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201.
The age related decrease in alpha1-adrenergic stimulated inositol 1,4,5 trisphosphate (IP3) production in parotid cells of aged rats can be partially restored by treatment with S-adenosylmethionine (SAM). This effect is completely blocked by S-adenosyl homocysteine (SAH) and occurs in association with an increase in the conversion of phosphatidylethanolamine to phosphatidylcholine and a decrease in membrane viscosity. In contrast, SAM treatment actually inhibits stimulated IP3 production in cells of young rats. The membrane viscosity of these cells is lower than that of those from aged rats. Although conversion of phosphatidylethanolamine to phosphatidylcholine is enhanced, no further decrease in membrane viscosity is elicited in young cell preparations. These findings suggest that age changes in the membrane environment may result in impaired alpha1-adrenergic signal transduction and that such alterations may be at least partially reversible by SAM treatment.  相似文献   
202.
Obtusifoliol 14β-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α-demethylases are orthologous enzymes. The sterol 14α-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α-demethylase catalyses the 14α-demethylation of obtusifoliol to 4α-methyl-5α-ergosta-8,14,24(28)-trien-3β-ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α-demethylases.  相似文献   
203.
A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.  相似文献   
204.
A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (Mr = 72 550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The Km for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.  相似文献   
205.
206.
We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.  相似文献   
207.
Small-subunit ribosomal RNA nucleotide sequences were inferred for Giraudyopsis stellifera Dangeard (Chrysomeridales), as well as for Pulvinaria sp. and Sarcinochrysis marina Geitler (Sarcinochrysidales,). Phylogenetic analyses of the molecular data indicate that the former is weakly related to the Phaeophyceae/Xanthophyceae clade, whereas the latter two have affinities to the Pelagophyceae, and the Sarcinochrysidales sensu stricto is transferred to this class. A recent study proposed that the Pelagophyceae belongs to a larger assemblage of chromophytic species characterized by reduced flagellar apparatuses. Although the flagellar apparatus characterizing the Sarcinochrysidales is reduced relative to the Chysomeridaels and some other chromophytes, it is the most complicated to be associated with “the reduced flagellar apparatus” lineage. Cladistic analyses of a traditional data set (largely ultrastructural features of the flagellar apparatus) and a combined traditional/molecular data set were used to assess the evolutionary trends of reduction in the flagellar apparatus within the heterokont chromophytes.  相似文献   
208.
209.
A large number ofPenicillium nalgiovense isolates from mould fermented sausages and the ex type culture were examined for characters of morphology, physiology and production of secondary metabolites. To separate biotypes within theP. nalgiovense species, the data obtained were evaluated using multivariate statistical methods. The macromorphological characters of the ex type culture and isolates from meat products appeared to be distinctive. The ex type culture is characterized by a brown reverse on both Czapek yeast extract and malt extract agar while the isolates from meat products have a yellow to orange reverse. Proteolytic and/or lipolytic activity was demonstrated by 75% of the examined cultures and all of them demonstrated ability to utilize lactate as sole carbon source. Growth on creatine sucrose agar was very inhibited and acid production was absent or very weak. TLC analysis showed production of three unknown secondary metabolites that constituted the characteristic profile. HPLC analysis showed production of only three known secondary metabolites; chrysogine (96%), nalgiolaxin and nalgiovensin (9%). The ex type culture produced nalgiolaxin and nalgiovensin but not chrysogine. The chemometric evaluation showed thatP. nalgiovense isolates from meat products from a homogenous species, which can not be divided into biotypes. The only indication of grouping, beside a separation of the ex type culture, was related to the conidium colour (white, turquoise or grey green). The examinedP. nalgiovense isolates showed some resemblance (morphologically and chemically) toP. chrysogenum.  相似文献   
210.
Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid  相似文献   
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