Progressive formation of DNA lesions during treatment with anti-metabolites without incorporation of the drugs into DNA

Anti-metabolites, such as methotrexate, 5-fluoropyrimidines or hydroxyurea, induced progressive formation of DNA lesions. 5-Fluoropyrimidines induce DNA lesions either by incorporation of the drug into DNA or by a mechanism not involving incorporation. The second mechanism, not involving incorporation, is also seen with methotrexate and hydroxyurea. The three anti-metabolites have in common their ability to reduce intracellular levels of nucleotides, resulting in reduced efficiency of repair of DNA lesions. The lesions probably appear spontaneously, independently of the drug treatment.     

S-adenosylmethionine decarboxylase from Leishmania donovani. Molecular, genetic, and biochemical characterization of null mutants and overproducers.

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Delta adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.     

Aspects of floral structure and phenology in the genusConophytum (Mesembryanthemaceae)

The complex floral structure in the southern African genusConophytum (Mesembryanthemaceae; 77 spp.) is described in detail and assigned to three basic floral types, two of which can be divided into two subtypes. Correlations between structural features and phenological patterns are demonstrated and discussed in the family context as well as in relation to the systematic subdivision of the genus.     

Physiological MR signal variations within the brain at 3 T.

The echoplanar technique in magnetic resonance (MR) imaging allows the acquisition of a series of images from a selected slice with a temporal resolution of 10/s. Simultaneous recording of physiological information on pulse and respiration allows correlation of the MR signal intensity with physiological signals, which can be obtained for each pixel examined. Such correlations can be found within the cerebrospinal fluid (CSF) spaces and within vessels if a flow-sensitive MR measurement technique is used. The use of an MR scanner with a field strength of 3 T improves the signal/noise ratio, but there is a stronger signal decay due to local magnetic inhomogeneities. This study shows that 3-T systems can be used for correlation of MR and physiological signals and that clear differentiation between signals from CSF and from vessels can be obtained due to their strongly different signal decays.     

Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis

The “cysteine string protein” (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a “J” domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the “tall cells” of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.     

Renal Excretion of N4-Acetyl Sulphanilamide and N4-Acetyl Sulphadimidine in Goats

The renal excretion of N4-acetyl sulphanilamide and N4-acetyl sulphadimidine was studied in 19 experiments with 6 goats during continuous intravenous administration of the 2 sulphonamide derivatives. Deacetylation of both compounds takes place to a small extent only. Further it is shown that both sulphonamide derivatives are bound to plasma proteins to a greater extent than sulphanilamide and sulphadimidine. The excretion of the N4-acetylated sulphonamides is compared with the renal excretion of creatinine. The non-protein-bound fraction of the 2 N4-acetylated sulphonamides is excreted by filtration and active tubular secretion. The renal clearances of the acetyl derivatives are higher than those of the parent compounds.     

Skeleton binding protein-1-mediated parasite sequestration inhibits spontaneous resolution of malaria-associated acute respiratory distress syndrome

Malaria is a hazardous disease caused by Plasmodium parasites and often results in lethal complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Parasite sequestration in the microvasculature is often observed, but its role in malaria pathogenesis and complications is still incompletely understood. We used skeleton binding protein-1 (SBP-1) KO parasites to study the role of sequestration in experimental MA-ARDS. The sequestration-deficiency of these SBP-1 KO parasites was confirmed with bioluminescence imaging and by measuring parasite accumulation in the lungs with RT-qPCR. The SBP-1 KO parasites induced similar lung pathology in the early stage of experimental MA-ARDS compared to wildtype (WT) parasites. Strikingly, the lung pathology resolved subsequently in more than 60% of the SBP-1 KO infected mice, resulting in prolonged survival despite the continuous presence of the parasite. This spontaneous disease resolution was associated with decreased inflammatory cytokine expression measured by RT-qPCR and lower expression of cytotoxic markers in pathogenic CD8⁺ T cells in the lungs of SBP-1 KO infected mice. These data suggest that SBP-1-mediated parasite sequestration and subsequent high parasite load are not essential for the development of experimental MA-ARDS but inhibit the resolution of the disease.