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pUC18 and pUC19 are well-known high copy-number plasmid vectors routinely used for DNA cloning purposes. We show here that, in Escherichia coli transformed by native pUC18, the α-complementation of β-galactosidase (i.e., mediated by the peptide LacZα18) is intrinsically weak and slow, but is greatly stimulated by the DnaK/DnaJ/GrpE chaperone system. In contrast, the α-complementation mediated by the peptide LacZα19 (in E. coli transformed by the native pUC19) is much more efficient and therefore does not require the assistance of the DnaK chaperone machinery. The marked difference between these two LacZα peptides is reproduced in a cell-free protein expression system coupled with α-complementation. We conclude that: (i) α-complementation of β-galactosidase is DnaK-mediated depending upon the LacZα peptide donor; (ii) DnaK, sensu stricto, is not necessary for α-complementation, but can enhance it to a great extent; (iii) this observation could be used to establish an easy and inexpensive method for screening small molecules libraries in search of DnaK inhibitors and also for deciphering the DnaK-mediated protein quality control mechanism.

The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β‐galactosidase, which we are using as a model for cold‐active enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady‐state kinetic analysis to have altered temperature‐dependent catalytic activity profiles via effects on K (m) and/or k (cat) compared to the wild‐type enzyme. In this follow‐up study, double‐mutated enzymes were constructed and tested for temperature effects, including two new tandem residue pairs (N180T/A181T and T383A/S384A), and pairwise combination of the single residue mutations (N251D, F387L, I476V, and V482L). All double‐mutated enzymes were found to be more catalytically active at moderate and/or less active at colder temperatures than wild‐type, with both K (m) and k (cat) effects observed for the two tandem mutations. For pairwise combinations, a K (m) effect was seen when the surface exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface exposed mutation I476V located in a coiled region of domain B 25 Å from the active site was paired with N251D or V482L, a k (cat) effect was observed. These results indicate that temperature‐dependent kinetic effects may be complex and subtle and are mediated by a combination of a small number of residues distant from the active site via changes to the hydration shell and/or perturbation of internal packing.

Enzyme immobilization on adequate carriers is a challenging strategy. Understanding the enzyme-carrier interactions and their effects on enzyme conformation and bioactivity is critical. In this study, a meso-macropores silica (MMS) was used to immobilize β-galactosidase from the yeast Kluyveromyces lactis (β-gal-KL) by physical adsorption. The bioactivity of the immobilized β-gal-KL was altered, evidenced by the increased K

BACKGROUND In this study, we aimed to evaluate the salivary β-galactosidase and Halimeter values (HMV), organoleptic scores (OLS) and Winkel tongue coating index (WTCI) in periodontal health and periodontitis (P), and also their changes after phase I periodontal therapy and tongue cleaning. METHODS The participants were separated as follows: 25 P with halitosis (Group 1), 25 P without halitosis (Group 2) and 25 healthy controls (Group 3). Periodontal recordings, HMV, OLS and WTCI scores were recorded, and whole saliva β-galactosidase levels were measured colorimetrically in the samples at baseline and 1 month after the therapy. RESULTS The baseline values of HMV, OLS, WTCI and salivary β-galactosidase levels were significantly higher in Group 1 than in Group 2 (P

Enzyme inhibitors play a crucial role in diagnosis of a wide spectrum of diseases related to bacterial infections. We report here the effect of a water-soluble self-assembled PdII8 molecular cage towards β-galactosidase enzyme activity. The molecular cage is composed of a tetrapyridyl donor (L) and cis-[(en)Pd(NO3 )2 ] (en=ethane-1,2-diamine) acceptor and it has a hydrophobic internal cavity. We have observed that the acceptor moiety mainly possesses the ability to inactivate the β-galactosidase enzyme activity. Kinetic investigation revealed the mixed mode of inhibition. This inhibition strategy was extended to control the growth of methicillin-resistant Staphylococcus aureus. The internalization of the Pd(II) cage inside the bacteria was confirmed when bacterial solutions were incubated with curcumin loaded cage. The intrinsic green fluorescence of curcumin made the bacteria glow when put under an optical microscope. Furthermore, this curcumin loaded molecular cage shows an enhanced antibacterial activity. Thus, PdII8 molecular cage is quite attractive due to its dual role as enzyme inhibitor and drug carrier.

Carbohydrate-active enzymes are a group of important enzymes playing a critical role in the degradation and synthesis of carbohydrates. Glycosidases can hydrolyze glycosides into oligosaccharides, polysaccharides, and glycoconjugates via a cost-effective approach. Lactase is an important member of β-glycosidases found in higher plants, animals, and microorganisms. β-Galactosidases can be used to degrade the milk lactose for making lactose-free milk, which is sweeter than regular milk and is suitable for lactose-intolerant people. β-Galactosidase is employed by many food industries to degrade lactose and improve the digestibility, sweetness, solubility, and flavor of dairy products. β-Galactosidase enzymes have various families and are applied in the food-processing industries such as hydrolyzed-milk products, whey, and galactooligosaccharides. Thus, this enzyme is a valuable protein which is now produced by recombinant technology. In this review, origins, structure, recombinant production, and critical modifications of β-galactosidase for improving the production process are discussed. Since β-galactosidase is a valuable enzyme in industry and health care, a study of its various aspects is important in industrial biotechnology and applied biochemistry.

Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, β-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues. Ministry of Education (MOE) Nanyang Technological University This work was supported by Nanyang Technological University (Start-up Grant: M4081627) and Singapore Ministry of Education Academic Research Fund Tier 1 (2019-T1-002-045,RG125/19) and Tier 2 (MOE2018-T2-2-042)

Carbofuran is a carbamate pesticide, widely used in agricultural practices to increase crop productivity. In mammals, carbofuran is known to cause several untoward effects, such as apoptosis in the hippocampal neuron, oxidative stress, loss of memory and chromosomal anomalies. Most of these effects are implicated with cellular senescence. Therefore, the present study aimed to determine the effect of carbofuran on cellular senescence and biological ageing. Spinster homolog 1 (Spns1) is a transmembrane transporter, regulates autolysosomal biogenesis and plays a role in cellular senescence and survival. Using senescence‐associated β‐galactosidase staining, we found that carbofuran accelerates the cellular senescence in spns1 mutant zebrafish. The yolk opaqueness, a premature ageing phenotype in zebrafish embryos, was accelerated by carbofuran treatment. In the survival study, carbofuran shortened the life span of spns1 mutant zebrafish. Autophagy is the cellular lysosomal degradation, usually up‐regulated in the senescent cells. To know the impact of carbofuran exposure on autophagy progress, we established a double‐transgenic zebrafish line, harbouring EGFP‐tagged LC3‐II and mCherry‐tagged Lamp1 on spns1 mutant background, whereas we found, carbofuran exposure synergistically accelerates autolysosome formation with insufficient lysosome‐mediated degradation. Our data collectively suggest that carbofuran exposure synergistically accelerates the cellular senescence and affects biological ageing in spns1 defective animals.

Utility of antibody as a therapeutic is by far limited to extracellular or membrane bound proteins, even though these molecules can be engineered to recognize many “undruggable” therapeutic targets. The primary impediments in developing large antibodies as drugs against intracellular targets involve their low transfection efficiency and suitable reversible encapsulation strategies for intracellular delivery with retention of biological activity. To address this, we outline an electrostatics-enhanced covalent self-assembly strategy to generate polymer-protein/antibody nanoassemblies. Through structure-activity studies, we down-select the best performing self-immolative pentafluorophenyl containing activated carbonate polymer for bioconjugation. With the help of an electrostatics-aided covalent self-assembly approach, we demonstrate efficient encapsulation of medium to large proteins (HRP, 44 kDa and β-gal, 465 kDa) and antibodies (~150 kDa). The designed polymeric nanoassemblies are shown to successfully traffic functional antibodies (anti-NPC and anti-pAkt) to cytosol to elicit their bioactivity towards binding intracellular protein epitopes and inducing apoptosis.

Cranberry pomace obtained from industrial juice production was characterized by proximate composition analysis and monosaccharide profile of the dietary fiber. Extraction of carbohydrates from pomace was investigated using microwave-assisted alkaline method and five commercial biocatalysts (pure endo-galactanase and four multienzyme biocatalysts). The extracts obtained from microwave-assisted approach had average total sugars yield of 21.3% and contained mostly oligosaccharides in the degree of polymerization range of 7 to 10. All multienzyme biocatalysts led to yields similar or higher than microwave-assisted approach (23.4% to 42.0%), but mainly generated shorter oligosaccharides with a degree of polymerization of 2 to 5. Compared to cranberry pomace dietary fiber, microwave-assisted extracts were enriched in pectic oligosaccharides, whereas the enzymatic extracts were enriched in glucans and had less rhamnose and galactose. Pomace ground for 5 min or more by ball mill assumed a powdery consistence. Longer milling did not affect particle size but increased their roughness. Such physical changes had no effect on the efficiency of multienzymatic treatment. PRACTICAL APPLICATION: The increased production of cranberries and cranberry products will continuously generate more pomace, a potentially valuable material for the generation of added-value products. Up to 60% to 70% of cranberry pomace is composed of plant cell wall material. The properties of naturally occurring plant cell wall polysaccharides and their corresponding oligosaccharides have been of a great interest, and many of them find application as functional food ingredients. Despite the fact that the cranberry pomace is rich in plant cell polysaccharides, it has been mainly explored as a source of phenolic antioxidants. This study reveals the efficiency of cranberry pomace as a source of nondigestible oligosaccharides. The use of microwave-assisted extraction and different biocatalysts for the enzymatic extraction led to oligosaccharides with well-defined monosaccharide composition and molecular weight distribution. The study of the effects of these extraction techniques on the yield and the characteristics of generated oligosaccharides would allow the modulation of their properties. As an overall, the findings of this study would contribute to lay the scientific ground for the development of innovative process for the isolation of nondigestible oligosaccharides as functional ingredients from cranberry pomace by products.

