Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation. (25/16624)

The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeria and Shigella infections. In contrast to the case for Listeria and Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the DeltaB5R and DeltaF13L deletion strains, but not DeltaA34R, were still able to induce actin tails. The DeltaA36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from DeltaA34R- and DeltaA36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation.  (+info)

Dynamic regulation of gastric surface pH by luminal pH. (26/16624)

In vivo confocal imaging of the mucosal surface of rat stomach was used to measure pH noninvasively under the mucus gel layer while simultaneously imaging mucus gel thickness and tissue architecture. When tissue was superfused at pH 3, the 25 microm adjacent to the epithelial surface was relatively alkaline (pH 4.1 +/- 0.1), and surface alkalinity was enhanced by topical dimethyl prostaglandin E2 (pH 4.8 +/- 0.2). Luminal pH was changed from pH 3 to pH 5 to mimic the fasted-to-fed transition in intragastric pH in rats. Under pH 5 superfusion, surface pH was relatively acidic (pH 4.2 +/- 0.2). This surface acidity was enhanced by pentagastrin (pH 3.5 +/- 0.2) and eliminated by omeprazole, implicating parietal cell H,K-ATPase as the dominant regulator of surface pH under pH 5 superfusion. With either pH 5 or pH 3 superfusion (a) gastric pit lumens had the most divergent pH from luminal superfusates; (b) qualitatively similar results were observed with and without superfusion flow; (c) local mucus gel thickness was a poor predictor of surface pH values; and (d) no channels carrying primary gastric gland fluid through the mucus were observed. The model of gastric defense that includes an alkaline mucus gel and viscous fingering of secreted acid through the mucus may be appropriate at the intragastric pH of the fasted, but not fed, animal.  (+info)

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin. (27/16624)

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.  (+info)

Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding. (28/16624)

p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. Here, we show that the two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but was not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictated the cell cycle-dependent functioning of the NLS of FER kinase. This process was governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences was not affected by kinase activity but was perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfered with S-phase progression in these cells. This was not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning.  (+info)

Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida. (29/16624)

The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]i increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+]i occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]i was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle approximately 20 s after the onset of the [Ca2+]i rise. High-resolution imaging revealed that the [Ca2+]i rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]i rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+]i rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR.  (+info)

Bacteroides fragilis toxin 2 damages human colonic mucosa in vitro. (30/16624)

BACKGROUND: Strains of Bacteroides fragilis producing a 20 kDa protein toxin (B fragilis toxin (BFT) or fragilysin) are associated with diarrhoea in animals and humans. Although in vitro results indicate that BFT damages intestinal epithelial cells in culture, the effects of BFT on native human colon are not known. AIMS: To examine the electrophysiological and morphological effects of purified BFT-2 on human colonic mucosa in vitro. METHODS: For resistance (R) measurements, colonic mucosa mounted in Ussing chambers was exposed to luminal or serosal BFT-2 (1.25-10 nM) and after four hours morphological damage was measured on haematoxylin and eosin stained sections using morphometry. F actin distribution was assessed using confocal microscopy. RESULTS: Serosal BFT-2 for four hours was four-, two-, seven-, and threefold more potent than luminal BFT-2 in decreasing resistance, increasing epithelial 3H-mannitol permeability, and damaging crypt and surface colonocytes, respectively (p<0.05). Confocal microscopy showed reduced colonocyte F actin staining intensity after exposure to BFT-2. CONCLUSIONS: BFT-2 increases human colonic permeability and damages human colonic epithelial cells in vitro. These effects may be important in the development of diarrhoea and intestinal inflammation caused by B fragilis in vivo.  (+info)

Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. (31/16624)

We show that several transcriptionally inactive genes localize to centromeric heterochromatin in the nucleus of cycling but not quiescent (noncycling) primary B lymphocytes. In quiescent cells, centromeric repositioning of inactive loci was induced after mitogenic stimulation. A dynamic repositioning of selected genes was also observed in developing T cells. Rag and TdT loci were shown to relocate to centromeric domains following heritable gene silencing in primary CD4+8+ thymocytes, but not in a phenotypically similar cell line in which silencing occurred but was not heritable. Collectively, these data indicate that the spatial organization of genes in cycling and noncycling lymphocytes is different and that locus repositioning may be a feature of heritable gene silencing.  (+info)

Chemical transformations in individual ultrasmall biomimetic containers. (32/16624)

Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.  (+info)