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1.
    
Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration‐dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin‐induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated β‐galactosidase activity. In DNA damage‐induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage‐induced senescence.  相似文献   

2.
    
Crystals of an extracellular β‐galactosidase from Penicillium sp. (MW = 120 ± 5 kDa) have been obtained from a sodium phosphate buffer using PEG as precipitant. The crystals belong to the tetragonal space group P41or P43, with unit‐cell parameters a = b = 110.82, c = 161.28 Å, and diffract to 1.85 Å resolution at a synchrotron source.  相似文献   

3.
    
In the quest to develop drugs against traveller's diarrhoea and cholera, the structure of the B pentamer of heat‐labile enterotoxin (LT) complexed with a new receptor‐binding antagonist, m‐carboxyphenyl‐α‐d ‐galactopyranoside, has been determined. The high resolution obtained for this structure allowed anisotropic refinement of the model. It was also now possible to confirm at a near‐atomic resolution the structural similarity between the B subunits of LT and the closely related cholera toxin (CT), including the similarity in deviations of planarity of the same peptide unit in LT and CT. The structure of the LT complex clearly revealed different conformations for the m‐­carboxyphenyl moiety of the ligand in the five B subunits of LT, while the binding modes of the well defined galactopyranoside moieties were identical. In two binding sites the m‐carboxyphenyl moiety displayed no significant electron density, demonstrating significant flexibility of this moiety. In a third binding site the m‐carboxyphenyl moiety could be modelled unambiguously into the density. The two remaining binding sites were involved in crystal packing contacts and the density for the ligands in these two binding sites clearly revealed different binding modes, of which one conformation was identical to and one completely different from the conformation of m‐carboxyphenyl‐galactopyranoside in the third subunit. The multiple binding modes observed in the crystal may represent the ensemble of conformations of m‐carboxyphenyl‐α‐d ‐galactopyranoside complexed to LT in solution.  相似文献   

4.
    
The conformational characteristics of protected homo‐oligomeric Boc‐[β3(R)Val]n‐OMe, n = 1, 2, 3, 4, 6, 9, and 12 have been investigated in organic solvents using nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) absorption spectroscopy and circular dichroism (CD) methods. The detailed 1H NMR analysis of Boc‐[β3(R)Val]12‐OMe reveals that the peptide aggregates extensively in CDCl3, but is disaggregated in 20%, (v/v) dimethyl sulfoxide (DMSO) in CDCl3 and in CD3OH. Limited assignment of the N‐terminus NH groups, together with solvent dependence of NH chemical shifts and temperature coefficients provides evidence for 14‐helix conformation in the 12‐residue peptide. FTIR analysis in CHCl3 establishes that the onset of folding and aggregation, as evidenced by NH stretching bands at 3375 cm−1 (intramolecular) and 3285 cm−1 (intermolecular), begins at the level of the tetrapeptide. The observed CD bands, 214 nm (negative) and 198 nm (positive), support 14‐helix formation in the 9 and 12 residue sequences. The folding and aggregation tendencies of homo‐oligomeric α‐, β‐, and γ‐ residues is compared in the model peptides Boc‐[ωVal]n‐NHMe, ω = α, β, and γ and n = 1, 2, and 3. Analysis of the FTIR spectra in CHCl3, establish that the tendency to aggregate at the di and tripeptide level follows the order β > α∼γ, while the tendency to fold follows the order γ > β > α.  相似文献   

5.
    
α‐Galactosidases catalyze the hydrolysis of galactooligosaccharides and galactopolysaccharides to α‐galactose residues and are widely distributed in microorganisms, plants and animals. α‐Galactosidase from rice (Oryza sativa L. ssp. japonica) was crystallized by the hanging‐drop vapour‐diffusion method. The crystals belong to space group P212121, with unit‐cell parameters a = 63.1, b = 71.3, c = 85.6 Å, and diffract beyond 1.9 Å resolution.  相似文献   

6.
    
β‐d ‐Xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units. In this study, the crystallization and preliminary X‐ray analysis of the β‐d ‐xylosidase (XynB1) from Geobacillus stearothermophilus T‐6, a family 39 glycoside hydrolase, are described. XynB1 is a tetrameric protein consisting of four identical subunits of 503 amino acids and with a calculated molecular weight of 58 001 Da. Both the native and the selenomethionine‐containing XynB1 were crystallized by the hanging‐drop vapour‐diffusion method and the crystals were found to belong to space group P212121, with unit‐cell parameters a = 92.7, b = 165.7, c = 311.0 Å. The native crystals diffracted X‐rays to a resolution of 2.1 Å.  相似文献   

7.
    
