Proteomic profiling of the silkworm skeletal muscle proteins during larval-pupal metamorphosis

The silkworm is a typical holometabolous insect going through drastic morphological changes upon metamorphosis from larvae to pupae. Comprehensive studies focusing on the changes help elucidate understanding of a biogenic mechanism. Here, we report the initial profile of the intersegmental muscle (ISM) proteins of the silkworm during larval-pupal metamorphosis. In total, 258 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifty-seven larval proteins were identified, where 3 proteins were exclusively detected in larval samples. Fifty-four other proteins were common in pupal samples. Of these, 12 proteins belonging to the contractile apparatus, metabolism, regulation, and signal transduction were altered in their contents during the metamorphosis from larvae to pupae. Three pupa-defective proteins were identified as isoforms of troponin I, followed by an immunoblotting validation. This data will be helpful in understanding the biochemistry of an insect ISM.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Retinoic acid improves a hybridoma culture in a fructose-based medium by up-regulation of fructose incorporation via retinoid nuclear receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.     

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.     

Exogenous sphingosine 1-phosphate and sphingosine kinase activated by endothelin-1 induced myometrial contraction through differential mechanisms

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid involved in diverse biological processes, is generated by sphingosine kinase (SphK) and acts via intracellular and/or extracellular mechanisms. We used biochemical, pharmacological, and physiological approaches to investigate in rat myometrium the contractile effect of exogenous S1P and the possible contribution of SphK in endothelin-1 (ET-1)-mediated contraction. S1P stimulated uterine contractility (EC₅₀ = 1 µM and maximal response = 5 µM) by a pertussis toxin-insensitive and a phospholipse C (PLC)-independent pathway. Phosphorylated FTY720, which interacts with all S1P receptors, except S1P₂ receptors, failed to mimic S1P contractile response, indicating that the effects of S1P involved S1P₂ receptors that are expressed in myometrium. Contraction mediated by S1P and ET-1 required extracellular calcium and Rho kinase activation. Inhibition of SphK reduced ET-1-mediated contraction. ET-1, via ET_A receptors coupled to pertussis toxin-insensitive G proteins, stimulated SphK1 activity and induced its translocation to the membranes. Myometrial contraction triggered by ET-1 is consecutive to the sequential activation of PLC, protein kinase C, SphK1 and Rho kinase. Prolonged exposure of the myometrium to S1P downregulated S1P₂ receptors and abolished the contraction induced by exogenous S1P. However, in these conditions, the tension triggered by ET-1 was not reduced, indicating that SphK activated by ET-1 contributed to its contractile effect via a S1P₂ receptor-independent process. Our findings demonstrated that exogenous S1P and SphK activity regulated myometrial contraction and may be of physiological relevance in the regulation of uterine motility during gestation and parturition. uterus; contraction     

Plant Homologues of Components of MAPK (Mitogen-Activated Protein Kinase) Signal Pathways in Yeast and Animal Cells

As they respond to numerous extracellular and intracellularstimuli, plants develop various morphological features and thecapacity for a large variety of physiological processes duringtheir growth. If we are to understand the molecular basis ofsuch developments, we must elucidate the way in which signalsgenerated by such stimuli can be transduced into plant cellsand transmitted by cellular components to induce the appropriateterminal events. In yeast and animal systems, signal pathwaysthat are known collectively as MAPK (mitogen-activated proteinkinase) cascades have been shown to play a central role in thetransmission of various signals. The components of these pathwaysinclude the MAPK family, the activator kinases of the MAPK family(the MAPKK family) and the activator kinases of the MAPKK family(the MAPKKK family). The members of each respective family arestructurally conserved and signals are transmitted by similarphosphotransfer reactions at corresponding steps that are mediatedby a specific member of each family in turn. Both cDNAs andgenes that encode putative homologues of these components haverecently been isolated from plant sources. Some of them havebeen shown to be related not only structurally but also functionallyto members of the MAPK cascades of other organisms. These findingssuggest that plants have signal pathways that are analogousto the MAPK cascades in yeast and animal cells but it remainsto be proven that plant homologues do in fact constitute kinasecascades. Given the presence of so many homologues of MAPKsand MAPKKKs in a single plant species, namely, Arabidopsis thaliana,we can be fairly confident that the putative MAPK cascades areinvolved in various physiological processes in plants. (Received March 28, 1995; )     

Integration of molecular and classical linkage groups of the silkworm, Bombyx mori (n = 28).

Previously published linkage groups (LGs) composed of molecular markers were assigned to classical LGs in the silkworm, Bombyx mori (n = 28). Four markers from the classical linkage map, og, w-1, Lp, and Pfl, were assigned to the molecular linkage maps using sequence tagged sites. In addition, linkage analysis was carried out using BF1 progeny between wild-type and mutant stocks carrying morphological phenotypic markers. As a result, the counterparts for 26 of 28 molecular LGs were identified with their counterparts of the classical LGs. Two visible markers, Sel and Xan, representing different classical LGs, were found to be linked.