Cellular senescence, a state of growth arrest, is involved in various age-related diseases. We previously found that carnitine palmitoyltransferase 1C (CPT1C) is a key regulator of cancer cell proliferation and senescence, but it is unclear whether CPT1C plays a similar role in normal cells. Therefore, this study aimed to investigate the role of CPT1C in cellular proliferation and senescence of human embryonic lung MRC-5 fibroblasts and the involved mechanisms. The results showed that CPT1C could reverse the cellular senescence of MRC-5 fibroblasts, as evidenced by reduced senescence-associated β-galactosidase activity, downregulated messenger RNA (mRNA) expression of senescence-associated secretory phenotype factors, and enhanced bromodeoxyuridine incorporation. Lipidomics analysis further revealed that CPT1C gain-of-function reduced lipid accumulation and reversed abnormal metabolic reprogramming of lipids in late MRC-5 cells. Oil Red O staining and Nile red fluorescence also indicated significant reduction of lipid accumulation after CPT1C gain-of-function. Consequently, CPT1C gain-of-function significantly reversed mitochondrial dysfunction, as evaluated by increased adenosine triphosphate synthesis and mitochondrial transmembrane potential, decreased radical oxygen species, upregulated respiratory capacity and mRNA expression of genes related to mitochondrial function. In summary, CPT1C plays a vital role in MRC-5 cellular proliferation and can reverse MRC-5 cellular senescence through the regulation of lipid metabolism and mitochondrial function, which supports the role of CPT1C as a novel target for intervention into cellular proliferation and senescence and suggests CPT1C as a new strategy for antiaging.

Cell senescence is due to the permanent cell cycle arrest that occurs as a result of the inherent limited replicative capacity toward the Hayflick limit (replicative senescence), or in response to various stressors (stress-induced premature senescence, SIPS). With the acquisition of the senescence-associated secretory phenotype (SASP), cells release several molecules (cytokines, proteases, lipids), and express the senescence-associated beta-galactosidase (SA-β-Gal). Here we tested whether vitamin E affects SA-β-Gal in an in vitro model of cell ageing. Skin fibroblasts from human subjects of different age (1, 13, 29, 59, and 88 years old) were cultured until they reached replicative senescence. At different passages (Passages 2, 9, 13, and 16), these cells were treated with vitamin E for 24 hr. Vitamin E reduced SA-β-Gal in all cells at passage 16, but at earlier passage numbers it reduced SA-β-Gal only in cells isolated from the oldest subjects. Therefore, short time treatment with vitamin E decreases SA-β-Gal in cells both from young and old subjects when reaching replicative senescence; but in cells isolated from older subjects, a decrease in SA-β-Gal by vitamin E occurs also at earlier passage numbers. The possible role of downregulation of CD36 by vitamin E, a scavenger receptor essential for initiation of senescence and SASP, is discussed.

Long noncoding RNAs (lncRNAs) have been investigated as novel regulatory molecules involved in diverse biological processes. Our previous study demonstrated that lncRNA-ES3 is associated with the high glucose-induced calcification/senescence of human aortic vascular smooth muscle cells (HA-VSMCs). However, the mechanism of lncRNA-ES3 in vascular calcification/aging remained largely unknown. Here, we report that the expression of basic helix-loop-helix family member e40 (Bhlhe40) was decreased significantly in HA-VSMCs treated with high glucose, whereas the expression of basic leucine zipper transcription factor (BATF) was increased. Overexpression of Bhlhe40 and inhibition of BATF alleviated calcification/senescence of HA-VSMCs, as confirmed by Alizarin Red S staining and the presence of senescence-associated β-galactosidase-positive cells. Moreover, we identified that Bhlhe40 regulates lncRNA-ES3 in HA-VSMCs by binding to the promoter region of the lncRNA-ES3 gene (LINC00458). Upregulation or inhibition of lncRNA-ES3 expression significantly promoted or reduced calcification/senescence of HA-VSMCs, respectively. Additionally, we identified that lncRNA-ES3 functions in this process by suppressing the expression of miR-95-5p, miR-6776-5p, miR-3620-5p, and miR-4747-5p. The results demonstrate that lncRNA-ES3 triggers gene silencing of multiple miRNAs by binding to Bhlhe40, leading to calcification/senescence of VSMCs. Our findings suggest that pharmacological interventions targeting lncRNA-ES3 may be therapeutically beneficial in ameliorating vascular calcification/aging.

Background Noncanonical NF-κB signaling through activation of the transcription factor RelB acts as key regulator of cell lineage determination and differentiation in various tissues including the immune system. To elucidate temporospatial aspects of Relb expression, we generated a BAC transgenic knock-in mouse expressing the fluorescent protein Katushka and the enzyme Cre recombinase under control of the murine Relb promoter (RelbCre-Kat mice). Results Co-expression of Katushka and Relb in fibroblast cultures and tissues of transgenic mice revealed highly specific reporter functions of the transgene. Crossing RelbCre-Kat mice with ROSA26R reporter mice that allow for Cre-mediated consecutive β-galactosidase or YFP synthesis identified various Relb expression domains in perinatal and mature mice. Besides thymus and spleen, highly specific expression patterns were found in different neuronal domains, as well as in other nonimmune organs including skin, skeletal structures and kidney. De novo Relb expression in the mature brain was confirmed in conditional knockout mice with neuro-ectodermal Relb deletion. Conclusion Our results demonstrate the usability of RelbCre-Kat reporter mice for the detection of de novo and temporarily restricted Relb expression including cell and lineage tracing of Relb expressing cells. Relb expression during mouse embryogenesis and at adulthood suggests, beyond immunity, important functions of this transcription factor in neurodevelopment and CNS function.

