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41.
Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis 总被引:8,自引:0,他引:8
Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis. 相似文献
42.
P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines
MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings. 相似文献
43.
S. Zeitlyn A. K. Rahman B. H. Nielsen M. Gomes P. E. Kofoed D. Mahalanabis 《BMJ (Clinical research ed.)》1992,304(6827):606-609
OBJECTIVE--To evaluate factors associated with non-compliance with having second vaccination against diphtheria, tetanus, and pertussis in a treatment centre in Dhaka to determine which children were most at risk of not completing immunisation. DESIGN--Cohort study of infants given first dose of the vaccine and followed up six weeks later to ascertain compliance with having second dose. Factors associated with non-compliance were evaluated. SETTING--Dhaka treatment centre of the International Centre for Diarrhoeal Disease Research, Bangladesh. SUBJECTS--136 unimmunised children aged 6 weeks to 23 months who lived within reach of the treatment centre. At time of the six week follow up 16 of the children could not be traced and seven had died. INTERVENTIONS--All children received their first dose of the vaccine. In each case health education workers had informed the mother about the value of immunisation, and she was given clear instructions to bring the child back after four weeks for the second dose. MAIN OUTCOME MEASURE--Rate of non-compliance with advice to return child for second vaccination. RESULTS--46 of 113 children (41%) received the second dose of the vaccine. Factors most closely associated with mothers'' failure to comply with the second dose were lack of education and low income. Children whose mothers knew most about immunisation at first interview were more likely to have their second dose. CONCLUSIONS--Preventive health care services such as immunisation are appropriately offered in treatment centres, but compliance among children varies with socioeconomic status and mother''s education. Further research should be aimed at ways to make health education more effective among uneducated parents. 相似文献
44.
U Pedersen-Bjergaard L B Nielsen K Jensen L Edvinsson I Jansen J Olesen 《Peptides》1991,12(2):333-337
Calcitonin gene-related peptide (CGRP) was injected alone and in combination with substance P (SP) or neurokinin A (NKA) into the forearm skin and temporal muscle of human volunteers. In the skin, 50 pmol of CGRP induced a wheal response and a delayed erythema. No pain was recorded. No interaction between CGRP and SP or NKA was observed. In the temporal muscle, 200 pmol of CGRP alone did not induce pain or tenderness but, in combination with SP or NKA, CGRP elicited a significant pain sensation. It is concluded that CGRP may be involved in neurogenic inflammation and that only SP, of the three peptides present in nociceptive C fibers, seems to be of major importance in relation to cutaneous nociception. Simultaneous neurogenic release of CGRP and other neuropeptides in skeletal muscle may induce myofascial pain. 相似文献
45.
Chromosome abnormalities found among 34910 newborn children: results from a 13-year incidence study in Århus,Denmark 总被引:2,自引:2,他引:0
Summary As part of a larger prospective study of the influence of environmental factors on pregnancy, birth and the fetus, chromosome examinations have been made in 34910 newborn children in Århus over a 13-year period. Klinefelter's syndrome was found in 1 per 576 boys, XYY in 1 per 851 boys, triple-X in 1 per 947 girls and Turner's syndrome in 1 per 1893 girls. Other sex chromosome aberrations were found in 1 per 11637 children. The total incidence of sex chromosome abnormalities was 1 per 426 children or 2.34 per 1000. The most frequent autosomal abnormalities were that of Down's syndrome with 1 per 592 children, and reciprocal translocations with 1 per 712 children. The total incidence of autosomal abnormalities was 1 per 164 children. Chromosome abnormalities were found in 276 liveborn children and in 19 fetuses, who were aborted after prenatal chromosome examination. The combined incidence of sex chromosomal and autosomal abnormalities was 1 per 118 children or 8.45 per 1000 children. 相似文献
46.
A Celis P Madsen H V Nielsen H H Rasmussen H Thiessen J B Lauridsen B van Deurs J E Celis 《FEBS letters》1988,227(1):14-20
A mouse monoclonal antibody (mAB 22-II-D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (Mr = 29,000) and 57a, respectively (Mr = 30,000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS-C-1 cells microinjected with mAB 22-II-D8B suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22-II-D8B of lymphoid human Molt-4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22-II-D8B may be masked in some cell types. 相似文献
47.
Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme. 相似文献
48.
Niels Peter Revsbech Lars Peter Nielsen Peter Bondo Christensen Jan Srensen 《Applied microbiology》1988,54(9):2245-2249
The construction of a microsensor which can be used to measure O2 and N2O simultaneously is described. The microsensor exhibited a linear response to both O2 and N2O, and the response to N2O was independent of the O2 concentration and vice versa. The N2O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter−1. The signals for O2 and N2O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O2 and N2O were measured in a biofilm which was exposed to acetylene to block the N2O reductase activity of denitrifying bacteria. O2 penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N2O concentration at 1.4 mm depth increased from 32 to 411 μmol liter−1 after the addition of the inhibitor. The shape of the N2O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm. 相似文献
49.
J Vervoort W J van Berkel S G Mayhew F Müller A Bacher P Nielsen J LeGall 《European journal of biochemistry》1986,161(3):749-756
Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin]. 相似文献
50.
Monoamine Neurotransmitter Metabolism and Locomotor Activity During Chemical Hypoxia 总被引:4,自引:1,他引:3
The effects of hypoxia on metabolism of 5-hydroxytryptamine (5-HT or serotonin) and 3,4-dihydroxyphenylethylamine (DA or dopamine) were compared with those on open-field activity in male CD-1 mice. Chemical hypoxia was induced with NaNO2. Hypoxia did not alter striatal concentrations of DA, 5HT, Trp, Tyr, 5-hydroxyindoleacetic acid, or homovanillic acid. However, NaNO2 (75 mg/kg) reduced the rates of conversion of [3H]Tyr to [3H]DA (-41%) and [3H]Trp to [3H]5-HT (-39%). Hypoxia also reduced dihydroxyphenylacetic acid (DOPAC) levels (-27%) and DOPAC/DA ratios (-20%). Open-field behavior, as measured in an automated activity monitor, decreased in a dose-dependent fashion with 75-150 mg/kg of NaNO2 (-35 to -90%). Comparison with previous studies suggests that the syntheses of dopamine, serotonin, and the amino acids are equally vulnerable to hypoxic insults but may be less sensitive than the synthesis of acetylcholine. 相似文献