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11.
Summary Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein. 相似文献
12.
Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types 总被引:1,自引:0,他引:1
G J Blackwell R W Bonser J Dawson L G Garland 《Biochemical and biophysical research communications》1985,127(3):950-955
In washed human platelets and in HL60 granulocytes phorbol myristate acetate (PMA, 1-2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL60 cells maintained a maximal response at PMA concentrations between 30-300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast cells. However, in the mast cell PMA concentrations above 10nM reduced maximum histamine release in a dose-dependent manner. 相似文献
13.
An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda 总被引:23,自引:0,他引:23
We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes. 相似文献
14.
Drugs are considered to produce their effects on biological tissues either by altering some physical property of cells or by interacting with specific cellular components, called receptors. Most drugs and endogenous neurotransmitters act on highly selective receptors located on the outer surface membrane of cells. These receptors were believed, until recently, to be stationary on the cell surface and to be present in unvarying numbers. Consequently, most early theorists modeled the drug-receptor interaction on the basis of stationary and static receptor molecules. The substantial advances in our understanding of drug action based on these models have partly justified this view. However, recent electron microscopic studies have revealed the presence of structures, including "coated" pits and vesicles, that appear to provide a mechanism by which cell surface receptors might be internalized in a process of endocytosis. The precise intracellular fate of these internalized receptors is unknown, but based on present understanding, it seems reasonable to believe that some are destroyed intracellularly whereas others are recycled to the cell surface. The importance of such processes to pharmacologic theory is a new awareness of a cellular pathway that is capable of internalizing drugs, receptors, or both. The implications of such a process to the theory of drug action extends to some unexplained drug phenomena such as down regulation, drug tolerance, tachyphyllaxis, and partial agonism. We present herein the theoretical framework for a model of drug action that incorporates the possibility of receptor internalization and subsequent degradation, recycling, or replacement. 相似文献
15.
In an attempt to show that the open field can still be used as a valid measure of fear, Jones (1983) has reported a failure to replicate some of our findings. The present studies show that this was due to procedural and methodological differences. For instance, we found that birds tested in a novel environment behaved quite differently from those, as in Jones' case, which were placed in one resembling the home cage. Moreover, birds housed in isolation for two days prior to testing reacted differently than those, as again in Jones' case, which were reared in isolation from hatching to the time of testing. The results were interpreted as being consistent with our view that open-field behaviour reflects a conflict between the need to reinstate contact with conspecifics on the one hand, and evade predation on the other. 相似文献
16.
Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences. 相似文献
17.
T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A 总被引:15,自引:0,他引:15
A B Rickinson M Rowe I J Hart Q Y Yao L E Henderson H Rabin M A Epstein 《Cellular immunology》1984,87(2):646-658
The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro. 相似文献
18.
Mathematical modelling of dynamics and control in metabolic networks. II. Simple dimeric enzymes 总被引:1,自引:0,他引:1
The dynamics of enzyme cooperativity are examined by studying a homotropic dimeric enzyme with identical reaction sites, both of which follow irreversible Michaelis-Menten kinetics. The problem is approached via scaling and linearization of the governing mass action kinetic equations. Homotropic interaction between the two sites are found to depend on three dimensionless groups, two for the substrate binding step and one for the chemical transformation. The interaction between the two reaction sites is shown capable of producing dynamic behavior qualitatively different from that of a simple Michaelis-Menten system; when the two sites interact to increase enzymatic activity over that of two independent monomeric enzymes (positive cooperativity) damped oscillatory behavior is possible, and for negative cooperativity in the chemical transformation step a multiplicity of steady states can occur, with one state unstable and leading to runaway behavior. Linear analysis gives significant insight into system dynamics, and their parametric sensitivity, and a way to identify regions of the parameter space where the approximate quasi-stationary and quasi-equilibrium analyses are appropriate. 相似文献
19.
Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
20.
We have examined the behavior of two reaction-diffusion models, originally proposed by Gierer & Meinhardt (1972) and by Kauffman, Shymko & Trabert (1978), for biological pattern formation. Calculations are presented for pattern formation on a disc (approximating the geometry of a number of embryonic anlagen including the frog eye rudiment), emphasizing the sensitivity of patterns to changes in initial conditions and to perturbations in the geometry of the morphogen-producing space. Analysis of the linearized equations from the models enabled us to select appropriate parameters and disc size for pattern growth. A computer-implemented finite element method was used to solve the non-linear model equations reiteratively. For the Gierer-Meinhardt model, initial activation (varying in size over two orders of magnitude) of one point on the disc's edge was sufficient to generate the primary gradient. Various parts of the disc were removed (remaining only as diffusible space) from the morphogen-producing cycle to investigate the effects of cells dropping out of the cycle due to cell death or malfunction (single point removed) or differentiation (center removed), as occur in the Xenopus eye rudiment. The resulting patterns had the same general shape and amplitude as normal gradients. Nor did a two-fold increase in disc size affect the pattern-generating ability of the model. Disc fragments bearing their primary gradient patterns were fused (with gradients in opposite directions, but each parallel to the fusion line). The resulting patterns generated by the model showed many similarities to results of "compound eye" experiments in Xenopus. Similar patterns were obtained with the model of Kauffman's group (1978), but we found less stability of the pattern subject to simulations of central differentiation. However, removal of a single point from the morphogen cycle (cell death) did not result in any change. The sensitivity of the Kauffman et al. model to shape perturbations is not surprising since the model was originally designed to use shape and increasing size during growth to generate a sequence of transient patterns. However, the Gierer-Meinhardt model is remarkably stable even when subjected to a wide range of perturbations in the diffusible space, thus allowing it to cope with normal biological variability, and offering an exciting range of possibilities for reaction-diffusion models as mechanisms underlying the spatial patterns of tissue structures. 相似文献