PubMed Health⌕ Search

Biomedical subjects

R D Klein

Publications and source records attributed to R D Klein.

At least 55 records · Page 3Linked to original sources
PubMed1993

Haemonchus contortus: ivermectin-induced paralysis of the pharynx.

How the avermectins cause the elimination of gastrointestinal nematodes from host animals has not yet been clearly identified. Using visual and radiometric parameters to measure oral ingestion in Haemonchus contortus, we showed that ivermectin (IVM) rapidly inhibited ingestion at concentrations > or = 10(-10) M. Motility, monitored quantitatively with an automated motility meter, was unaffected by IVM at concentrations < or = 10(-8) M, while ATP levels were unaffected at concentrations < or = 10(-6) M. Since motility and ATP levels, independent measures of short-term viability, are unaffected by concentrations of IVM that effectively block oral ingestion, the drug can be used as a chemical ligature. Although H. contortus was shown to be dependent upon an exogenous supply of glucose for survival in culture, IVM (10(-9) and 10(-7) M) altered neither the uptake of 3-O-[3H]methylglucose nor the metabolism of [13C]glucose by the parasite. These data suggest that H. contortus depends upon the transcuticular uptake of glucose in culture. If oral ingestion of other nutrients is essential for long-term survival in vivo, disruption of this process may represent the primary mechanism of IVM action.

3-O-Methylglucose↗
PubMed1993

Ascaris suum: cloning of a cDNA encoding phosphoenolpyruvate carboxykinase.

A cDNA encoding phosphoenolpyruvate carboxykinase (PEPCK) from Ascaris suum was cloned by complementation of a strain of Escherichia coli deficient in PEPCK and malic enzyme. The product of this cDNA was enzymatically similar to a recombinant PEPCK obtained from Haemonchus contortus by the same method. Comparison of the predicted amino acid sequence of A. suum PEPCK with other PEPCKs showed that this enzyme is most closely related to the H. contortus enzyme. The two nematode enzymes share considerable homology in regions thought to be functionally involved in substrate binding and catalysis, some of which distinguish the nematode enzymes from PEPCKs from other organisms. This analysis suggests a structural explanation for the kinetic differences seen between nematode and vertebrate PEPCKs and supports the hypothesis that nematode PEPCK is a target for selective inhibition.

Amino Acid Sequence↗
PubMed1993

Expression of cloned beta-tubulin genes of Haemonchus contortus in Escherichia coli: interaction of recombinant beta-tubulin with native tubulin and mebendazole.

Two distinct beta-tubulin cDNA isotypes (beta 8-9 and beta 12-16) from Haemonchus contortus were expressed for the first time in Escherichia coli and characterised by their specific mebendazole (MBZ) binding and polymerization properties. Beta-tubulin was expressed without translational fusion to an E. coli sequence under the regulation of the tryptophan promoter in the pTrp2 vector. Beta-tubulin was produced in large amounts in insoluble 'inclusion bodies'. The inclusion bodies were purified and solubilised and the beta-tubulin renatured by treatment with urea followed by dilution with alkaline buffer and a shift to physiological pH. The yield was more than 10 mg of beta-tubulin per litre of cell culture. The recombinant tubulin produced was recognized in Western blot by specific anti-beta-tubulin antibodies. Tritiated MBZ binding to the recombinant H. contortus beta-tubulin was measured in the presence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [3H]MBZ high-affinity binding (HB) to 'pure' (no other eukaryotic protein present) beta 8-9 or beta 12-16 was observed. Enhanced high-affinity binding was observed when recombinant beta 8-9 or beta 12-16 were mixed and pre-incubated with whole supernatants or tubulin-enriched extracts from H. contortus. The enhancement was more than additive. Beta 12-16 bound more MBZ and caused a greater enhancement than beta 8-9. Mixing recombinant beta 8-9 or beta 12-16 with whole supernatants or tubulin-enriched fractions from H. contortus promoter polymerization at 37 degrees C. Use of 35S-labelled protein showed that the polymer contained recombinant tubulin. Western blot using specific anti-alpha-tubulin monoclonal antibodies showed that the polymer contained alpha-tubulin. Similarly the recombinant nematode beta-tubulin co-polymerized with tubulin from chicken brain. Our data suggest that the recombinant beta-tubulin can interact and copolymerize with parasite or chicken tubulin. Furthermore the interaction of recombinant nematode beta-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-binding sites. However, interaction of recombinant beta-tubulin with microtubule proteins from chicken brain did not result in the formation of high-affinity MBZ-binding sites.

Animals↗
PubMed1993

Feedback control of sex determination by dosage compensation revealed through Caenorhabditis elegans sdc-3 mutations.

