Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype.

A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.     

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.     

Production of chemotactic factor for lymphocytes by neutral SH-dependent protease of rabbit PMN leukocytes from immunoglobulins,especially IgM

In inflammation, PMN leukocyte emigration is followed by lymphocyte emigration. Two neutral proteases were isolated from lysosomal fraction of rabbit PMN leukocytes and purified by chromatography. The SH-dependent protease converted in vitro a naturally occurring IgM and specific antibody IgM to a chemotactic factor for lymphocytes; its molecular size was assumed to be around 14,000. Lymphocytes were collected from the thoracic duct lymph of rats. The chemotactic generation was induced by a short treatment with small amount of the enzyme, but the chemotactic factor produced was inactivated by a prolonged digestion with the enzyme. The chemotactic generation by the enzyme of rabbit IgG was apparently less marked. On the other hand, the SH-independent protease was ineffective for such chemotactic generation, suggesting different enzymatic characteristics of these proteases.     

Antigenic Difference Between Polar Monotrichous and Peritrichous Flagella of Vibrio parahaemolyticus

Polar monotrichous and peritrichous flagella of Vibrio parahaemolyticus were isolated and purified separately. On hydroxylapatite column chromatography, the flagellins of polar monotrichous flagella were eluted with a higher concentration of phosphate than those of peritrichous flagella. Gel diffusion tests showed an antigenic difference between the flagellins of polar monotrichous and peritrichous flagella. Electron microscope observations on cells stained with ferritin-conjugated antibodies demonstrated that polar monotrichous and peritrichous flagella reacted specifically with antimonotrichous flagellin and antiperitrichous flagellin antisera, respectively.     

Stereochemistry of the methoxymercuration of glycal acetates

The methoxymercuration of the acetates of d-glucal, d-galactal, l-arabinal and d-xylal was studied by n.m.r. spectroscopy, and by stereoselective demercuration with thiourea, as well as by reductive demercuration with sodium borohydride. It is considered to be a trans-addition to the double bond to yield isomeric methyl glycosides having mercury attatched to C-2. The proportion of diaxial to diequatorial addition is strongly affected by the protecting groups at C-4 and C-5.