Biomedical subjects

M Ernst Schweingruber

Publications and source records attributed to M Ernst Schweingruber.

4 recordsLinked to original sources

PubMed2005

Biosynthesis and uptake of thiamine (vitamin B1) in bloodstream form Trypanosoma brucei brucei and interference of the vitamin with melarsen oxide activity.

Bloodstream forms of Trypanosoma brucei brucei were cultivated in the presence and absence of thiamine (vitamin B1) and pyridoxine (vitamin B6). The vitamins do not change growth behaviour, indicating that Trypanosoma brucei is prototrophic for the two vitamins even though in silico no bona-fide thiamine-biosynthetic genes could be identified in the T. brucei genome. Intracellularly, thiamine is mainly present in its diphosphate form. We were unable to detect significant uptake of [3H]thiamine and structural thiamine analogues such as pyrithiamine, oxithiamine and amprolium were not toxic for the bloodstream forms of T. brucei, indicating that the organism does not have an efficient uptake system for thiamine and its analogues. We have previously shown that, in the fission yeast Saccharomyces pombe, the toxicity of melarsen oxide, the pharmacologically active derivative of the frontline sleeping sickness drug melarsoprol, is abolished by thiamine and the drug is taken up by a thiamine-regulated membrane protein which is responsible for the utilization of thiamine. We show here that thiamine also has weak effects on melarsen oxide-induced growth inhibition and lysis in T. brucei. These effects were consistent with a low affinity of thiamine for the P2 adenosine transporter that is responsible for uptake of melaminophenyl arsenicals in African trypanosomes.

Animals↗

PubMed2004

The melaminophenyl arsenicals melarsoprol and melarsen oxide interfere with thiamine metabolism in the fission yeast Schizosaccharomyces pombe.

The melaminophenyl arsenical melarsoprol is the main drug used against late-stage sleeping sickness caused by Trypanosoma brucei subspecies. Its active metabolite in the human body is melarsen oxide. Here, it is shown that this metabolite inhibits growth of the fission yeast Schizosaccharomyces pombe and that its toxicity can be abolished efficiently by thiamine (vitamin B(1)), thiamine analogues, and the pyrimidine moiety of the thiamine molecule. Uptake of melarsen oxide is mediated by a membrane protein (car1p), which is involved in the uptake of thiamine and its pyrimidine moiety. Melarsoprol is taken up by cells in a thiamine- and car1p-dependent manner but is not toxic to cells.

Arsenicals↗

PubMed2003

Ksg1, a homologue of the phosphoinositide-dependent protein kinase 1, controls cell wall integrity in Schizosaccharomyces pombe.

It has previously been shown that the Schizosaccharomyces pombe mutant ksg1-358 has a mating and sporulation defect at 30 degrees C and that it is temperature sensitive for growth at 35 degrees C. However the molecular basis for these phenotypes remained largely unknown. In this study we show that ksg1-358 mutant cells lysed at the non-permissive temperature, which could be prevented by sorbitol. Overexpression of ksg1 using the nmt1-promoter showed slow growth and cells became swollen when incubated at 35 degrees C under low inositol conditions. Interestingly, in a two-hybrid assay we found that the ksg1-protein interacted with Pck1p, a protein implicated in regulating cell wall integrity in S. pombe. Genetic complementation assays showed that an overexpression of pck2, the homologue of pck1 involved in the regulation of cell wall synthesis, could partially rescue ksg1-358 phenotypes. We digested the ksg1-358 cell wall using beta-glucanase. We found that the ksg1-358 mutant was more resistant to cell lysis at 30 degrees C than the wildtype strain h972, which was similar to a pck1-deletion strain. A ksg1-overexpressing strain was hypersensitive towards beta-glucanase treatment similar to a pck2-deletion strain. The pck1-deletion partially rescued beta-glucanase hypersensitivity of the ksg1-overexpressing strain but the pck2-deletion increased it. The ksg1-358 mutation increased beta-glucanase resistance of a pck1-overexpressing strain but it had no effect on a pck2-overexpressing strain. Our results provide evidence that ksg1 is a novel regulator of cell wall integrity in the fission yeast Schizosaccharomyces pombe. They further suggest that Ksg1p acts in a pathway with Pck1p, possibly upstream and through direct interaction.

Cell Wall↗

PubMed2002

Two non-complementing genes encoding enzymatically active methylenetetrahydrofolate reductases control methionine requirement in fission yeast Schizosaccharomyces pombe.

By transforming two methionine auxotrophic mutants from fission yeast Schizosaccharomyces pombe with a wild-type gene library, we defined two genes, met9 and met11, which both encode a methylenetetrahydrofolate reductase. The genes cannot complement each other. We detected single transcripts for both. In vitro measurements of enzymatic activities showed that the met11-encoded enzyme was responsible for only 15-20% of the total methylenetetrahydrofolate reductase activity. A strain in which gene met9 was disrupted required significantly more methionine for full growth and efficient mating and sporulation than the strain disrupted for gene met11. The in vitro and in vivo data thus indicated that met9 was the major expressed gene. Our results are in accordance with the assumption that the two methylenetetrahydrofolate reductases generate the methyl groups necessary for methionine synthetase to convert homocysteine to methionine, and suggest that expression of the two genes is an important parameter in the control of methionine biosynthesis.

Blotting, Northern↗