Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells. (33/2135)

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.  (+info)

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I. (34/2135)

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.  (+info)

The Schizosaccharomyces pombe hst4(+) gene is a SIR2 homologue with silencing and centromeric functions. (35/2135)

Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4(+) gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Delta mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4(+) appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Delta mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4(+) is most similar to HST4. Furthermore, hst4(+) is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4(+) rescues the temperature-sensitivity and telomeric silencing defects of an hst3Delta hst4Delta double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.  (+info)

Endosome fusion in living cells overexpressing GFP-rab5. (36/2135)

CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Time-lapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion events occur mostly by a 'bridge' fusion mechanism in which the initial opening between vesicles does not expand; instead, membrane flows slowly and continuously from the smaller to the larger endosome in the fusing pair, through a narrow, barely perceptible membranous 'bridge' between them. The unique aspect of rab5 mediated 'bridge' fusion is the persistence of a tight constriction at the site where vesicles merge and we hypothesize that this constriction results from the relatively slow disassembly of a putative docking/fusion complex. To determine the relation of rab5 to the fusion 'bridge', we used confocal fluorescence microscopy to monitor endosome fusion in cells overexpressing GFP-rab5 fusion proteins. Vesicle docking in these cells is accompanied by recruitment of the GFP-rab5 into a brightly fluorescent spot in the 'bridge' region between fusing vesicles that persists throughout the entire length of the fusion event and which often persist for minutes following endosome fusion. Other endosomal membrane markers, including FM4-64, are not concentrated in fusion 'bridges'. These results support the idea that the GFP-rab5 spots represent the localized accumulation of GFP-rab5 between fusing endosomes and not simply overlap of adjacent membranes. The idea that the GFP-rab5 spots do not represent membrane overlap is further supported by experiments using photobleaching techniques and confocal imaging which show that GFP-rab5 localized in spots between fusion couplets is resistant to diffusion while GFP-rab5 on endosomal membranes away from these spots rapidly diffuses with a rate constant of about 1.0 (+/-0.3) x10(-)(9)cm(2)/second.  (+info)

Competition of various beta-lactam antibiotics for the major penicillin-binding proteins of Helicobacter pylori: antibacterial activity and effects on bacterial morphology. (37/2135)

The penicillin-binding proteins (PBPs) of helical (log-phase) Helicobacter pylori ATCC 43579 were identified by using biotinylated ampicillin. The major PBPs had apparent molecular masses of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various beta-lactams for these PBPs were tested by competitive-binding assays. Only PBP63 appeared to be significantly bound to each of the competing antibiotics, whereas PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and ceftriaxone. Whereas most of the beta-lactams significantly bound two or more PBPs, aztreonam specifically targeted PBP63. The influence of sub-MICs of these beta-lactams on the morphologies of log-phase H. pylori was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight beta-lactams examined induced blebbing and sphere formation, whereas aztreonam was the only antibiotic studied which induced pronounced filamentation in H. pylori. Finally, studies comparing the PBPs of helical (log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old) cultures of H. pylori revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the coccoid forms, whereas PBP66 remained the major PBP in the coccoid forms, although somewhat reduced in level compared to the helical form. PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of H. pylori and compared the relative affinities of seven different beta-lactams for the PBPs in the helical forms and their effects on bacterial morphology.  (+info)

Disassembly of nuclear inclusions in the dividing cell--a novel insight into neurodegeneration. (38/2135)

Spinocerebellar ataxias and Huntington's disease are examples of neurodegenerative diseases caused by a trinucleotide repeat expansion. One hallmark of such diseases is the formation of inclusion bodies (IBs) within neuronal tissue. Although these inclusions may play a pivotal role in the disease process, the reasons underlying their specific accumulation remain obscure. By studying intranuclear IBs in dividing cells we demonstrate for the first time that inclusions such as those of ataxin-1 disperse during mitosis, thus reducing the nuclear aggregate burden. IBs reform in the interphase nucleus. By high-resolution confocal microscopy we also show that inclusions comprise ordered structures capable of homotypic interactions. Unlike those of a non-pathologic protein, ataxin-1 inclusions were shown to be capable of non-specific protein sequestration. Our studies indicate that the specific accumulation of inclusions in terminally differentiated cells such as neurons is a direct consequence of their inability to divide and therefore provides a key to explaining their persistence in neurodegenerative disease.  (+info)

