on protein turnover. The massive endocrine influences during lactation suggest that the metabolism of other tissues besides these and mammary tissue would be influenced, but evidence is rather sparse. Ion transport and protein metabolism in some muscles may indeed be increased. Although substrate cycles characteristically account for a substantially smaller portion of the energy expenditure in the intact animal than do ion transport and protein turnover, stage of lactation influences some of these cycles, particularly the triacylglycerol fatty acid cycle. The needs for additional quantitative in vivo measurements of metabolic conversions and for mechanistic model description of metabolic events in nonmammary tissues are discussed.
Midwest wrestling study: prediction of minimal weight for high school wrestlers.
This study determined the validity of previously published or newly derived equations to predict fat-free body mass (FFB) in high school wrestlers from the midwestern United States. Five laboratories participated in the data-pooling study (total sample of 860 subjects). Measures included body composition by underwater weighing and anthropometric measurements of body mass, stature, and selected circumferences, diameters, and skinfolds. Cross-validation of selected equations to predict FFB revealed the lowest levels of error from the equations of Lohman, Thorland et al., Katch and McArdle, and Behnke and Wilmore. Modification of the constants in these equations or generation of new equations did not substantially reduce prediction error. Overall, total error for these top equations ranged from 2.44 to 2.59 kg. However, based on observed trends, this error was of lower magnitude with the younger and lighter subjects and of higher magnitude with the older and heavier subjects. We conclude that these equations could be used singularly or collectively to determine FFB, and a minimal weight could then be derived and assigned to a scholastic wrestler.
Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family.
A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.
Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the 'hotspot' topogenic signal model.
The genes which encode glycosomal glyceraldehyde-phosphate dehydrogenase (gGAPDH) of Trypanosoma cruzi are arranged as a tandemly repeated pair on a single chromosome and are identical at the level of nucleotide sequence. They are separated by an intergenic region which contains a 317 base pair sequence with the properties of a retroposon. The genes express a 1.5 kb mRNA and a 38 kd protein. The amino acid sequence contains features characteristic of glycosomal enzymes such as peptide insertions and a C-terminal extension. However, T. cruzi gGAPDH lacks one of the positively charged 'hotspot' motifs which have been proposed as topogenic signals for import into the glycosome, a unique microbody-like organelle. Molecular modelling of the T. cruzi and T. brucei enzymes suggests that neither structure would fulfil the requirements of the 'hotspot' glycosomal import model.
Alcohol dehydrogenase III in Aspergillus nidulans is anaerobically induced and post-transcriptionally regulated.
An alcohol dehydrogenase was shown to be induced in Aspergillus nidulans by periods of anaerobic stress. This alcohol dehydrogenase was shown to correspond to the previously described cryptic enzyme, alcohol dehydrogenase III (McKnight et al. 1985), by analysis of a mutation in the structural gene of alcohol dehydrogenase III, alcC, created by gene disruption. Survival tests on agar plates showed that this enzyme is required for long-term survival under anaerobic conditions. Northern blot analysis and gene fusion studies showed that the expression of the alcC gene is regulated at both the transcriptional and translational levels. Thus there are mechanisms in this filamentous fungus allowing survival under anaerobic stress that are similar to those described in higher plants.
Acyclovir treatment of varicella in otherwise healthy children.
STUDY OBJECTIVE: To determine whether acyclovir administered orally affects the duration and severity of varicella in otherwise normal children. DESIGN: Randomized, placebo-controlled, double-blind trial. SETTING: Patients' residence and university hospital clinic. PATIENTS: One hundred five children between 5 and 16 years of age with laboratory-confirmed varicella entered the study. Of the 102 who were included in the final analysis, 50 received acyclovir and 52 received placebo. INTERVENTION: Placebo or acyclovir was given orally four times daily, for 5 to 7 days. The acyclovir dose was adjusted as follows: 5 to 7 years of age, 20 mg/kg; 7 to 12 years, 15 mg/kg; and 12 to 16 years, 10 mg/kg. MEASUREMENTS AND MAIN RESULTS: Acyclovir recipients, compared with the placebo group, defervesced sooner (median, 1 day vs 2 days; p = 0.001), experienced onset of cutaneous healing sooner, as reflected by a decrease in number of lesions (median, 3 days vs 2 days; p = 0.002), and had fewer skin lesions (median, 500 vs 336; p = 0.02). Acyclovir did not significantly change the rate of complications of varicella (10% in the acyclovir group vs 13.5% among placebo subjects). Adverse drug effects were not observed. Acyclovir recipients had lower geometric mean serum antibody titers to varicella-zoster virus than their placebo counterparts 4 weeks after the onset of illness, but antibody titers in both groups were similar 1 year later. CONCLUSIONS: These results provide evidence that acyclovir is useful and well tolerated for treatment of varicella in otherwise healthy children.
Mapping of B-cell epitopes on the Leishmania donovani 70 kDa heat-shock protein.
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Pepscan and circular-dichroism analysis of a repetitive antigen of Trypanosoma cruzi.
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The trypanothione reductase gene of Leishmania donovani.
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The sodium pump and other mechanisms of thermogenesis in selected tissues.
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Ozone, acidic rain and soil magnesium effects on growth and foliar pigments of Pinus taeda L.
Height and diameter growth, biomass accumulation and leaf pigment concentrations were measured in loblolly pine (Pinus taeda L.) seedlings grown in soil containing 12 or 35 microg Mg g(-1) and exposed from May to October to subambient, ambient, or twice-ambient ozone (O(3)), and to simulated acidic rain with a pH of either 4.0 or 5.3. At the end of one growing season, height and diameter growth of seedlings exposed to twice-ambient O(3) were not statistically different from those of seedlings exposed to subambient O(3). Biomass of all plant parts was reduced by 7 to 16% in response to increasing O(3) concentration. No statistically significant growth responses to rain chemistry or soil magnesium status were observed, and there were no statistically significant interactive treatment effects. Needle pigment concentrations were not significantly affected by rain chemistry or soil Mg status and there were no visible signs of injury to needles that could be attributed to O(3) stress or Mg deficiency. Concentrations of chlorophyll a and b, and carotenes were 23, 30 and 21% higher (P </= 0.05) respectively, in seedlings exposed to twice-ambient O(3) than in seedlings from the other ozone treatments. There were no interactive treatment effects on needle pigment concentrations.