Short nucleotide sequence analysis of seven restriction fragments of murine herpesvirus 68 (MHV-68) DNA has been undertaken and used to determine the overall genome organization and relatedness of this virus to other well characterized representatives of the alpha-, beta-and gammaherpesvirus subgroups. Nine genes have been identified which encode amino acid sequences with greater similarity to proteins of the gammaherpesvirus Epstein-Barr virus (EBV) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simples virus type 1 or the betaherpesvirus human cytomegalovirus. In addition, the genome organization of MHV-68 is shown to have an overall collinearity with that of the gammaherpesviruses EBV and herpesvirus saimiri. In common with these viruses, dinucleotide frequency analysis of MHV-68 coding sequences reveals a marked reduction in CpG dinucleotide frequency thus implicating a dividing cell population as the site of latency in vivo.

Vitamin C is an important antioxidant and cofactor which is involved in the regulation of development, function and maintenance of several cell types in the body. Deficiencies in vitamin C can lead to conditions such as scurvy, which, among other ailments, causes gingivia, bone pain and impaired wound healing. This review examines the functional importance of vitamin C as it relates to the development and maintenance of bone tissues. Analysis of several epidemiological studies and genetic mouse models regarding the effect of vitamin C shows a positive effect on bone health. Overall, vitamin C exerts a positive effect on trabecular bone formation by influencing expression of bone matrix genes in osteoblasts. Recent studies on the molecular pathway for vitamin C actions that include direct effects of vitamin C on transcriptional regulation of target genes by influencing the activity of transcription factors and by epigenetic modification of key genes involved in skeletal development and maintenance are discussed. With an understanding of mechanisms involved in the uptake and metabolism of vitamin C and knowledge of precise molecular pathways for vitamin C actions in bone cells, it is possible that novel therapeutic strategies can be developed or existing therapies can be modified for the treatment of osteoporotic fractures.

Infectious HPIV3 was produced by the intracellular coexpression of four plasmid-borne cDNAs. These separately encoded a complete HPIV3 genome (negative-sense), the HPIV3 nucleocapsid protein N, the phosphoprotein P, and the polymerase protein L. The cDNA-encoded HPIV3 genome differed from the JS wildtype (wt) strain of HPIV3 used in its construction by seven point mutations: four of these are silent mutations in the HN or L gene coding regions that serve as markers of a cDNA-derived virus, two were introduced to create an amino acid substitution that ablates an epitope recognized by the HN-specific monoclonal neutralizing antibody 423/6, and the remaining point mutation results in an incidental amino acid substitution in the HN protein at amino acid position 263. The four plasmids were transfected into HEp-2 cell monolayers and their expression was driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The titer of virus present in the harvested transfection supernatant was low (<5 PFU/ml), and the recovered recombinant virus (rJS) retained each of the seven mutations present in the cDNA from which it was derived. Despite the introduced and incidental mutations, rJS retained the wt phenotypes as regards replication at elevated temperature in vitro and efficient replication in the upper and lower respiratory tract of hamsters. rJS was also recovered from a cDNA encoding a complete antigenome (positive-sense) with slightly greater efficiency than from the negative-sense construct. The ability to generate infectious HPIV3 from cDNA should greatly enhance our ability to develop new live-attenuated parainfluenza virus vaccines, including chimeric PIV1 and PIV2 vaccines, and to understand the genetic basis of attenuation of PIV3 candidate vaccines.

The frequencies of HCoV and LDI illnesses were similar. HCoV illness was less severe than LDI illness, was accompanied by multiple respiratory and systemic symptoms, and was associated with hospitalization.

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal ␣-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.