Lgr5-LacZ mice harbor the Escherichia coli LacZ gene encoding β-galactosidase (β-gal) under the control of the Lgr5 promoter, a stem/progenitor cell marker. In injured livers of Lgr5-LacZ mice, cells expressing β-galactosidase (β-gal) are considered as potential bipotent liver progenitors; however, their origin and identity remain unknown. Unexpectedly, using lineage tracing, we demonstrate that the β-gal+ cells do not originate from liver parenchymal cells. Instead, β-gal+ cells, isolated from injured livers of both Lgr5-LacZ and wild-type mice, are positive for markers of Kupffer cells, liver-resident macrophages. The β-gal expression in these cells is a result of elevated expression of the endogenous beta-galactosidase Glb1. In injured livers of Lgr5-LacZ mice, bacterial β-gal expression is very low, suggesting transgene silencing. The gene expression profile of the β-gal+ Kupffer cells from injured livers suggests a role in liver regeneration.

Our lab has developed a new series of self-immolative MR agents for the rapid detection of enzyme activity in mouse models expressing β-galactosidase (β-gal). We investigated two molecular architectures to create agents that detect β-gal activity by modulating the coordination of water to Gd(III). The first is an intermolecular approach, wherein we designed several structural isomers to maximize coordination of endogenous carbonate ions. The second involves an intramolecular mechanism for q modulation. We incorporated a pendant coordinating carboxylate ligand with a 2, 4, 6, or 8 carbon linker to saturate ligand coordination to the Gd(III) ion. This renders the agent ineffective. We show that one agent in particular (6-C pendant carboxylate) is an extremely effective MR reporter for the detection of enzyme activity in a mouse model expressing β-gal.

3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2'-fucosyllactose (2'-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function. Whereas the biological production of 2'-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of α-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2'-FL, and lower digestibility of 3-FL than 2'-FL by bifidobacteria. In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in kcat and the decrease in Km . We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP- L-fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D-lactose. Production using the lacZΔm15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g·L-1 ·hr-1 and the specific 3-FL yield of 0.5 g/g dry cell weight.

Background Senescence-associated β-galactosidase (SA-β-GAL) histochemistry is the most commonly used biomarker of cellular senescence. These SA-β-GAL-positive cells are senescent embryonic cells that are usually removed by apoptosis from the embryo, followed by macrophage-mediated clearance. Results Some authors have proposed that SA-β-GAL activity in differentiated neurons from young and adult mammals cannot be uniquely attributed to cell senescence, whether in vivo or in vitro. Using the developing visual system of the chicken as a model, the present study found that SA-β-GAL detected in the developing retina corresponded to lysosomal β-galactosidase activity, and that SA-β-GAL activity did not correlate with the chronotopographical distribution of apoptotic cells. However, SA-β-GAL staining in the undifferentiated retina coincided with the appearance of early differentiating neurons. In the laminated retina, SA-β-GAL staining was concentrated in the ganglion, amacrine, and horizontal cell layers. The photoreceptors and pigment epithelial cells also exhibited SA-β-GAL activity throughout retinal development. We have also found that SA-β-GAL staining strongly correlated p21 immunoreactivity. Conclusion In conclusion, the results clearly show that SA-β-GAL activity cannot be regarded as a specific marker of senescence during retinal development, and that it is mainly expressed in subpopulations of postmitotic neurons, which are nonproliferative cells, even at early stages of cell differentiation.

Calmodulin-like skin protein (CLSP) is a secreted peptide that is produced by skin keratinocytes and some related epithelial cells. It has previously been shown that CLSP is recruited via the bloodstream into the central nervous system where it likely exerts a neuroprotective effect against toxicity related to Alzheimer's disease (AD) by binding to the heterotrimeric humanin receptor and activating intracellular survival signaling. However, it remains to be elucidated whether secreted CLSP shows a protective effect in the skin tissues. In the current study, using primary keratinocytes treated with hydrogen peroxide (H2 O2 ) or exposed to ultraviolet (UV) irradiation as senescence models of keratinocytes, we addressed whether CLSP affects senescence in skin keratinocytes. We found that CLSP expression was upregulated by H2 O2 or UV in keratinocytes. Furthermore, co-incubation with recombinant CLSP reduced the increase in senescence-associated β-galactosidase-positivity in keratinocytes that were induced by H2 O2 or UV. These results suggest that CLSP may function as a senescence-suppressing factor in keratinocytes.

