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11.
12.
Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces bayanus and Alcoholic Fermentation by Yeasts 总被引:1,自引:0,他引:1
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Regina C. Nabais Isabel S-Correia Cristina A. Viegas Júlio M. Novais 《Applied microbiology》1988,54(10):2439-2446
The addition of Ca2+ (as CaCl2) in optimal concentrations (0.75 to 2.0 mM) to a fermentation medium with a trace contaminating concentration of Ca2+ (0.025 mM) led to the rapid production of higher concentrations of ethanol by Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus. The positive effect of calcium supplementation (0.75 mM) on alcoholic fermentation by S. bayanus was explained by the increase in its ethanol tolerance. The ethanol inhibition of growth and fermentation followed the equation μxi = μoi [1 - (X/Xmi)]ni, where μoi and μxi are, respectively, the specific growth (i = g) and fermentation (i = f) rates in the absence or presence of a concentration (X) of added ethanol, and Xmi is the maximal concentration of ethanol which allows growth or fermentation. The toxic power is given by ni. In Ca2+ - supplemented medium (0.75 mM), ng = 0.42 for growth and nf = 0.43 for fermentation compared with 0.52 and 0.55, respectively, in unsupplemented medium; for both media, Xmg = 10% (vol/vol) and Xmf = 13% (vol/vol). For lethal concentrations of ethanol, the specific death rates were minimal for cells that were grown and incubated with ethanol in medium with an optimal concentration of Ca2+, maximal for cells grown and incubated with ethanol in unsupplemented medium, and intermediate for cells grown in unsupplemented medium and incubated with ethanol in calcium-supplemented medium. The effect of Ca2+ on the acidification curve of energized cells in the presence of ethanol was found to be closely associated with its protective effect on growth, fermentation, and viability. 相似文献
13.
Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes. 相似文献
14.
15.
Birgit Conrad Regina S. Savchenko Roland Breves Jürgen Hofemeister 《Molecular & general genetics : MGG》1996,250(2):230-236
The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation 相似文献
16.
Luciana A. Haddad Regina C. Mingroni-Netto Angela M. Vianna-Morgante Sérgio D. J. Pena 《Human genetics》1996,97(6):808-812
Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence,
several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population
screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification
of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels.
The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for
the (CGG)29 allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity
compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer,
internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147
bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described
showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis,
it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.
Received: 31 May 1995 / Revised: 4 October 1995 相似文献
17.
Márcia Camargo-De-Morais Marta De Freitas Angela G. De Mattos Nádia Schröder Ana C. Zilles Carla S. F. Lisboa Nice Arteni Armando Barlem Rejane Schierholt Guilherme Zwetsch Carlos A. B. Souza Regina Pessoa-Pureur Carlos A. Netto 《Neurochemical research》1996,21(5):595-602
Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus
of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the
rat. In vitro [14Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament
subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased
while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of32P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP
levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult.
These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the
hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L. 相似文献
18.
James M. Chen Spero Manolatos Paul W. Brandt-Rauf Randall B. Murphy Regina Monaco Matthew R. Pincus 《Journal of Protein Chemistry》1996,15(6):511-518
The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun. 相似文献
19.
Regina Monaco James M. Chen Denise Chung Paul Brandt-Rauf Matthew R. Pincus 《The protein journal》1995,14(6):457-466
Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein. 相似文献
20.
The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for anα-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to beα-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energyα-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in theirα-helical and bend conformations shows that the all-α-helical dimer is clearly favored energetically over the bend dimer. 相似文献