Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.     

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.     

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.     

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.     

Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons

Necdin is expressed predominantly in postmitotic neurons and serves as a growth suppressor that is functionally similar to the retinoblastoma tumor suppressor protein. Using primary cultures of dorsal root ganglion (DRG) of mouse embryos, we investigated the involvement of necdin in the terminal differentiation of neurons. DRG cells were prepared from mouse embryos at 12.5 days of gestation and cultured in the presence of nerve growth factor (NGF). Immunocytochemistry revealed that necdin accumulated in the nucleus of differentiated neurons that showed neurite extension and expressed the neuronal markers microtubule-associated protein 2 and synaptophysin. Suppression of necdin expression in DRG cultures treated with antisense oligonucleotides led to a marked reduction in the number of terminally differentiated neurons. The antisense oligonucleotide-treated cells did not attempt to reenter the cell cycle, but underwent death with characteristics of apoptosis such as caspase-3 activation, nuclear condensation, and chromosomal DNA fragmentation. Furthermore, a caspase-3 inhibitor rescued antisense oligonucleotide-treated cells from apoptosis and significantly increased the population of terminally differentiated neurons. These results suggest that necdin mediates the terminal differentiation and survival of NGF-dependent DRG neurons and that necdin-deficient nascent neurons are destined to caspase-3-dependent apoptosis.     

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.     

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.