Possible involvement of ecdysteroids in photoperiodically induced suppresion of ovarian development in a Japanese strain of the migratory locust,Locusta migratoria

In a Japanese population of Locusta migratoria, adult females become reproductively inactive under crowding and long days (LD) and reproductively active under crowding and short days (SD). The identity and titre of ecdysteroids in the haemolymph and ovaries from adult females reared under SD and LD were investigated by RIA/HPLC. The effects of exogenous juvenile hormone (JH) III treatments on the termination of such reproductive arrest and ecdysteroid contents in LD females were also examined. In general, ecdysteroid titres in both haemolymph and ovaries were significantly higher in reproductively active SD females than in reproductively inactive LD females. A clear difference was also observed in oocyte growth between SD and LD individuals. JH III applications (three consecutive topical applications, 150 μg per insect per day from day 3) stimulated ovarian development in LD females and significantly increased the haemolymph and ovarian ecdysteroids to a level comparable to that of reproductively active SD adult females.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.     

Regulatory expression of MDP77 protein in the skeletal and cardiac muscles

The mdp77 gene was first cloned from the cDNA library of denervated chick muscles, while its role(s) in vivo was unknown. In the present study, using specific polyclonal antibodies against MDP77, we show that MDP77 was expressed specifically in the skeletal and cardiac muscle, and confirm its presence in the cytoplasm of the extrafusal muscle fibers. In mature muscles, MDP77 immunoreactivity was observed in a repetitive manner along the sarcomere. The onset of MDP77 expression occurred just after myotube formation both in vivo and in vitro. Furthermore, MDP77 was enriched in the intrafusal muscle fibers. Our findings suggest that MDP77 plays an important role(s) in the differentiation, maturation and function of both the skeletal and cardiac muscles.     

Importance of N-linked sugar residues in the development of Auerbach's plexus in the rat colon: a lectin histochemical study

The lectin-binding patterns in Auerbach's plexus in the distal portions of the rat colon from 15- to 21-day-old foetuses, newborns, and adults were examined by light and electron microscopy using 16 different lectins (ConA, RCA-1, WGA, PNA, SBA, UEA-1, DBA, LCA, PHA-L, DSA, GS-1, VVA, MPA, BPA, MAA, and PSA). The binding of ConA was shown to increase after day 19 of gestation in parallel with differentiation of Auerbach's plexus, whereas the staining intensity for DSA and RCA-1 increased after day 17 of gestation in accordance with the appearance of the plexus. At the electron microscopical level, DSA binding sites were observed to be localized mainly in the plasma membrane, Golgi apparatus, and nuclear membrane of nerve cells. Positive sites were also observed in the axolemma and in the plasma membrane of nerve cell processes, Schwann cells, and the surrounding smooth muscle cells. PSA, PHA-L, LCA, and WGA showed constant staining during the development after day 15 of gestation. Other lectins, most of which are specific for O-glycosidic mucin-type sugar residues, were essentially negative throughout the developmental stages. Moreover, N-glycanase digestion significantly diminished the positive reactions. N-linked oligosaccharides may thus play important roles in the development and maturation of the Auerbach's plexus, and may be involved in the developmental defect of the plexus, e.g. as occurs in Hirschsprung's disease.     

Direct, Ca2+-dependent interaction between tubulin and synaptotagmin I: a possible mechanism for attaching synaptic vesicles to microtubules

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.     

Antibacterial activity of isoflavonoids isolated from Erythrina variegata against methicillin-resistant Staphylococcus aureus

AIMS: To screen 16 isoflavonoids isolated from Erythrina variegata (Leguminosae) for their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: The roots of E. variegata were macerated with acetone. The chloroform-soluble fraction of the residue was subjected to repeated silica gel column chromatography followed by elution with various solvents. Structures of the isolated compounds were determined by extensive spectroscopic studies. Each compound was dissolved in dimethyl sulphoxide and added to agar plates (final concentration 1.56-100 microg ml(-1) and suspensions of MRSA spotted onto the agar plates to determine the minimum inhibitory concentration (MIC). Repeated silica gel chromatography yielded 16 compounds and spectroscopic studies revealed that all were isoflavonoids. Whilst 14 compounds showed antibacterial activity in this concentration range, the MIC values varied significantly among them. Of the active compounds, 3,9-dihydroxy-2,10-di(gamma,gamma-dimethylallyl)-6a,11a-dehydropterocarpan (erycristagallin) and 9-hydroxy-3-methoxy-2-gamma,gamma-dimethylallylpterocarpan (orientanol B) exhibited the highest activity with MIC values of 3.13-6.25 microg ml(-1). CONCLUSIONS: Erycristagallin and orientanol B showed the highest anti-MRSA activity (3.13-6.25 microg ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: Erycristagallin and orientanol B could be leading compounds for phytotherapeutic agents against MRSA infections.