LMP-1, a LIM-domain protein, mediates BMP-6 effects on bone formation

Glucocorticoids can promote osteoblast differentiation from fetal calvarial cells and bone marrow stromal cells. We recently reported that glucocorticoid specifically induced bone morphogenetic protein-6 (BMP-6), a glycoprotein signaling molecule that is a multifunctional regulator of vertebrate development. In the present study, we used fetal rat secondary calvarial cultures to determine genes induced during early osteoblast differentiation as initiated by glucocorticoid treatment. Glucocorticoid, and subsequently BMP-6, was found to induce a novel rat intracellular protein, LIM mineralization protein-1 (LMP-1), that in turn resulted in synthesis of one or more soluble factors that could induce de novo bone formation. Blocking expression of LMP-1 using antisense oligonucleotide prevented osteoblast differentiation in vitro. Overexpression of LMP-1 using a mammalian expression vector was sufficient to initiate de novo bone nodule formation in vitro and in sc implants in vivo. These data demonstrate that LMP-1 is an essential positive regulator of the osteoblast differentiation program as well as an important intermediate step in the BMP-6 signaling pathway.     

Factors responsible for computed electrogastrographic parameters in humans

OBJECTIVES: Clinical knowledge about myoelectrical frequency is well known, but the factors responsible for recorded myoelectrical amplitude remain less clear. METHODS: We assembled an electrogastrographic system that could automatically compute the dominant myoelectrical frequency and power and power ratio. We enrolled 50 healthy volunteers (25 men and 25 women) to study their myoelectrical characteristics. Three surface electrodes were placed in the fundic, stomach body, and antral positions for two 30-min recordings in the fasting and postprandial states. RESULTS: The three different electrodes recorded similar dominant frequencies of about 3 cpm before and after a meal. The fasting dominant powers from these electrodes were 52.9 +/- 14.7, 44.6 +/- 11.5, and 50.1 +/- 15.1 dB, respectively (p < 0.01), whereas the postprandial dominant powers were 61.6 +/- 28.8, 54.3 +/- 26.6, and 61.9 +/- 27.8 dB, respectively (p < 0.01). Meal ingestion did increase the power (p < 0.05). Women had a lower dominant power than men (p < 0.001). Moreover, the dominant powers of each electrode were significantly correlated with body mass index (r = 0.3-0.36, p < 0.05) regardless of meal ingestion. The postprandial power ratio was not influenced by electrode position, gender, or body mass index. CONCLUSIONS: Myoelectrical dominant frequencies and power ratios are similar throughout the whole stomach area, whereas a lower power area exists on the stomach body. Gender-related variation in dominant power seems to be an effect of body size. The power ratio is the only reliable parameter for expressing myoelectrical amplitude.     

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.     

Clock-spiking cells not only in the eye of the fly, but also in the antenna!

Progressive pseudorheumatoid dysplasia (spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDTPA) or progressive pseudorheumatoid arthropathy of childhood (PPAC) (MIM 208.230)) is an autosomal recessively inherited skeletal dysplasia with changes in the spine similar to spondyloepiphyseal dysplasia tarda. The disease begins mostly between the ages of 2 to 8 years with progressive joint stiffness and pain, soft tissue swelling and deformities of multiple joints including the proximal interphalangeal joints of the hands. We describe a patient where a symmetric rhizomelic shortening of the extremities and a bilateral severe pes equinovarus was present at birth.     

Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans

The Candida albicans PLB1 gene was cloned using a polymerase chain reaction-based approach relying on degenerate oligonucleotide primers designed according to the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (Mirbod, F., Banno, Y., Ghannoum, M. A., Ibrahim, A. S., Nakashima, S., Yasuo, K., Cole, G. T., and Nozawa, Y. (1995) Biochim. Biophys. Acta 1257, 181-188). Sequence analysis of a 6.7-kilobase pair EcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a pre-protein of 605 residues. Comparison of the putative candidal phospholipase with those of other proteins in data base revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae (45%), Penicillium notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe (38%). Thus, we have cloned the gene encoding a C. albicans phospholipase B homolog. This gene, designated caPLB1, was mapped to chromosome 6. Disruption experiments revealed that the caplb1 null mutant is viable and displays no obvious phenotype. However, the virulence of strains deleted for caPLB1, as assessed in a murine model for hematogenously disseminated candidiasis, was significantly attenuated compared with the isogenic wild-type parental strain. Although deletion of caPLB1 did not produce any detectable effects on candidal adherence to human endothelial or epithelial cells, the ability of the caplb1 null mutant to penetrate host cells was dramatically reduced. Thus, phospholipase B may well contribute to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.     

Tyrosine phosphorylation regulates the SH3-mediated binding of the Wiskott-Aldrich syndrome protein to PSTPIP, a cytoskeletal-associated protein

Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivo results in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASP in vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton.     

Salivary function in patients with reflux esophagitis: effect of cisapride

Saliva plays an important role in esophageal acid clearance. Reduction in salivary function has been considered in the pathogenesis of reflux esophagitis. Cisapride, a prokinetic agent, has been reported effective for treating mild-to-moderate grade gastroesophageal reflux disease. Some studies have shown that cisapride increases saliva volume and acid-buffering capacity. The aim of this study was to evaluate the effect of cisapride on salivary gland function by means of dynamic salivary scintigraphy. METHODS: Fifty-five patients with endoscopic reflux esophagitis (Savary-Miller Grades I-II) were enrolled in this study. In Group 1 (n = 29), patients were evaluated during the fasting state, both before and after cisapride treatment (5 mg, 3 times/day, before meals, for 2 wk). In Group 2 (n = 26), patients were evaluated during the postprandial state, both before and after cisapride treatment. Uptake ratio (UR) and excretion ratio (ER) of the salivary gland in each group were compared using the paired Student's t-test. RESULTS: In Group 1, no significant differences were found in UR or ER after cisapride treatment. However, in Group 2, ER increased significantly after treatment (p < 0.01), but UR did not show any significant change. CONCLUSION: Cisapride can increase the secretion function of salivary glands during the postprandial phase but not the fasting phase.