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Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α 2 interferon produced by Escherichia coli containing the human gene for interferon α 2 . All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV). 相似文献
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Naoto Ohnishi Hiroaki Kodania Satoshi Ando Atsushi Komamine 《Physiologia plantarum》1990,80(1):95-101
The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G1 phase by anisoniycio or cycloheximide caused the arrest of cells in the G1 phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G1 phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G1 phase caused the arrest of cells in the G1 phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G1 phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G1 phase, and that the mRNA(s) that encode these proteins are also synthesized at the G1 phase. 相似文献
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Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2. 相似文献
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RNA polymerase activity and incorporation of 3 H-orotic acid into RNA in livers of young mice were investigated. Total RNA polymerase activity of isolated liver nuclei increased approximately three-fold during the first 30 days after birth. Assays with α-amanitin revealed that both RNA polymerase I and II activity increased over this period. Chromatographic fractionation of solubilized RNA polymerases confirmed that the ratio of polymerase I to polymerase II activity was similar at 1 day and at 30 days of age. Incorporation of 3 H-orotic acid into total liver RNA in intact animals also increased during this period. The results are compared with the increase in cell number and cell size in postnatal liver development. 相似文献
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Abstract: "High 5" cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ-opioid receptor. The maximal binding capacity for the narcotic antagonist [3 H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [3 H][ d -penicillamine2 , d -penicillamine5 ]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ-receptors of NG108-15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100-fold less effective in competing with [3 H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose-dependent manner by exposure of High 5 membranes to the exogenous G protein Go , which increased the binding capacity for DPDPE. Functional studies revealed a dose-dependent inhibition (up to 30%) by opioids on forskolin-stimulated cyclic AMP synthesis, and this effect was potentiated by Go . Quantification of Gαo and Gαi disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ-opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gαo /mg of membrane protein, and a 20-fold higher concentration for Gαi . The distinct G-protein expression in insect cells may be considered an advantage for studying functions of G protein-coupled receptors. 相似文献
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Coupling of the Human Y2 Receptor for Neuropeptide Y and Peptide YY to Guanine Nucleotide Inhibitory Proteins in Permeabilized SMS-KAN Cells 总被引:2,自引:0,他引:2
Christian Freitag Anja B. Svendsen Nya Feldthus Kristine Løssl Søren P. Sheikh 《Journal of neurochemistry》1995,64(2):643-650
Abstract: Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH-terminals of the α-subunits of Gi1/2 , Gi3 , and Go , the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, K D = 76 p M for intact cells and K D = 906 p M for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in K D and a decrease in apparent number of binding sites ( B max ) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in B max . In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC50 = 420 n M ) compared with 94% inhibition (IC50 = 380 n M ) in permeabilized cells. In permeabilized cells, preincubation with antisera against αi1/2 and αi3 blocked the functional response of PYY, with anti-αi3 being the most potent; whereas anti-αo failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different Gi s (but not Go ). 相似文献
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Rie Hosoi Toshio Matsuda Shoichi Asano Hiroaki Nakamura Hitoshi Hashimoto Kazuhiro Takuma Akemichi Baba 《Journal of neurochemistry》1997,69(5):2189-2196
Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na+ ,K+ -ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K+ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na+ ,K+ -ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na+ , Ca2+ , and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes. 相似文献
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SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10−5 M-10−3 M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10−3 M) inhibited the incorporation of [3 H]thymidine, [3 H]uridine and [3 H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [3 H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10−3 M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane. 相似文献
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Behera AK Durand E Cugini C Antonara S Bourassa L Hildebrand E Hu LT Coburn J 《Cellular microbiology》2008,10(2):320-331
Borrelia burgdorferi , the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α3 β1 on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin α3 β1 and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg–Gly–Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α3 β1 and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin α3 β1 . This interaction is specific, as P66, another integrin ligand of B. burgdorferi , does not activate signalling through α3 β1 . In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin α3 β1 and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells. 相似文献
