Fumonisin B1: Isolation from corn culture,and purification by high performance liquid chromatography

A method is described to isolate fumonisin B₁ (FB₁) from corn cultured for 18 days at 25°C withFusarium moniliforme. Cultured corn was extracted with aqueous methanol and purified with XAD-2 column chromatography and high performance liquid chromatography (HPLC). About 450 mg of FB₁ were obtained from 800g cultured corn. Its identity was established by fast-atom bombardment (FAB) mass spectrometry, and infrared spectrum and nuclear magnetic spectrum. Its purity was estimated to be 95% by gas chromatography/mass spectrometry (GC/MS).References     

Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Paraná, Brazil

Natural mycoflora associated with fumonisins were analyzed in 150 samples of freshly harvested corn from Central-Southern, Central-Western and Northern regions of the State of Paraná, Brazil and correlated to climatic conditions. The corn samples were frequently contaminated with Fusarium sp.(98.7 to 100%) and Penicillium sp. (93 to 100%), when compared to Aspergillus sp. (not detected to 27.7%). The highest contamination with potentially mycotoxigenic fungi occurred in corn harvested in the Central-Western region, where total mould and yeast counts ranged from 5.5 × 10³ to 5.2 × 10⁶ CFU/g, with 98.7% contaminated byFusarium sp. and 93% by Penicillium sp. In this region F. moniliforme (F. verticillioides) was the predominant Fusariumsp., and was isolated in 85.9% of the samples. Aspergillus sp. was isolated from 27.7% samples. FB₁ was detected in 100% of the samples (mean of 2.39 g/g) and FB₂ in 97.7% (mean of 1.09 g/g). Fumonisins were also detected in all samples from Northern region, with mean of 4.56 g/g (FB₁) and 2.20 g/g (FB₂).Considering 1.0 g/g as the threshold, 72% of the corn samples from the Central-West and 92% from the North were contaminated with concentrations above this value, in contrast to a 18.5% contamination rate from Central-Southern samples. Between corn planting to harvesting season, the average maximum temperature and relative humidity were 26 °C and 77.1%(Central-Southern), 27 °C and 69% (Northern)and 29.9 °C and 89.1% (Central-Western).Therefore, the higher fumonisins contamination of corn from Northern region when compared to the Central-South were due to the differences in rainfall levels (92.8 mm in Central-Southern, 202 mm in Northern) during the month preceding harvest.This revised version was published online in October 2005 with corrections to the Cover Date.     

Sterigmatocystin and aflatoxin B<Subscript>1</Subscript> contamination of corn,soybean meal,and formula feed in Japan

Sterigmatocystin (STC) and aflatoxin B₁ (AFB₁) were analyzed in 246 corn samples, 126 soybean meal samples, and 861 formula feed samples from the Japanese market between April 2010 and March 2015. The detection rate, the highest concentration, and the mean concentration of STC were respectively 14%, 6.4 μg/kg, and 1.2 μg/kg for corn; 14%, 1.1 μg/kg, and 0.63 μg/kg for soybean meal; and 43%, 9.1 μg/kg, and 0.97 μg/kg for formula feed. The detection rate, the highest concentration, and the mean concentration of AFB₁ were respectively 46%, 24 μg/kg, and 3.9 μg/kg for corn; 30%, 6.7 μg/kg, and 1.1 μg/kg for soybean meal; and 47%, 20 μg/kg, and 1.6 μg/kg for formula feed. A weak negative correlation between the STC and AFB₁ concentrations was observed: there was a high concentration of AFB₁ in samples that contained a lower concentration of STC and vice versa. Spearman’s rank correlation coefficient showed a weak negative correlation of ? 0.30 (p < 0.001, n = 128) for corn and ? 0.23 (p < 0.001, n = 575) for formula feed. In conclusion, no correlation was observed between the mean concentrations of STC contamination in formula feed (0.97 μg/kg) and in corn (1.2 μg/kg) and the blending rate (approximately 50%). The rate of STC contamination in the formula feed (43%) was higher than that in corn (14%). Therefore, it is likely that ingredients other than corn contribute to the contamination of formula feed with STC. In this study, regarding STC, problematic samples were not found.     

