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1.
Nozomi Yoshinari Kazunori Ando Akira Kudo Masato Kinoshita Atsushi Kawakami 《Development, growth & differentiation》2012,54(9):818-828
Conditional cell labeling, cell tracing, and genetic manipulation approaches are becoming increasingly important in developmental and regenerative biology. Such approaches in zebrafish research are hampered by the lack of an ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the medaka fish (Oryzias latipes) β‐actin (Olactb) promoter, which drives constitutive transgene expression during all developmental stages, and the analysis of adult organs except blood cell types. Taking advantage of the compact medaka promoter, we succeeded in generating a zebrafish transgenic (Tg) line with unprecedentedly strong and widespread transgene expression from embryonic to adult stages. Moreover, the Tg carries a pair of loxP sites, which enables the reporter fluorophore to switch from DsRed2 to enhanced green fluorescent protein (EGFP). We induced Cre/loxP recombination with Tg(hsp70l: mCherry‐t2a‐CreERt2) in the double Tg embryo and generated a Tg line that constitutively expresses EGFP. We further demonstrate the powerful application of Olactb‐driven Tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of regenerating fin. Thus, the use of promoter elements from medaka is an alternative approach to generate Tgs with stronger and even novel expression patterns in zebrafish. The Olactb promoter and the Tg lines presented here represent an important advancement for the broader use of Cre/loxP‐based Tg applications in zebrafish. 相似文献
2.
《Genesis (New York, N.Y. : 2000)》2017,55(4)
p57Kip2 (p57 ) is a maternally expressed imprinted gene regulating growth arrest which belongs to the CIP/KIP family of cyclin‐dependent kinase inhibitors. While initially identified as a cell cycle arrest protein through inhibition of cyclin and cyclin‐dependent kinase complexes, p57 activity has also been linked to differentiation, apoptosis, and senescence. In addition, p57 has recently been shown to be involved in tumorigenesis and cell fate decisions in stem cells. Yet, p57 function in adult tissues remains poorly characterized due to the perinatal lethality of p57 knock‐out mice. To analyze p57 tissue‐specific activity, we generated a conditional mouse line (p57FL‐ILZ/+ ) by flanking the coding exons 2–3 by LoxP sites. To track p57‐expressing or mutant cells, the p57FL‐ILZ allele also contains an IRES‐linked β‐galactosidase reporter inserted in the 3′ UTR of the gene. Here, we show that the β‐galactosidase reporter expression pattern recapitulates p57 tissue specificity during development and in postnatal mice. Furthermore, we crossed the p57FL‐ILZ/+ mice with PGK‐Cre mice to generate p57cKO‐ILZ/+ animals with ubiquitous loss of p57. p57cKO‐ILZ/+ mice display developmental phenotypes analogous to previously described p57 knock‐outs. Thus, p57FL‐ILZ/+ is a new genetic tool allowing expression and functional conditional analyses of p57. 相似文献
3.
G.J. Speksnijder R.J. Etches A.M. Verrinder Gibbins 《Molecular reproduction and development》1999,52(1):33-42
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
4.
K. N. Neustroev E. A. De Sousa A. M. Golubev J. R. Brando Neto E. V. Eneyskaya A. A. Kulminskaya I. Polikarpov 《Acta Crystallographica. Section D, Structural Biology》2000,56(11):1508-1509
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
5.
Zui Fujimoto Osamu Kobayashi Satoshi Kaneko Mitsuru Momma Hideyuki Kobayashi Hiroshi Mizuno 《Acta Crystallographica. Section D, Structural Biology》2002,58(8):1374-1375
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.
Kimihito Usui Umeharu Ohto Toshinari Ochi Toshiyuki Shimizu Yoshinori Satow 《Acta Crystallographica. Section F, Structural Biology Communications》2012,68(1):73-77
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
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Ryo Uehara Riki Iwamoto Sayaka Aoki Takuya Yoshizawa Kazufumi Takano Hiroyoshi Matsumura Shun‐ichi Tanaka 《Protein science : a publication of the Protein Society》2020,29(9):2000-2008
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
9.
Ikue Kikuchi Yuji Nakayama Takao Morinaga Yasunori Fukumoto Naoto Yamaguchi 《Cell biology international》2010,34(6):645-653
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
10.
Agustín Rico‐Díaz ngel Vizoso Vzquez M. Esperanza Cerdn Manuel Becerra Julia Sanz‐Aparicio 《Acta Crystallographica. Section F, Structural Biology Communications》2014,70(11):1529-1531
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
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Douglas H. Juers Beatrice Rob Megan L. Dugdale Nastaron Rahimzadeh Clarence Giang Michelle Lee Brian W. Matthews Reuben E. Huber 《Protein science : a publication of the Protein Society》2009,18(6):1281-1292
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
13.
Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study 
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下载免费PDF全文 Flor González‐Cataño Luz Tovar‐Castro Eduardo Castaño‐Tostado Carlos Regalado‐Gonzalez Blanca García‐Almendarez Anaberta Cardador‐Martínez Silvia Amaya‐Llano 《Biotechnology progress》2017,33(6):1568-1578
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
14.
Hodaya V. Solomon Orly Tabachnikov Hadar Feinberg Lata Govada Naomi E. Chayen Yuval Shoham Gil Shoham 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(10):1114-1119
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
15.
Nicolas Currier Kathleen Chea Mirka Hlavacova Daniel J. Sussman David C. Seldin Isabel Dominguez 《Genesis (New York, N.Y. : 2000)》2010,48(3):183-194
We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF‐1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3β inhibition. Mammary tumor cell lines derived from female LEF‐EGFP transgenic mice treated with the carcinogen 7, 12‐dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer. genesis 48:183–194, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
16.
Fahimeh Mirakhori Bahman Zeynali Azita Parvaneh Tafreshi Ameneh Shirmohammadian 《Molecular reproduction and development》2013,80(4):286-296
Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23‐day‐old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl‐treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17β‐estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)‐positive cells and conversely increased the number of TUNEL‐positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase‐3β, and unexpectedly decreased the expression of active (stabilized) β‐catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized β‐catenin and 17β‐estradiol synthesis. Mol. Reprod. Dev. 80: 286–296, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
17.
《Peptide Science》2017,108(3)
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
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Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (Km) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (Vmax) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015. 相似文献
20.
Masahiro Nakajima Ryuta Yoshida Akimasa Miyanaga Hayao Taguchi 《Acta Crystallographica. Section F, Structural Biology Communications》2014,70(10):1398-1401
Lin1840 is a putative β‐glucosidase that is predicted to be involved in 1,2‐β‐glucan metabolism since the lin1839 gene encoding a 1,2‐β‐oligoglucan phosphorylase and the lin1840 gene are located in the same gene cluster. Here, Lin1840 was crystallized. The crystals of Lin1840 diffracted to beyond 1.8 Å resolution. The crystal belonged to space group I121, with unit‐cell parameters a = 89.75, b = 95.10, c = 215.00 Å, α = 90.00, β = 96.34, γ = 90.00°. 相似文献