Bacterial resistance to antibiotics is a serious medical and public health concern worldwide. Such resistance is conferred by a variety of mechanisms, but the extensive variability in levels of resistance across bacteria is a common finding. Understanding the underlying evolutionary processes governing this functional variation in antibiotic resistance is important as it may allow the development of appropriate strategies to improve treatment options for bacterial infections. The main objective of this study was to examine the functional evolution of β‐lactamases, a common mechanism of enzymatic resistance that inactivates a widely used class of antibiotics. We first obtained β‐lactamase protein sequences and minimal inhibitory concentration (MIC), a measure of antibiotic function, from previously published literature. We then used a molecular phylogenetic framework to examine the evolution of β‐lactamase functional activity. We found that the functional activity of antibiotic resistance mediated by β‐lactamase has evolved in a convergent manner within molecular classes, but is not associated with any single amino acid substitution. This suggests that the dynamics of convergent evolution in this system can vary between the functional and molecular (sequence) levels. Such disassociation may hamper bioinformatic approaches to antibiotic resistance determination and underscore the need for (less efficient but more effective) activity assays as an essential step in evaluating resistance in a given case.  相似文献   

8.
    
The X‐ray crystal structure of AmpC β‐lactamase (AmpCD) with a tripeptide deletion (Gly286‐Ser287‐Asp288) produced by Escherichia coli HKY28, a ceftazidime‐resistant strain, was determined at a resolution of 1.7 Å. The structure of AmpCD suggests that the tripeptide deletion at positions 286–288 located in the H10 helix causes a structural change of the Asn289–Asn294 region from the α‐helix present in the native AmpC β‐lactamase of E. coli to a loop structure, which results in a widening of the substrate‐binding site.  相似文献   

9.
    
β‐d ‐Galactosidase (β‐Gal) is an exoglycosidase that cleaves β‐galactosides from glycoproteins, sphingolipids and keratan sulfate. This study reports the expression, purification, crystallization and preliminary X‐ray crystallographic analysis of human lysosomal β‐Gal. The sitting‐drop vapour‐diffusion method was used to crystallize β‐Gal in complexes with its product galactose and with the inhibitor 1‐deoxygalactonojirimycin. The resulting crystals were isomorphous and belonged to space group P21. The crystals of the β‐Gal–galactose and the β‐Gal–inhibitor complexes had unit‐cell parameters a = 94.8, b = 116.1, c = 140.3 Å, β = 92.2° and a = 94.8, b = 116.0, c = 140.3 Å, β = 92.2°, respectively. Diffraction data were collected to 1.8 Å resolution for both crystals.  相似文献   

10.
    
β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β3‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β3h‐D ‐Ala in position 2 or β3hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

11.
    
Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, 1H‐NMR and 13C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

12.
    
Germline chimeric chickens can be constructed by injecting donor chicken blastodermal cells (CBCs) into recipient embryos and incubating to hatch. Transgenic chickens can be produced through chimeric intermediates if the donor cells are genetically manipulated; the chance of producing a transgenic chimera would be increased by enriching the donor population in transfected cells. To demonstrate that donor CBCs can be sorted according to the expression of a foreign gene, CBCs in suspension were subjected to transfection with plasmid DNA encoding bacterial β‐galactosidase (β‐gal). Following an overnight incubation, the CBCs were loaded with 5‐dodecanoylaminofluorescein di‐β‐D‐galactopyranoside (C12FDG), which is fluorescent after cleavage by β‐gal. The treated cells were subjected to fluorescence activated cell sorting (FACS) to give “positive” (fluorescent) and “negative” (non‐fluorescent) populations. Almost 100% of the “positive” population showed β‐gal activity. “Positive” cells were cultured on mouse SNL 76/7 fibroblast feeder cells and formed colonies, most of which still stained positively for β‐gal activity after three days. FACS‐sorted cells of Barred Plymouth Rock origin were injected into recipient White Leghorn embryos, resulting in chimeric embryos. Of the 298 embryos injected with sorted cells, 23 (8%; 18 injected with “positive cells, five with “negative”) survived to rearing. Somatic chimerism was seen in 12 of 18 (67%) “positive” and three of five (60%) “negative” birds with the proportion of black pigmentation averaging 19% overall. Twenty birds reached sexual maturity, of which 12 (60%) were somatically chimeric; seven (35%) of these produced donor‐derived chicks. Donor CBCs can, therefore, be sorted by FACS according to the expression of a selectable marker gene without impairing their ability to contribute to germline chimeras; this procedure could be incorporated into a practicable method by which to increase the chances of producing a transgenic chicken. Mol. Reprod. Dev. 52:33–42, 1999. © 1999 Wiley‐Liss, Inc.  相似文献   

13.
    
