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71.
Coupling of voltage-dependent gating and Ba++ block in the high- conductance, Ca++-activated K+ channel 总被引:11,自引:8,他引:3
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Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed. 相似文献
72.
The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies 总被引:14,自引:0,他引:14
I A Quakyi R Carter J Rener N Kumar M F Good L H Miller 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(12):4213-4217
Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine. 相似文献
73.
Suppression of anti-mouse immunoglobulin antibodies in subhuman primates receiving murine monoclonal antibodies against T cell antigens 总被引:1,自引:0,他引:1
J N Lowder R A Miller R Hoppe R Levy 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(2):401-406
Murine monoclonal antibodies stimulate the production of human anti-mouse immunoglobulin antibodies (AMIA) when administered to patients. This limits their long-term usefulness as therapeutic and diagnostic agents. We report the use of three maneuvers to suppress AMIA against T cell-specific monoclonal antibodies in cynomolgus monkeys. Twelve monkeys received daily i.v. infusions of 1 mg each of anti-Leu-2a, -3a, and -5 on days 1 through 10. One group (control) received no suppressive regimen. The second group received cyclosporine, 12.5 mg/kg daily on days -7 to +14. The third group (PI) were passively immunized with 0.4 ml of hyperimmune monkey AMIA serum on days -7, -1, 2, 4, 6, and 8. The fourth group (TLI) received 1700 rad fractionated total lymphoid irradiation ending on day -1. The animals treated with TLI were markedly delayed in the onset of AMIA, which was suppressed to less than 1% of the control group. The AMIA specific for the constant region of animals receiving PI was also suppressed to one-third of control. The majority of the AMIA in all the animals was anti-idiotypic and wholly anti-idiotypic in the TLI animals. 相似文献
74.
Studies on the mechanism of T cell inhibition by the Pseudomonas aeruginosa phenazine pigment pyocyanine 总被引:10,自引:0,他引:10
J Nutman M Berger P A Chase D G Dearborn K M Miller R L Waller R U Sorensen 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(10):3481-3487
Pseudomonas aeruginosa and its products have been shown to inhibit mitogen-induced human lymphocyte blastogenesis as measured by [3H]TdR uptake. The phenazine pigment pyocyanine has been identified as one of the inhibitors present in cellfree culture supernatants. To determine the mechanism of the inhibitory action of pyocyanine, we studied its effect on the early stages of T cell activation. Pyocyanine inhibited lymphocyte stimulation induced by specific antigens, the lectin concanavalin A and the calcium ionophore, ionomycin, suggesting that its inhibitory effect is not dependent on interference with the T cell antigen receptor complex itself. Using quin-2, we showed that pyocyanine did not interfere with the mitogen-induced increase in cytosolic-free Ca2+. We also showed that pyocyanine did not interfere with the function of calmodulin stimulated Ca2+-Mg2+ ATPase activity, indicating that the mechanism of action of pyocyanine differs from that of the structurally related phenothiazine compounds. Analysis of IL 2 production and IL 2 receptor expression clearly showed that pyocyanine inhibits the production of this essential lymphokine as well as the expression of IL 2 receptors on the T cell membrane. This inhibition is dose dependent and not due to cellular toxicity. There was parallel inhibition of growth in cell volume as well as [3H]TdR uptake. Thus, our results demonstrate that pyocyanine inhibits T cell proliferation by decreasing the production of the critical lymphokine IL 2 and by decreasing the expression of the IL 2 receptor. Local suppression of lymphocyte stimulation by phenazine pigments such as pyocyanine may interfere with cellular immune responses that may be necessary for eradication of chronic infection with P. aeruginosa. 相似文献
75.
