Polyaniline-assisted silver nanoparticles: a novel support for the immobilization of α-amylase

The size distribution of nanomaterials and their similarity in size with enzyme molecules together with other advantageous properties such as thermal stability, high surface-to-volume ratio, and irradiation resistance have revolutionized nanobiocatalytic approaches in various areas of enzyme technology. In the present study, polyaniline-assisted Ag nanocomposites were synthesized using ammonium peroxydisulfate as oxidant. These nanocomposites were used as a support for the covalent conjugation of α-amylase, one of the important industrial enzymes. X-ray diffraction study showed that the crystalline nature of nanocomposites was increased in the presence of Ag nanoparticles. Thermogravimetric and differential thermal analysis revealed that the synthesized nanocomposites retained significantly very high thermal stability. Scanning electron micrograph showed that Ag nanoparticles were homogeneously dispersed in polyaniline film providing large surface area and microenvironment for enzyme loading. Fourier transform infrared spectroscopy confirmed the conjugation of α-amylase to the functionalized nanocomposites. The conjugated α-amylase exhibited better tolerance to variations in the medium pH and temperature compared with the native enzyme. Immobilized α-amylase hydrolyzes starch more efficiently as compared to the free enzyme in batch process.     

Use of β-galactosidase (lacZ) gene α-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis

The mutagenic potential of titanium dioxide nanoparticles (TiO(2)-NPs) of an average size 30.6nm was investigated using β-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-)Escherichia coli DH5α system. Plasmid pUC19 was treated with varying concentrations of TiO(2)-NPs and allowed to transfect the CaCl(2)-induced competent DH5α cells. The data revealed loss in transformation efficiency of TiO(2)-NPs treated plasmids as compared to untreated plasmid DNA in DH5α host cells. Induction of multiple mutations in α-fragment of lacZ gene caused synthesis of non-functional β-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO(2)-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO(2)-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO(2)-NPs induced mutations. This study suggested the pUC19/E. coli DH5αlacZ gene α-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials.     

Identification of a novel mutation of the gene for gap junction protein α3 (GJA3) in a Chinese family with congenital cataract

Cataract, defined as any opacity of the crystallin lens, can be divided into early onset (congenital or infantile) and age-related. It is the leading cause of visual disability in children, and mutations in many genes have currently been linked with this disorder. In the present study, we identified a genetic defect in a Chinese family with congenital cataract. Genomic DNA was extracted from the venous blood of the family and 100 normal controls. To screen for the disease-causing mutation, we sequenced eight candidate genes, and to predict the functional consequences of the mutation, a structural model of the protein was developed using the Protein Data Bank and PyMOL 1.1r1. We found a novel variant (c.163 A > G transition) in the gene for gap junction protein α3, or the connexin46 gene. This mutation resulted in the substitution of a highly conserved asparagine at codon 55 by aspartic acid (p.N55D). There were no nucleotide polymorphisms in the other candidate genes sequenced.     

Specific stimulation of α2-6 sialyltransferase activity by a novel cytosolic factor from rat colon

A factor present in the 100 000 g supernatant from the homogenate of rat colon stimulated the activity of purified GaIβ1-4GlcNAc α2,6 sialyltransferase [α2-6ST(N)] from rat liver and α2-6ST(N) from either liver microsomes or Golgi membrane. The stimulation of α2-6ST(N) activity by the colon factor using protein acceptors was about four-fold and highly reproducible when the reaction product of the α2-6ST(N) was assayed by either precipitation or affinity chromatography. In contrast, the colon factor did not stimulate the GaIβ1-4GlcNAc α2,3 sialyltransferase [α2-3ST (N)], from rat jejunum microsomes or purified Galβ1-3GalNAc α2,3 sialyltransferase [α2-3ST (O)] from porcine liver, or purified β1,4 galactosyltransferase (GT) from bovine milk. In addition to rat colon, the 100 000 g supernatant from the homogenates of rat brain and kidney also stimulated the α2-6ST(N) activity. The stimulation of α2-6ST(N) by the colon factor resulted in a decrease in the K_m (by about two-fold) and an increase in V_max (about 2- to 3-fold) for desialylated α₁ acid glycoprotein and CMP-[¹⁴C]N-acetylneuraminic acid. The stimulation of α2-6ST(N) activity by the colon factor was temperature dependent, protease sensitive and was inhibited by CTP, but did not need the presence of either metal ions or detergent. The cytosolic factor was partially purified by ion-exchange chromatography with the retention of the activator activity in the peaks containing low molecular weight proteins, but the activity was lost on attempts to further purification. A specific marked stimulation of the α2-6ST(N) activity by cytosolic factors in certain tissues might suggest a physiological role for these factors in the regulation of α2-6ST(N) activity.     

Pharmacophore-based discovery of a novel cytosolic phospholipase A(2)α inhibitor

The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)α (cPLA(2)α). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA(2)α inhibitor in cell-free and cell-based in vitro assays.     