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Lichenan a β(1→ 3,1→ 4)β-glucan exhibited a broad antiviral activity against mechanically transmitted viruses of different taxonomic groups in different Nicoliana spp. As shown for tobacco mosaic virus in vitro . lichenan did not interfere with cell-free protein synthesis programmed with naked RNA or virus particles cotranslationally disassembled. Thus, inhibition of virus replication by lichenan is probably not attributed to direct interaction of lichenan and viral structural components, but rather to reduced sensitivity of the host tissue. Treatment of a suspension of cultured cells of Nicotiana tabacum with lichenan was accompanied by a slight increase in phenylalanine ammonia-lyase (PAL) activity. However, lichenan applied to leaf tissue did not significantly stimulate PAL-activity, therefore, antiviral action apparently did not depend on the induction of the phenylpropanoid pathway. According to experiments with lichenan and digitonin (used as a callose elicitor) restriction of viruses to initially-infected cells by means of callose deposition also appears to be an improbable mechanism of viral inhibition. In cell suspensions treated with lichenan, slight changes in the extracellular concentration of free K+ were observed that did not reflect a marked K+ leakage, as caused by digitonin. This finding requires further examination. According to the present results, it is likely that lichenan affects the early virus-cell interactions that occur after virus disassembly. 相似文献
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Zolpidem is a positive allosteric modulator of GABAA receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABAA receptors containing the α1 subunit, it has a lower affinity to GABAA receptors containing α2 , α3 , or α5 subunits and has a very weak efficacy at receptors containing the α5 subunit. Here, we show that replacing histidine in position 105 in the α5 subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α5 subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α5 H105 in α1 , α2 , and α3 are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α1 H101, α2 H101, and α3 H126 by arginine, as naturally present in α4 and α6 , leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α5 containing receptors also for the action of zolpidem. 相似文献
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Abstract: Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [3 H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 µ M . Concanavalin A also reduced the release triggered by extracellular Ca2+ in neurons depolarized by 25 m M K+ or rendered Ca2+ -permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the α2 -adrenergic agonist UK 14,304. Inactivation of Gs and Gi /Go type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K+ and Ca2+ currents. In contrast, the lectin efficiently blocked both the Ca2+ -dependent and -independent α-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as α-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl α- d -mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to α-latrotoxin, concanavalin A failed to alter K+ -evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of α-latrotoxin receptors may reduce action potential-dependent transmitter release. 相似文献
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Amina El Jamali Nadia Rachdaoui Claude Jacquemin Claude Corrèze 《Journal of neurochemistry》1996,67(6):2532-2539
Abstract: Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(βγ-imido)diphosphate, guanosine 5'- O -(3-thiotriphosphate) [GTP(S)], and AIF4 − in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in Gs α, Gi α, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc− cells, lacking Gs α protein. Gs α proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted Gs α protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via Gs α, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin. 相似文献
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Abstract: Excitatory amino acids, acting at both N methyl- d -aspartate (NMDA) and non-NMDA receptors, release the inhibitory neuromodulator adenosine from superfused rat cortical slices. This study was initiated to investigate the possible purinergic sources and mechanisms of release for the adenosine release evoked by NMDA and non-NMDA receptor activation. Inhibition of the bidirectional nucleo-side transporter with dipyridamole greatly enhanced adenosine release evoked by glutamate, NMDA, kainate, and ( RS -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Inhibition of ecto -5'-nucleotidase with α,β-methylene ADP and GMP had no effect on either kainateor AMPA-evoked adenosine release, but it decreased glutamate- and NMDA-evoked adenosine release by 23 and 68%, respectively. A similar inhibition of NMDA-evoked adenosine release was observed with α,β-methylene ADP alone, indicating that the inhibitory effect was not due to the reported competitive inhibition of NMDA receptors by GMP. Finally, NMDA-evoked adenosine release, but not kainate- or AMPA-evoked release, was Ca2+ dependent. These results indicate that activation of non-NMDA receptors releases adenosine per se in a Ca2+ -independent manner. In contrast, NMDA receptor activation releases primarily a nucleotide that is subsequently converted extracellularly to adenosine; in this case, release is Ca2+ dependent. Although neither NMDA- nor non-NMDA-evoked adenosine release occurs via the nucleoside transporter, this transporter does appear to be a major route for removal of adenosine from the extracellular space. 相似文献
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Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After
Spinal Cord Injury in Rats 总被引:5,自引:0,他引:5
Takeharu Tonai Yutaka Taketani Natsuo Ueda Takehiko Nishisho Yasukazu Ohmoto† Yasuyo Sakata† Masahiro Muraguchi† Kaori Wada† & Shozo Yamamoto 《Journal of neurochemistry》1999,72(1):302-309
Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A2 (detected as thromboxane B2 ) and prostaglandin I2 (detected as 6-ketoprostaglandin F1α . Thromboxane B2 was predominant during the first 1 h, whereas the 6-ketoprostaglandin F1α level exceeded that of thromboxane B2 at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α. 相似文献