Stable association of PYK2 and p130(Cas) in osteoclasts and their co-localization in the sealing zone

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.     

Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans

For many profoundly hearing-impaired listeners (hearing loss > 90 dB HL) speechreading is the most important means of communication; amplified speech may provide, at best, additional information to speechreading. In order to improve audiovisual communication, three speech pattern elements comprising voice-fundamental frequency (f0), the first formant (F1), and the first and the second formant (F1F2) were presented as supplements to speechreading. A fourth condition consisted of a natural speech supplement, a fifth of speechreading only. Twenty subjects were tested; all audiovisual speech scores were significantly higher than the purely visual scores. Audiovisual scores for amplified, natural speech were significantly higher than those for f0 and F1F2 coded speech. Scores for natural speech and for F1 coded speech were not significantly different. The relations between the increase in audiovisual speech scores over the visual scores and measures of difference limen for frequency (DLf) and gap detection were not clear. The most prominent correlations with the speech scores were found for the DLf at 125 Hz and for gap detection.     

The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in bakers'' yeast

An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375,000-400,000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3',5'-ADP and 4'-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzes a reaction between 4'-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase, to reform CoA. This sequence of reactions, referred to as the CoA/4'-phosphopantetheine cycle, provides a mechanism by which the 4'-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3',5'-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4'-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment segment is referred to as the 10,000-15,000 M(r) subunit. The 5'-ADP-4'-pantothenic acid-synthetase, 5'-ADP-4'-pantothenylcysteine-synthetase, 5'-ADP-4'-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10,000-15,000 M(r) subunit. Thus, the 10,000-15,000 M(r) subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.     

Ultrastructural evidence for intra- and extranuclear projections of GABAergic neurons of the suprachiasmatic nucleus

GABAergic projections of the suprachiasmatic nucleus (SCN) were demonstrated in a double-labelling ultrastructural study which visualised the efferents of the SCN by PHA-L tracing, diaminobenzidine (DAB) immunocytochemistry, and GABA with immunogold postembedding staining. The results show a strong contralateral projection of the SCN that is partly GABA-containing. In addition, ipsilateral SCN projections to the dorsomedial hypothalamus and periventricular part of the paraventricular nucleus and sub-paraventricular nucleus were shown to contain GABA. The present results indicate that the SCN may utilize this inhibitory neurotransmitter to regulate and organize its own circadian rhythm as well as using GABA to transmit its diurnal information to other regions of the brain.