The galactosylation of xyloglucan blocks many of the enzymatic processes targeting this oligosaccharide. We found that the expression of a gene encoding Aspergillus oryzae β-galactosidase (LacA) is induced in the presence of xyloglucan oligosaccharides. With detailed analyses of the substrate specificity of purified recombinant LacA, we show that LacA cleaves galactopyranosyl residues from xyloglucan oligosaccharides, but not from xyloglucan polysaccharide, and plays a vital role in xyloglucan degradation. LacA acts cooperatively with the isoprimeverose-producing oligoxyloglucan hydrolase IpeA to hydrolyze xyloglucan oligosaccharides. Galactosylation of the xylopyranosyl side chain at the nonreducing end of oligoxyloglucan saccharides completely abolishes IpeA activity while LacA efficiently removes the galactopyranosyl residue. Conversely, an isoprimeverose unit at the nonreducing end of the main chain of xyloglucan oligosaccharides blocks LacA activity, while IpeA can still remove the isoprimeverose moiety. This is the first study reporting the cooperative action of β-galactosidase and isoprimeverose-producing oligoxyloglucan hydrolase on xyloglucan oligosaccharide degradation. Our findings shed light on the true role of LacA and the enzymatic coordination between β-galactosidase and other hydrolases on xyloglucan degradation.

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age‐related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence‐associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal β‐galactosidase, and this has been exploited as a marker for senescence (senescence‐associated β‐galactosidase activity). Consequently, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose‐modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal β‐galactosidase (GLB1)‐dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole‐body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of β‐catenin‐positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose‐modified duocarmycin prodrugs to treat senescence‐related pathologies., Galactose‐modified duocarmycin prodrugs get preferentially processed in senescent cells and behave as senolytics.

Hydrolysis efficiency of β-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial β-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnβGal), Aspergillus oryzae (AoβGal), Bacillus circulans (BcβGal), Bifidobacterium bifidum (BbβGal), and Kluyveromyces lactis (KlβGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnβGal), Asn140Ala and Asn199Ala (AoβGal), Arg111Ala and Glu355Ala (BcβGal), Arg122Ala and Phe358Ala (BbβGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlβGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcβGal, BbβGal, and KlβGal. The double mutations in BcβGal and BbβGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlβGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of β-galactosidases and rendering them promising for industrial applications.

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H2O2‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16INK 4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H2O2‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H2O2‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H2O2 treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.

Lysosomal storage diseases (LSDs) are a group of genetic disorders, resulting from deficiencies of lysosomal enzyme. Genotype-phenotype correlation is essential for timely and proper treatment allocation. Recently, by integrating prediction outcomes of 7 bioinformatics tools, we developed a SAAMP algorithm to predict the impact of individual amino-acid substitution. To optimize this approach, we evaluated the performance of these bioinformatics tools in a broad array of genes. PolyPhen and PROVEAN had the best performances, while SNP&GOs, PANTHER and I-Mutant had the worst performances. Therefore, SAAMP 2.0 was developed by excluding 3 tools with worst performance, yielding a sensitivity of 94% and a specificity of 90%. To generalize the guideline to proteins without known structures, we built the three-dimensional model of iduronate-2-sulfatase by homology modeling. Further, we investigated the phenotype severity of known disease-causing mutations of the GLB1 gene, which lead to 2 LSDs (GM1 gangliosidosis and Morquio disease type B). Based on the previous literature and structural analysis, we associated these mutations with disease subtypes and proposed a theory to explain the complicated genotype-phenotype correlation. Collectively, an updated guideline for phenotype prediction with SAAMP 2.0 was proposed, which will provide essential information for early diagnosis and proper treatment allocation, and they may be generalized to many monogenic diseases.

In proteins, the indole side chain of tryptophan can interact with water molecules either in-plane, forming hydrogen bonds, or out-of-plane, with the water molecule contacting the aromatic π face. The latter interaction can be either of the lone pair⋅⋅⋅π (lp⋅⋅⋅π) type or corresponds to the O-H⋅⋅⋅π binding mode, an ambiguity that X-ray structures usually do not resolve. Here, we report molecular dynamics (MD) simulations of a solvated β-galactosidase monomer, which illustrate how a water molecule located at the π face of an indole side chain of tryptophan can adapt to the position of proximate residues and "select" its binding mode. In one such site, the water molecule is predicted to rapidly oscillate between the O-H⋅⋅⋅π and lp⋅⋅⋅π binding modes, thus gaining entropic advantage. Our MD simulations provide support for the role of lp⋅⋅⋅π interactions in the stabilization of protein structures.

Ageing is a strong independent risk factor for disability, morbidity and mortality. Post‐mitotic cells including those in the heart are a particular risk to age‐related deterioration. As the occurrence of heart disease is increasing rapidly with an ageing population, knowledge regarding the mechanisms of age‐related cardiac susceptibility and possible therapeutic interventions needs to be acquired to prevent advancing levels of heart disease. To understand more about the ageing heart, numerous aged animal models are being used to explore the underlying mechanisms. Due to time‐consuming for investigations involving naturally aged animals, mimetic ageing models are being utilized to assess the related effects of ageing on disease occurrence. d‐galactose is one of the substances used to instigate ageing in various models, and techniques involving this have been widely used since 1991. However, the mechanism through which d‐galactose induces ageing in the heart remains unclear. The aim of this review was to comprehensively summarize the current findings from in vitro and in vivo studies on the effects of d‐galactose‐induced ageing on the heart, and possible therapeutic interventions against ageing heart models. From this review, we hope to provide invaluable information for future studies and based on the findings from experiments involving animals, we can inform possible therapeutic strategies for the prevention of age‐related heart diseases in clinical settings.