Rotational diffusion of the α(2a) adrenergic receptor revealed by FlAsH labeling in living cells

The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α(2A) adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of ～50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557-573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113) and revealed a discrepancy of a factor between 10 and 100.     

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 2: α-Branched quaternary amines

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 μg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC. As such, pyrazinoyl quaternary amines represent a promising new class of ENaC blockers for the treatment of cystic fibrosis that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride.     

Optical resolution of 2-(3-indolyl)propionic acid with Mucor javanicus and α-chymotrypsin

Resolution of 2-(3-indolyl)propionic acid was achieved via biocatalytic hydrolysis of its chloroethyl ester. Of the enzymes tested, Mucor javanicus lipase (R selectivity) and -chymotrypsin (S selectivity) had high reactivity and enantioselectivity (E value > 50). Neither enzyme showed enantioselectivity (E value = 1) for 2-phenylpropionic acid.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Modulation of α(2C) adrenergic receptor temperature-sensitive trafficking by HSP90

Decreasing the temperature to 30°C is accompanied by significant enhancement of α(2C)-AR plasma membrane levels in several cell lines with fibroblast phenotype, as demonstrated by radioligand binding in intact cells. No changes were observed on the effects of low-temperature after blocking receptor internalization in α(2C)-AR transfected HEK293T cells. In contrast, two pharmacological chaperones, dimethyl sulfoxide and glycerol, increased the cell surface receptor levels at 37°C, but not at 30°C. Further, at 37°C α(2C)-AR is co-localized with endoplasmic reticulum markers, but not with the lysosomal markers. Treatment with three distinct HSP90 inhibitors, radicicol, macbecin and 17-DMAG significantly enhanced α(2C)-AR cell surface levels at 37°C, but these inhibitors had no effect at 30°C. Similar results were obtained after decreasing the HSP90 cellular levels using specific siRNA. Co-immunoprecipitation experiments demonstrated that α(2C)-AR interacts with HSP90 and this interaction is decreased at 30°C. The contractile response to endogenous α(2C)-AR stimulation in rat tail artery was also enhanced at reduced temperature. Similar to HEK293T cells, HSP90 inhibition increased the α(2C)-AR contractile effects only at 37°C. Moreover, exposure to low-temperature of vascular smooth muscle cells from rat tail artery decreased the cellular levels of HSP90, but did not change HSP70 levels. These data demonstrate that exposure to low-temperature augments the α(2C)-AR transport to the plasma membrane by releasing the inhibitory activity of HSP90 on the receptor traffic, findings which may have clinical relevance for the diagnostic and treatment of Raynaud Phenomenon.     

Discovery of 3,3-di(indolyl)indolin-2-one as a novel scaffold for α-glucosidase inhibitors: In silico studies and SAR predictions

3,3-Di(indolyl)indolin-2-ones 4a-4n were synthesized and evaluated for their in vitro α-glucosidase inhibitory activity. These newly synthesized compounds showed moderate to potent α-glucosidase inhibitory activity with IC₅₀ range from 5.98 ± 0.11 to 145.95 ± 0.46 μM, when compared to the standard drug acarbose. Among this series of 3,3-di(indolyl)indolin-2-ones, compound 4j (5.98 ± 0.11 μM) having a 2-fluorobenzyl group on the indole ring was found to be the most active compound. Molecular docking studies showed that compound 4j have high binding affinities with the active site of α-glucosidase enzyme through hydrogen bonds, arene-cation, π-π stacking and hydrophobic interactions. This study showed these 3,3-di(indolyl)indolin-2-ones as a new class of α-glucosidase inhibitors.     

Extensive variation at the α-glycerophosphate dehydrogenase locus in species of waterstriders (Gerridae: Hemiptera)

Variation at the -glycerophosphate dehydrogenase (-Gpdh; EC 1.1.1.8) locus was surveyed in 11 species of waterstriders (Gerridae: Hemiptera) and five other species of aquatic Hemiptera. Species of waterstriders exhibited considerable inter- and intraspecific variation in degree of winglessness. Average heterozygosity (0.401±0.090) and average number of observed electromorphs (5.36±0.96) for the 11 gerrid species were well above values reported for nearly all other insect species surveyed to date. Wing-monomorphic and wing-polymorphic species did not differ in average -Gpdh heterozygosity. Of the three wing-polymorphic species surveyed geographically, two species exhibited marked variation in wing-morph frequencies but homogeneous -Gpdh allele frequencies. The third species exhibited geographically homogeneous -Gpdh and wing-morph frequencies, but no significant association between -Gpdh phenotype and wing morph was observed in any surveyed population. These results are consistent with hypotheses evoking either relaxed purifying selection at the -Gpdh locus in species of Gerridae due to the apparent reduced importance of flight, or selective maintenance of common -Gpdh electromorphs.This work was supported by NSF Grant DEB 76-20967 to Alan H. Brush, funds from the Research Foundation of the University of Connecticut to Carl W. Schaefer, and USPHS Grant GM 21133 to Richard K. Koehn.     