Ascorbic acid is a required cofactor in the hydroxylations of lysine and proline necessary for collagen formation; its role in bone cell differentiation and formation is less well characterized. This study examines the cross-sectional relation between dietary vitamin C intake and bone mineral density (BMD) in women from the Postmenopausal Estrogen/Progestin Interventions Trial. BMD (spine and hip) was measured using dual energy X-ray absorptiometry (DXA). The PEPI participants (n = 775) included in this analysis were Caucasian and ranged in age from 45 to 64 years. At the femoral neck and total hip after adjustment for age, BMI, estrogen use, smoking, leisure physical activity, calcium and total energy intake, each 100 mg increment in dietary vitamin C intake, was associated with a 0. 017 g/cm2 increment in BMD (P = 0.002 femoral neck; P = 0.005 total hip). After adjustment, the association of vitamin C with lumbar spine BMD was similar to that at the hip, but was not statistically significant (P = 0.08). To assess for effect modification by dietary calcium, the analyses were repeated, stratified by calcium intake (>500 mg/day and

The safety, infectivity, immunogenicity, transmissibility, and phenotypic stability of an intranasal bovine parainfluenza virus type 3 (BPIV-3) candidate vaccine was evaluated in a randomized, double-blind, placebo-controlled trial. Of human parainfluenza virus type 3 (HPIV-3)-seronegative children, 92% were infected, and 92% developed a serum hemagglutination-inhibiting (HAI) antibody response to BPIV-3 and 61% to HPIV-3. Geometric mean HAI titers were 1:40 to BPIV-3 and 1:16 to HPIV-3. In studies to evaluate vaccine transmissibility, none of 14 placebo recipients in close contact with 14 vaccinees shed BPIV-3. BPIV-3 isolates from seronegative vaccinees retained the attenuation phenotype when tested in rhesus monkeys. Although it is difficult to evaluate the safety and immunogenicity of such a vaccine in an open population of children who frequently become infected with HPIV-3, it appears that the live BPIV-3 vaccine is attenuated, infectious, immunogenic, poorly transmissible, and phenotypically stable and warrants further evaluation as a candidate vaccine in infants and children.

Inorganic phosphate (Pi) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells by stabilizing the enzyme (without affecting transcription, ALP release from the cell surface, or the amount of ALP protein). These observations suggest that Pi determines the level of ALP activity by modulating a process of irreversible inactivation. The current studies were intended to examine the hypothesis that this inactivation of ALP activity is caused by the dissociation of an active center Zn and that Pi inhibits that dissociation. Initial studies showed that Zn, like Pi, could increase ALP specific activity in human osteosarcoma SaOS-2 cells in a time- and dose-dependent manner (e.g., a 50% increase at 0.2 micromol/liter Zn, P < 0.005). This effect was specific for Zn (i.e., no similar effect was seen with Ca, Fe, Co, Mg, Mn, or Cu), but not for SaOS-2 cells. Zn also increased ALP specific activity in (human osteosarcoma) MG-63 cells and in cells derived from normal human vertebrae (P < 0.001 for each). The effect of Zn to increase ALP activity was not associated with parallel increases in total protein synthesis, collagen production, or tartrate-resistant acid phosphatase activity (no change in any of these indices), net IGF-2 synthesis (a Zn-dependent decrease, P < 0.005), or PTH-dependent synthesis of cAMP (a biphasic increase, P < 0.02). Kinetic studies of Pi and Zn as co-effectors of ALP activity showed that Zn was a mixed-type effector with respect to Pi, whereas Pi was competitive with respect to Zn. Mechanistic studies showed that (1) Zn reversed the effect of Pi withdrawal to decrease ALP activity, but not by reactivating inactive ALP protein (the process required protein synthesis, without increases in ALP mRNA or the level of ALP immunoreactive protein); (2) Zn increased the half-life of ALP activity in intact cells and after a partial purification; and (3) Pi inhibited the process of ALP inactivation by EDTA (which chelates active center Zn). All these findings are consistent with the general hypothesis that Pi increases the half-life of skeletal ALP by preventing the dissociation of active center Zn and with a mechanistic model of skeletal ALP activity in which active center Zn participates in Pi-ester binding and/or hydrolysis.