Estradiol enhances liposome-mediated uptake, preferential nuclear accumulation and functional expression of exogenous genes in MDA-MB231 breast tumor cells. (39/2135)

Exposure of p53 mutated estrogen-receptor-negative MDA-MB231 human breast tumor cells to a pharmacological concentration of estradiol enhances liposome-mediated uptake and expression of SV-40 luciferase. Unexpectedly, the effect of estradiol on SV-40 expression is evident even when estradiol exposure occurs after the initial uptake phase; this suggests that estradiol may influence gene expression by mechanisms other than increasing gene uptake alone, such as altering the intracellular distribution of the gene. We determined that while uptake of SV-40 luciferase is increased only three-fold by estradiol, there is a 30-fold increase in the nuclear/cytoplasmic ratio of the gene. In order to demonstrate that the influence of estradiol on gene uptake and expression is translated into a functional response, the effects of estradiol on the function of an exogenous gene, in this case the apoptotic function of p53, were assessed in the p53 mutated MDA-MB231 breast tumor cell. While liposome-mediated delivery of CMV-p53 alone was ineffective in promoting cell death, incubation with estradiol and the liposomal p53 complex resulted in a two-fold increase in cell killing over that observed in cells transfected with the corresponding mock vector (empty vector for p53). Evidence that cell killing was occurring through apoptosis included apoptotic body formation, cell shrinkage and an increase in fluorescence after terminal transferase end-labeling. The capacity of estradiol to promote apoptosis in MDA-MB231 cells by a p53-liposome complex is likely to be related to the preferential redistribution of the gene from the cytoplasm to the nucleus which could occur during both the uptake and post-uptake phases. Consequently, although direct effects on gene expression, and the stability of message and protein cannot be ruled out, the predominant effect of estradiol in this experimental system appears to be to influence DNA translocation from the cytoplasm to the cell nucleus.  (+info)

Renal function and Ca(2+) currents after dye-labeling identification of renal sympathetic neurons. (40/2135)

The present study was performed to determine whether renal efferent sympathetic neurons could be identified using a retrograde neuronal tracer without compromising renal function and whether the labeling and identification procedure alters Ca(2+) currents and neuromodulation of those neurons. Renal sympathetic and superior cervical ganglion (SCG) neurons were labeled with the fluorescent retrograde tracer fast blue. Renal function studies made 1 wk after labeling revealed that renal hemodynamics and fluid and electrolyte excretion were similar between the dye-injected (left) kidney and the control (right) kidney under control conditions and after hemorrhage. After volume expansion, urine flow in the dye-injected kidney was slightly, but significantly, less than that of the control kidney, whereas urinary sodium excretion increased by approximately ninefold in both kidneys. Patch-clamp studies of SCG neurons in 10 mM external Ca(2+) revealed that peak currents were not affected by the presence of the dye or a 1-min exposure to ultraviolet (UV) light. Neither maximal norepinephrine-induced Ca(2+) current inhibition nor the sensitivity to norepinephrine was affected by the dye or 1-min UV exposure. Facilitation protocols revealed that G protein modulation of Ca(2+) currents remained intact in dye-labeled UV-exposed neurons. This study demonstrates that a retrograde fluorescent dye technique to identify renal sympathetic neurons does not compromise renal function and the presence of the dye label or UV exposure has no effect on Ca(2+) currents and neuromodulation in these neurons. Isolation of single identified renal sympathetic neurons coupled with patch-clamp techniques represents a tool to investigate the role of individual current systems in the modulation of excitability in these neurons, which play an important role in the control of renal hemodynamics and excretory function and in the pathogenesis of hypertension.  (+info)