3-(3-Phenoxybenzyl)amino-β-carboline: a novel antitumor drug targeting α-tubulin

3-(3-Phenoxybenzyl)amino-β-carboline 2h showed extremely-high activity; the IC₅₀ value was 0.074 μM. To verify 2h-induced cell death types, we observed the chromatin condensation, the DNA fragmentation and activated caspase-3 using Hoechst 33342, agarose electrophoresis and western blot, and suggesting 2h-induced cell death type was apoptosis. Flow cytometry showed that 2h-treated cell was induced SubG1 cell population after G2/M cell cycle arrest. In addition, using affinity chromatography and peptide mass fingerprinting, we found that interacting protein with this compound was α-tubulin protein.     

Genetic polymorphism of human plasma α1-B-glycoprotein: phenotyping by immunoblotting or by a simple method of 2-D electrophoresis

Summary Genetic polymorphism of human plasma (serum) ₁B-glycoprotein (₁B) was observed using one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 of plasma samples followed by Western blotting with specific antiserum to ₁B. A simple method of two-dimensional agarose gel electrophoresis (pH 5.4) — horizontal PAGE (pH 9.0) of plasma samples, followed by general protein staining, was reported as an alternative method for ₁B typing. The there different phenotypes of ₁B observed (designated 1-1, 1-2, and 2-2) were apparently identical to those reported by Altland et al. (1983), who used double one-dimensional electrophoresis. Family data supported the hypothesis that the three ₁B phenotypes are determined by two codominant alleles at an autosomal locus, designated A1B. Allele frequencies in a Swedish population were: A1B ^*1, 0.937; A1B ^*2, 0.063; PIC, 0.111. For clues on linkage relationships of human A/B, the previously known linkages of A1B in pigs and horses, including the one between A1B and the gene that determines susceptibility to malignant hyperthermia in pigs were discussed.     

Tetrastatin, the NC1 domain of the α4(IV) collagen chain: a novel potent anti-tumor matrikine

Background

NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far.

Methodology/Principal Findings

We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (K_D = 148±9.54 nM).

Conclusion/Significance

Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.     

Lanthionine synthetase C–like protein 2 (LanCL2) is a novel regulator of Akt

The serine/threonine protein kinase Akt controls a wide range of biochemical and cellular processes under the modulation of a variety of regulators. In this study, we identify the lanthionine synthetase C–like 2 (LanCL2) protein as a positive regulator of Akt activation in human liver cells. LanCL2 knockdown dampens serum- and insulin-stimulated Akt phosphorylation, whereas LanCL2 overexpression enhances these processes. Neither insulin receptor phosphorylation nor the interaction between insulin receptor substrate and phosphatidylinositide 3-kinase (PI3K) is affected by LanCL2 knockdown. LanCL2 also does not function through PP2A, a phosphatase of Akt. Instead, LanCL2 directly interacts with Akt, with a preference for inactive Akt. Moreover, we show that LanCL2 also binds to the Akt kinase mTORC2, but not phosphoinositide-dependent kinase 1. Whereas LanCL2 is not required for the Akt-mTORC2 interaction, recombinant LanCL2 enhances Akt phosphorylation by target of rapamycin complex 2 (mTORC2) in vitro. Finally, consistent with a function of Akt in regulating cell survival, LanCL2 knockdown increases the rate of apoptosis, which is reversed by the expression of a constitutively active Akt. Taken together, our findings reveal LanCL2 as a novel regulator of Akt and suggest that LanCL2 facilitates optimal phosphorylation of Akt by mTORC2 via direct physical interactions with both the kinase and the substrate.     

Extensive hydrolysis of raw rice starch by a chimeric α-amylase engineered with α-amylase (AmyP) and a starch-binding domain from Cryptococcus sp. S-2

Recombinant chimeric α-amylase (AmyP-Cr) was constructed by a catalytic core of α-amylase (AmyP) from a marine metagenomic library and a starch-binding domain (SBD_Cr) of α-amylase from Cryptococcus sp. S-2. The molecular fusion did not alter optimum pH, optimum temperature, hydrolysis products, and an ability of preferential and rapid degradation towards raw rice starch, but catalytic efficiency and thermostability were remarkably improved compared with those of the wild-type AmyP. AmyP-Cr achieved the final hydrolysis degree of 61.7 ± 1.2% for 10% raw rice starch and 47.3 ± 0.8% for 15% raw rice starch after 4 h at 40 °C with 1.0 U per mg of raw starch. The catalytic efficiency was very high, with 3.6–4.0 times higher than that of AmyP. The enhanced catalytic efficiency was attributed to the better thermostability and the higher adsorption and disruption to raw rice starch caused by SBD_Cr. The properties of AmyP-Cr open a new way in terms of a new design of raw rice starch processing.