Mariniflexile ostreae sp. nov., a member of the family Flavobacteriaceae isolated from an oyster

A Gram-negative, aerobic, non-flagellated and rod-shaped bacterial strain able to move by gliding, designated TYO-10^T, was isolated from an oyster collected at Tongyoung on the South Sea, South Korea. Strain TYO-10^T was found to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain TYO-10^T joined the cluster comprising the type strains of Mariniflexile species. Strain TYO-10^T exhibited 16S rRNA gene sequence similarity values of 95.4–95.7 % to the type strains of Mariniflexile species and of less than 95.4 % to the type strains of other recognized species. Strain TYO-10^T was found to contain MK-6 as the predominant menaquinone and iso-C_15:0, iso-C_15:1 G, anteiso-C_15:0, iso-C_17:0 3-OH and iso-C_15:0 3-OH as the major fatty acids. The major polar lipids of strain TYO-10^T were identified as phosphatidylethanolamine and an unidentified lipid, which is similar to those of the type strains of Mariniflexile species, but different from those of other phylogenetically related species. The DNA G+C content of strain TYO-10^T was determined to be 35.9 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain TYO-10^T is separated from other Mariniflexile species. On the basis of the data presented, strain TYO-10^T is considered to represent a novel species of the genus Mariniflexile, for which the name Mariniflexile ostreae sp. nov. is proposed. The type strain is TYO-10^T (= KCTC 42113^T = CECT 8622^T).     

Genome-wide association analysis to identify SNP markers affecting teat numbers in an F2 intercross population between Landrace and Korean native pigs

Most reproductive traits have low heritability and are greatly affected by environmental factors. Teat number and litter size are traits related to the reproduction ability of pigs. To identify quantitative trait loci (QTLs) for teat number traits, a genome-wide association study (GWAS) was conducted using an F₂ intercross between Landrace and Korean native pigs. Genotype analysis was performed using the porcine SNP 60 K beadchip. The GWAS was performed using a mixed-effects model and linear regression approach. When a genome-wide threshold was determined using the Bonferroni method (P = 1.61 × 10^?6), 38 single nucleotide polymorphism (SNP) markers in pig chromosome 7 (SSC7) were significantly associated with three teat number traits (total teat number, left teat number, and right teat number). Among these, SNPs in 5 genes (HDDC3, LOC100156276, LOC100155863, ANPEP, SCAMP2) were selected for further study based primarily on their statistical significance. A significant association was detected in SCAMP2 g.25280 G>A for total teat number (P = 2.0 × 10^?12), HDDC3 g.1319 G>A SNP for left teat number (P = 2.3 × 10^?7), and SCAMP2 g.14198 G>A for right teat number (P = 4.7 × 10^?12). These results provide valuable information about the selective breeding for desirable teat numbers in pigs.     

A micro-culture technique for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus)

A micro-culture technique was developed to determine the optimum culture conditions for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus). The optimum concentrations of PHA and Con A ranged from 10–50 µg/culture, those of lymphocytes from 1?2×10⁵ cells/culture, and those of serum from 10–20%. Tritiated thymidine was used at a concentration of .04µCi/culture.     

Sulphate uptake and metabolism in the chrysomonad,Monochrysis lutheri

The intracellular concentration of inorganic ³⁵SO₄ in Monochrysis lutheri cells exposed to 0.513 mM Na₂ ³⁵SO₄ for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this ³⁵SO₄ with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 μmoles/g cells/hr). The flux of free ³⁵SO₄ to organic ³⁵S was 0.029 μmoles/g cells/hr. Assuming an internal electrical potential in the cells of-70 mV, this intracellular concentration of inorganic ³⁵SO₄ was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a K _m of 3.2×10^-5 M and a V _max of 7.9×10^-5 μmoles sulphate/hr/10⁵ cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the proteins(s) associated with sulphate transport.     

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes k ₁ for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients k ₁ probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/L) and DMSO (2250 mOsm/L) the filtration coefficient L _p in the presence of a penetrating cryoprotectant (L _pDMSO = (4.45 ± 0.04) · 10^?14 m³/Ns) is 3 orders lower compared to the case with electrolyte (L _pNaCl = (2.25 ± 0.25) · 10^?11 m³/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.     