The poor mechanical stability of chitosan has long impeded its industrial utilization as an immobilization carrier. In this study, the mechanical properties of chitosan beads were greatly improved through utilizing the slow rate of the sodium bicarbonate-induced chitosan gelation and combining it with the chemical cross-linking action of glutaraldehyde (GA). The GA-treated sodium bicarbonate-gelled chitosan beads exhibited much better mechanical properties and up to 2.45-fold higher observed activity of the immobilized enzyme (β-D-galactosidase (β-gal)) when compared to the GA-treated sodium tripolyphosphate (TPP)-gelled chitosan beads. The differences between the sodium bicarbonate-gelled and the TPP-gelled chitosan beads were proven visually and also via scanning electron microscopy, elemental analysis, and differential scanning calorimetry. Moreover, the optimum pH, the optimum temperature, the apparent Km , and the apparent Vmax of the β-gals immobilized onto the two aforementioned types of chitosan beads were determined and compared. A reusability study was also performed. This study proved the superiority of the sodium bicarbonate-gelled chitosan beads as they retained 72.22 ± 4.57% of their initial observed activity during the 13th reusability cycle whereas the TPP-gelled beads lost their activity during the first four reusability cycles, owing to their fragmentation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:347-361, 2018.

We report a spore-based biocatalysis platform capable of producing and self-assembling active multimeric enzymes on a spore surface with a high loading density. This was achieved by co-expressing both a spore surface-anchoring scaffoldin protein containing multiple cohesin domains and a dockerin-tagged enzyme of interest in the mother cell compartment during Bacillus subtilis sporulation. Using this method, tetrameric β-galactosidase was successfully displayed on the spore surface with a loading density of 1.4 × 104 active enzymes per spore particle. The resulting spore biocatalysts exhibited high conversion rates of transgalactosylation in water/organic emulsions. With easy manufacture, enhanced thermostability, excellent reusability, and long-term storage stability at ambient temperature, this approach holds a great potential in a wide range of biocatalysis applications especially involving organic phases.

Inherited metabolic disorders are traditionally diagnosed using broad and expensive panels of screening tests, often including invasive skin and muscle biopsy. Proponents of next-generation genetic sequencing have argued that replacing these screening panels with whole exome sequencing (WES) would save money. Here, we present a complex patient in whom WES allowed diagnosis of GM1 gangliosidosis, caused by homozygous GLB1 mutations, resulting in β-galactosidase deficiency. A 10-year-old girl had progressive neurologic deterioration, macular cherry-red spot, and cornea verticillata. She had marked clinical improvement with initiation of the ketogenic diet. Comparative genomic hybridization microarray showed mosaic chromosome 3 paternal uniparental disomy (UPD). GM1 gangliosidosis was suspected, however β-galactosidase assay was normal. Trio WES identified a paternally-inherited pathogenic splice-site GLB1 mutation (c.75+2dupT). The girl had GM1 gangliosidosis; however, enzymatic testing in blood was normal, presumably compensated for by non-UPD cells. Severe neurologic dysfunction occurred due to disruptive effects of UPD brain cells.

Rhodospirillum rubrum L‐asparaginase mutant E149R, V150P, F151T (RrA) down‐regulates telomerase activity due to its ability to inhibit the expression of telomerase catalytic subunit hTERT. The aim of this study was to define the effect of short‐term and long‐term RrA exposure on proliferation of cancer Jurkat cell line and normal human CD4+ T lymphocytes. RrA could inhibit telomerase activity in dose‐ and time‐dependent manner in both Jurkat and normal CD4+ T cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell cycle inhibition, replicative senescence, and development of apoptosis. Complete death of Jurkat cells was observed at the day 25 of RrA exposure while normal CD4+ T cells died at the day 50 due to the initial longer length of telomeres. Removal of RrA from senescent cells led to a reactivation of hTERT expression, restoration telomerase activity, re‐elongation of telomeres after 48 h of cultivation, and survival of cells. These findings demonstrate that proliferation of cancer and normal telomerase‐positive cells can be limited by continuous telomerase inhibition with RrA. Longer telomeres of normal CD4+ T lymphocytes make such cells more sustainable to RrA exposure that could give them an advantage during anti‐telomerase therapy. These results should facilitate further investigations of RrA as a potent anti‐telomerase therapeutic protein.

Myelination is a late developmental process regulated by a set of inhibitory and stimulatory factors, including extracellular matrix components. Accordingly, chondroitin sulfate proteoglycans (CSPGs) act as negative regulators of myelination processes. A disintegrin and metalloproteinase with thrombospondin motifs type 4 (ADAMTS-4) is an extracellular protease capable of degrading CSPGs. Although exogenous ADAMTS-4 has been proven to be beneficial in several models of central nervous system (CNS) injuries, the physiological functions of endogenous ADAMTS-4 remain poorly understood. We first used Adamts4/LacZ reporter mice to reveal that ADAMTS-4 is strongly expressed in the CNS, especially in the white matter, with a cellular profile restricted to mature oligodendrocytes. Interestingly, we evidenced an abnormal myelination in Adamts4-/- mice, characterized by a higher diameter of myelinated axons with a shifting g-ratio. Accordingly, lack of ADAMTS-4 is accompanied by motor deficits and disturbed nervous electrical activity. In conclusion, we demonstrate that ADAMTS-4 is a new marker of mature oligodendrocytes contributing to the myelination processes and thus to the control of motor capacities.

