The cationic amphipathic alpha-helix of HIV-1 viral protein R (Vpr) binds to nucleic acids,permeabilizes membranes,and efficiently transfects cells

Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52-96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a alpha-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.     

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.     

Evidence of regio-specific glycosylation in human intestinal mucins: presence of an acidic gradient along the intestinal tract

Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, transverse and sigmoid colon, and rectum) of two individuals with ALeb blood group. A systematic study of the monosaccharides and oligosaccharide alditols released by reductive beta-elimination from mucins was performed using gas chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy techniques. Important variations were observed in the mucin-associated oligosaccharide content with an increasing gradient of sialic acid from the ileum to the colon associated with a reverse gradient of fucose. Moreover, a comparative study of the Sda/Cad and ABH blood group determinants along the gastrointestinal tract showed the same reverse distribution in the two kinds of antigens. In addition, besides their heterogeneity, sialic acids presented considerable variations in the degree of O-acetylation in relation to glycan sialylation level. These data are discussed in view of recent concepts suggesting that the oligosaccharide composition of the gut constitutes a varied ecosystem for microorganisms that are susceptible to adapt there and possess the specific adhesion system and specific enzymes able to provide a carbohydrate nutrient.     

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.     

Cloning of a pine germin-like protein (GLP) gene promoter and analysis of its activity in transgenic tobacco Bright Yellow 2 cells

Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant cell wall proteins. These proteins seem to be involved in many developmental stages and stress-related processes, but their exact participation in these processes generally remains obscure. In Pinus caribaea Morelet, the PcGER1 gene is expressed uniquely in embryo tissues, and encodes a GLP ionically bound to the walls of pine embryo cells maintained in 2,4-D-containing medium. We have cloned a genomic fragment including the 1520 bp 5'-upstream promoter region of PcGER1 . This sequence contains, in its 1200 bp distal part, several cis elements (e.g. SEF4, 60 kDa protein, ABA RE and Dof recognition sites) present in genes responding to hormones and/or expressed in embryo or seed tissues, or during germination. The PcGER1 promoter sequence was cloned upstream of the GUS ( β -glucuronidase) reporter gene and transferred to tobacco Bright Yellow 2 (BY-2) cells via Agrobacterium tumefaciens -mediated transformation. Promoter activity and growth performances of transgenic asynchronous cell suspensions were analysed in the presence or absence of 2,4-D and/or BA. Optimal growth, maximum cell-wall yield and PcGER1 promoter activity were observed in the presence of 2,4-D and BA at day 4, the end of the exponential growth phase where 70–75% cells have a 2C DNA content. Analysis of promoter activity during the cell cycle in an aphidicoline-synchronized culture suggested that the expression is maximum in G1 cells. We also showed that under optimal growth conditions, 5' promoter deletions decreased the activity of the reporter gene. We discuss the function of this gene with regards to cell growth.
Accession number : The PcGER1 promoter sequence was submitted to the genbank database under the accession number AY077704 .     

Production of recombinant xenotransplantation antigen in Escherichia coli

The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug. As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection. An E. coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA. This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.     

Origin and diffusion of mtDNA haplogroup X

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as “X1” and “X2.” The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.     

NMR studies of the phosphorylation motif of the HIV-1 protein Vpu bound to the F-box protein beta-TrCP

A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.