Phylogeography of the cactophilic species Drosophila gouveai: demographic events and divergence timing in dry vegetation enclaves in eastern Brazil

Aim  The aim of this study was to assess the causal mechanisms underlying populational subdivision in Drosophila gouveai , a cactophilic species associated with xeric vegetation enclaves in eastern Brazil. A secondary aim was to investigate the genetic effects of Pleistocene climatic fluctuations on these environments.
Location  Dry vegetation enclaves within the limits of the Cerrado domain in eastern Brazil.
Methods  We determined the mitochondrial DNA haplotypes of 55 individuals (representing 12 populations) based on sequence data of a 483-bp fragment from the cytochrome c oxidase subunit II (COII) gene. Phylogenetic and coalescent analyses were used to test for the occurrence of demographic events and to infer the time of divergence amongst genetically independent groups.
Results  Our analyses revealed the existence of two divergent subclades (G1 and G2) plus an introgressed clade restricted to the southernmost range of D. gouveai . Subclades G1 and G2 displayed genetic footprints of range expansion and segregated geographical distributions in south-eastern and some central highland regions, east and west of the Paraná River valley. Molecular dating indicated that the main demographic and diversification events occurred in the late to middle Pleistocene.
Main conclusions  The phylogeographical and genetic patterns observed for D. gouveai in this study are consistent with changes in the distribution of dry vegetation in eastern Brazil. All of the estimates obtained by molecular dating indicate that range expansion and isolation pre-dated the Last Glacial Maximum, occurring during the late to middle Pleistocene, and were probably triggered by climatic changes during the Pleistocene. The current patchy geographical distribution and population subdivision in D. gouveai is apparently closely linked to these past events.     

Evaluation of DNA damage induced by norfloxacin in liver and kidney of adult rats and in fetal tissues after transplacental exposure

Norfloxacin, a recently developed antimicrobial fluoroquinolone, was investigated for DNA-damaging activity in rat liver and kidney. After oral administration of single doses ranging from 1 to 8 mmole/kg, DNA fragmentation was absent in liver and kidney both 2 and 6 h after treatment. However, when administered to pregnant rats, the highest doses produced a detectable amount of DNA damage in fetal tissues. This damage appears to be an aspecific consequence of maternal and fetal toxicity rather than a specific genotoxic effect.     

Differential susceptibility to recombinant interferon-gamma-induced HLA-DQ antigen modulation among clones from a human metastatic melanoma

Twenty-one clones from an early culture of a histocompatibility leukocyte antigen (HLA) class II negative human metastatic melanoma (Me 9229) were screened for susceptibility to phenotypic modulation induced by recombinant interferon-gamma (rIFN-gamma) by using SPV-L3, a monoclonal antibody to HLA-DQ antigens, in indirect immunofluorescence followed by fluorescence-activated cell sorter analysis. After treatment with 500 U/ml of rIFN-gamma for 3 days one of the clones (9229/18) expressed high levels of DQ antigens, in terms of percentage of positive cells, whereas many other clones were much less susceptible or remained DQ negative. Scatchard analysis of the data of specific binding of 125-I-labeled rIFN-gamma revealed that one clone susceptible (9229/18) and one clone resistant (9229/5) to HLA-DQ modulation expressed similar numbers of interferon-gamma binding sites per cell; dose-response experiments showed that all clones could be induced to express HLA-DR and -DP antigens after exposure to rIFN-gamma. However, the DQ-negative profile of clone 9229/5 was not modified even after incubation with up to 1 X 10(4) U/ml of rIFN-gamma or by extending the culture time in the presence of this lymphokine up to 120 hr. Furthermore, Northern blot analysis indicated a direct correlation between changes in the levels of HLA-DR and -DQ-specific mRNA after rIFN-gamma treatment, and the lack or expression of HLA class II antigens at the cell surface of the two different clones. Karyotype studies did not reveal differences between clones 9229/5 and 9229/18 and Southern blot analysis indicated that both clones had similar EcoRI and HindIII restriction patterns for DR and DQ gene sequences. Finally, strong DQ-specific mRNA signal and antigen expression at the cell surface could be induced even on clone 9229/5 by treating the cells with supernatants from mixed lymphocyte cultures, recently shown to contain a class II-inducing factor different from interferon-gamma. Taken together these results indicate that DQ antigens can be modulated even in clones resistant to rIFN-gamma induction and suggest that the differential susceptibility observed in response to this lymphokine could play a role in the genesis of the phenomenon of intratumor heterogeneity.     

Naloxone doesn't determine the response of growth hormone to clonidine in obese patients

The effect of naloxone (opioid receptor blocker) on the impairment of growth hormone (GH) release after clonidine (alfa 2-adrenergic agonist) was investigated in 10 volunteer obese subjects. The patients (4 males and 6 females, 16-22 year old) with fat excess (15 +/- 2 kg) estimated by bioelectrical impedance analysis (BIA) were studied repeatedly. The patients, were perfused by a slow saline infusion. 30 min later they received a bolus dose of clonidine (150 micrograms p.o.), followed 30 min later by a bolus dose of naloxone (10 mg i.v.) or a corresponding volume of isotonic sodium cloride (I.S.) for control. No significant changes occurred in blood GH concentration after clonidine administration and naloxone did not induce GH response at clonidine. These results suggest that in obese subjects the impairment of GH release after clonidine is not mediated via receptors sensitivity to naloxone.     

Effect of a hyperlipidic diet on lipid composition, fluidity, and (Na+-K+)ATPase activity of rat erythrocyte membranes

Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.     

DNA damage in stomach, kidney, liver and lung of rats treated with atrazine

The genotoxic activity of atrazine, a widely used triazine herbicide, was assayed by the DNA alkaline elution technique in rats given orally a single high dose or repeated daily doses. DNA breaks (and/or alkali-labile lesions) were detected in cell suspensions obtained from stomach, kidney and liver, but not in those from lung.     

Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length,chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.     

Presynaptic regulation of acetylcholine release in the CNS

The release of ACh appears to be under the control of autoreceptors localized on cholinergic nerve terminals. Moreover, the process can be regulated by transmitters other than ACh or by modulators either through receptor-mediated or carrier-mediated mechanisms. In this chapter we report on our recent results concerning the regulation of the release of ACh by ACh itself, 5-HT and GABA in the rat hippocampus. In particular it will be shown: 1) that the release of the cholinergic transmitter can be inhibited through muscarinic receptors of the M3 subtype; 2) that 5-HT can interact with ACh by depressing ACh release through the activation of receptors of the 5-HT1B subtype; 3) that the release of ACh can be enhanced by GABA by a novel mechanism involving a selective penetration of the amino acid into the cholinergic terminals.     

Secretion of β-lactamase from K. lactis using pEPS1, a convenient episomal vector designed for the secretion of foreign proteins

Summary A convenient shuttle vector that enables high level secretion of proteins from Kluyveromyces lactis has been developed. The vector, pEPS1, contains a unique cloning site that allows the construction, in a single ligation step, of episomal plasmids capable of directing secretion of foreign gene products from K. lactis. As an example we demonstrate the production of -lactamase and determine optimal conditions for its secretion into the culture media.     

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: A nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5′-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2′,3′-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5′-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD₅₀ = 0.32 μM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5′-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5′-nucleotidase is not essential for the activation of this nucleoside analog.