Endothelin-1 binding sites and inositol-monophosphate formation in kidney medulla from adult and juvenile SHR and WKY rats

High resolution autoradiography at the cellular level localized endothelin-1 binding sites to the collecting ducts in the rat renal papilla. These receptors were functionally coupled to the phosphatidyl-inositol system since endothelin-1 stimulated the accumulation of IP₁ in a concentration-dependent manner in cross chopped slices from renal papillae. The concentration-effect curves in 4 week old normotensive Wistar-Kyoto rats (WKY) lay to the right of curves from 4 week old and adult spontaneously hypertensive rats (SHR) and adult WKY (20–24 week old) animals; the EC₅₀ values in the 4 week old animals were 17 ± 5 and 8 ± 1 nM (P < 0.05, N = 5; mean ± SE) for the normotensive and hypertensive animals, respectively. Autoradiographic studies showed that the density and distribution of binding sites for [¹²⁵I]endothelin-1 in the kidneys did not differ between the groups; receptor densities in the renal papillae were 461 ± 37 (fmol/mg protein) in the 4 week old WKY, and 443 ± 27 (fmol/mg protein) in the 4 week old SHR. The plasma levels of immunoreactive endothelin-1 were also similar between groups; 4 week old SHR (39 ± 3 pg/ml) and 4 week old WKY (36 ± 1 pg/ml). The increased response to endothelin-1 may be related to the development of the hypertensive state in the SHR.     

Epidermal growth factor disrupts gap-junctional communication and induces phosphorylation of connexin43 on serine.

Growth factors regulate cellular proliferation and differentiation by activating plasma membrane tyrosine kinase receptors and triggering a cascade of events mediated by intracellular signaling proteins. The mechanism underlying growth factor modification of cellular functions, such as gap-junctional communication (gjc), has not been established clearly. Addition of epidermal growth factor (EGF) to T51B rat liver epithelial cells resulted in the rapid activation of EGF receptor tyrosine kinase activity followed by a transient dose-dependent disruption of gjc. This change did not result from the gross disturbance of membrane gap junction plaques as measured by immunofluorescence microscopy, but instead correlated with markedly elevated phosphorylation of the connexin43 (cx43) gap junction protein, a profound shift to predominantly phosphorylated forms of cx43, and the appearance of a novel phosphorylated cx43 protein. These changes in cx43 phosphorylation involved only serine residues. On restoration of gjc, these alterations in cx43 phosphorylation reverted to the pre-EGF treatment state. Both events were inhibited by the serine/threonine protein phosphatase inhibitor, okadaic acid. Therefore, unlike the case for pp60v-src, EGF-induced disruption of gjc is not associated with tyrosine phosphorylation of cx43, but instead may result from phosphorylation of cx43 by activated intracellular signaling serine protein kinase(s).     

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.     

Evaluation of the sedative effect of fragrance on Filipinas using a biochemical marker

The purpose of this study is to evaluate the usefulness of salivary amylase activity as an indicator of the acute psychological sedative effects of fragrances used in household products such as clothing softeners. Twenty seven healthy Philippine female subjects in their late 30 s were enrolled (38.7+/-5.2 yr). This study was undertaken to investigate the favorite fragrance of Asian and Pacific Islander women. Our results indicated that (i) a mixed floral fragrance might be a favorite fragrance for Filipinas; (ii) fragrances contained in a softener significantly induced a sedative effect in humans, as assessed by both analysis of the biochemical marker and subjective evaluation; (iii) salivary amylase activity has the potential to be an excellent indicator for the evaluation of the acute psychological sedative effects of fragrance.     

Contributions of crust proteins to spore surface properties in Bacillus subtilis

Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.     

Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli O157: H 7

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.     

Halotolerant Cyanobacterium Aphanothece halophytica Contains a Betaine Transporter Active at Alkaline pH and High Salinity

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT_{A. halophytica}) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis, is basic, 9.54, that of BetT_{A. halophytica} is acidic, 4.58. BetT_{A. halophytica} specifically catalyzed the transport of betaine. Choline, γ-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT_{A. halophytica}. Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT_{A. halophytica} is a Na⁺-betaine symporter. Betaine uptake activities of BetT_{A. halophytica} were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT_{A. halophytica} showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetT_{A. halophytica} were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na⁺-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT_{A. halophytica} is the first identified transporter for compatible solutes in cyanobacteria.