Evidence for two Ca2(+)-mobilizing purinoceptors on rat hepatocytes.

Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration.     

Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites

Abstract: This study examined the hypothesis that chronic ethanol consumption results in significant abnormalities in both the dopaminergic and the serotonergic system of aged rats. Levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) were determined in brain areas of both the nigrostriatal and mesocorticolimbic DA systems in 5-, 14-, and 24-month-old male Fischer 344 rats. Aging was associated with a reduced concentration of DA in the striatum (ST), ventral tegmental area (VTA), and ventral pallidum (VP) and an increased concentration of 5-HIAA in the ST, globus pallidus, nucleus accumbens, frontal cortex, and VP. In addition, there was an increase in the 5-HIAA/5-HT ratio in all brain areas analyzed. Six weeks of ethanol consumption was accompanied by significant changes in mesocorticolimbic brain areas. In the VTA of 5-month-old ethanol-fed rats DA content was decreased to the levels found in aged rats, e.g., 24 months of age. Ethanol also significantly lowered 5-HT and 5-HIAA contents in the VTA and reduced DOPAC and 5-HIAA levels in the VP. In addition, ethanol blunted the normal age-related increase in 5-HIAA/5-HT ratio in the VTA, VP, and substantia nigra. It is interesting that although the age-related changes were found in both nigrostriatal and mesocorticolimbic brain areas, the ethanol-associated effects were found only in brain areas of the mesocorticolimbic system. The changes in DA and 5-HT function that accompany aging and ethanol consumption may contribute to the problems in motor function and ethanol abuse found in the aged.     

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.     

Regiospecificity of chlorophenol reductive dechlorination by vitamin b(12s)

Vitamin B(12), reduced by titanium (III) citrate to vitamin B(12s), catalyzes the reductive dechlorination of chlorophenols. Reductive dechlorination of pentachlorophenol and of all tetrachlorophenol and trichlorophenol isomers was observed. Reaction of various chlorophenols with vitamin B(12) favored reductive dechlorination at positions adjacent to another chlorinated carbon, but chlorines ortho to the hydroxyl group of a phenol were particularly resistant to reductive dechlorination, even if they were also ortho to a chlorine. This resulted in a reductive dechlorination pattern favoring removal of para and meta chlorines, which differs substantially from the pattern exhibited by anaerobic microbial consortia.     

Autonomous replication of foreign DNA in Histoplasma capsulatum: role of native telomeric sequences.

Genetic transformation of the dimorphic pathogenic fungus Histoplasma capsulatum can result in chromosomal integration of the transforming DNA or the generation of multicopy linear plasmids carrying the transforming DNA. We showed previously that Escherichia coli plasmids do not replicate autonomously in H. capsulatum without significant modifications, one of which is the in vivo addition of Histoplasma telomeres at the termini of linear DNA. To address the requirements for autonomous replication in H. capsulatum, we constructed a circular E. coli plasmid containing adjacent inverted stretches of Histoplasma telomeric repeats separated by a unique restriction site. The linearized plasmid bearing telomeric termini was maintained in H. capsulatum without modification other than the addition of more telomeric sequence. We recovered the original plasmid in E. coli after removal of the telomeric termini by using engineered restriction sites. Thus, no special Histoplasma modification or sequence other than the telomeres was needed for autonomous replication in H. capsulatum. Additionally, this plasmid provides a shuttle vector that replicates autonomously in E. coli (as a circular plasmid) and in H. capsulatum (as a linear plasmid).     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Bioactivity and molecular modelling of diphenylsulfides and diphenylselenides

Bis(2-bromo-4,5-dimethoxyphenyl)sulfide (5) and bis(2-bromo-4,5-dimethoxyphenyl) selenide (7) have been shown to block cells in the G2/M phase of the cell cycle, whereas the debromo (4, 6) equivalents do not. The dibromoselenide (7) is cytotoxic to tumour cells in vitro and has been shown to increase the mitotic index of treated cells. These biological effects are consistent with disruption of the mitotic apparatus. This agent does not inhibit microtubule assembly in vitro, but does bind to tubulin. Molecular modelling of these structures indicates that their spatial and electronic structures may make an important contribution to the biological activity.     

Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae

In an attempt to unify the genetic and biological research on Mycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.B Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the cloned M. leprae genes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.     

Construction and analysis of a recombination-deficient (radA) mutant of Haloferax volcanii

By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii , we created and characterized a recombination-deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA / RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination-deficient background is desired.