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71.
Joachim Messing 《Molecular biotechnology》1996,5(1):39-47
DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning
sites. A chronology of the original concepts and experiments is reviewed. 相似文献
72.
Cholinomimetics Increase Glutamate Outflow via an Action on the Corticostriatal Pathway: Implications for Alzheimer's Disease 总被引:3,自引:0,他引:3
Sas N. Dijk Paul T. Francis Gary C. Stratmann David M. Bowen 《Journal of neurochemistry》1995,65(5):2165-2169
Abstract: Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 µ M ) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M1 receptor, the selective M1 agonist PD 142505-0028 (10 µ M ) was topically applied to the frontal cortex. Like physostigmine, PD 142505-0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M1 antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function. 相似文献
73.
E. E. Hiatt L. Georgi S. Huston D. C. Harshman S. A. Lewis A. G. Abbott 《Journal of nematology》1995,27(2):143-152
The genetic heterogeneity of two M. arenaria race 2 populations (designated Pelion and Govan) was examined using RFLP analysis of 12 clonal lines established from single egg masses (six distinct clonal lines from each population). These populations are essentially identical by traditional biochemical and race identification schemes; however, the Govan population is more aggressive than the Pelion population, producing larger galls and exhibiting greater reproductive capabilities on many soybean cultivars and experimental accessions. Variation at the genomic DNA level was examined using probes representative of expressed DNA sequences present in the eukaryotic genome. Ribosomal DNA, interspersed repeated sequences, and cDNA probes were tested for detection of polymorphism within and between single egg mass lines of each population. Cloned cDNAs and ribosomal intergenic spacer sequences detect polymorphism both within and between populations, demonstrating the usefulness of these sequence classes for molecular genetic analysis of population structure and genome evolution. 相似文献
74.
YANG Jianbo WU Lijun LI Li WU JiadaoYU Zengliang XU Zhihong 《中国科学:生命科学英文版》1997,40(1):107-112
While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ- mutation frequency at 20% survival could reach (3.6–16.8) × 104, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region
examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants
contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined);
this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our
experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no
addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G.
The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In
comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily
substituted. 相似文献
75.
Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus 总被引:3,自引:0,他引:3
Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. 相似文献
76.
Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na+ current, the M current and the low-voltage-activated Ca2+ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na+ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na+ current (PFpeak and PFss) as a function of voltage. Whereas PFpeak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PFss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na+ current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K+ current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased IM, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca2+ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a ICa. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a ICa. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed. 相似文献
77.
Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203 总被引:2,自引:0,他引:2
Kyongsu Hong Yoshikazu Komurasaki Hideyuki Kobayashi Hiromichi Ishikawa Kozo Inoue 《FEMS immunology and medical microbiology》1995,12(1):73-82
Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M+ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M+ and M− bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M+ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M− bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells. 相似文献
78.
利用灵敏度高,专一性强的放射免疫法(RIA)测定了苦瓜和瓜萎不同器官中皮质醇的含量。结果表明:苦瓜和瓜萎各器官均含有皮质醇,且含量在50~450ng/g.FW;顶芽中含量最高,其次是花器官;苦瓜和瓜篓雌花皮质醇含量明显高于雄花,且雌花在授粉后当天含量最高,说明皮质醇可能在植物的生长发育过程中起某种作用。 相似文献
79.
A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [3H]adenosine, [3H]inosine, [3H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [3H]adenosine and [3H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [3H]adenosine and [3H]guanosine was predominantly into glial cells, but also into neurons. [3H]Inosine labelled glia almost exclusively. However, the adenosine analog, [3H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [3H]adenosine, [3H]guanosine or [3H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor. 相似文献
80.
(1) By treating Mycoplasma capricolum cells with phospholipase A2 about 80% of membrane phospholipids were rapidly hydrolyzed. The rate and extent of hydrolysis (at 37°C) were the same in intact cells and in isolated unsealed membranes. (2) Due to the low endogenous lysophospholipase activity detected in M. capricolum, phospholipase A2 treatment resulted in the accumulation of lysophospholipids and free fatty acids. The free fatty acids were efficiently extracted from the cells by 1% bovine serum albumin whereas the lysophospholipids were almost fully retained within the cell membrane. (3) Following phospholipase A2 treatment in the presence of 1% bovine serum albumin, cell intactness was preserved as indicated by the constant absorbance of the cell suspension and the retention of nucleic acids and NADH dehydrogenase activity within the cells. The treated cells showed, however, a slight decrease in K+ content and a decrease in cell viability. Viability was fully preserved after phospholipase A2 treatment of cells grown with exogenous sphingomyelin. (4) Adapting M. capricolum to a cholesterol-poor medium resulted in a marked decrease in the cholesterol to phospholipid molar ratio (from about 1.1 to 0.3). Phospholipase A2 treatment of the cholesterol-poor cells resuted in cell lysis. Cell lysis was induced in the cholesterol-rich cells by hydrolysing the lysophospholipids accumulated following phospholipase A2 treatment. (5) It is suggested that after phospholipase A2 treatment of M. capricolum cells, a relatively stable cell membrane is maintained and cell intactness is preseved due to the interaction of cholesterol, present in high amount in this membrane, with the lysophospholipids formed. 相似文献