Reaction of T lymphocytes with anti-T3 induces translocation of C-kinase activity to the membrane and specific substrate phosphorylation 总被引:3,自引:0,他引:3
A E Nel P Bouic G R Lattanze H C Stevenson P Miller W Dirienzo G F Stefanini R M Galbraith 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(10):3519-3524
Reaction of the T cell membrane with monoclonal antibodies to T3 can initiate cellular activation, and this is associated with increased intracellular Ca2+ and inositol-trisphosphate (IP3) release. We therefore studied the possible involvement of Ca2+/phospholipid-dependent kinase (C-kinase) in these phenomena. Quantitative assays of exogenous substrate phosphorylation in unstimulated cells showed Ca2+/phospholipid-dependent kinase activity in the cytosol, but no comparable activity in the particulate fractions corresponding to membrane and cytoskeleton material. At concentrations of soluble anti-T3 that partially activate T cells in the absence of macrophages, there was a 50 to 60% decrease in C-kinase activity in the cytosol, with a comparable increase in activity in the membrane fraction. A similar transfer of activity was also induced with the known C-kinase activator, 12-O-tetradecanoyl-phorbol-13-acetate, although redistribution was more rapid in onset, more complete, and more sustained. Redistribution of enzyme activity was additionally confirmed by qualitative assays of endogenous substrate phosphorylation. Labeling of intact cells followed by immunoprecipitation analysis with anti-T3 indicated signal-dependent phosphorylation of two components of the T3 complex and an unidentified 94,000 substrate that was resistant to reduction and alkylation. These findings are consistent with an important role for C-kinase in transduction of membrane events by the T3-Ti complex. 相似文献
76.
Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein 总被引:9,自引:0,他引:9
E S Miller J Karam M Dawson M Trojanowska P Gauss L Gold 《Journal of molecular biology》1987,194(3):397-410
The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs. 相似文献
77.
In the mouse neuroblastoma x dorsal root ganglion hybrid cell line F-11, bradykinin receptor stimulation induced the release of inositol-1,4,5-trisphosphate (IP3) and inositol-1,4-bisphosphate (IP2). Maximal stimulation of [2-3H]IP3 and [2-3H]IP2 release by bradykinin in the absence of LiCl occurred at 7 (or less) and 15 s, respectively, with average levels of 5.7-(IP3) and 3.4-(IP2) fold of control values. The EC50 for bradykinin was 33 +/- 5 nM. IP3 and IP2 concentrations returned to basal levels approximately 1 min after bradykinin addition. Bradykinin-induced IP3 release was blocked by several novel bradykinin analogues. In particular, [D-Arg0]-Hyp3-Thi5,8-[D-Phe7]-bradykinin [Hyp, hydroxyproline; Thi, beta-(2-thienyl)-L-alanine] blocked IP3 production in a dose-dependent fashion. Several of these analogues alone showed little or no agonist activity. The bradykinin receptor may be coupled to phospholipase C via a GTP-sensitive protein (Gi or Go), as preincubation for 18-20 h with pertussis toxin decreased IP3 concentrations by 45%. Bradykinin is also known to modulate the concentrations of other second messengers in neurons, increasing the concentrations of Ca2+, diacylglycerol (DG), and cyclic GMP and decreasing the concentration of cyclic AMP. These second messengers modulated bradykinin-dependent IP3 release to varying degrees. A23187, a Ca2+ ionophore, produced a 37% decrease in IP3 concentration. 12-O-Tetradecanoylphorbol-13-acetate, which mimics the effects of DG and activates protein kinase C, inhibited IP3 release by 80%. Dibutyryl cyclic GMP produced little or no inhibition of IP3. [D-Ala2,D-Leu5]Enkephalin (DADLE), an opioid peptide that decreases cyclic AMP concentrations, likewise had no effect.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
78.
Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase 总被引:1,自引:0,他引:1
H N Bramson N E Thomas W T Miller D C Fry A S Mildvan E T Kaiser 《Biochemistry》1987,26(14):4666-4670
Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B. 相似文献
79.
Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues 总被引:19,自引:0,他引:19
K J Miller V N Reinhold A C Weissborn E P Kennedy 《Biochimica et biophysica acta》1987,901(1):112-118
In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli. 相似文献
80.
Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. 总被引:62,自引:11,他引:51
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R B DuBridge P Tang H C Hsia P M Leong J H Miller M P Calos 《Molecular and cellular biology》1987,7(1):379-387
We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer. 相似文献