Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

5-Aryl-4-carboxamide-1,3-oxazoles: potent and selective GSK-3 inhibitors

5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.     

Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning

Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains.     

HIV-1 continues to replicate and evolve in patients with natural control of HIV infection

Elucidating mechanisms leading to the natural control of HIV-1 infection is of great importance for vaccine design and for understanding viral pathogenesis. Rare HIV-1-infected individuals, termed HIV-1 controllers, have plasma HIV-1 RNA levels below the limit of detection by standard clinical assays (<50 to 75 copies/ml) without antiretroviral therapy. Although several recent studies have documented persistent low-grade viremia in HIV-1 controllers at a level not significantly different from that in HIV-1-infected individuals undergoing treatment with combination antiretroviral therapy (cART), it is unclear if plasma viruses are undergoing full cycles of replication in vivo or if the infection of new cells is completely blocked by host immune mechanisms. We studied a cohort of 21 HIV-1 controllers with a median level of viremia below 1 copy/ml, followed for a median of 11 years. Less than half of the cohort carried known protective HLA types (B*57/27). By isolating HIV-1 RNA from large volumes of plasma, we amplified single genome sequences of both pro-rt and env longitudinally. This study is the first to document that HIV-1 pro-rt and env evolve in this patient group, albeit at rates somewhat lower than in HIV-1 noncontrollers, in HLA B*57/27-positive, as well as HLA B*57/27-negative, individuals. Viral diversity and adaptive events associated with immune escape were found to be restricted in HIV-1 controllers, suggesting that replication occurs in the face of less overall immune selection.     

Impact of process conditions on the centrifugal recovery of a disabled herpes simplex virus

Despite continuous improvements in culturing and recovery techniques, high-titer stocks of purified disabled herpes simplex virus type-1 (HSV-1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing-out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV-1 DIS. 80% virus recovery was achieved after 90 min at 26000g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed-angle centrifuge rotor operating at 26000g. Plaque assays of recovered HSV-1 DIS gave values on the order of 10(6) pfu/mL, compared to values typically above 10(9) pfu/mL obtained for the replication-competent HSV-1 viron.     

Mouse Mammary Tumor Virus Superantigen Expression in B Cells Is Regulated by a Central Enhancer within the pol Gene

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. The mechanism of superantigen expression from the viral sag gene in B cells is largely unknown, due to problems with detection and quantification of these low-abundance proteins. We have established a sensitive superantigen-luciferase reporter assay to study the expression and regulation of the MMTV sag gene in B-cell lymphomas. The regulatory elements for retroviral gene expression are generally located in the 5′ long terminal repeat (LTR) of the provirus. However, we found that neither promoters nor enhancers in the MMTV 5′ LTR play a significant role in superantigen expression in these cells. Instead, the essential regulatory regions are located in the pol and env genes of MMTV. We report here that maximal sag expression in B-cell lines depends on an enhancer within the viral pol gene which can be localized to a minimal 183-bp region. Regulation of sag gene expression differs between B-cell lymphomas and pro-B cells, where an enhancer within the viral LTRs is involved. Thus, MMTV sag expression during B-cell development is achieved through the use of two separate enhancer elements.     

Structure and Distribution of Endogenous Nonecotropic Murine Leukemia Viruses in Wild Mice

Virtually all of our present understanding of endogenous murine leukemia viruses (MLVs) is based on studies with inbred mice. To develop a better understanding of the interaction between endogenous retroviruses and their hosts, we have carried out a systematic investigation of endogenous nonecotropic MLVs in wild mice. Species studied included four major subspecies of Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus) as well as four common inbred laboratory strains (AKR/J, HRS/J, C3H/HeJ, and C57BL/6J). We determined the detailed distribution of nonecotropic proviruses in the mice by using both env- and long terminal repeat (LTR)-derived oligonucleotide probes specific for the three different groups of endogenous MLVs. The analysis indicated that proviruses that react with all of the specific probes are present in most wild mouse DNAs tested, in numbers varying from 1 or 2 to more than 50. Although in common inbred laboratory strains the linkage of group-specific sequences in env and the LTR of the proviruses is strict, proviruses which combine env and the LTR sequences from different groups were commonly observed in the wild-mouse subspecies. The “recombinant” nonecotropic proviruses in the mouse genomes were amplified by PCR, and their genetic and recombinant natures were determined. These proviruses showed extended genetic variation and provide a valuable probe for study of the evolutionary relationship between MLVs and the murine hosts.