A GH1 β‐glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4′‐galactosyllactose, a tri‐galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.  相似文献   

14.
    
The bis‐functionalized diamondoid α‐amino acid 2‐aminoadamantane‐2‐carboxylic acid (Adm) has been used as the building block of four Nα‐formyl homo‐dipeptide alkylamide sequences via a solution‐phase Ugi multicomponent reaction approach. The conformers of these peptides have been determined in the crystalline state by X‐ray diffraction to distinguish the influences of the C‐terminal substituent. One of the Adm peptides folds into an open and a hydrogen‐bonded γ‐turn geometry. Moreover, 3D‐structures have been observed featuring two consecutive γ‐turns in an incipient γ‐helical structure, a significantly distorted nonhelical β‐turn, as well as an S‐shaped conformation with opposite helical screw senses. A significant topological variety is thus exhibited by the ‐Adm‐Adm‐ sequences contingent on their C‐terminal substituents, illustrating both the broad conformational potential and the need for further characterization of this sterically bulky residue in explorations of its ϕ, ψ space.  相似文献   

15.
    
β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH2 (TAPP). Each amino acid was replaced with an l ‐ or d ‐β3‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β3h‐ d ‐Ala‐Phe‐PheNH2 was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

16.
    
β‐Galactosidase from Aspergillus niger (An‐β‐Gal), belonging to the family 35 glycoside hydrolases, hydrolyzes the β‐galactosidase linkages in lactose and other galactosides. It is extensively used in industry owing to its high hydrolytic activity and safety. The enzyme has been expressed in yeasts and purified by immobilized metal‐ion affinity chromatography for crystallization experiments. The recombinant An‐β‐Gal, deglycosylated to avoid heterogeneity of the sample, has a molecular mass of 109 kDa. Rod‐shaped crystals grew using PEG 3350 as the main precipitant agent. A diffraction data set was collected to 1.8 Å resolution.  相似文献   

17.
    
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18.
    
The active site of ß‐galactosidase (E. coli) contains a Mg2+ ion ligated by Glu‐416, His‐418 and Glu‐461 plus three water molecules. A Na+ ion binds nearby. To better understand the role of the active site Mg2+ and its ligands, His‐418 was substituted with Asn, Glu and Phe. The Asn‐418 and Glu‐418 variants could be crystallized and the structures were shown to be very similar to native enzyme. The Glu‐418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg2+ site also reduce Na+ binding affinity. The Phe variant had reduced stability, bound Mg2+ weakly and could not be crystallized. All three variants have low catalytic activity due to large decreases in the degalactosylation rate. Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native. The results indicate that His‐418, together with the Mg2+, modulate the central role of Glu‐461 in binding and as a general acid/base catalyst in the overall catalytic mechanism. Glucose binding as an acceptor was also dramatically decreased, indicating that His‐418 is very important for the formation of allolactose (the natural inducer of the lac operon).  相似文献   

19.
Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL?1 initial lactose concentration and 6 U mL?1 enzyme concentration, obtaining 25.46 ± 0.01 gL?1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL?1 of trisaccharides and 13.79 ± 0.21 gL?1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568–1578, 2017  相似文献   

20.
    
Geobacillus stearothermophilus T‐6 is a Gram‐positive thermophilic soil bacterium that contains a multi‐enzyme system for the utilization of plant cell‐wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of endo‐acting extracellular enzymes that break down the high‐molecular‐weight polysaccharides into decorated oligosaccharides. These oligosaccharides enter the cell and are further hydrolyzed into sugar monomers by a set of intracellular glycoside hydrolases. One of these intracellular degrading enzymes is GanB, a glycoside hydrolase family 42 β‐galactosidase capable of hydrolyzing short β‐1,4‐galactosaccharides to galactose. GanB and related enzymes therefore play an important part in the hemicellulolytic utilization system of many microorganisms which use plant biomass for growth. The interest in the biochemical characterization and structural analysis of these enzymes stems from their potential biotechnological applications. GanB from G. stearothermophilus T‐6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory as part of its complete structure–function study. The best crystals obtained for this enzyme belong to the primitive orthorhombic space group P212121, with average crystallographic unit‐cell parameters of a = 71.84, b = 181.35, c = 196.57 Å. Full diffraction data sets to 2.45 and 2.50 Å resolution have been collected for both the wild‐type enzyme and its E323A nucleophile catalytic mutant, respectively, as measured from flash‐cooled crystals at 100 K using synchrotron radiation. These data are currently being used for the full three‐dimensional crystal structure determination of GanB.  相似文献   

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