A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products.
- MeSH
- bakteriální proteiny genetika metabolismus chemie MeSH
- beta-galaktosidasa * genetika metabolismus chemie izolace a purifikace MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- glykosylace MeSH
- klonování DNA * MeSH
- koncentrace vodíkových iontů MeSH
- laktosa * metabolismus MeSH
- mléko mikrobiologie MeSH
- molekulová hmotnost MeSH
- oligosacharidy metabolismus MeSH
- Pedobacter * enzymologie genetika MeSH
- rekombinantní proteiny genetika metabolismus chemie izolace a purifikace MeSH
- sekvence aminokyselin MeSH
- stabilita enzymů * MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Mucopolysaccharidosis type IVB (MPS IVB) is a very rare lysosomal storage disorder characterized by skeletal dysplasia, hearing disorder, and cardiac valvular disease. Herein, we report an extremely rare manifestation of MPS IVB in a 60-year-old female patient who underwent a successful aortic valve replacement. The patient presented with mild coarse facial features, short stature, mild dyspnea, sternal protrusion, mild lumbar hyperlordosis, and waddling gait owing to bilateral femoral head necroses and bilateral arthrosis of the knees. The patient also suffered from dyspnea, NYHA II-III. Echocardiography revealed severe stenosis of a calcified aortic valve (AVA 0.67 cm2, AVAi 0.45 cm2/m2, PG max/mean 130/80 mmHg), left ventricular hypertrophy with predominant septal thickening (18 mm) and mild left ventricle outflow tract obstruction at rest, mild mitral valve regurgitation, and dilated ascending aorta (36 mm, 26.5 mm/m2). Dyspnea resolved after septal myectomy and replacement of the aortic valve with bioprosthesis. Excretion levels and spectrum of glycosaminoglycans (GAGs) in urine were normal in the patient. We confirmed the diagnosis of MPS IVB by identifying decreased beta-galactosidase activity in isolated leukocytes (6 nmol/h/mg; controls 95-272) and by molecular genetic analyses (c.438_440delTCT and c.817_818TG>CT mutations in the GLB1 gene). Primary lysosomal storage of glycosaminoglycans was detected in fibroblasts of the aortic valve. Additional pathologies included valvular fibrosis, calcification, neovascularization, and mild chronic inflammation. In conclusion, the diagnosis of MPS IVB should be considered in older patients with cardiac valvular disease and progressive skeletal abnormality even if urinary excretion levels of GAGs are normal.
- MeSH
- aortální chlopeň diagnostické zobrazování patologie patofyziologie chirurgie transplantace MeSH
- aortální stenóza diagnostické zobrazování etiologie patofyziologie chirurgie MeSH
- beta-galaktosidasa genetika MeSH
- bioprotézy MeSH
- biopsie MeSH
- časové faktory MeSH
- chirurgická náhrada chlopně * přístrojové vybavení MeSH
- echokardiografie MeSH
- kalcinóza diagnostické zobrazování etiologie patofyziologie chirurgie MeSH
- lidé středního věku MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- mukopolysacharidóza IV komplikace diagnóza genetika MeSH
- mutace MeSH
- mutační analýza DNA MeSH
- opožděná diagnóza MeSH
- srdeční chlopně umělé MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter-derived vectors in cancers with Mdm2 gene amplification.
