Fyn and Cdk5 mediate semaphorin-3A signaling,which is involved in regulation of dendrite orientation in cerebral cortex

Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.     

Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells

Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3beta-HSD type II (3beta-HSD II), P450c17, P450c21 and P45011beta, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 microM of the flavonoids 6-hydroxyflavone, 4'-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 microM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 microg/ml (24 microM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3beta-HSD II activity at concentrations from 1 to 25 microM, and I(50) values were estimated to be 1.3, 2, 1, 0.5 and 2.7 microM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 microM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011beta at 12.5 and/or 25 microM. Results of Lineweaver-Burk's plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3beta-HSD II and P450c21. K(m) and V(max) values of 3beta-HSD II for DHEA were estimated to be 6.6 microM and 328pmol/minmg protein, respectively. K(m) and V(max) values of P450c21 for progesterone were estimated to be 2.8 microM and 16pmol/minmg protein, respectively. K(i) values of 3beta-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 microM, respectively.     

Estrogens inhibit l-glutamate uptake activity of astrocytes via membrane estrogen receptor alpha

We investigated the effects of estrogen-related compounds including xenoestrogens [17beta-estradiol (E2), 17alpha-ethynylestradiol (EE), diethylstilbestrol (DES), p-nonylphenol (PNP), bisphenol A (BPA) and 17alpha-estradiol (17alpha)] on l-glu uptake by cultured astrocytes via glutamate-aspartate transporter (GLAST). After 24 h treatment, E2 inhibited the l-glu uptake at 1 micro m and higher concentrations. EE and DES also inhibited the l-glu uptake at 1 nm and higher concentrations. The other four compounds had no effect. The effects of E2, EE and DES were completely blocked by 10 nm of ICI182 780 (ICI). beta-Estradiol 17-hemisuccinate : bovine serum albumin (E2-BSA), a membrane-impermeable conjugate of E2, also elicited the inhibition of l-glu uptake at 1 nm and higher concentrations, and the effect was blocked by ICI. 16alpha-Iodo-17beta-estradiol (16alphaIE2), an estrogen receptor alpha (ERalpha) selective ligand, revealed an inhibitory effect at 10 nm, while genistein, an ERbeta selective ligand, failed to reveal such an effect at this concentration. Western blot analysis showed that the predominant ER of cultured astrocytes was ERalpha. The colocalization of ERalpha with GLAST on plasma membranes was immunohistochemically detected in these cells. From these results, we concluded that estrogens down-regulate l-glu uptake activity of astrocytes via membrane ERalpha.     

Interisland Evolution of <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom Phospholipase A<Subscript>2</Subscript> Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A₂ (PLA₂), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA₂ from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA₂, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp⁴⁹]PLA₂ isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA₂, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA₂ isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA₂ isozymes are clearly separated into three groups, PLA2 type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Basic [Asp⁴⁹]PLA₂-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys⁴⁹]PLA₂ type.     

Oligomerization and pore formation of a sphingomyelin-specific toxin,lysenin

Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of approximately 3 nm. Antibody scanning analysis suggested that the C-terminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.     

The effects of reactive nitrogen intermediates on gene expression in Mycobacterium tuberculosis

Nitric oxide (NO) and related reactive nitrogen intermediates (RNI) are effective antimycobacterial agents and signal-transducing molecules. The present study uses microarray analysis to examine the effects of RNI on Mycobacterium tuberculosis gene expression. A common set of 53 genes was regulated by two chemically distinct nitric oxide donors. For a subset of the RNI-inducible genes, evidence exists suggesting that they may play a role in promoting survival of the tubercle bacillus in the host. Results obtained from studies based on a murine experimental tuberculosis model involving nos2-deficient mice suggest that RNI could regulate M. tuberculosis gene expression in vivo. Finally, there is a remarkable overlap between the RNI-inducible regulon and that previously reported to be regulated by hypoxia; and both reactive nitrogen species and anaerobicity upregulate the expression of one and the same putative two-component regulatory response system. Together, the results of this study provide evidence suggesting that (i) RNI play a role in regulating M. tuberculosis gene expression in vivo; (ii) the reactive nitrogen species upregulate genes that may be conducive to the survival of the tubercle bacillus in the infected host; and (iii) RNI and hypoxia may regulate mycobacterial gene expression via overlapping signal transduction pathways.     

Effect of moderate acute exercise on expression of mRNA involved in the calcineurin signaling pathway in human skeletal muscle

Calcineurin, a calcium-regulated protein phosphatase, activates gene expression specific to slow muscle fibers by dephosphorylating a family of the nuclear factor of activated T cells (NFAT), which cooperates with myocyte enhancer factor-2 (MEF2) and AP-1. However, it remains unknown how acute exercise influences this signaling pathway and leads to the development of slow muscle fibers. In the present study, we investigated the effect of moderate acute exercise on mRNA expression of genes in the calcineurin signaling pathway in human skeletal muscle. Five healthy volunteers underwent 1 h bicycle ergometer at 50%VO2max, and vastus lateralis muscle biopsies were collected before and after exercise. Four hours after exercise, alterations in mRNA expression of NFAT 1-3 were observed with a wide variety among subjects, while c-fos mRNA was significantly induced in all subjects. By contrast, the expression of calcineurin, MEF2, and myocyte-enriched calcineurin-interacting protein 1 (MCIP1) remained unchanged. These results suggest that even moderate acute exercise may change mRNA expression of genes in the calcineurin-signaling pathway.     

Selective genotyping with epistasis can be utilized for a major quantitative trait locus mapping in hypertension in rats

Epistasis used to be considered an obstacle in mapping quantitative trait loci (QTL) despite its significance. Numerous epistases have proved to be involved in quantitative genetics. We established a backcross model that demonstrates a major QTL for hypertension (Ht). Seventy-eight backcrossed rats (BC), derived from spontaneously hypertensive rats (SHR) and normotensive Fischer 344 rats, showed bimodal distribution of systolic blood pressure (BP) values and a phenotypic segregation ratio consistent with 1:1. In this backcross analysis, sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (Serca) II heterozygotes showed widespread bimodality in frequency distribution of BP values and obviously demonstrated Ht. First, in genome-wide screening, Mapmaker/QTL analysis mapped Ht at a locus between D1Mgh8 and D1Mit4 near Sa in all 78 BC. The peak logarithm of the odds (LOD) score reached 5.3. Second, Serca II heterozygous and homozygous BC were analyzed separately using Mapmaker/QTL. In the 35 Serca II heterozygous BC, the peak LOD score was 3.8 at the same locus whereas it did not reach statistical significance in the 43 Serca II homozygotes. Third, to map Ht efficiently, we selected 18 Serca II heterozygous BC with 9 highest and 9 lowest BP values. In these 18 BC, the peak LOD score reached 8.1. In 17 of the 18, D1Mgh8 genotypes (homo or hetero) qualitatively cosegregated with BP phenotypes (high or low) (P < 0.0001, by chi-square analysis). In conclusion, selective genotyping with epistasis can be utilized for a major QTL mapping near Sa on chromosome 1 in SHR.