In Caenorhabditis elegans, sex determination and dosage compensation are coordinately controlled through a group of genes that respond to the primary sex determination signal. Here we describe a new gene, sdc-3, that also controls these processes. In contrast to previously described genes, the sex determination and dosage compensation activities of sdc-3 are separately mutable, indicating that they function independently. Paradoxically, the sdc-3 null phenotype fails to reveal the role of sdc-3 in sex determination: sdc-3 null mutations that lack both activities disrupt dosage compensation but cause no overt sexual transformation. We demonstrate that the dosage compensation defect of sdc-3 null alleles suppresses their sex determination defect. This self-suppression phenomenon provides a striking example of how a disruption in dosage compensation can affect sexual fate. We propose that the suppression occurs via a feedback mechanism that acts at an early regulatory step in the sex determination pathway to promote proper sexual identity.

Alleles↗
PubMed1992

Cloning of a cDNA encoding phosphoenolpyruvate carboxykinase from Haemonchus contortus.

Biochemical and metabolic data have led to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. To facilitate the determination of the molecular basis for host vs parasite differences in PEPCK, we have cloned a cDNA encoding this enzyme from a parasitic nematode of ruminants, Haemonchus contortus. H. contortus PEPCK was cloned by functional complementation of a PEPCK-, malic enzyme- strain of Escherichia coli (E1786) using an egg stage H. contortus cDNA library in lambda ZAPII. Selection was for growth on malate as the sole carbon source (malate+ phenotype). We isolated a plasmid, pPEPCK, which reproducibly confers a malate+ phenotype in E1786. The sequence of the 2.0-kb EcoRI insert of pPEPCK predicts a 612-amino acid protein which shows about 74% similarity to Drosophila melanogaster and chicken PEPCK. Extracts of E1786[pPEPCK], but not E1786, contain IDP- or GDP-dependent PEPCK enzyme activity. Sequence analysis revealed that the open reading frame (ORF) in pPEPCK lacked a 5' initiation codon and was probably expressed as an in-frame fusion protein with beta-galactosidase. A strategy combining library screening with PCR analysis of positive clones led to the identification of a clone encoding 6 additional NH2-terminal amino acids, including a Met, which, by comparison with known PEPCK amino acid sequences, is likely to be the translation initiation site.

Animals↗
PubMed1992

Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus.

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of beta-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian beta-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of beta-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three beta-tubulin cDNAs from this organism, beta 12-16, beta 12-164, and beta 8-9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes. beta 8-9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode beta-tubulins with mammalian beta-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to beta 8-9 and beta 12-16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to beta 8-9.

Amino Acid Sequence↗
PubMed1992

The nervous systems of helminths as targets for drugs.

Processes that critically differentiate parasitic helminths and their hosts are obvious candidates for chemotherapeutic intervention. The recognition that neurobiology distinguishes helminths from their vertebrate hosts is due in part to the fact that several efficacious anthelmintics, derived generally from empirical screening, have been found to act selectively on the neuromuscular system of these parasites. In addition, basic physiological and pharmacological research has revealed considerable differences in the ways in which helminths and their hosts transmit information in the nervous system and respond to it in innervated tissues. Unfortunately, most of these differences have yet to be exploited in chemotherapy. The topics for this review include an analysis of mechanistic aspects of the pharmacology of anthelmintics that act on neuromuscular systems and a consideration of the prospects for discovery of novel drugs that act on this system.

Animals↗
PubMed1991

A DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis can be maintained as an extrachromosomal element.

A 4.05-kb DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis was cloned into the pUC19 vector. When an ade2 strain of Sc. occidentalis was transformed with this plasmid, pADE-2 was found to integrate into the host chromosome and was also present in a variety of extrachromosomal species. These extrachromosomal elements were present in multiple copies, varied in molecular mass and were composed of polymerized forms of pADE-2. A fragment containing the ADE2 gene was used to transform a Sc. occidentalis ade2 mutant, as either a linear or circularized molecule. The linear form integrated into the host genome, whereas the circularized form was found as a stably maintained extrachromosomal element with no evidence of integration or detectable loss of the Ade+ phenotype upon subculturing of transformed yeast under nonselective conditions for 60 generations. The ratio of the number of extrachromosomal ADE2 genes to genomic ADE2 ranged from 3.8 to 6.6.

Blotting, Southern↗
PubMed1991

Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.

Amino Acid Sequence↗
PubMed1991

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Amino Acid Sequence↗
PubMed1991

Quantitative review of studies of dietary fat and rat colon carcinoma.

