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
Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine-PAGE, gene ontology (GO), and LC-MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 μM) became senescent; UPla (10 μg/mL), ULu (10 μg/mL), as well as positive controls lipoic acid (10 μg/mL) and transferrin (10 μg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-β-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine-PAGE revealed prominent detectable bands between 10 and 100 kDa. LC-MS/MS identified 150-180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with "extracellular space" and "proteasome core complex". UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.
- MeSH
- beta-galaktosidasa metabolismus MeSH
- buňky Hep G2 MeSH
- chromatografie kapalinová MeSH
- HEK293 buňky MeSH
- králíci MeSH
- kyselina lipoová * metabolismus MeSH
- lidé MeSH
- oxidační stres MeSH
- peroxid vodíku * farmakologie metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- stárnutí buněk MeSH
- tandemová hmotnostní spektrometrie MeSH
- transferriny metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Staining mice tissues for β-galactosidase activity is a fundamental tool to detect age- or disease-associated cellular senescence. However, reported analyses of positivity for senescence-associated β-galactosidase activity or for other markers of senescence in post-mitotic cells of healthy murine tissues have been fragmentary or inconclusive. Here, we attempted to independently deepen this knowledge using multiple senescence markers within the same cells of wild type mice entering middle age (9 months of age). A histochemistry protocol for the pH-dependent detection of β-galactosidase activity in several tissues was used. At pH 6, routinely utilized to detect senescence-associated β-galactosidase activity, only specific cellular populations in the mouse body (including Purkinje cells and choroid plexus in the central nervous system) were detected as strongly positive for β-galactosidase activity. These post-mitotic cells were also positive for other established markers of senescence (p16, p21 and DPP4), detected by immunofluorescence, confirming a potential senescent phenotype. These data might contribute to understanding the functional relation between the senescence-associated β-galactosidase activity and senescence markers in post-mitotic cells in absence of disease or advanced aging.
- MeSH
- beta-galaktosidasa metabolismus MeSH
- biologické markery metabolismus MeSH
- imunohistochemie MeSH
- koncentrace vodíkových iontů MeSH
- mitóza fyziologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- stárnutí buněk fyziologie MeSH
- stárnutí fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A new β-galactosidase-producing bacterium KGP, isolated from the Bay of Bengal, was identified as Paracoccus marcusii through morphology, biochemistry and 16S rRNA sequencing. This study is the first report on the production of β-galactosidase from P. marcusii. The medium components for the high yield of β-galactosidase were optimised using response surface methodology (RSM). A set of 17 experiments consisting of three independent variables, viz. yeast extract, galactose and MgSO4, was employed. A second-order polynomial equation was used for the analysis of the response, and the optimum β-galactosidase yield was achieved using 12.5 g/L yeast extract, 12.5 g/L galactose and 12.5 mmol/L MgSO4. The predicted quadratic model was inferred to be significant from the F-value, P value and the lack of fit value. Optimisation of the media components resulted in a ninefold increase (560 Miller units) in β-galactosidase production. Furthermore, the hydrolysis and transgalactosylation efficiency of the crude β-galactosidase was assessed and the results showed that the lactose was successfully hydrolysed and transgalactosylated at an optimum temperature of 40 °C and 50 °C, respectively. Considering the overall yield and productivity, P. marcusii can be considered a candidate for the industrial production of β-galactosidase. This study provides an essential basis for the future production and use of the alkali-tolerant β-galactosidase from P. marcusii KGP.
- MeSH
- beta-galaktosidasa metabolismus MeSH
- bioreaktory MeSH
- fylogeneze MeSH
- galaktosa metabolismus MeSH
- hydrolýza MeSH
- koncentrace vodíkových iontů MeSH
- laktosa metabolismus MeSH
- oligosacharidy biosyntéza MeSH
- Paracoccus klasifikace genetika izolace a purifikace metabolismus MeSH
- RNA ribozomální 16S genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
N-Acetyllactosamine (LacNAc; Galβ4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metastatic formation. In this study, we synthesized a series of fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts and presentations and evaluated the impact of their architecture on the binding affinity to Gal-1 and Gal-3. The controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors with comparable molecular weight (Mn ≈ 22,000 g mol-1) and narrow dispersity (D̵ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc disaccharide (4-22 mol % content in glycopolymer) prepared by enzymatic synthesis under catalysis by β-galactosidase from Bacillus circulans. The structure-affinity relationship study based on the enzyme-linked immunosorbent assay revealed that the type of LacNAc presentation, individual or clustered on bi- or trivalent linkers, brings a clear discrimination (almost 300-fold) between Gal-1 and Gal-3, reaching avidity to Gal-1 in the nanomolar range. Whereas Gal-1 strongly preferred a dense presentation of individually distributed LacNAc epitopes, Gal-3 preferred a clustered LacNAc presentation. Such a strong galectin preference based just on the structure of a multivalent glycopolymer type is exceptional. The prepared nontoxic, nonimmunogenic, and biocompatible glycopolymers are prospective for therapeutic applications requiring selectivity for one particular galectin.
