Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

Association of polymorphisms in genes involved in enamel formation,taste preference and immune response with early childhood caries in Saudi pre-school children

Dental caries is primarily elicited by modifiable factors such as inadequate oral hygiene, poor dietary practices and deficient fluoride exposure. However, there is a growing body of evidence suggesting the profound influence of genetic factors in dental caries susceptibility. The present study aimed to evaluate the association between single nucleotide polymorphisms (SNPs) in ENAM (rs12640848), MMP20 (rs1784418), TAS2R38 (rs713598), and LTF (rs4547741) genes and early childhood caries (ECC) in Saudi preschool children. This case-control study enrolled 360 Saudi preschool children (262 with ECC and 98 caries-free). Data on environmental factors were collected through a questionnaire. However, caries experience and oral hygiene data were obtained during clinical examination. Buccal swab samples were collected for DNA extraction and SNPs were genotyped using PCR and DNA sequencing. Children with ECC were compared to caries free children (control), then they were categorized into two categories based on ECC severity as follows; non-severe ECC (NS-ECC), and severe-ECC (S-ECC). Association between the SNPs, ECC, NS-ECC, and S-ECC was reported as an odds ratio (OR) with a 95% confidence interval (CI). The majority of the children (72.8%) exhibited ECC (31.7% NS-ECC and 41.1% S-ECC) with mean dmft of 4.20 ± 4.05. Multivariate analyses of environmental factors showed that nocturnal feeding was a risk factor for ECC (P = 0.008). Poor oral hygiene was also a risk factor for both NS-ECC and S-ECC (ECC: P < 0.0001, NS-ECC: P = 0.032 and S-ECC: P < 0.0001). Univariate analysis showed that the AG genotype of rs1784418 of MMP20 gene was protective against ECC (OR = 0.532; 95% CI = 0.316–0.897, P = 0.018) and against NS-ECC (OR = 0.436; 95% CI = 0.238–0.798, P = 0.007). When environmental risk factors for ECC were included as covariates during multivariate analysis, AG variant in rs1784418 of MMP20 gene remained less frequent in NS-ECC cases compared to controls with borderline significance (OR = 0.542; 95% CI = 0.285–1.033, P = 0.063). Our findings concluded that MMP20 rs1784418 SNP might be associated with protection against ECC in Saudi preschool children.     

The effect of light on oxygen consumption in two amphipod crustaceans–the hypogean Niphargus stygius and the epigean Gammarus fossarum

The effects of light on the metabolic rates of the hypogean amphipod Niphargus stygius and the epigean amphipod Gammarus fossarum were compared by measuring oxygen consumption and respiratory electron transport system (ETS) activity. They were exposed to light intensities of 720 and 4700?lx at 10°C. Oxygen consumption increased significantly in N. stygius exposed to both low and high intensities of light, but no significant increase was observed in G. fossarum at either intensity. The increase of oxygen consumption in N. stygius was significantly greater at the higher light intensity. This indicates a stress response in which exploitation of half the metabolic potential for energy production in N. stygius during exposure to high light intensity constitutes an adverse effect on its metabolism, since this species usually uses less than 25% of its total metabolic potential for standard metabolic demands.     

Production of a new terpenoid from biotransformation of (-)-isolongifolanol by Aspergillus niger and suppression of SOS-inducing activity

Abstract

The stereoselective oxidation of (—)-isolongifolanol (1) with a longifolene skeleton by Aspergillus niger (NBRC 4414) as a biocatalyst and suppressive effect on umuC gene expression by chemical mutagens furylfuramid and AFB1 of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Compound 1 was converted to a new terpenoid, (-)-(2S,8R)-8,12-dihydroxy-isolongifolanol (2). Its structure was determined by NMR, IR, specific rotation and mass spectrometry. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB₁ in the umu test. Compound 1 suppressed 51% of the SOS-inducing activity against furylfuramid at < 1.0 mM. Compound 2 suppressed 15% and 24% of the SOS-inducing activity against furylfuramid and AFB1 at < 1.0 mM respectively.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

Age dependent expression of melatonin membrane receptor (MT1, MT2) and its role in regulation of nitrosative stress in tropical rodent Funambulus pennanti

Age-dependent declining level of melatonin induces free radical load and thereby deteriorates immune function. However, reports are lacking about age-dependent melatonin membrane receptor (MT1 & MT2) expression, their role in regulation of reactive nitrogen species (RNS) and eventually how they affect immunity of a tropical rodent F. pennanti. We checked MT1R, MT2R and iNOS expression in lymphoid organs of young middle and old aged squirrels. Nitrite and nitrate ion concentration (NOx) in lymphoid organs, testes and plasma, lymphocyte proliferation and IL-2 level was recorded. Age-dependent decrease in MT1 and MT2 receptor expression, lymphocyte proliferation, IL-2 level and increased RNS in lymphoid organs, testes and plasma was observed with decreased circulatory melatonin. Androgen and AR expression was increased in middle-aged while declined in old-aged squirrels. Present study suggests that age associated immunosenescence is consequence of increased RNS which might have important relationship with melatonin membrane receptors in F. pennanti.     

DETERMINATION OF METHYLENE BLUE BIOSORPTION BY Rhizopus arrhizus IN THE PRESENCE OF SURFACTANTS WITH DIFFERENT CHEMICAL STRUCTURES

Methylene blue (MB) biosorption properties of Rhizopus arrhizus were investigated in the presence of surfactants. The effects of cationic and anionic surfactants on MB removal by dead biomass (1 g L^?1) were determined. MB removal was tested as a function of initial pH (2–12), contact time (5–1440 min), and dye (37.4–944.7 mg L^?1) and surfactant (0–10 mM) concentrations. The opposite charged anionic surfactant dodecylbenzenesulfonic acid sodium salt (DBS) enhanced sorption of cationic MB by biomass dramatically. Maximum biosorption capacity was 471.5 mg g^?1 at pH 8 with 0.5 mM DBS at 944.7 mg L^?1 MB concentration. The surfactant-stimulated fungal decolorization method may provide a highly efficient, inexpensive, and time-saving procedure in biological wastewater treatment technologies.     

Review ArticleNaphthalene Combretastatin Analogues: Synthesis,Cytotoxicity and Antitubulin Activity

Synthesis and evaluation of new combretastatin analogues with varied modifications on the bridge and the aromatic rings, have shown that the 2-naphthyl moiety is a good surrogate for the 3-hydroxy-4-methoxyphenyl (B-ring) of combretastatin A-4. Other bicyclic systems, such as 6(7)-quinolyl and 5-indolyl, can replace the B-ring, but they produce less potent analogues in the cytotoxicity and tubulin polymerization inhibition assays. Other modifications are detrimentral for the potency of the studied analogues. The 2-naphthyl combretastatin 53 and the related 6-quinolyl combretastatin 106 analogues are the most potent among the derivatives of this type, whereas 92 and 95 are the most potent among the naphthalene derivatives with a heterocycle in the bridge. Previous and new results in this family of combretastatin analogues are discussed.