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101.
Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO3-inducible. NH4+ was the initial repressor signal for the uptake process which was involved in the control of the NO3inducible reductase system.  相似文献   
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NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5′ or 3′ sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.  相似文献   
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d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries. Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998  相似文献   
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Background aimsWe have previously described a xeno-free scalable system to generate transplantable dopaminergic neurons from human pluripotent stem cells. However, several important questions remain to be answered about our cell therapy efforts. These include determining the exact time at which cells should be transplanted and whether cells at this stage can be frozen, shipped, thawed and injected without compromising their ability to mature and survive the transplantation procedure. We also needed to determine whether further optimization of the culture process could shorten the development time and reduce variability and whether a current Good Manufacture Practice (CGMP) facility could manufacture cells with fidelity.MethodsWe developed an optimized protocol that included modulating the sonic hedgehog homolog gradient with bone morphogenetic proteins (BMP2) and addition of activin to the culture medium, which shortened the time to generate Lmx1A and FoxA2 immunoreactive cells by 4–6 days.ResultsWe showed that cells at this stage could be safely frozen and thawed while retaining an excellent ability to continue to mature in vitro and survive transplant in vivo. Importantly, we successfully adapted this process to a CGMP facility and manufactured two lots of transplant-ready dopaminergic neurons (>250 vials) under CGMP-compatible conditions. In vitro characterization, including viability/recovery on thawing, whole genome expression as well as expression of midbrain/dopaminergic markers, showed that the cells manufactured under GMP-compatible conditions were similar to cells produced at lab scale.ConclusionsOur results suggest that this optimized protocol can be used to generate dopaminergic neurons for Investigational New Drug enabling studies.  相似文献   
106.
The P5CS ({Delta} 1-Pyrroline–5-Carboxylate Synthetase) gene encodes for a bifunctional enzyme that catalyzes the rate limiting reaction in proline biosynthesis in living organisms. A wide range of multifunctional roles of proline have now been shown in stress defense. The proline biosynthetic genes, especially, P5CS is commonly used in metabolic engineering for proline overproduction conferring stress tolerance in plants. The gene is functionally well characterized at the molecular level, but there is more to learn about its evolutionary path in the plant kingdom, particularly the drive behind functional (osmoprotective and developmental) divergence of duplication of P5CS genes. In this review, we present the current understanding of the evolutionary trail of plant P5CS gene which plays a key role in stress tolerance.  相似文献   
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Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.  相似文献   
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Inositol hexaphosphate (IP6) is a natural constituent found in almost all cereals and legumes. It is known to cause numerous antiangiogenic manifestations. Notwithstanding its great potential, it is underutilized due to the chelation and rapid excretion from the body. Jacalin is another natural constituent obtained from seeds of jackfruit and can target disaccharides overexpressed in tumor cells. The current study was in-quested to develop and evaluate a surface-modified gold nanoparticulate system containing IP6 and jacalin which may maximize the apoptotic effect of IP6 against HCT-15 cell lines. IP6 loaded jacalin-pectin-gold nanoparticles (IJP-GNPs) were developed through reduction followed by incubation method. The developed formulation was tested for various in vitro and in silico studies to investigate its potential. HCT-15 cells when exposed to IJP-GNP resulted in significant apoptotic effects in dose as well as time-dependent manner, as measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, micronucleus, and reactive oxygen species assay. IJP-GNP displayed cell cycle arrest at the G0/G1 phase. To further explore the mechanism of chemoprevention, in silico studies were performed. The docking results revealed that the interactive behavior of IP6, P-GNP, and jacalin could target and inhibit the tumor formation activity, supported by in vitro studies. Taken together, all the findings suggested that IP6 loaded nanoparticles may increase the hope of future drug delivery strategy for targeting colon cancer.  相似文献   
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