- MeSH
- akrylamidy chemie MeSH
- aminocukry chemie MeSH
- Bacillus enzymologie MeSH
- beta-galaktosidasa metabolismus MeSH
- disacharidy chemická syntéza MeSH
- ELISA MeSH
- epitopy MeSH
- galektin 1 analýza metabolismus MeSH
- galektiny analýza metabolismus MeSH
- katalýza MeSH
- krevní proteiny analýza metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- polymerizace MeSH
- polymery chemie metabolismus farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Galactooligosaccharides (GOS) are currently attracting considerable interest as prebiotic substances and can be prepared by transgalactosylation reactions from lactose using β-galactosidase. We applied various combinations of the commercial β-galactosidases, such as Nola Fit 5500, Saphera 2600 L, Maxilact LGI 5000 and Maxilact A4 MG to achieve the highest yield of GOS and reduced lactose content. The combination of the Maxilact LGI 5000 and Nola Fit 5500 resulted in amount of GOS 105 g L-1 with lactose content lower than 5 g L-1, whilst the combination of the Maxilact A4 MG and Maxilact LGI 5000 enzymes led to an increase in GOS to 141,1 g L-1 and decrease of the lactose content to 46,9 g L-1. The combination of enzymes produced a higher yield of GOS, reduced the concentration of lactose, eventually, increases the efficiency of galactooligosaccharides purification that could be potentially used in the further investigations.
BACKGROUND: Human milk oligosaccharides (HMOs) are one of the major glycan source of the infant gut microbiota. The two species that predominate the infant bifidobacteria community, Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, possess an arsenal of enzymes including α-fucosidases, sialidases, and β-galactosidases to metabolise HMOs. Recently bifidobacteria were obtained from the stool of six month old Kenyan infants including species such as Bifidobacterium kashiwanohense, and Bifidobacterium pseudolongum that are not frequently isolated from infant stool. The aim of this study was to characterize HMOs utilization by these isolates. Strains were grown in presence of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3'-sialyl-lactose (3'-SL), 6'-sialyl-lactose (6'-SL), and Lacto-N-neotetraose (LNnT). We further investigated metabolites formed during L-fucose and fucosyllactose utilization, and aimed to identify genes and pathways involved through genome comparison. RESULTS: Bifidobacterium longum subsp. infantis isolates, Bifidobacterium longum subsp. suis BSM11-5 and B. kashiwanohense strains grew in the presence of 2'-FL and 3'- FL. All B. longum isolates utilized the L-fucose moiety, while B. kashiwanohense accumulated L-fucose in the supernatant. 1,2-propanediol (1,2-PD) was the major metabolite from L-fucose fermentation, and was formed in equimolar amounts by B. longum isolates. Alpha-fucosidases were detected in all strains that degraded fucosyllactose. B. longum subsp. infantis TPY11-2 harboured four α-fucosidases with 95-99 % similarity to the type strain. B. kashiwanohense DSM 21854 and PV20-2 possessed three and one α-fucosidase, respectively. The two α-fucosidases of B. longum subsp. suis were 78-80 % similar to B. longum subsp. infantis and were highly similar to B. kashiwanohense α-fucosidases (95-99 %). The genomes of B. longum strains that were capable of utilizing L-fucose harboured two gene regions that encoded enzymes predicted to metabolize L-fucose to L-lactaldehyde, the precursor of 1,2-PD, via non-phosphorylated intermediates. CONCLUSION: Here we observed that the ability to utilize fucosyllactose is a trait of various bifidobacteria species. For the first time, strains of B. longum subsp. infantis and an isolate of B. longum subsp. suis were shown to use L-fucose to form 1,2-PD. As 1,2-PD is a precursor for intestinal propionate formation, bifidobacterial L-fucose utilization may impact intestinal short chain fatty acid balance. A L-fucose utilization pathway for bifidobacteria is suggested.
