Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.     

Preparation of platinum(II) complexes with L-serine using KI. X-ray crystal structure, HPLC and 195Pt NMR spectra

The preparation of platinum(II) complexes containing L-serine using K(2)[PtCl(4)] and KI as raw materials was undertaken. The cis-trans isomer ratio of the complexes in the reaction mixture differed significantly depending on whether KI was present or absent in the reaction mixture. One of the two [Pt(L-ser-N,O)(2)] complexes (L-ser=L-serinate anion) prepared using KI crystallizes in the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions a=8.710(2) A, b=9.773(3) A, c=11.355(3) A, Z=4. The crystal data revealed that this complex has a cis configuration. The other [Pt(L-ser-N,O)(2)] complex also crystallizes in the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions a=7.0190(9) A, b=7.7445(6) A, c=20.946(2) A, Z=4. The crystal data revealed that this complex has a trans configuration. The 195Pt NMR chemical shifts of trans-[Pt(L-ser-N,O)(2)] and cis-[Pt(L-ser-N,O)(2)] complexes are -1632 and -1832 ppm, respectively. 195Pt NMR and HPLC measurements were conducted to monitor the reactions of the two [Pt(L-ser-N,O)(2)] complexes with HCl. Both 195Pt NMR and HPLC showed that the reactivities of cis- and trans-[Pt(L-ser-N,O)(2)] toward HCl are different: coordinated carboxyl oxygen atoms of trans-[Pt(L-ser-N,O)(2)] were detached faster than those for cis-[Pt(L-ser-N,O)(2)].     

Acid Sphingomyelinase of Human Brain: Purification to Homogeneity

Abstract: Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human brain by extraction with 0.1% Triton X-100, followed by sequential chromatography on Concanavalin A-Sepharose, octyl-Sepharose, hydroxylapatite, DEAE-cellulose, red A-agarose, Sephadex G-200, and DEAE-cellulose with ampholyte elution. Sphingomyelinase activity was purified more than 20,000-fold from the starting homogenate with a 1% yield. Specific activity of up to 800 μmol/h/mg protein could be achieved. Gel electrophoresis with 6% polyacrylamide containing sodium dodecyl sulfate gave a single protein band with a molecular weight of 70,000, in good agreement with the molecular weight previously estimated from sucrose density gradient centrifugation in 0.1% Triton X-100. Triton X-100 could be readily removed from the enzyme by sucrose density gradient centrifugation. The Triton-free enzyme showed the same K _m and pH optimum. Heat stability of the enzyme was reversibly affected by Triton X-100, in that removal of the detergent made the enzyme more heat labile. The K _m of purified enzyme for sphingomyelin was 36 μ M . It was unaffected by sulfhydryl reagents, but was inhibited by dithiothreitol at high concentrations. The preparation was free of all lysosomal hydrolase activities tested, including galactosylceramidase and α-mannosidase, which tended to copurify in our previous procedure. The enzyme was inactive toward sphingosylphosphorylcholine. It was active with bis[ p -nitrophenyll- and bis[4-methylumbelliferyl]phosphate and the chromogenic and fluorogenic sphingomyelin analogues.     

Reverse hydroxamate-based selective TACE inhibitors

Reverse hydroxamate-based selective TACE inhibitors are described. They have potent TACE inhibitory activities and excellent selectivities against MMP-1, 2, 3, 8, 9, 13, 14, and 17. One representative compound, 18 has demonstrated an excellent oral inhibitory activity of the lipopolysaccharide (LPS)-stimulated TNF-alpha production in rats.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.     

2,3-Diphenylpropionic acids as potent VLA-4 antagonists

The discovery and SAR of 2,3-diphenylpropionic acid derivatives as highly potent VLA-4 antagonists are described. One representative compound, 9cc has inhibited intercellular adhesion by a VCAM-1/VLA-4 interaction with an IC(50) of 1.7 nM, and has good pharmacokinetics and oral bioavailability.     

Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions

Nectins, Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, induce the activation of Cdc42 and Rac small G proteins, enhancing the formation of cadherin-based adherens junctions (AJs) and claudin-based tight junctions. Nectins recruit and activate c-Src at the nectin-based cell-cell contact sites. c-Src then activates Cdc42 through FRG, a Cdc42-GDP/GTP exchange factor. We showed here that Rap1 small G protein was involved in the nectin-induced activation of Cdc42 and formation of AJs. Rap1 was recruited to the nectin-based cell-cell contact sites and locally activated through the c-Src-Crk-C3G signaling there. The activation of either c-Src or Rap1 alone was insufficient for and the activation of both molecules was essential for the activation of FRG. The activation of Rap1 was not necessary for the c-Src-mediated phosphorylation or recruitment of FRG. The inhibition of the Crk, C3G, or Rap1 signaling reduced the formation of AJs. These results indicate that Rap1 is activated by nectins through the c-Src-Crk-C3G signaling and involved in the nectin-induced, c-Src- and FRG-mediated activation of Cdc42 and formation of AJs.     

Glutamate triggers elevation of intracellular Ca2+ concentration in neural precursor cells

Both neurons and glial cells are derived from neuralprecursor cells in the ventricular zone during braindevelopment. The fate of the neural precursor cells isaffected by neurotransmitters such as glutamate. Inthis study, we examined glutamate-triggeredintracellular Ca²⁺ signaling in neural precursorcell lines by the calcium digital imaging method. Whenimmortalized primary-cultured neural precursor cellswere treated with glutamate, a subpopulation of thesecells showed an increase in intracellular Ca²⁺concentration. In an effort to determine the role ofthe glutamate-triggered intracellular Ca²⁺ signalin neural precursor cells, we tried to cultureimmortalized basal ganglial and hippocampal neuralprecursor cell lines in glutamate-free medium. Thehippocampal (MHP-2) cells became adapted to theglutamate-free medium, and when treated with glutamatethe adapted subline (MHP-2-E1) showed an increase inintracellular Ca²⁺ concentration. In contrast,the basal ganglial neural precursor cell lines failedto become adapted to the glutamate-free medium. Theseresults suggest that hippocampal and basal ganglialneural precursor cells differ in their cellularresponse to glutamate as an exogenous stimulus.     

MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline

MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.