Fluoroaluminate induces activation and association of Src and Pyk2 tyrosine kinases in osteoblastic MC3T3-E1 cells

Fluoride is known to increase bone mass in vivo, probably through stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (Scaronuscarona, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res. Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2, and p70(S6K) were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase). The tyrosine phosphorylation of Pyk2 increased in a dose- and time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src. The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src. Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells. These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src. Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.     

Pyruvate improves myocardial tolerance to reperfusion injury by acting as an antioxidant: a chemiluminescence study

The utilization of various substrates by sperm from the cauda epididymidis of the tammar was examined because the major naturally occurring sugar in the semen of this species is N-acetyl-D-glucosamine (NAG) and not furctose, as in eutherian mammals. The sperm displayed a high level of endogenous respiration that supported motility for relatively prolonged periods of time in vitro. They also metabolised exogenous 14C-labelled glucose, NAG, sucrose, and acetate through glycolytic and/or oxidative processes to produce lactate and 14CO2 at varying rates. The rate of uptake of NAG by tammar sperm was about four times greater than that of other substrates. Glucose and/or NAG stimulated the rate of oxygen consumption by about 20%, but acetate stimulated oxygen consumption by more than 40%. The most striking findings were that NAG almost completely inhibited the oxidation of glucose and sucrose by the sperm and depressed the uptake of glucose, 3-O-methylglucose, and sucrose. Acetate oxidation also was inhibited by NAG, but only by about 50%. Tammar sperm generated substantial amounts of free glucose during incubation with NAG, but this and the inhibitory effects of NAG on glucose oxidation were not mimicked by rat sperm. It is proposed that tammar sperm fail to oxidise glucose in the presence of NAG because of the rapid cellular uptake of NAG relative to glucose. Also, the intracellular glucose and acetate liberated from NAG would compete with exogenous glucose for processing in the Embden-meyerhof and tricarboxylic acid (TCA) cycle pathways. It is also suggested that tammar sperm oxidise sucrose after extracellular hydrolysis into its glucose and fructose components. The biological implications of these metabolic and transport properties of tammar sperm have as yet to be determined.     

Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include beta2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.     

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.     

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Insulin-like growth factor binding protein-1 at mouse neuromuscular synapses

The insulin-like growth factor (IGF) signaling system includes the growth factors and their cell surface receptors, along with circulating IGF binding proteins (IGFBPs) that may alter and modulate the action of these neurotrophic hormones. These IGFBPs, along with IGFs and receptors, have been detected in various tissues including the brain. In this study, using polyclonal antibody to human IGFBP-1 or bovine IGFBP-2, we found that mouse muscle extracts contain similar-sized proteins that cross-react with these antibodies on Western immunoblots. After establishing that these antibodies reacted with the homologous murine IGFBPs, we performed immunocytochemistry to demonstrate the localization of IGFBP-1 at the neuromuscular junction, a model nicotinic, cholinergic synapse, as well as within intramuscular nerves. IGFBP-2, a distinct macromolecule, is present on the surface of muscle fibers and is not present within synapses or nerves.     

Antimetabolite-induced increases in the invasive capacity of murine leukaemia L1210 cells

Pretreatment of murine leukaemia L1210 cells with non-lethal concentrations of various antimetabolites increased the in vitro invasive capacity of these cells into monolayers of rat embryo fibroblasts. The increase in invasive capacity was partly correlated with the induced cell cycle arrest. The concomitant increase in cell surface fucosylation and inhibition of invasion with sulphate indicate a role for glycoproteins in this process. Our results suggest that treatment with antimetabolites may lead to a more aggressive phenotype by altering cell surface properties.     

Sodium alginate–poly(hydroxyethylmethacrylate) interpenetrating polymeric network membranes for the pervaporation dehydration of ethanol and tetrahydrofuran

Interpenetrating polymeric network (IPN) membranes of sodium alginate (NaAlg) and various amounts of poly(hydroxyethylmethacrylate) (PHEMA) have been prepared and tested for the pervaporation dehydration of ethanol and tetrahydrofuran (THF). The presence of hydrophilic PHEMA in the IPN matrix was found to be responsible for increase in membrane selectivity to water. NaAlg–PHEMA IPN membrane containing 20 wt % of PHEMA exhibited a selectivity of 571 to water for the water–ethanol mixture and 857 for water–THF mixture. These data are much better than those observed for the pristine NaAlg membrane. However, flux of the IPN membranes was smaller than that of pristine NaAlg membrane. Comparatively higher flux values were observed for water–THF mixture than for water–ethanol mixture. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3324–3329, 2006