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.

Summary Transcriptional coactivator with PDZ‐binding motif (TAZ) directly interacts with transcription factors and regulates their transcriptional activity. Extensive functional studies have shown that TAZ plays critical regulatory roles in stem cell proliferation, differentiation, and survival and also modulates the development of organs such as the lung, kidney, heart, and bone. Despite the importance of TAZ in stem cell maintenance, TAZ function has not yet been evaluated in spermatogenic stem cells of the male reproductive system. Here, we investigated the expression and functions of TAZ in mouse testis. TAZ was expressed in spermatogenic stem cells; however, its deficiency caused significant structural abnormalities, including atrophied tubules, widened interstitial space, and abnormal Leydig cell expansion, thereby resulting in lowered sperm counts and impaired fertility. Furthermore, TAZ deficiency increased the level of apoptosis and senescence in spermatogenic cells and Leydig cells upon aging. The expression of senescence‐associated β‐galactosidase (SA‐βgal), secretory phenotypes, and cyclin‐dependent kinase inhibitors (p16, p19, and p21) significantly increased in the absence of TAZ. TAZ downregulation in testicular cells further increased SA‐βgal and p21 expression induced by oxidative stress, whereas TAZ overexpression decreased p21 induction and prevented senescence. Mechanistic studies showed that TAZ suppressed DNA‐binding activity of p53 through a direct interaction and thus attenuated p53‐induced p21 gene transcription. Our results suggested that TAZ may suppress apoptosis and premature senescence in spermatogenic cells by inhibiting the p53‐p21 signaling pathway, thus playing important roles in the maintenance and control of reproductive function.

A bioactive synthetic porous shell was engineered to enable cells to survive in an oligotrophic environment. Eukaryotic cells (yeast) were firstly coated with a β-galactosidase (β-gal), before crystallization of a metal-organic framework (MOF) film on the enzyme coating; thereby producing a bioactive porous synthetic shell. The β-gal was an essential component of the bioactive shell as it generated nutrients (that is, glucose and galactose) required for cell viability in nutrient-deficient media (lactose-based). Additionally, the porous MOF coating carried out other vital functions, such as 1) shielding the cells from cytotoxic compounds and radiation, 2) protecting the non-native enzymes (β-gal in this instance) from degradation and internalization, and 3) allowing for the diffusion of molecules essential for the survival of the cells. Indeed, this bioactive porous shell enabled the survival of cells in simulated extreme oligotrophic environments for more than 7 days, leading to a decrease in cell viability less than 30 %, versus a 99 % decrease for naked yeast. When returned to optimal growth conditions the bioactive porous exoskeleton could be removed and the cells regained full growth immediately. The construction of bioactive coatings represents a conceptually new and promising approach for the next-generation of cell-based research and application, and is an alternative to synthetic biology or genetic modification.

Summary Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)‐induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF‐α‐induced cellular senescence in EPCs, as indicated by reduced senescence‐associated β‐galactosidase activity and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked TNF‐α‐induced superoxide levels, NADPH oxidase activity, and microRNA‐21 (miR‐21) and p16INK 4a synthesis. Kallistatin prevented TNF‐α‐mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR‐34a synthesis, whereas miR‐34a overexpression abolished kallistatin‐induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR‐34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ‐induced aortic senescence, oxidative stress, and miR‐34a and miR‐21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild‐type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR‐34 or sir‐2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR‐34, but stimulated sir‐2.1 and sod‐3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR‐34a‐SIRT1 pathway.

Background Pollution, especially cigarette smoke, is a major cause of skin damage. Objectives To assess the effects of the small molecule polyphenol, honokiol, on reversing cigarette smoke-induced damage in vitro to relevant skin cells. Methods Keratinocytes (HaCat) cultures were exposed to cigarette smoke and, after 48 hours, IL-1α and IL-8 were measured in cell supernatants. Moreover, TIMP-2 production, apoptosis rate, and senescence β-galactosidase expression were evaluated in primary human foreskin fibroblasts (HFF-1) cultures. Results Honokiol at 10 μm reduced IL-1α production by 3.4 folds (P < 0.05) and at 10 and 20 μm reduced IL-8 by 23.9% and 53.1% (P < 0.001), respectively, in HaCat keratinocytes. In HFF-1, honokiol restored TIMP-2 production by 96.9% and 91.9% (P < 0.001), respectively, at 10 and 20 μm, as well as reduced apoptosis by 47.1% (P < 0.001) and 41.3% (P < 0.01), respectively. Finally, honokiol reduced senescence-associated β-galactosidase expression in HFF-1. Conclusion Honokiol protects both HFF-1 and HaCat against cigarette smoke-induced inflammation, collagenolysis, apoptosis, and senescence.