- MeSH
- alfa-L-fukosidasa klasifikace genetika metabolismus MeSH
- beta-galaktosidasa metabolismus MeSH
- Bifidobacterium longum enzymologie genetika metabolismus MeSH
- Bifidobacterium enzymologie genetika metabolismus MeSH
- DNA bakterií genetika MeSH
- feces mikrobiologie MeSH
- fukosa metabolismus MeSH
- genom bakteriální MeSH
- kojenec MeSH
- kyseliny mastné těkavé metabolismus MeSH
- kyseliny sialové metabolismus MeSH
- laktosa analogy a deriváty metabolismus MeSH
- lidé MeSH
- mateřské mléko metabolismus MeSH
- metabolické sítě a dráhy MeSH
- oligosacharidy metabolismus MeSH
- propylenglykol metabolismus MeSH
- RNA ribozomální 16S genetika MeSH
- sekvence nukleotidů MeSH
- střeva mikrobiologie MeSH
- trisacharidy metabolismus MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- Publikační typ
- časopisecké články 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
OBJECTIVE: Senescence is an important biological phenomenon involved in both physiologic and pathologic processes. We propose that chorioamniotic membrane senescence is a mechanism associated with human parturition. The present study was conducted to explore the association between senescence and normal term parturition by examining the morphologic and biochemical evidences in chorioamniotic membranes. STUDY DESIGN: Chorioamniotic membranes were collected from normal term deliveries; group 1: term labor and group 2: term, not in labor. Senescence-related morphologic changes were determined by transmission electron microscopy and biochemical changes were studied by senescence-associated (SA) β-galactosidase staining. Amniotic fluid samples collected from both term labor and term not in labor were analyzed for 14 SA secretory phenotype (SASP) markers. RESULTS: Morphologic evidence of cellular senescence (enlarged cells and organelles) and a higher number of SA β-galactosidase-stained amnion and chorion cells were observed in chorioamniotic membranes obtained from women in labor at term, when compared to term not in labor. The concentration of proinflammatory SASP markers (granulocyte macrophage colony-stimulating factor, interleukin-6 and -8) was significantly higher in the amniotic fluid of women in labor at term than women not in labor. In contrast, SASP factors that protect against cell death (eotaxin-1, soluble Fas ligand, osteoprotegerin, and intercellular adhesion molecule-1) were significantly lower in the amniotic fluid samples from term labor. CONCLUSION: Morphologic and biochemical features of senescence were more frequent in chorioamniotic membranes from women who experienced term labor. Senescence of chorioamniotic membranes were also associated with amniotic fluid SASP markers.
- MeSH
- amnion cytologie metabolismus ultrastruktura MeSH
- beta-galaktosidasa metabolismus MeSH
- chemokin CCL11 metabolismus MeSH
- chorion cytologie metabolismus ultrastruktura MeSH
- dospělí MeSH
- faktor stimulující granulocyto-makrofágové kolonie metabolismus MeSH
- interleukin-6 metabolismus MeSH
- interleukin-8 metabolismus MeSH
- lidé MeSH
- ligand Fas metabolismus MeSH
- mezibuněčná adhezivní molekula-1 metabolismus MeSH
- mitochondrie ultrastruktura MeSH
- mladý dospělý MeSH
- osteoprotegerin metabolismus MeSH
- plodová voda cytologie metabolismus MeSH
- porod v termínu metabolismus MeSH
- porodní děj metabolismus MeSH
- průřezové studie MeSH
- stárnutí buněk * MeSH
- studie případů a kontrol MeSH
- těhotenství MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A new enzymatic method for the synthesis of β-galactosides of nucleosides and acyclic nucleoside analogues has been developed, using β-galactosidase from Escherichia coli as a catalyst and lactose as a sugar donor. The method is very rapid, feasible and last but not least inexpensive. Its applicability has been proven for a broad variety of possible substrates with respect to its scaling up for preparative use. Five new compounds from a series of nucleoside and acyclic nucleoside analogues have been prepared on a scale of several hundred milligrams, in all cases revealing very good results of the method concerning the reproducibility of the reaction yields and simplicity of the purification process.
