Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.     

Phosphatidylserine induces apoptosis in adherent cells

Phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane in apoptotic cell death. However, the roles of PS in apoptotic signaling are still unclear. In this study, we found that exogenous PS, but not other phospholipids, induced cell death in adherent cells, but not in suspension culture. The cell death exhibited typical features of apoptosis such as cell shrinkage, nuclear fragmentation and abnormal chromatin condensation. When PS was added to CHO-K1 cells in monolayer culture, they began to show changes in cell shape and actin cytoskeleton and protein kinase C (PKC) activity, followed by cell detachment, caspase activation, cleavage of focal adhesion kinase (FAK) and finally loss of viability. These results suggested that PS causes apoptosis through actin disorganization, cell detachment and cleavage of FAK.     

Potential of selected strains of lactic acid bacteria to induce a Th1 immune profile

Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.     

Increased posthatching mortality and loss of sexually dimorphic gene expression in alligators (Alligator mississippiensis) from a contaminated environment

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.     

Elucidating the multiple genetic lineages and population genetic structure of the brooding coral <Emphasis Type="Italic">Seriatopora</Emphasis> (Scleractinia: Pocilloporidae) in the Ryukyu Archipelago

The elucidation of species diversity and connectivity is essential for conserving coral reef communities and for understanding the characteristics of coral populations. To assess the species diversity, intraspecific genetic diversity, and genetic differentiation among populations of the brooding coral Seriatopora spp., we conducted phylogenetic and population genetic analyses using a mitochondrial DNA control region and microsatellites at ten sites in the Ryukyu Archipelago, Japan. At least three genetic lineages of Seriatopora (Seriatopora-A, -B, and -C) were detected in our specimens. We collected colonies morphologically similar to Seriatopora hystrix, but these may have included multiple, genetically distinct species. Although sexual reproduction maintains the populations of all the genetic lineages, Seriatopora-A and Seriatopora-C had lower genetic diversity than Seriatopora-B. We detected significant genetic differentiation in Seriatopora-B among the three populations as follows: pairwise F _ST = 0.064–0.116 (all P = 0.001), pairwise G′′_ST = 0.107–0.209 (all P = 0.001). Additionally, only one migrant from an unsampled population was genetically identified within Seriatopora-B. Because the peak of the settlement of Seriatopora larvae is within 1 d and almost all larvae are settled within 5 d of spawning, our observations may be related to low dispersal ability. Populations of Seriatopora in the Ryukyu Archipelago will probably not recover unless there is substantial new recruitment from distant populations.     

Unexpected ceiling of genetic differentiation in the control region of the mitochondrial DNA between different subspecies of the ayu Plecoglossus altivelis

Sequence analyses of the non-coding, control region (CR) and coding region of the ND4-tRNA(Ser) genes in the mitochondrial DNA (mtDNA) were conducted for populations of the ayu Plecoglossus altivelis altivelis and the Ryukyu-ayu P. a. ryukyuensis. The level of genetic differentiation between the two subspecies evaluated from the CR data was substantially low, when comparing with that estimated from ND4-tRNA(Ser) gene region data, as well as those from nuclear genome data sets. By contrast, the differentiation between subspecies in the ND4-tRNA(Ser) gene region was substantial, being consistent with the results from the previous nuclear genome analyses. Results of UPGMA and minimum spanning network analyses also implied the unexpected ceiling of genetic differentiation in the CR. These results suggest that the CR does not reflect accurately the level of overall genetic differentiation between the populations of the ayu, but other coding regions of the mtDNA do reflect it so that the mtDNA on the whole may function as a rich source of useful markers for genetic assessment of populations of this species.     

An improved enrichment broth for isolation of Escherichia coli O157, with specific reference to starved cells,from radish sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

The role of circulating ghrelin in growth hormone (GH) secretion in freely moving male rats

To examine the physiological significance of plasma ghrelin in generating pulsatile growth hormone (GH) secretion in rats, plasma GH and ghrelin levels were determined in freely moving male rats. Plasma GH was pulsatilely secreted as reported previously. Plasma ghrelin levels were measured by both N-RIA recognizing the active form of ghrelin and C-RIA determining total amount of ghrelin. Mean +/- SE plasma ghrelin levels determined by N-RIA and C-RIA were 21.6 +/- 8.5 and 315.5 +/- 67.5 pM, respectively, during peak periods when plasma GH levels were greater than 100 ng / ml. During trough periods when plasma GH levels were less than 10 ng / ml, they were 16.5 +/- 4.5 and 342.1 +/- 29.8 pM, respectively. There were no significant differences in plasma ghrelin levels between two periods. Next, effect of a GH secretagogue antagonist, [D-Lys-3]-GHRP-6, on plasma GH profiles was examined. There were no significant differences in both peak GH levels and area under the curves of GH (AUCs) between [D-Lys-3]-GHRP-6-treated and control rats. These findings suggest circulating ghrelin in peripheral blood does not play a role in generating pulsatile GH secretion in freely moving male rats.     

T-588 Protects Motor Neuron Death Against Glutamate-Induced Neurotoxicity

To examine the possible neuroprotective effect of T-588 against glutamate-induced neurotoxicity, we analyzed the pharmacological utility of T-588 in a postnatal organotypic culture model of motor neuron degeneration. Treatment with 10^–5 M of glutamate resulted a motor neuron loss and decreased activity of choline acetyltransferase (ChAT). Cotreatment of 10^–5 M of glutamate and T-588 revealed a protective effect against motor neuron death and decreased ChAT activity. We concluded that T-588 may play important roles in the survival and maintenance of spinal motor neurons in its neuroprotection against glutamate-induced neurotoxicity. Our data may provide a rationale for designing a therapeutic strategy for protection against pathologically induced motor neuron damage or cell death such as amyotrophic lateral sclerosis and motor neuropathy.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.