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At least 505 records · Page 28Linked to original sources
PubMed2004

CLIC-5A functions as a chloride channel in vitro and associates with the cortical actin cytoskeleton in vitro and in vivo.

CLIC-5A is a member of the chloride intracellular channel protein family, which is comprised of six related human genes encoding putative chloride channels. In this study, we found that reconstitution of purified recombinant CLIC-5A into artificial liposomes resulted in a dose-dependent chloride efflux that was sensitive to the chloride channel blocker IAA-94. CLIC-5A was originally isolated as a component of an ezrin-containing cytoskeletal complex from human placental microvilli. Here we show that similar protein complexes can be isolated using either immobilized CLIC-5A or the C-terminal F-actin-binding domain of ezrin and that actin polymerization is required for de novo assembly of these complexes. To investigate the behavior of CLIC-5A in vivo, JEG-3 placental choriocarcinoma cells were stably transfected with epitope-tagged CLIC-5A. In fixed cells, CLIC-5A displayed a polarized distribution and colocalized with ezrin in apical microvilli. Microvillar localization of CLIC-5A was retained after Triton X-100 extraction and was disrupted by treatment with latrunculin B. In transient transfections assays, we mapped a region between residues 20 and 54 of CLIC-5A that is required for targeting of CLIC-5A to microvilli in JEG-3 cells. Interestingly, expression of CLIC-5A in JEG-3 cells did not enhance the rate of iodide efflux in intact cells, suggesting that if CLIC-5A is a chloride channel, its channel activity may be restricted to intracellular membrane compartments in these cells. Regardless of its role in ion transport, CLIC-5A, like ezrin, may play an important role in the assembly or maintenance of F-actin-based structures at the cell cortex.

Actins↗
PubMed1973

Genetic control of immune responses in vitro. II. Cellular requirements for the development of primary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro.

The cellular requirements for the development of primary IgG GAT-specific PFC responses in cultures of spleen cells from responder, C57Bl/6, mice stimulated with GAT and GAT-MBSA and in cultures of spleen cells from nonresponder, SJL and B10.S, mice stimulated with GAT-MBSA were investigated. Macrophages were required for development of responses to GAT and GAT-MBSA in cultures of spleen cells from responder mice and for responses to GAT-MBSA in cultures of spleen cells from nonresponder mice. Macrophages from nonresponder mice supported the development of responses to GAT by nonadherent responder spleen cells, indicating that the failure of nonresponder mice to respond to GAT is not due to a macrophage defect. Furthermore, responder macrophages supported the responses of nonadherent, nonresponder spleen cells to SRBC and GAT-MBSA, but not to GAT. This indicates that the capacity to respond to GAT is a function of the nonadherent population which is composed of thymus-derived (T) helper cells and precursors of antibody-producing cells. Treatment of spleen cells with anti-theta serum and complement before culture initiation abolished PFC responses to GAT and GAT-MBSA thus establishing the requirement for T cells in the development of PFC responses to these antigens. Since precursors of antibody-producing cells in nonresponder mice are capable of synthesizing antibody specific for GAT after stimulation with GAT-MBSA and since the response to GAT is thymus-dependent, it appears that nonresponder mice lack GAT-specific helper T cell function.

Alanine↗
PubMed1974

Genetic control of immune responses in vitro. IV. Conditions for cooperative interactions between nonresponder parental B cells and primed (responder plus nonresponder) F1 T cells in the development of an antibody response under Ir gene control in vitro.

The conditions for cooperative interactions between nonresponder B10.S B cells and GAT-primed irradiated (C57BL/6 x SJL)F(1) T cells in the response by cultures of mouse spleen cells to GAT were investigated. GAT-specific antibody responses could be elicited by soluble GAT in cultures of GAT-primed irradiated (C57BL/6 x SJL)F(1) T cells with C57BL/6 B cells but not with B10.S B cells. In contrast, when GAT was presented to the cultures on F(1) macrophages or as aggregates of GAT with MBSA, GAT-specific PFC responses were observed with both B10.S or C57BL/6 B cells. Irradiated GAT-primed T cells were nevertheless essential for the development of these responses. The GAT-specific response of B10.S B cells in these cultures was inhibited by the addition of soluble GAT at culture initiation. These results indicate that genetic disparity at Ir loci is not an absolute barrier to T-B-cell cooperative interactions in the response to antigens under Ir gene control. The significance of these data for the function of Ir gene products in immunocompetent cells is discussed.

