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131.
Chinese hamster lung (CHL) cells were susceptible to Herpes Simplex type-1 and Chandipura viruses; which induced chromosomal abnormalities in these cells. Chromosomal changes induced in these cells were specific. The cells were refractory to measles virus and chromosomal abnormalities were not detected after inoculation of the virus. On the other hand human peripheral blood (HPB) leukocytes were susceptible to all the 3 viruses studied and exhibited chromosomal abnormalities upon infection. The aberrations induced in HPBL cultures were random. The results suggest that a virus could induce chromosomal changes only in susceptible cells. This is the first report of comparative in vitro study on chromosomes. 相似文献
132.
133.
Identification of the components controlling inactivation of voltage-gated Ca2+ channels 总被引:1,自引:0,他引:1
Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker. 相似文献
134.
Williams JB Mallorga PJ Lemaire W Williams DL Na S Patel S Conn PJ Conn JP Pettibone DJ Austin C Sur C 《Analytical biochemistry》2003,321(1):31-37
Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors. 相似文献
135.
136.
Paranjape S Patil BR Kadam VD 《In vitro cellular & developmental biology. Animal》2003,39(5-6):193-195
The optimum temperature for the growth of porcine stable (PS) kidney cell line is 37 degrees C. We have adapted the cell line to grow at 40 degrees C. The original cell line grown at 37 degrees C has been denoted as PS-37, and the adapted new strain has been denoted as PS-40. Both the cell lines were screened for mycoplasma by Hoechst staining and tritiated uridine-uracil uptake and were found to be negative. Comparative characterization of PS-40 and its progenitor PS-37 cell line was done by using various parameters. The antigenic studies indicated that the new cell strain was not cross-contaminated with any other cell lines. It was observed that PS-40 cells were more fibroblastic with clean cytoplasm and appeared healthy. The growth of PS-40 cells was faster than the original cell line. The karyological study showed heteroploid chromosome number in PS-40 cells. The modal chromosome number of PS-40 cells was 58, whereas that of PS-37 cell line was 38. The lactic dehydrogenase isoenzyme pattern showed a cathodal shift of bands. The PS-40 cell strain could be cryopreserved and revived. The viability of PS-37 as well as PS-40 cell lines is in the range of 90-95%, and the growth characteristics of thawed cells showed six- to eightfold multiplications within 5 d. The virus susceptibility study revealed that the cytopathic effect was more profound and observed 1 d earlier in PS-40 cell line. Increased yields of Japanese encephalitis, Sindbis, and Semliki forest viruses were obtained by 1.8, 1.75, and 1.5 log plaque-forming units/ml, respectively. The yield of West Nile virus was, however, comparable to that in PS-37 cell line. Both the cell lines were refractory to Dengue viruses. 相似文献
137.
Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [(18)O]P(i) exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P(i)-release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P(i)-release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase (dTTPase) reaction is the release of dTDP. Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer. The results of this study are therefore consistent with those reported earlier (Hingorani, M. M., Washington, M. T., Moore, K. C., and Patel, S. S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5012-5017), and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F(1)-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites. 相似文献
138.
139.
Suresh R Vig M Bhatia S Goodspeed EP John B Kandpal U Srivastava S George A Sen R Bal V Durdik JM Rath S 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(8):4262-4272
Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants. 相似文献
140.
Immunotherapy of cancer with dendritic-cell-based vaccines 总被引:32,自引:0,他引:32
Animal studies have shown that vaccination with genetically modified tumor cells or with dendritic cells (DC) pulsed with
tumor antigens are potent strategies to elicit protective immunity in tumor-bearing animals, more potent than “conventional”
strategies that have been tested in clinical settings with limited success. While both vaccination strategies are forms of
cell therapy requiring complex and costly ex vivo manipulations of the patient’s cells, current protocols using dendritic
cells are considerably simpler and would be more widely available. Vaccination with defined tumor antigens presented by DC
has obvious appeal. However, in view of the expected emergence of antigen-loss variants as well as natural immunovariation,
effective vaccine formulations must contain mixtures of commonly, if not universally, expressed tumor antigens. When, or even
if, such common tumor antigens will be identified cannot be, predicted, however. Thus, for the foreseeable future, vaccination
with total-tumor-derived material as source of tumor antigens may be preferable to using defined tumor antigens. Vaccination
with undefined tumor-derived antigens will be limited, however, by the availability of sufficient tumor tissue for antigen
preparation. Because the mRNA content of single cells can be amplified, tumor mRNA, or corresponding cDNA libraries, offer
an unlimited source of tumor antigens. DC transfected with tumor RNA were shown to engender potent antitumor immunity in animal
studies. Thus, immunotherapy using autologous DC loaded with unfractionated tumor-derived antigens in the form of RNA emerges
as a potentially powerful and broadly useful vaccination strategy for cancer patients.
Received: 10 October 1997 / Accepted: 12 January 1998 相似文献