(TG:CA)(n) repeats in human housekeeping genes

The unravelling of human genome sequence gives a new opportunity to investigate the role of repetitive sequences in gene regulation. Among the various types of repetitive sequences, the dinucleotide (TG:CA)(n) repeats are one of the most abundant in human genome and exhibit polymorphism. Early on, it was observed that the (TG:CA)(n) repeats could modulate gene expression and has the propensity to undergo conformational transitions in in vivo conditions. Recent reports describe the role of polymorphic (TG:CA)(n) repeats in gene regulation in several genes. In this work, we have analysed the distribution of (TG:CA)(n) (n >or= 6) repeats in human 'housekeeping genes' on which recently released Gene Chip data is available. Our results indicate that (i). The number of short intragenic (TG:CA)(n) repeats is significantly higher than the number of long repeats (ii). the proportion of genes with (TG:CA)(n) repeats (n >or= 12 units) had lower mean expression levels compared to those without these repeats, (iii). the genes belonging to the functional class of 'signalling and communication' had a positive association with repeats in contrast to the genes belonging to the 'information' class that were negatively associated with repeats.     

2″-p-coumaroylvitexin 7-glucoside from Mollugo oppositifolia

Besides vitexin, two compounds have been isolated from Mollugo oppositifolia and identified as vitexin 7-glucoside and 2″-p-coumaroylvitexin 7-glucoside. The latter is a new natural compound. Some features common to the electron-impact mass spectra of permethyl vitexin 7-glucoside and permethyl isovitexin 7-glucoside are discussed.     

Emerging tools for real-time label-free detection of interactions on functional protein microarrays

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.     

Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment

Cholesterol-dependent cytolysins are bacterial protein toxins that bind to cholesterol-containing membranes, form oligomeric complexes and insert into the bilayer to create large aqueous pores. Membrane-dependent structural rearrangements required to initiate the oligomerization of perfringolysin O monomers have been identified, as have the monomer-monomer interaction surfaces, using site-specific mutagenesis, disulfide trapping and multiple fluorescence techniques. Upon binding to the membrane, a structural element in perfringolysin O moves to expose the edge of a previously hidden beta-strand that forms the monomer-monomer interface and is required for oligomer assembly. The beta-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack, thereby aligning the transmembrane beta-hairpins of adjacent monomers in the proper register for insertion. Collectively, these data reveal a novel membrane binding-dependent mechanism for regulating cytolysin monomer-monomer association and pore formation.     

RNA interference demonstrates a novel role for H2A.Z in chromosome segregation

The histone variant H2A.Z plays an essential role in metazoans but its function remains to be determined. Here, we developed a new inducible RNAi strategy to elucidate the role of H2A.Z in mammalian cell lines. We show that in the absence of H2A.Z, the genome becomes highly unstable and that this instability is caused by defects in the chromosome segregation process. Analysis of H2A.Z localization reveals that in these cells it is enriched at heterochromatic foci with HP1alpha on the arms of chromosomes but not at centromeric regions. When H2A.Z is depleted, normal HP1alpha-chromatin interactions are disrupted on the chromosomal arms and, notably, also at pericentric regions. Therefore, H2A.Z controls the localization of HP1alpha. We conclude that H2A.Z is essential for the accurate transmission of chromosomes.     

Growth and production of biomass of Rhodovulum sulfidophilum in sardine processing wastewater

AIMS: Rhodovulum sulfidophilum was grown in sardine processing wastewater to assess growth characteristics for the production of bacterial biomass with simultaneous reduction of chemical oxygen demand. METHODS AND RESULTS: Growth characteristics were compared in diluted and undiluted, settled and non-settled wastewater growing in anaerobic light and aerobic dark conditions; and also at different agitation speeds. The highest biomass (8.75 g l(-1)) and a reduction in chemical oxygen demand of 71% were obtained in unsettled, undiluted wastewater after 120 h culture with 15% inoculum. In settled wastewater, highest biomass (7.64 g l(-1)) and a COD reduction of 77% was also obtained after 120 h. Total biomass was higher (4.34 g l(-1)) after 120 h culture in anaerobic light compared to (3.23 g l(-1)) in aerobic dark growth. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Better performance, mean of total biomass (6.97 g l(-1) after 96 h), total carotenoids (4.24 mg g(-1) dry cell from 24 h) and soluble protein (431 microg ml(-1) after 96 h) were obtained from aerobic dark culture at 300 rev min(-1). The COD reduction, however, was lower (69%) after 96 h culture. Thus, the benefits in the production of bacterial biomass in non-sterilized sardine processing wastewater with the reduction of chemical oxygen demand could be achieved.     

Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells

Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.     

Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane

The GTPase dynamin is a mechanochemical enzyme involved in membrane fission, but the molecular nature of its membrane interactions and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using site-directed fluorescence labeling and several independent fluorescence spectroscopic techniques, we have developed robust assays for the detection and real-time monitoring of dynamin-membrane and dynamin-dynamin interactions. We show that dynamin interacts preferentially with highly curved, PIP2-dense membranes and inserts partially into the lipid bilayer. Our kinetic measurements further reveal that cycles of GTP binding and hydrolysis elicit major conformational rearrangements in self-assembled dynamin that favor dynamin-membrane association and dissociation, respectively. Sorting nexin 9, an abundant dynamin partner, transiently stabilizes dynamin on the membrane at the onset of stimulated GTP hydrolysis and may function to couple dynamin's mechanochemical conformational changes to membrane destabilization. Amphiphysin I has the opposite effect. Thus, dynamin's mechanochemical properties on a membrane surface are dynamically regulated by its GTPase cycle and major binding partners.