Alanine↗
PubMed1991

Effects of in-vitro exposure to HCG on subsequent HCG-responsiveness of human granulosa cells obtained following treatment with GnRH analogue and gonadotrophins: an in-vitro model for luteal phase support.

The pregnancy rate in patients undergoing assisted conception treatment following pituitary desensitization with GnRH analogue and ovarian stimulation with gonadotrophins has been reported to be higher when ovarian function is supported in the luteal phase by exogenous human chorionic gonadotrophin (HCG). In the present study, we have examined the effects of culturing monolayers of granulosa cells, collected from such patients at oocyte retrieval, for various time intervals in the presence or absence of HCG on their subsequent ability to secrete progesterone (P4) either spontaneously or in response to further challenge with HCG. When cultured in the absence of HCG, granulosa cells demonstrated a rapid decline in both the spontaneous P4 secretion rate and the ability to secrete P4 in response to HCG. Maintenance in the presence of HCG inhibited the rapid decline in ability to secrete P4 spontaneously and also significantly enhanced the ability to respond to subsequent HCG stimulation. These results suggest that HCG support in the luteal phase in GnRH analogue-treated patients may have a cellular basis for its action both in maintenance of P4 secretion and also in rendering the corpus luteum more sensitive to rescue by conceptus-derived HCG.

Buserelin↗
PubMed1994

Use of low-salt culture medium for in vitro maturation of porcine oocytes is associated with elevated oocyte glutathione levels and enhanced male pronuclear formation after in vitro fertilization.

The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supplements; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl (59.3 +/- 8.1 and 70.9 +/- 2.0%, respectively) than in medium containing 68.49 mM NaCl (39.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

Animals↗
PubMed1995

111In-oxine labelling of tumour-cytotoxic macrophages generated in vitro from circulating blood monocytes: an in vitro evaluation.

The labelling of ex-vivo activated macrophages with doses of 111In-oxine sufficient for gamma camera imaging does not damage cell viability or the functional competence of cells (secretion of tumour necrosis factor-alpha and interleukin-6). The mean labelling efficiency was about 84%. Release of 111In equivalent to 15% occurred over 24 h under cell culture conditions, indicating good stability of the radioactive cell label. 111In-oxine-labelled macrophages are a suitable tool for biokinetic studies of adoptive immunotherapy.

Cell Survival↗
PubMed2005

Cardiovascular actions of the venom from the Irukandji (Carukia barnesi) jellyfish: effects in human, rat and guinea-pig tissues in vitro and in pigs in vitro.

1. We have investigated the cardiovascular pharmacology of the crude venom extract (CVE) from the potentially lethal, very small carybdeid jellyfish Carukia barnesi, in rat, guinea-pig and human isolated tissues and anaesthetized piglets. 2. In rat and guinea-pig isolated right atria, CVE (0.1-10 microg/mL) caused tachycardia in the presence of atropine (1 micromol/L), a response almost completely abolished by pretreatment with tetrodotoxin (TTX; 0.1 micromol/L). In paced left atria from guinea-pig or rat, CVE (0.1-3 microg/mL) caused a positive inotropic response in the presence of atropine (1 micromol/L). 3. In rat mesenteric small arteries, CVE (0.1-30 microg/mL) caused concentration-dependent contractions that were unaffected by 0.1 micromol/L TTX, 0.3 micromol/L prazosin or 0.1 micromol/L omega-conotoxin GVIA. 4. Neither the rat right atria tachycardic response nor the contraction of rat mesenteric arteries to CVE were affected by the presence of box jellyfish (Chironex fleckeri) antivenom (92.6 units/mL). 5. In human isolated driven right atrial trabeculae muscle strips, CVE (10 microg/mL) tended to cause an initial fall, followed by a more sustained increase, in contractile force. In the presence of atropine (1 micromol/L), CVE only caused a positive inotropic response. In separate experiments in the presence of propranolol (0.2 micromol/L), the negative inotropic effect of CVE was enhanced, whereas the positive inotropic response was markedly decreased. 6. In anaesthetized piglets, CVE (67 microg/kg, i.v.) caused sustained tachycardia and systemic and pulmonary hypertension. Venous blood samples demonstrated a marked elevation in circulating levels of noradrenaline and adrenaline. 7. We conclude that C. barnesi venom may contain a neural sodium channel activator (blocked by TTX) that, in isolated atrial tissue (and in vivo), causes the release of transmitter (and circulating) catecholamines. The venom may also contain a 'direct' vasoconstrictor component. These observations explain, at least in part, the clinical features of the potentially deadly Irukandji syndrome.

Animals↗