Mutational analysis of amiloride sensitivity of the NhaA Na+/H+ antiporter from Vibrio parahaemolyticus.

The activity of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus is inhibited by amiloride. We found an amino acid sequence in the NhaA that was identical to a putative amiloride binding domain of the Na+/H+ exchanger in mammalian cells. We constructed mutant NhaAs that had amino acid substitutions in the putative amiloride binding domain by site-directed mutagenesis. These include V62L (Val62 replaced by Leu), F63Y, F64Y, and L65F. Most mutant NhaAs showed decreased sensitivity for amiloride. Among these, the F64Y mutant NhaA showed the least amiloride sensitivity, with a Ki value 7 to 10 times greater than that in the wild type. Thus, the sequence between residues V62 and L65 in NhaA, especially F64, is very important for the inhibitory effect of amiloride on the antiporter.     

Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes

Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacaine相似文献   

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.     

Vertical Distribution and Estimated Doses from Artificial Radionuclides in Soil Samples around the Chernobyl Nuclear Power Plant and the Semipalatinsk Nuclear Testing Site

For the current on-site evaluation of the environmental contamination and contributory external exposure after the accident at the Chernobyl Nuclear Power Plant (CNPP) and the nuclear tests at the Semipalatinsk Nuclear Testing Site (SNTS), the concentrations of artificial radionuclides in soil samples from each area were analyzed by gamma spectrometry. Four artificial radionuclides (²⁴¹Am, ¹³⁴Cs, ¹³⁷Cs, and ⁶⁰Co) were detected in surface soil around CNPP, whereas seven artificial radionuclides (²⁴¹Am, ⁵⁷Co, ¹³⁷Cs, ⁹⁵Zr, ⁹⁵Nb, ⁵⁸Co, and ⁶⁰Co) were detected in surface soil around SNTS. Effective doses around CNPP were over the public dose limit of 1 mSv/y (International Commission on Radiological Protection, 1991). These levels in a contaminated area 12 km from Unit 4 were high, whereas levels in a decontaminated area 12 km from Unit 4 and another contaminated area 15 km from Unit 4 were comparatively low. On the other hand, the effective doses around SNTS were below the public dose limit. These findings suggest that the environmental contamination and effective doses on the ground definitely decrease with decontamination such as removing surface soil, although the effective doses of the sampling points around CNPP in the present study were all over the public dose limit. Thus, the remediation of soil as a countermeasure could be an extremely effective method not only for areas around CNPP and SNTS but also for areas around the Fukushima Dai-ichi Nuclear Power Plant (FNPP), and external exposure levels will be certainly reduced. Long-term follow-up of environmental monitoring around CNPP, SNTS, and FNPP, as well as evaluation of the health effects in the population residing around these areas, could contribute to radiation safety and reduce unnecessary exposure to the public.     

Correlations in one-dimensional fully developed chaos

Correlations in the baker map and the tent map as examples of one-dimensional, fully developed chaos are considered. It is shown, utilizing symbolic dynamical systems derived from these maps, that the vanishing second-order correlation function is not sufficient to guarantee uncorrelatedness. Importance of the higher-order, especially third-order, correlation functions is emphasized for chaotic systems. In search of the quantities that grasp correlational behaviors as a whole in chaotic systems, it is proposed to use the fixed-separation correlation integral, which is a modified quantity of the usual correlation integral devised to calculate the fractal dimension of strange attractors, for these maps. It is shown that the new quantity contains all the even-number orders of autocorrelation function that are commonly considered.     

Suppression of anoikis by v-Src but not by activated c-H-ras in human gallbladder epithelial cells

Detachment of anchorage-dependent normal epithelial cells from their substratum causes the type of apoptosis known as anoikis, whereas malignant cells can proliferate independently of anchorage. Because src and ras oncogenes are activated in many human cancers, we investigated their role and downstream signaling pathways in anoikis resistance, using HAG-1 human epithelial cells transfected with v-src or activated H-ras. Consequently, anchorage-dependent mock- or ras-transfected cells underwent anoikis. In contrast, anchorage-independent v-Src-transformed cells did not exhibit such apoptotic features. Focal adhesion kinase (FAK), a transducer of integrin, was only activated in v-Src-transformed cells. Herbimycin A, an Src kinase inhibitor, reduced tyrosyl phosphorylation of FAK and reversed resistance to anoikis. However, both protein kinase C (PKC) and phophatidylinositol-3 (PI-3) kinase inhibitors failed to induce anoikis. These data suggest that the ability of activated Src to prevent anoikis may be mediated by Src to a downstream signaling pathway involving FAK, but not Ras, PI-3 kinase, or PKC.     

Characteristic Decrease of Nuclear Protein Kinase Activity in G1-Arrested Cells of Saccharomyces cerevisiae cdc28

An alkalopsychrotrophic strain, Micrococcus sp. 207, inducibly and extracellularly produced amylase and pullulanase. The main hydrolysis product from amylose, with a crude enzyme preparation, was maltotetraose. The optimum temperature for activity of the amylase was 60°C and that for pullulanase 55°C. The activities at 0 to 30°C exhibited similar activation energy values. In an optimized production medium at pH 9.7, the highest yields of these enzymes were obtained after cell growth at 18°C for 4 days. At pH 8.5, the yields of amylase and pullulanase became maximum after 3 days cultivation. With more prolonged cultivation, the yield of amylase but not that of pullulanase activity decreased. These enzymes were not produced at temperatures above 30°C. Sucrose was not effective as an inducer, but it stimulated cell growth and enhanced the enzyme productivities with soluble starch.