A quantitative overview of 14 studies of rat colon carcinogenesis was undertaken to examine the relationship between fat intake, and fat intake by degree of saturation, on the incidence of colon carcinoma while controlling for calorie consumption. Calorie consumption was not recorded in 11 of the 14 studies. Hence, two types of analyses were conducted. The first examines carcinoma incidence as a function of percent fat (by weight), with calories controlled for by including weight gain per week in the analysis. The second estimates calories per day, for studies not providing such information, using weight gain per week and age at death, followed by a joint analysis of estimated fat calories and estimated total calories. With either approach, a rather strong positive relationship between colon carcinoma incidence and fat intake is indicated for Fischer 344 rats, but no association is apparent for Sprague-Dawley rats. This situation is somewhat clarified when the degree of saturation is taken into account: both strains gave results that suggest a negative relationship between colon cancer incidence and omega-3 fatty acids intake and a positive relationship with non-omega-3 polyunsaturated fat intake among Fischer 344 rats. These analyses suggest an important and specific role for dietary fat in the promotion of rat colon carcinoma.

Animals↗
PubMed1989

Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis.

The gene encoding invertase (INV) has been cloned from Schwanniomyces occidentalis. The enzyme consists of 533 amino acids, 8 potential glycosylation sites and has a 45% identity with the invertase from Saccharomyces cerevisiae. The proenzyme has a 22 amino acid signal sequence that has a high alpha-helical transmembrane potential which differs significantly from that predicted for the Saccharomyces cerevisiae enzyme.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis.

The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a beta-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/- 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/- 0.025 units, a Km of 21 +/- 1.5 mM, and temperature and pH optima of 55 degrees C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.

Amino Acid Sequence↗
PubMed1988

Cloning of the orotidine 5'-phosphate decarboxylase (ODC) gene of Schwanniomyces occidentalis by complementation of the ura3 mutation in S. cerevisiae.

A DNA fragment containing the gene encoding orotidine 5'-phosphate decarboxylase (ODC) from the yeast, Schwanniomyces occidentalis (formerly castellii) has been isolated from a genomic library constructed in the S. cerevisiae expression vector, pYcDE8. A recombinant plasmid, p2-1A, containing a 2.47 kb insert was shown to complement the ura3-52 mutation of several strains of S. cerevisiae. This DNA insert was shown to be from Schwanniomyces occidentalis by Southern hybridization analysis. A restriction enzyme cleavage map of the insert has been derived and the ODC gene localized to a 1.1 kb region by deletion analysis. In addition, we have demonstrated that expression of ODC is not dependent on the ADH1 promoter carried on pYcDE8. This is the first report of the cloning of a gene from a member of the genus Schwanniomyces.

Carboxy-Lyases↗
PubMed1988

Secreted amylolytic enzymes from Schwanniomyces occidentalis: purification by fast protein liquid chromatography (FPLC) and preliminary characterization.

Amylolytic enzyme preparations are used extensively for the liquefaction and saccharification of starch in the production of ethanol and SCP (single cell protein). We report the first purification of two amylolytic enzymes from the yeast Schwanniomyces occidentalis using fast protein liquid chromatography (FPLC) in a two step process: size exclusion (Superose 12) followed by anion exchange (Mono Q). The procedure is amenable to direct scale up processes. The enzymes glucoamylase (E.C. 3.2.1.2) and alpha-amylase (E.C. 3.2.1.1) were found in the cell free supernatant of S. occidentalis when grown on a variety of carbon sources. The enzymes are substrate induced and catabolite repressed. Both amylolytic enzymes were purified from three separate culture broths containing either starch, maltose or cellobiose and their physical properties compared. Native molecular masses of glucoamylase and alpha-amylase were determined to be 122,000 +/- 28,000 daltons and 47,000 +/- 11,000 daltons, respectively, while subunit size was approximated at 143,000 +/- 2,000 daltons and 54,500 +/- 1,000 daltons, respectively. Both proteins are N-glycosylated with carbohydrate representing 10-15% of the total mass. The correlation of native mass and denatured subunit structure, while not identical due to slight aberrant behavior on gels and columns as a result of glycosylation, suggest that both proteins exist as monomeric polypeptides. Isoelectric points for both proteins under native conditions could not be determined since alpha-amylase failed to enter native polyacrylamide gels. However, a pI for glucoamylase of 6.2 +/- 0.2 (native) and a pI for alpha-amylase of 6.3 +/- 0.3 (in 6M urea) were determined. Glucoamylase and alpha-amylase specific activities (for the homogeneous proteins) were determined to be 48-67 x 10(3) units/mg and 214-457 x 10(3) units/mg respectively. We could find no apparent differences in either glucoamylase or alpha-amylase proteins obtained from three separate cultures which had been grown on different carbon sources. The purification method we have utilized is easily scaled up to larger protein concentrations, and provides a rapid procedure for analyzing and purifying these amylolytic enzymes.

Amylases↗