Intracellular delivery of proteins is a promising strategy of intervention in disease, which relies heavily on the development of efficient delivery platforms due to the cell membrane impermeability of native proteins, particularly for negatively charged large proteins. This work reports a vesicle supra-assembly approach to synthesize novel amine-functionalized hollow dendritic mesoporous silica nanospheres (A-HDMSN). An amine silica source is introduced into a water-oil reaction solution prior to the addition of conventional silica source tetraethylorthosilicate. This strategy favors the formation of composite vesicles as the building blocks which further assemble into the final product. The obtained A-HDMSN have a cavity core of ≈170 nm, large dendritic mesopores of 20.7 nm in the shell and high pore volume of 2.67 cm3 g-1 . Compared to the calcined counterpart without amine groups (C-HDMSN), A-HDMSN possess enhanced loading capacity to large negative proteins (IgG and β-galactosidase) and improved cellular uptake performance, contributed by the cationic groups. A-HDMSN enhance the intracellular uptake of β-galactosidase by up to 5-fold and 40-fold compared to C-HDMSN and free β-galactosidase, respectively. The active form of β-galactosidase delivered by A-HDMSN retains its intracellular catalytic functions.

Fil: Venica, Claudia Ines. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Lactologia Industrial. Universidad Nacional del Litoral. Facultad de Ingenieria Quimica. Instituto de Lactologia Industrial; Argentina

A strategy for labeling native enzymes in a manner that preserves their activity is reported: capture-tag-release (CTR). Key to this approach is the small molecule CTR probe that contains an enzyme inhibitor, benzophenone crosslinker, and aryl phosphine ester. After UV-derived capture of the enzyme, addition of an azide-containing tag triggers a Staudinger ligation that labels the enzyme. A further consequence of the Staudinger ligation is fragmentation of the CTR probe, thus releasing the inhibitor and restoring enzymatic activity. As a proof-of-principle, the CTR strategy was applied to the hydrolase β-galactosidase. The enzyme was efficiently labeled with biotin, and the kinetic data for the biotinylated enzyme were comparable to those for unlabeled β-galactosidase. The CTR probe exhibits excellent targeting specificity, as it selectively labeled β-galactosidase in a complex protein mixture.

This article describes recent developments in C3 -symmetric tris-urea low-molecular-weight gelators and their applications. The C3 -symmetric tris-ureas are excellent frameworks to form supramolecular polymers through noncovalent interactions. In organic solvents, hydrophobic tris-ureas form supramolecular gels. Amphiphilic tris-ureas form supramolecular gels in aqueous media. Functional supramolecular gels were prepared by introducing appropriate functional groups into the outer sphere of tris-ureas. Supramolecular hydrogels obtained from amphiphilic tris-ureas were used in the electrophoresis of proteins. These electrophoreses results showed several unique characteristics compared to typical electrophoreses results obtained using polyacrylamide matrices.

The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water-based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well-defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass-transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme-laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.

In the n-alkane-assimilating yeast Yarrowia lipolytica, the transcription of ALK1, encoding cytochrome P450, that catalyses n-alkane hydroxylation is activated by a complex composed of Yas1p and Yas2p via a promoter element, ARE1, in response to n-alkanes. An Opi1-family transcription factor, Yas3p, represses the transcription by binding to Yas2p in the nucleus when cultured in glucose-containing medium, but it is localized to the ER, presumably through interaction with acidic phospholipids, phosphatidic acid and/or phospho inositides, when cultured in n-alkane-containing medium. Here, to elucidate the mechanisms regulating the localization of Yas3p, point and deletion mutants of Yas3p were constructed and analysed. The substitution of Trp(360) and Cys(361) by Arg abrogated the localization of Yas3p to the ER and decreased ARE1-mediated transcriptional activation by n-alkane. A Yas3p truncation mutant consisting of residues 259-422 did not bind to acidic phospholipids, but it was localized to the ER in the presence of n-alkane, implying the acidic-phospholipid-independent recruitment of this mutant to the ER in response to n-alkane. The W360R and C361R substitutions in this truncation mutant abolished its localization to the ER. The results suggest that these residues are implicated in the acidic phospholipid-independent interaction of Yas3p to the ER.

Various methods have been developed for gene disruption in bacteria; however, extra in vitro manipulation steps or the residual presence of a scar in the host chromosome limits the use of such methods. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The λ Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. We demonstrated lacZ gene point mutation repair, two precise disruptions of the lacZ gene and constructed a library of lacZ variants having variable β-galactosidase activity by changing its ribosome binding site sequences using the ISHp608 system. This technique can be used in E. coli genome modification and could be extended for use in other bacteria.

A series of eight stereoisomeric N-(tetrahydroxy bicyclo-[5.1.0]oct-2S*-yl)phthalimides were prepared in one to four steps from N-(bicyclo[5.1.0]octa-3,5-dien-2-yl)phthalimide (±)-7, which is readily available from cyclooctatetraene (62 % yield). The structural assignments of the stereoisomers were established by (1) H NMR spectral data as well as X-ray crystal structures for certain members. The outcomes of several epoxydiol hydrolyses, particularly ring contraction and enlargement, are of note. The isomeric phthalimides as well as the free amines did not exhibit β-glucosidase inhibitory activity at a concentration of less than 100 μM.

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of a-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum a-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins.