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21.
Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum 总被引:1,自引:0,他引:1
Hendrik Wolf Alfred Pühler Eberhard Neumann 《Applied microbiology and biotechnology》1989,30(3):283-289
Summary Intact and osmotically sensitive cells of Corynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse
with time constants
and with initial field intensities of E
0=35–40 kV cm-1; prepulse temperature 20°C. Cell regeneration (survival) was 100%–80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electroporation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of E
0=25–30 kV cm-1. Cell regeneration under these conditions was found to be 20–30%. The optimum yield of transformants/g plasmid-DNA was 3×103 for intact cells, 2×104 for intact cells which were frozen and thawed twice and 7×104 for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1–3×108/ml and at a DNA concentration of 0.2 g/ml up to 2 g/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is 20°C, although regeneration of electroporated C. glutamicum cells starts to decrease at temperatures20°C. 相似文献
22.
Identification of dexamethasone-binding sites on male-rat liver plasma membranes by affinity labelling. 总被引:2,自引:0,他引:2
Binding studies with [3H]dexamethasone identified two binding sites on plasma membranes prepared from the male rat liver, a low-capacity site with a KD of 7.0 nM and a higher-capacity site with a KD of 90.1 nM. Both sites exhibited glucocorticoid responsiveness and specificity for glucocorticoids and progestins. Triamcinolone acetonide, which competes well for the binding of dexamethasone to the cytosolic glucocorticoid receptor, did not compete well for the binding of [3H]dexamethasone to the plasma-membrane binding sites. The binding sites were sensitive to protease and neuraminidase treatment, and resistant to extraction with NaCl, but were extracted with the detergent Triton X-100. As these experiments indicated the presence of plasma-membrane protein components which bind glucocorticoids at physiological concentrations, affinity-labelling experiments with dexamethasone mesylate were conducted. Two peptides were specifically labelled, one at approx. Mr 66,000 and one at Mr 45,000. The Mr-66,000 peptide was not sensitive to glucocorticoids, and was extracted by NaCl, and so did not correspond to either of the sites identified in the dexamethasone-binding studies. The Mr-45,000 entity, on the other hand, resembled the dexamethasone-binding sites in its response to glucocorticoid manipulation of the animal and in its resistance to salt extraction. This peptide was not present in rat serum. Thus we have identified a plasma-membrane peptide which binds dexamethasone. Whether this peptide is involved in transport of the glucocorticoid across the plasma membrane remains to be determined. 相似文献
23.
Summary CommercialViscum album extract Helixor-M contains a dialysable oligosaccharide (HM-BP) that activates natural killer (NK) cytotoxicity against K562 tumour cells when preincubated with human peripheral blood mononuclear cells (PBMC) for 72 h. The activated effector cells were exclusively found in the monocyte/macrophage subpopulation. However, when peripheral non-adherent cells (PNAC) were preincubated with HM-BP for 72 h the NK cytotoxicity of CD56+CD3– NK cells was activated. This discrepancy was found to be due to the release of prostaglandin E2 from activated monocytes/macrophages, which blocked activation of the cytotoxicity of NK cells. Analysis of the supernatant culture medium after 72 h preincubation demonstrated that HM-BP induced release of interferon (IFN) from T cells (preferentially from CD3+CD4+ cells) and of tumour necrosis factor (TNF) from monocytes/macrophages. Release of IFN was the crucial step for activation of NK cytotoxicity since enhancement of NK cytotoxicity during pretreatment of PBMC or PNAC with HM-BP was completely blocked in the presence of anti-IFN antibodies. Anti-interleukin-2, anti-TNF or anti-IFN antibodies had no effect on the HM-BP-induced enhancement of NK cytotoxicity. The activation of the NK cytotoxicity of nonadherent cells by interleukin-2 treatment was found to be synergistic to the enhancement of NK cytotoxicity by treatment with HM-BP. 相似文献
24.
Corinne Vander Wauven Alfred Jann Dieter Haas Thomas Leisinger Victor Stalon 《Archives of microbiology》1988,150(4):400-404
Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N2-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N2-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N2-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N2-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT
ornithine 5-aminotransferase
- SOAT
N2-succinylornithine 5-aminotransferase
- Oru
ornithine utilization
- Aru
arginine utilization 相似文献
25.
Armando Tovar Jean K. Tews Nimbe Torres Alfred E. Harper 《Journal of neurochemistry》1988,51(4):1285-1293
Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood-brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n-butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2-(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p less than 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system. 相似文献
26.
Interaction of fatigue and hypercapnia in the canine diaphragm 总被引:1,自引:0,他引:1
Schnader J.; Howell S.; Fitzgerald R. S.; Roussos C. 《Journal of applied physiology》1988,64(4):1636-1643
We studied 10 open-chest dogs and measured the pressure across the diaphragm (Pdi) in each period of the protocol during stimulation at frequencies of 1, 20, 50, and 80 Hz. Three ranges of arterial PCO2 (PaCO2) were examined: less than or equal to 26, 36-50, and greater than or equal to 89 Torr. The diaphragm was fatigued with repetitive phrenic stimulation (30 Hz). During the fatiguing activity, five of the animals were subjected to hypercapnia and the other five to hypocapnia. A frequency-Pdi curve was generated for each period in the protocol. The data show that 1) fatiguing to 50% of the initial Pdi value during hypercapnia was significantly more rapid than during hypocapnia; 2) both the prefatigue and postfatigue mean Pdi values over all interactions of frequency, fatigue, and PaCO2 were unaffected by the fatiguing environment (hypercapnia vs. hypocapnia); 3) the percent reduction of Pdi by hypercapnia was the same at all four frequencies; 4) hypocapnia did not alter either the pre- or postfatigue frequency-Pdi curve; and 5) one-half relaxation time, unaffected by PaCO2, was prolonged by fatigue. We conclude that the hypercapnic diaphragm has less endurance than the hypocapnic diaphragm and that although both fatigue and hypercapnia decrease Pdi, they appear to be separate entities working through different mechanisms. 相似文献
27.
We have reported previously that, when exposed to hypercapnia of various intensities, the diaphragm reduces its force of twitch and tetanic contractions in the in vitro rat preparation as well as in the in vivo dog preparation. The experiments reported here with 31P nuclear magnetic resonance (31P-NMR) spectroscopy attempt to examine cellular mechanisms that might be responsible for this deterioration in mechanical performance. Specifically they describe certain characteristics of this preparation and cautions needed to study the resting in vitro rat diaphragm with such techniques. Second, they report the response of intracellular pH (pHi), phosphocreatine (PCr), ATP, and inorganic phosphate (Pi) in the resting in vitro rat diaphragm exposed to long-term normocapnia or to long-term hypercapnia. The results show that 1) to maintain a viable preparation, it was necessary to keep the diaphragm extended to an area approximating that at functional residual capacity, 2) the diaphragm seemed quite capable of maintaining a constant pHi and constant contents of ATP and Pi during normocapnia, but there was a gradual decline in PCr, and 3) during hypercapnia there was a significant decrease in pHi, but the behavior of the phosphate metabolites was exactly as during normocapnia. The results suggest that the decrease in mechanical performance of the diaphragm is probably not due to a decrease in the availability of the high-energy phosphates, although they do not completely exclude this possibility or possibilities related to regional compartmentation. 相似文献
28.
29.
Ferricrocin functions as the main intracellular iron-storage compound in mycelia ofNeurospora crassa
Summary
Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores. 相似文献
30.
William P. Norred Elaine Wang Hwansoo Yoo Ronald T. Riley Alfred H. Merrill Jr. 《Mycopathologia》1992,117(1-2):73-78
The effects of fumonisins B1FB1, B2(FB{2}), and the backbone of fumonisin B1 remaining after hydrolysis of the tricarballylic groups with base (HFB1) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK1). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC50 of FB1=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC50 for FB1=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation. 相似文献