Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

The relation between hip impact velocity and hip impact force differs between sideways fall techniques.

Fall techniques that reduce fall severity may decrease the risk of hip fractures. A fundamental variable for fall severity is impact force, but impact velocity is also used. The purpose of the study was to determine whether impact velocity is valid to determine differences in fall severity between different techniques. Five young adults with martial arts (MA) experience performed sideways falls from kneeling height using three techniques: Block with arm (Block) and MA techniques with and without use of the arm to break the fall. In addition, one subject also performed MA falls from standing height. Linear regression analysis showed a moderate relation between hip impact velocity and force, which was depended on technique. In falls with comparable impact velocities, forces in MA falls were lower than forces in Block falls. Hence, differences in impact force could not be predicted by velocity. In conclusion, hip impact velocity may be useful to make an approximate prediction of impact force within fall techniques. However, to determine differences between techniques it was not always a valid predictor. When direct impact force measurements are not possible, methods combining impact velocity with energy estimates before and after impact might be more valid.     

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

Treatment of intestinal dysbacteriosis in children with diabetes mellitus]

The use of the probiotic Acylact in combination with Viferone containing human recombinant interferon-alpha 2 in the treatment of intestinal dysbacteriosis in children with insulin-dependent diabetes mellitus (IDDM) was shown to be highly efficient. Elimination of microecologic disorders in the intestine was accompanied in all the children by immune rearrangements. The quantity of secretory and serum immunoglobulins and complement increased and the white blood cell chemiluminescence proved to be higher. The correction of the immune status of the children with IDDM resulted in a certain decrease of glycemia. The data are indicative of the fact that such a treatment of intestinal dysbacteriosis in children with IDDM is promising.     

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.     

Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2,4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30-40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G1----S). Since the latter type of effect most probably is Ca2+ dependent and since the spindle is sensitive to Ca2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase. All compounds were found to cause an increase of ATP with some of the applied concentrations. The results with diethyl maleate suggest that an increase of ATP is favored by an attack on mitochondrial reduced glutathione. The possible analogy between this effect and an increase of ATP and Ap4A in bacteria during oxidative stress is considered.