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101.
Arvizu-Flores AA Sugich-Miranda R Arreola R Garcia-Orozco KD Velazquez-Contreras EF Montfort WR Maley F Sotelo-Mundo RR 《The international journal of biochemistry & cell biology》2008,40(10):2206-2217
Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group. 相似文献
102.
A multitarget approach is needed for effective gene silencing that combines more than one antiviral strategy. With this in mind, we designed a wild-type (wt) and selectively disabled chimeric mutant (mt) constructs that consisted of small hairpin siRNA joined by a short intracellular cleavable linker to a known hammerhead ribozyme, both targeted against the full-length X RNA of hepatitis B. These chimeric RNAs possessed the ability to cleave the target RNA under in vitro conditions and were efficiently processed at the cleavable site. When this wt chimeric RNA construct was introduced into a liver-specific mammalian cell line, HepG2, along with the HBx substrate encoding DNA, very significant (approximately 70%) intracellular downregulation in the levels of target RNA was observed. When the siRNA portion of this chimeric construct was mutated, keeping the ribozyme (Rz) region unchanged, it caused only approximately 25% intracellular reduction. On the contrary, when only the Rz was made catalytically inactive, about 55% reduction in the target RNA was observed. Construct possessing mt Rz and mt siRNA caused only 10% reduction. This wt chimeric construct also resulted in almost complete knockdown of intracellular HBx protein production, and the mt versions were less effective. The intracellular reduction of target RNA with either wt or mt constructs also interfered with the known functions of HBx protein with varying efficiencies. Thus, in this proof of concept study we show that the levels of the target RNA were reduced potently by the wt chimeric siRNA-Rz construct, which could be modulated with mt versions of the same. 相似文献
103.
Nath N Hurst R Hook B Meisenheimer P Zhao KQ Nassif N Bulleit RF Storts DR 《Journal of proteome research》2008,7(10):4475-4482
For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks. 相似文献
104.
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106.
Kumar V Mudgal MM Rani N Jha A Jaggi M Singh AT Sanna VK Singh P Sharma PK Irchhaiya R Burman AC 《Journal of enzyme inhibition and medicinal chemistry》2009,24(3):763-770
A new series of functionalized amino acid derivatives N-substituted 1-N-(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazolidine carboxamide (1-17) and 1-N-substituted-3-amino-2-hydroxy-3-phenylpropane-1-carboxamide (18-34) were synthesized and evaluated for their in vitro cytotoxicity against human cancer cell lines. Compound 6 has shown interesting cytotoxicity (IC(50) = 5.67 microm) in ovarian cancer, while compound 10 exhibited promising cytotoxicity in ovarian (IC(50) = 6.1 microm) and oral (IC(50) = 4.17 microm) cancers. These compounds could be of use in designing new anti-cancer agents. 相似文献
107.
Haiyan Liu Ali Javaheri Rebecca J. Godar John Murphy Xiucui Ma Nidhi Rohatgi 《Autophagy》2017,13(11):1952-1968
Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes. 相似文献
108.
Vishnoi N Flaherty K Hancock LC Ferreira ME Amin AD Prochasson P 《Biochimica et biophysica acta》2011,1809(10):557-566
The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63-79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452-480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex. 相似文献
109.
A new family of mononuclear Zn(II) complexes [Zn(Pyimpy)2](ClO4)2 (1), [Zn(Pyimpy)(Cl)2] (2), [Zn(Pyimpy)(SCN)2] (3) and [Zn(Pyimpy)(N3)2] (4) were synthesized using designed tridentate ligand Pyimpy having NNN donors (Pyimpy: (2-((2-phenyl-2-(pyridin-2-l)hydazono)methyl)pyridine)). Complexes were characterized by different spectroscopic studies and it has been found out that all complexes exhibited strong fluorescent emission at room temperature. Molecular structures of [Zn(Pyimpy)2](ClO4)2·C6H5CH3·0.5H2O (1·C6H5CH3·0.5H2O) and [Zn(Pyimpy)(Cl)2]·CH3CN (2·CH3CN) were determined by X-ray crystallography and ligand coordinated Zn(II) ions was described as distorted octahedral and distorted square pyramidal, respectively. DNA binding properties of these complexes were investigated by absorption spectral, fluorescence quenching and circular dichroism spectral studies. 相似文献
110.