Two distinct mechanisms localise cyclin B transcripts in syncytial Drosophila embryos

We demonstrate that two independent mechanisms act on maternally derived cyclin B transcripts to concentrate the transcripts at the posterior pole of the Drosophila oocyte and at the cortex of the syncytial embryo. The cortical accumulation occurs because the cyclin B transcript is concentrated around nuclei and comigrates with them to the cortex. The perinuclear localisation of the transcript is blocked by inhibitors of microtubule polymerisation and the transcript colocalises with microtubular structures during the cell cycle, suggesting that the transcript is associated either directly or indirectly with microtubules. Neither microtubules nor actin filaments are required to maintain the posterior concentration of cyclin B transcripts. Instead, this seems to depend on the association of the transcripts with a component of the posterior cytoplasm. The distribution pattern of the transcript at the posterior pole throughout embryogenesis and in a variety of mutant embryos suggests that this component is associated with polar granules

The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes

Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with antibodies specific to CP190, indicate that the protein is associated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX₂CysX₁₂HisX₄His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci

The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes

Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with antibodies specific to CP190, indicate that the protein is associated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX₂CysX₁₂HisX₄His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10³ and 66 x 10³, on Western blots. We have used this antibody to isolate five clones (λcs1, -2, -3, -4 and λj63) from λgt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcs1-4) share at least two epitopes with the 185 x 10³ M_r centrosomal antigen. This indicates that clones λcs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10³ bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10³ and 66 x 10³, on Western blots. We have used this antibody to isolate five clones (λcs1, -2, -3, -4 and λj63) from λgt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcs1-4) share at least two epitopes with the 185 x 10³ M_r centrosomal antigen. This indicates that clones λcs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10³ bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle

The Putative Role of Resveratrol in SIRT-1 Mediated Modulation of the Vitamin D Pathway

The nuclear vitamin D receptor (VDR) modulates gene transcription in 1,25-dihydroxyvitamin D (1,25D) target tissues such as kidney, colon, and bone. The 1,25D hormone is derived from vitamin D in the skin or from the diet, and binds to and activates the VDR. We have previously shown that resveratrol, an antioxidant found in the skin of red grapes, has the ability to activate the VDR signaling pathway. Moreover, cells treated with both resveratrol and 1,25D resulted in an additive or even synergistic stimulation of VDR-mediated transcription compared to cells treated with 1,25D alone. Based on these initial results, experiments were designed to test the significance of mutations in the hormone-binding domain of VDR. Identical hormone treatments were applied to “wild-type” (non-mutated) and single point VDR mutations. 1,25D displayed a significant drop in activity caused by these mutations, while the ability of resveratrol to activate VDR was only modestly attenuated. One possible interpretation of these results is that resveratrol may affect VDR activity indirectly, perhaps via the ability of resveratrol to activate SIRT1, an enzyme which has been shown to deacetylate (and thereby activate) other nuclear receptors such as the liver X receptor (LXR). In support of this hypothesis, radiolabeled 1,25D displacement assays revealed an increase in bound radiolabeled 1,25D only in the presence of resveratrol, suggesting that direct binding of resveratrol to VDR is unlikely. Additionally, we observed increased transcriptional activity in response to resveratrol in a subset of other nuclear receptors, including the liver X receptor (LXR), which is closely related to VDR and is known to be deacetylated by SIRT1. Finally, we tested receptor-mediated transcriptional activity in a system containing VDR in the absence and presence of overexpressed SIRT1. Transcriptional activity was higher in cells expressing SIRT1, and synergistic activity of 1,25D combined with resveratrol was observed. We are currently conducting additional experiments employing the VDR/SIRT1 assay in multiple cellular contexts. In conclusion, this study elucidates, for the first time, a potential novel pathway for crosstalk between two nutritionally derived lipids, vitamin D and resveratrol

Two distinct mechanisms localise cyclin B transcripts in syncytial Drosophila embryos

We demonstrate that two independent mechanisms act on maternally derived cyclin B transcripts to concentrate the transcripts at the posterior pole of the Drosophila oocyte and at the cortex of the syncytial embryo. The cortical accumulation occurs because the cyclin B transcript is concentrated around nuclei and comigrates with them to the cortex. The perinuclear localisation of the transcript is blocked by inhibitors of microtubule polymerisation and the transcript colocalises with microtubular structures during the cell cycle, suggesting that the transcript is associated either directly or indirectly with microtubules. Neither microtubules nor actin filaments are required to maintain the posterior concentration of cyclin B transcripts. Instead, this seems to depend on the association of the transcripts with a component of the posterior cytoplasm. The distribution pattern of the transcript at the posterior pole throughout embryogenesis and in a variety of mutant embryos suggests that this component is associated with polar granules

Free carrier effects in gallium nitride epilayers: the valence band dispersion

The dispersion of the A-valence-band in GaN has been deduced from the observation of high-index magneto-excitonic states in polarised interband magneto-reflectivity and is found to be strongly non-parabolic with a mass in the range 1.2-1.8 m_{e}. It matches the theory of Kim et al. [Phys. Rev. B 56, 7363 (1997)] extremely well, which also gives a strong k-dependent A-valence-band mass. A strong phonon coupling leads to quenching of the observed transitions at an LO-phonon energy above the band gap and a strong non-parabolicity. The valence band was deduced from subtracting from the reduced dispersion the electron contribution with a model that includes a full treatment of the electron-phonon interaction.Comment: Revtex, 4 pages, 5 figure

Evidence of Differential Allelic Effects between Adolescents and Adults for Plasma High-Density Lipoprotein

A recent meta-analysis of genome-wide association (GWA) studies identified 95 loci that influence lipid traits in the adult population and found that collectively these explained about 25–30% of heritability for each trait. Little is known about how these loci affect lipid levels in early life, but there is evidence that genetic effects on HDL- and LDL-cholesterol (HDL-C, LDL-C) and triglycerides vary with age. We studied Australian adults (N = 10,151) and adolescents (N = 2,363) who participated in twin and family studies and for whom we have lipid phenotypes and genotype information for 91 of the 95 genetic variants. Heterogeneity tests between effect sizes in adult and adolescent cohorts showed an excess of heterogeneity for HDL-C (pHet<0.05 at 5 out of 37 loci), but no more than expected by chance for LDL-C (1 out of 14 loci), or trigycerides (0 out 24). There were 2 (out of 5) with opposite direction of effect in adolescents compared to adults for HDL-C, but none for LDL-C. The biggest difference in effect size was for LDL-C at rs6511720 near LDLR, adolescents (0.021±0.033 mmol/L) and adults (0.157±0.023 mmol/L), pHet = 0.013; followed by ZNF664 (pHet = 0.018) and PABPC4 (pHet = 0.034) for HDL-C. Our findings suggest that some of the previously identified variants associate differently with lipid traits in adolescents compared to adults, either because of developmental changes or because of greater interactions with environmental differences in adults

Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition