Morphological variety of intracellular microcolonies of Legionella species in Vero cells

Intracellular microcolonies of six Legionella species growing in Vero cells showed distinctly varied morphologies. The varieties were observed by light microscopy of Gimenez-stained, Legionella-infected Vero cells and by electron microscopy (EM). Legionella pneumophila Philadelphia-1 formed needle-shaped crystal-like microcolonies. Legionella bozemanii WIGA formed microcolonies like wool balls containing filamentous cells. In EM, these organisms proliferated in endosomes, which were adjacent to swollen rough endoplasmic reticula. Legionella oakridgensis OR-10 showed serpentine chains. Many mitochondria were observed around the microcolonies. Legionella jordanis BL-540 formed spherical moss-like microcolonies which were or were not surrounded by endoplasmic membranes. Legionella feeleii WO-44C spread throughout the cytoplasm without making clusters. Legionella dumoffii Tex-KL made big clusters that spread in the cytoplasm, a portion of which was outside the endosome membranes. These different morphologies imply diversity in modes of intracellular multiplication of Legionella spp.     

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na⁺]_i in these cells loaded with the Na⁺ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na⁺]_i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA₂ activity (10 μm AACOCF₃) obviated EGF-induced increases in [Na⁺]_i. In contrast, PGE₂ (10 μg/ml) and cAMP (2 mm) increased [Na⁺]_i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na⁺]_i. Similarly, EGF failed to increase [Na⁺]_i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na⁺]_i followed by a decline towards its baseline value. EGF-induced increases in [Na⁺]_i were unaltered by inhibition of K⁺ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA₂ activity. Their stimulation increases PGE₂ and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001     

PHOSPHORYLATION OF ENDOGENOUS PROTEINS IN MYELIN OF RAT BRAIN

Abstract— The phosphorylation of endogenous proteins occurring in the myelin of rat brain was examined using the method of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two myelin basic proteins and at least five more proteins were phosphorylated after incubation of myelin fraction in the presence of ATP + Mg²⁺. The apparent molecular weights of the proteins other than the myelin basic proteins were 120,000, 76,000, 60,000, 41,000 and 38,000, respectively. The proteins of mol wt 60,000. 41,000 and 38,000 were extracted by treatment with hydrochloric acid, whereas those of mol wt 120,000 and 76,000 were insoluble in hydrochloric acid and chloroform-methanol. Folch-Lees proteolipid protein was not found to be phosphorylated under the conditions studied. The endogenous phosphorylation of the proteins was not stimulated by adenosine 3',5'-monophosphate.     

Probability of quantal transmitter release from nerve terminals: theoretical considerations in the determination of spatial variation

The release of transmitter occurs in discrete quantal units, such that the number released (m) is equal to the number available (n) times the average probability of release (p). Although a common method of estimating these parameters is to use simple binomial statistics, results may be biased if there is spatial or temporal variation in n and p (vars p, vart n, vart p). The problem arises in the simultaneous analysis of five variables, which is impractical due to the complexity and margin of error involved. The proposed solution is to eliminate two variables (vart n, vart p) by assuming stationarity and to obtain the required information from the first three moments of m. The resulting quadratic equation gives two solutions, p1 and p2. Computer simulation of quantal output as a function of vars p indicates that p1 is the better estimator of p when vars p is small, but that p2 is better when vars p is large. This changeover or "inflection" occurs at points which correspond to the maximum vars p obtainable by unimodal distributions of p (larger vars p being obtained by bimodal distributions). Comparison of the simulated histogram of m with those predicted by p1 and p2 shows that p1 provides the better fit, whether vars p is large or small. This discrepancy indicates that histogram analysis is unable to distinguish the appropriate estimate. The major limitations in the procedure can be met by assuming (1) stationarity (which can be attained and tested experimentally), and (2) normal distribution of p (since vars p is then less than "inflection" point, p1 will always be the correct estimate). The overall findings demonstrate that vars p and unbiased estimates of n and p may be calculated, provided reasonable assumptions are made. This in turn should allow the continued use of quantal parameters for describing transmitter release.     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.     

Polyunsaturated hydrocarbons in the hemolymph: biosynthetic precursors of epoxy pheromones of geometrid and arctiid moths

Female moths of many species in Geometridae, Arctiidae and some other macrolepidopteran families produce epoxy pheromones, which are probably derived from polyunsaturated hydrocarbons. In order to understand a biosynthetic site, hemolymph from both sexes of two geometrid species, Ascotis selenaria cretacea and Hemerophila artilineata, and one arctiid species, Spilosoma imparilis, was shaken with n-hexane and the solvent extracts were analyzed by GC-MS. Each extract of the female hemolymph sex-specifically included polyunsaturated hydrocarbons corresponding to the pheromonal epoxy components in addition to many saturated hydrocarbons, but no epoxy compounds were detected in it. Based on this analysis, deuterated polyunsaturated hydrocarbons were injected into the abdomens of two geometrid females, and the labeled epoxy components were successfully yielded from the pheromone glands. This result indicated that the polyunsaturated hydrocarbons occurring in the female hemolymph were direct pheromone precursors, which might be produced outside the pheromone gland, probably in oenocytes associated with abdominal epidermal cells or in the fat body, and transported to the pheromone gland via the hemolymph for their epoxydation and emission into the atmosphere.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.