- MeSH
- beta-galaktosidasa genetika metabolismus MeSH
- Cytomegalovirus genetika metabolismus MeSH
- genetická transkripce MeSH
- HEK293 buňky MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- proliferace buněk MeSH
- promotorové oblasti (genetika) MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- regulace genové exprese u nádorů * MeSH
- reportérové geny MeSH
- signální transdukce MeSH
- transkripční faktory TFII genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cell mobilization, a process that influences circulation, margination, and finally, homing play key roles in the regeneration processes mediated by stem cells. Recent studies as well as prior studies from our group indicate an important role of the spleen in hematopoietic reconstitution, but to date the role of the spleen in hematopoietic reconstitution has been unclear and it has not been precisely documented in ablated animals. Therefore, we undertook the present study to define more closely the role of the spleen in hematopoietic reconstitution in lethally irradiated mice. After transplantation of irradiated mice with lacZ+ -marked lin- / CD117+ bone marrow cells, we compared splenectomized mice (T(S), splenectomy performed prior to irradiation) to nonsplenectomized, irradiated mice (T(N)) as well as to normal (unirradiated) mice. Impaired hematopoietic reconstitution was observed in T(S) mice. Splenectomy markedly altered the distribution of hematopoietic stem cells, as demonstrated by fluorescence-activated cell sorting analysis of endogenous CD117+ cells in the thymus and bone marrow of recipients. Cell engraftment was demonstrated by histochemical and polymerase chain reaction analyses of recipient tissues. These experiments demonstrated that in T(S) animals, transplanted hematopoietic stem cells mobilized to extravascular tissues, particularly the gastrointestinal tract. The number of donor cells in recipient tissues continued to increase for 30 days after transplantation with the highest numbers observed in the T(S) group. DNA marking analysis led to the conclusion that engrafted cells were not only integrated into recipient tissues but were also capable of performing complex cellular processes, including proliferation and repair. Our results are consistent with the novel possibility that cellular repair markedly affects stem cell regenerative functions and that repair is markedly influenced by the integrity and presence of organs not directly involved in specific tissue regeneration processes, particularly the spleen.
- MeSH
- beta-galaktosidasa genetika metabolismus MeSH
- časové faktory MeSH
- exprese genu MeSH
- hematopoetické kmenové buňky cytologie metabolismus MeSH
- hematopoéza účinky záření MeSH
- imunohistochemie MeSH
- kostní dřeň metabolismus účinky záření MeSH
- myši kmene 129 MeSH
- myši transgenní MeSH
- myši MeSH
- počet lymfocytů MeSH
- pohyb buněk MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proliferační antigen buněčného jádra metabolismus MeSH
- protoonkogenní proteiny c-kit metabolismus MeSH
- průtoková cytometrie MeSH
- splenektomie metody MeSH
- thymus cytologie metabolismus MeSH
- transplantace kostní dřeně MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins, like other phytohormones, act in plants as signaling molecules at very low concentrations. The system that mediates between their chemical recognition and the responses that they induce requires a hormone receptor that, together with down-stream located elements, forms a signaling network, converting the signal into a specific response. Identification of the cytokinin-binding histidine kinases CRE1/AHK4, AHK3, and AHK2 as cytokinin receptors in Arabidopsis was an important milestone in the elucidation of cytokinin signal transduction pathways. Their molecular characterization through the use of transgenic E. coli strains revealed that a variety of cytokinin compounds may have signaling functions, but only with specific receptors. This indicates that differential ligand specificities of the receptors may be a mechanism to fine-tune the various cytokinin responses. This chapter describes the detailed protocol of a method employing transgenic E. coli which substantially contributes to our understanding of cytokinin perception, a crucial step in the cytokinin regulation of diverse plant growth and development processes.
- MeSH
- Arabidopsis enzymologie MeSH
- beta-galaktosidasa genetika metabolismus MeSH
- biotest metody MeSH
- cytokininy metabolismus farmakologie MeSH
- Escherichia coli genetika metabolismus MeSH
- proteinkinasy genetika metabolismus MeSH
- regulace genové exprese u bakterií účinky léků MeSH
- regulátory růstu rostlin metabolismus farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A genomic library of bacterial strain Paenibacillus thiaminolyticus was constructed and the plasmid DNA of the clone, containing the gene encoding beta-d-galactosidase with beta-d-fucosidase activity, detected by 5-bromo-4-chloro-3-indoxyl beta-d-galactopyranoside, was sequenced. Cells of Escherichia coli BL21 (DE3) were used for production of the enzyme in the form of a histidine-tagged protein. This recombinant fusion protein was purified using Ni-NTA agarose affinity chromatography and characterized by using p-nitrophenyl beta-d-fucopyranoside (K(m) value of (1.18 +/- 0.06) mmol/L), p-nitrophenyl beta-d-galactopyranoside (K(m) value of (250 +/- 40) mmol/L), p-nitrophenyl beta-d-glucopyranoside (K(m) value of (77 +/- 6) mmol/L), and lactose (K(m) value of (206 +/- 5) mmol/L) as substrates. Optimal pH and temperature were estimated as 5.5 and 65 degrees C, respectively. According to the amino acid sequence, the molecular weight of the fusion protein was calculated to be 68.6 kDa and gel filtration chromatography confirmed the presence of the enzyme in a monomeric form. In the following step, its ability to catalyze transfucosylation reactions was tested. The enzyme was able to catalyze the transfer of fucosyl moiety to different p-nitrophenyl glycopyranosides (producing p-nitrophenyl beta-d-fucopyranosyl-(1,3)-beta-d-fucopyranoside, p-nitrophenyl beta-d-fucopyranosyl-(1,3)-alpha-d-glucopyranoside, p-nitrophenyl beta-d-fucopyranosyl-(1,3)-alpha-d-mannopyranoside, and p-nitrophenyl beta-d-fucopyranosyl-(1,6)-alpha-d-galactopyranoside) and alcohols (producing methyl beta-d-fucopyranoside, ethyl beta-d-fucopyranoside, 1-propyl beta-d-fucopyranoside, 2-propyl beta-d-fucopyranoside, 1-octyl beta-d-fucopyranoside, and 2-octyl beta-d-fucopyranoside). These results indicate the possibility of utilizing this enzyme as a promising tool for enzymatic synthesis of beta-d-fucosylated molecules.
