Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl-2, Bcl-x(L)/Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We demonstrate that caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose-response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre-treatment with the caspase inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment.     

Menthol tolerant clones of Mentha arvensis: approach for in vitro selection of menthol rich genotypes

In vitro raised shoots of Mentha arvensis L. were screened for menthol tolerance level by growing them in media containing 0–100 g ml^–1 menthol. A total of 2850 regenerated shoots were step wise screened for menthol tolerance at the concentrations of 50 g ml^–1 followed by 60 and 70 g ml^–1. In this screening, only 30 individual regenerated shoots were able to survive. The clones from the primary screen were inoculated into rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied and grown in the field in replicated plots of 2.5×2.5 m sizes. Twigs of 30 clones from the replicated trials were rechecked for tolerant phenotypes at a concentration of 70 g ml^–1 menthol wherein, these survived even after 7 days (secondary screening). These clones were checked for oil and menthol content and were found to be better than the control plants. Out of these 30 plants, five tolerated 80 g ml^–1 menthol (tertiary level screening) and were found to contain the highest amount of menthol per g leaf biomass. Molecular analysis through RAPD showed distinct variation in the profiles of these five plants, in comparison to the control. Using this method the relationship between the primer OPT 04, menthol tolerance and high menthol content character of the genotype was established. Further, a cultivar `Saksham' was released from the selections by CIMAP for superior performance.     

RGS9 modulates dopamine signaling in the basal ganglia

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.     

Evolutionary conservation of a 2-kb intronic sequence flanking a tissue-specific alternative exon in the PTBP2 gene

nPTB is a member of the polypyrimidine tract-binding (PTB) protein family, which participates in alternative pre-mRNA processing. Tissue-specific splicing of exon 10 in nPTB (HGMW-approved symbol PTBP2) may play an important role in regulating the functional activity of nPTB in neuronal versus nonneuronal cells. In this study, we found that 297 consecutive intronic nucleotides flanking this alternatively spliced exon 10 were identical between human, green monkey, mouse, rat, and pig, while 207 consecutive intronic nucleotides were identical between human and bird DNA. In addition, a 2-kb sequence spanning this intron region showed 85 and 70% conservation in mammal and bird DNA, respectively. Unexpected intergenic sequence conservation between human and mouse genomes has recently been identified. We have now identified intragenic (intronic) sequence conservation from mammals to birds. The striking conservation of this large segment of flanking intronic sequence suggests an important role in tissue-specific splice site selection and may function in regulating the production of functional nPTB.     

Effects of moonlight exposure on plasma melatonin rhythms in the seagrass rabbitfish, Siganus canaliculatus

Influences of light-dark (LD) cycle and moonlight exposure on plasma melatonin rhythms in the seagrass rabbitfish, Siganus canaliculatus, a lunar synchronized spawner, were determined by time-resolved fluoroimmunoassay (TR-FIA). When the fish were exposed to a natural LD (12:12) cycle, plasma melatonin levels exhibited a clear daily rhythm, with higher levels at midnight and lower levels during the day. These rhythms were not evident under either constant light (LL) or constant dark (DD) conditions. Plasma melatonin levels under LL condition were low and high under DD condition. These results indicate that plasma melatonin rhythms are driven by LD cycle in this species. When the fish were exposed to the 4 lunar phases, plasma melatonin levels around the new moon were significantly higher than during the first quarter moon and the full moon. Exposure to experimental new moon and full moon conditions caused significant increases and decreases of plasma melatonin levels, respectively. The synchronous rhythmicity of melatonin levels in the plasma support the hypothesis that the seagrass rabbitfish perceives moonlight intensity and responds with secretion of melatonin into the bloodstream.     

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.