Differential expression and localization of calmodulin-dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion

The level and characteristics of 3'-5'-cyclic nucleotide phosphodiesterase (PDE) activity in chick dorsal root ganglion (DRG) extracts of 5-day posthatching chicken (P5) and E10 and E18 embryos were studied. At all stages, PDE activity is stimulated by calcium and calmodulin. A 5-fold increase in basal cAMP and cGMP PDE activity is evident from E10 to E18, while from E18 to P5 basal PDE activity remains constant. Ion exchange chromatography elution profile indicates that PDE1 isoforms represent the bulk of the PDE activity present. Inhibition studies were performed in order to distinguish the activity due to PDE1A, B and C. Western blot analysis using anti-mammalian PDE1A, B and C specific antibodies was also performed. Densitometric analysis of the stained bands reveals that PDE1B and PDE1C display a prominent increase between day 10 and day 18 of development (eight- and 3.6 fold, respectively) while a more limited increase (1.6- and 1.5-fold) is observed between E18 and P5; on the other hand PDE1A shows continuously increasing levels throughout development. Immunohistochemical analysis was performed with isoform specific antibodies used for western blot analysis. PDE1A immunoreactivity is found in the cytoplasm and fibers of several neurons differing in size and distributed throughout the ganglion. PDE1B staining is evident on all neurons, however, fibers appear very faintly labelled. All neurons appear stained by PDE1C antibody, although the intensity of immunostaining is always heterogeneous in different neuronal populations: no staining was evident on fibers or in non-neural cells. The distinct spatial and temporal expression patterns of PDE1 isoforms may indicate their different physiological roles in developing and mature chick DRG.     

Orally bioavailable highly potent HIV protease inhibitors against PI-resistant virus

Efforts directed to identifying potent HIV protease inhibitors (PI) have yielded a class of compounds that are not only very active against wild-type (NL4-3) HIV virus but also very potent against a panel of PI-resistant viral isolates. Chemistry and biology are described.     

Correlation and prediction of gene expression level from amino acid and dipeptide composition of its protein

Background  

A large number of papers have been published on analysis of microarray data with particular emphasis on normalization of data, detection of differentially expressed genes, clustering of genes and regulatory network. On other hand there are only few studies on relation between expression level and composition of nucleotide/protein sequence, using expression data. There is a need to understand why particular genes/proteins express more in particular conditions. In this study, we analyze 3468 genes of Saccharomyces cerevisiae obtained from Holstege et al., (1998) to understand the relationship between expression level and amino acid composition.     

Dynamics of Deinococcus radiodurans under controlled growth conditions

Deinococcus radiodurans is a potent radiation resistant bacterium with immense potential in nuclear waste treatment. In this investigation, the translational and rotational dynamics of dilute suspensions of D. radiodurans cultured under controlled growth conditions was studied by the polarized and depolarized dynamic light-scattering (DLS) techniques. Additionally, confocal laser scanning microscopy was used for characterizing the cultured samples and also for identification of D. radiodurans dimer, tetramer, and multimer morphologies. The data obtained showed translational diffusion coefficients (DT) of 1.2 x 10(-9), 1.97 x 10(-9), and 2.12 x 10(-9) cm2 /s, corresponding to an average size of 3.61, 2.22, and 2.06 microm, respectively, for live multimer, tetramer, and dimer forms of D. radiodurans. Depolarized DLS experiments showed very slow rotational diffusion coefficients (DR) of 0.182/s for dimer and 0.098/s for tetramer morphologies. No measurable rotational diffusion was observed for multimer form. Polarized DLS measurements on live D. radiodurans confirmed that the bacterium is nonmotile in nature. The dynamics of the dead dimer and tetramer D. radiodurans were also studied using polarized and depolarized DLS experiments and compared with the dynamics of live species. The dead cells were slightly smaller in size when compared to the live cells. However, no additional information could be obtained for dead cells from the polarized and depolarized dynamic light-scattering studies.     

Palmitate activates autophagy in INS-1E β-cells and in isolated rat and human pancreatic islets

We have investigated the in vitro effects of increased levels of glucose and free fatty acids on autophagy activation in pancreatic beta cells. INS-1E cells and isolated rat and human pancreatic islets were incubated for various times (from 2 to 24 h) at different concentrations of glucose and/or palmitic acid. Then, cell survival was evaluated and autophagy activation was explored by using various biochemical and morphological techniques. In INS-1E cells as well as in rat and human islets, 0.5 and 1.0 mM palmitate markedly increased autophagic vacuole formation, whereas high glucose was ineffective alone and caused little additional change when combined with palmitate. Furthermore, LC3-II immunofluorescence co-localized with that of cathepsin D, a lysosomal marker, showing that the autophagic flux was not hampered in PA-treated cells. These effects were maintained up to 18-24 h incubation and were associated with a significant decline of cell survival correlated with both palmitate concentration and incubation time. Ultrastructural analysis showed that autophagy activation, as evidenced by the occurrence of many autophagic vacuoles in the cytoplasm of beta cells, was associated with a diffuse and remarkable swelling of the endoplasmic reticulum. Our results indicate that among the metabolic alterations typically associated with type 2 diabetes, high free fatty acids levels could play a role in the activation of autophagy in beta cells, through a mechanism that might involve the induction of endoplasmic reticulum stress.     

Vascular circadian rhythms in a mouse vascular smooth muscle cell line (Movas-1)

The circadian system in mammals is a hierarchy of oscillators throughout the organism that are coordinated by the circadian clock in the hypothalamic suprachiasmatic nucleus. Peripheral clocks act to integrate time-of-day information from neural or hormonal signals, regulating gene expression, and, subsequently, organ physiology. However, the mechanisms by which the central clock communicates with peripheral oscillators are not understood and are likely tissue specific. In this study, we establish a mouse vascular cell model suitable for investigations of these mechanisms at a molecular level. Using the immortalized vascular smooth muscle cell line Movas-1, we determined that these cells express the circadian clock machinery with robust rhythms in mRNA expression over a 36-h period after serum shock synchronization. Furthermore, norepinephrine and forskolin were able to synchronize circadian rhythms in bmal1. With synchronization, we observed cycling of specific genes, including the tissue inhibitor of metalloproteinase 1 and 3 (timp1, timp3), collagen 3a1 (col3a1), transgelin 1 (sm22alpha), and calponin 1 (cnn1). Diurnal expression of these genes was also found in vivo in mouse aortic tissue, using microarray and real-time RT-PCR analysis. Both of these revealed ultradian rhythms in genes similar to the cycling observed in Movas-1 in vitro. These findings highlight the cyclical nature of structurally important genes in the vasculature that is similar both in vivo and in vitro. This study establishes the Movas-1 cells as a novel cell model from which to further investigate the molecular mechanisms of clock regulation in the vasculature.