- MeSH
- alfa-L-fukosidasa genetika metabolismus MeSH
- beta-galaktosidasa genetika metabolismus MeSH
- chromatografie afinitní MeSH
- Escherichia coli enzymologie genetika metabolismus MeSH
- gelová chromatografie MeSH
- katalýza MeSH
- molekulová hmotnost MeSH
- Paenibacillus enzymologie MeSH
- rekombinantní proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Investigations of environmental pollution by endocrine-disrupting chemicals are now in progress. Up to now, several in vitro bioassays have been developed for evaluation of the endocrine disruptive activity; however, there is still a lack of comparative studies of their sensitivity. In this work comparison of the estrogen screening assay based on beta-galactosidase expression and a bioluminescent estrogen screen revealed differences in the sensitivity and specificity of the two tests. With the beta-galactosidase screen a slight estrogen-like activity of Delor 103, a commercial mixture of PCB congeners, and a fungicide triclosan was measured whereas no activity was detected using the bioluminescent assay. A bioluminescent androgen test negated previously suggested androgenic potential of triclosan. Further, this work demonstrates the androgenic activity of Delor 103, with an EC(50) value of 2.29 x 10(-2)mg/L. On the other hand, chlorobenzoic acids (CBAs), representing potential PCB degradation metabolites, exhibited no androgenic activity but were slightly estrogenic. Their estrogenicity varied with their chemical structure, with 2,3-CBA, 2,3,6-CBA, 2,4,6-CBA and monochlorinated compounds exhibiting the highest activity. Thus the results indicated possible transitions of the hormonal activity of PCBs during bacterial degradation.
- MeSH
- androgeny farmakologie metabolismus MeSH
- beta-galaktosidasa analýza genetika MeSH
- biotest metody MeSH
- endokrinní disruptory farmakologie metabolismus MeSH
- estrogeny farmakologie metabolismus MeSH
- kvasinky genetika účinky léků MeSH
- látky znečišťující životní prostředí farmakologie metabolismus MeSH
- polychlorované bifenyly farmakologie metabolismus MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The X-ray structure of cold-active beta-galactosidase (isoenzyme C-2-2-1) from an Antarctic bacterium Arthrobacter sp. C2-2 was solved at 1.9A resolution. The enzyme forms 660 kDa hexamers with active sites opened to the central cavity of the hexamer and connected by eight channels with exterior solvent. To our best knowledge, this is the first cold-active beta-galactosidase with known structure and also the first known beta-galactosidase structure in the form of compact hexamers. The hexamer organization regulates access of substrates and ligands to six active sites and this unique packing, present also in solution, raises questions about its purpose and function. This enzyme belongs to glycosyl hydrolase family 2, similarly to Escherichia coli beta-galactosidase, forming tetramers necessary for its enzymatic function. However, we discovered significant differences between these two enzymes affecting the ability of tetramer/hexamer formation and complementation of the active site. This structure reveals new insights into the cold-adaptation mechanisms of enzymatic pathways of extremophiles.
- MeSH
- Arthrobacter enzymologie MeSH
- bakteriální proteiny genetika chemie metabolismus MeSH
- beta-galaktosidasa genetika chemie metabolismus MeSH
- financování organizované MeSH
- ionty chemie MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- nízká teplota MeSH
- rozpouštědla chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
