The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning-defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR-3 and PAR-6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR-3 associates with the tight-junction-associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin-1, -4 or -5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3 localization at intercellular junctions and delays ASIP/PAR-3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR-3-aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.     

Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.

Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.     

EL-4 tumor cell-induced human and rabbit platelet aggregations

EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.     

Improved immobilization of DNA to microwell plates for DNA-DNA hybridization.

An improved and simplified protocol for DNA immobilization was developed to enhance DNA-DNA hybridization on microwell plates. Target DNA was immobilized by simple dry-adsorption. Efficiencies of DNA immobilization and retention were enhanced 1.4-6.5 times and 4.2-19.6 times, respectively, compared with a conventional method. The overall hybridization efficiency was increased 3.1-5.2 times. This simple new protocol can reduce the consumption of scarce DNA samples.     

Localization of α-Galactosidase in an Alkalophilic Strain of Micrococcus†

Localization of α-galactosidase in an alkalophilic strain of Micrococcus was investigated in relation to the cell membrane as a permeability barrier. The most α-galactosidase appered to be intracellular; only about 4% of α-galactosidase was released by lysozyme or freeze-thaw treatments of the whole cells. The enzyme activity was not inhibited by treatment of the whole cells with diazo-7-amino-1,3-naphthalene disulfonic acid (NDS) which penetrated the cell wall but not the cytoplasmic membrane. The enzyme activity of the whole cells increased about four-fold by toluene-acetone treatment which caused an alteration in the membrane permeability. The enzyme in such cells became to be relatively sensitive to pH. These results showed that cell membrane played a protective role as a permeability barrier against alkaline environment.     

K+/H+ antiporter in alkaliphilic Bacillus sp. no. 66 (JCM 9763)

A K⁺/H⁺ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. no. 66 (JCM 9763). An outwardly directed K⁺ gradient (intravesicular K⁺ concentration, K_in, 100 mM; extravesicular K⁺ concentration, K_out, 0.25 mM) stimulated uphill H⁺ influx into right-side-out vesicles and created the inside-acidic pH gradient (ΔpH). This H⁺ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4. Addition of 100 μM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ eflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but Na⁺ and Li⁺ could not. The H⁺ efflux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles. Received: March 20, 1997 / Accepted: May 22, 1997     

Pressure-regulated metabolism in microorganisms.

There has been a renewal of interest in the survival strategies employed by deep-sea, high-pressure-adapted (piezophilic) microorganisms as well as in the effects of high pressure on mesophilic, 1-atmosphere-pressure-adapted microorganisms. This is partly the result of a greater appreciation of the adaptations of microorganisms to life in extreme environments and partly the result of the development of new techniques for examining physiological and molecular processes as a function of pressure.     

Purification of two pressure-regulated c-type cytochromes from a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

From a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F, a membrane-bound cytochrome c-551 and a cytoplasmic cytochrome c-552 were purified. The cytochrome c-551 contained 44.2 nmol of heme c mg protein⁻¹ and cytochrome c-552 contained 31.3 nmol of heme c mg protein⁻¹. The CO difference spectrum of cytochrome c-551 showed a peak at 413.7 nm and troughs at 423.2, 522 and 552 nm which indicated that this cytochrome combined with CO. Cytochrome c-551 was found to consist of two subunits with molecular masses of 29.1 kDa and 14.7 kDa, respectively, and each subunit contained one heme c molecule. Cytochrome c-552 also consisted of two subunits with molecular masses of 16.9 kDa and 14.7 kDa, respectively, and only one of these subunits contained heme c. Cytochrome c-551 was constitutively synthesized when the cells were grown at pressures of either 0.1 MPa or 60 MPa, whereas cytochrome c-552 was synthesized only at 0.1 MPa. These results together with the results of analysis of membrane-associated catalytic activities suggest that the respiratory system of DB-172F is regulated by pressure and may be intimately related to the baroadaptability mechanism of this deep-sea bacterium.