Aflatoxin in human and camel milk in Abu Dhabi,United Arab Emirates

During an initial survey, using thin layer chromatography, 10 of 64 samples of mothers’ breast milk, collected from donors at the Corniche Maternity Hospital and the Al-Nehyan Clinic for Maternity and Childhood, were found to contain Aflatoxin M₁ at concentrations ranging from 0.3 to 1.3 ng mL^?1. A second survey using HPLC showed Aflatoxin M₁ at concentrations ranging from 7 to 23 pg mL^?1 in all of the 15 samples collected. 6 of 20 samples of camel milk collected from several sources in Abu Dhabi were also found to contain Aflatoxin M₁ at levels ranging from 0.25 to 0.8 ng mL^?1.     

Mechanisms of Na+ uptake,ammonia excretion,and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi,Hemigrammus rhodostomus,and Moenkhausia diktyota)

Mechanisms of Na⁺ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J _in), efflux (J _out), and net flux rates (J _net) of Na⁺ and Cl^?, and the net excretion rate of ammonia (J _Amm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na⁺, Cl^? ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l^?1). J _in ^Na , J _in ^Cl , and J _Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J _in ^Na was unaffected by amiloride (10^?4 M, NHE and Na⁺ channel blocker), but both J _in ^Na and J _in ^Cl were virtually eliminated (85–99 % blockade) by AgNO₃ (10^?7 M). A time course study on cardinal tetras demonstrated that J _in ^Na blockade by AgNO₃ was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10^?4 M) caused a 50 % reduction in J _in ^Na _.. Additionally, J _in ^Na was unaffected by phenamil (10^?5 M, Na⁺ channel blocker), bumetanide (10^?4 M, NKCC blocker), hydrochlorothiazide (5 × 10^?3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J _in ^Na (by acetazolamide and AgNO₃ respectively), had any inhibitory effect on J _Amm. Therefore, Na⁺ uptake in Rio Negro tetras depends on an internal supply of H⁺ from CA, but does not fit any of the currently accepted H⁺-dependent models (NHE, Na⁺ channel/V-type H⁺-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na⁺/NH₄ ⁺ exchange metabolon” paradigm. Na⁺, K⁺-ATPase and V-type H⁺-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH₄Cl, 10^?3 M) significantly increased J _in ^Na , and NH₄ ⁺ was equally or more effective than K⁺ in activating branchial Na⁺,(K⁺) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na⁺ uptake, but that Na⁺ uptake (by an as yet unknown H⁺-dependent apical mechanism) depends on ammonia excretion, driven by active NH₄ ⁺ entry via basolateral Na⁺,(K⁺)-ATPase.     

Identification of methylation quantitative trait loci (mQTLs) influencing promoter DNA methylation of alcohol dependence risk genes

Interaction of DNA methylation and sequence variants that are methylation quantitative trait loci (mQTLs) may influence susceptibility to diseases such as alcohol dependence (AD). We used genome-wide genotype data from 268 African Americans (AAs: 129 AD cases and 139 controls) and 143 European Americans (EAs: 129 AD cases and 14 controls) to identify mQTLs that were associated with promoter CpGs in 82 AD risk genes. 282 significant mQTL–CpG pairs (9.9 × 10^?100 ≤ P _nominal ≤ 7.7 × 10^?8) in AAs and 313 significant mQTL–CpG pairs (2.7 × 10^?53 ≤ P _nominal ≤ 9.9 × 10^?8) in EAs were identified [i.e., mQTL–CpG associations survived multiple-testing correction, q values (false discovery rate) ≤ 0.05]. The most significant mQTL was rs1800759, which was strongly associated with CpG cg12011299 in both AAs (P _nominal = 9.9 × 10^?100; q = 6.7 × 10^?91) and EAs (P _nominal = 2.7 × 10^?53; q = 1.4 × 10^?44). Rs1800759 (previously known to be associated to AD) and CpG cg12011299 (distance: 37 bp) are both located in alcohol dehydrogenase (ADH) 4 gene (ADH4) promoter region. In general, the strength of association between mQTLs and CpGs was inversely correlated with the distance between them. Association was also influenced by race and AD. Additionally, 48.3 % of the mQTLs identified in AAs and 65.6 % of the mQTLs identified in EAs were predicted to be expression QTLs. Three mQTLs (rs2173201, rs4147542, and rs4147541 in ADH1B-AHD1C gene cluster region) found in AAs were previously identified by our genome-wide association studies as being significantly associated with AD in AAs. Thus, DNA methylation, which can be influenced by sequence variants and is implicated in gene expression regulation, appears to at least partially underlie the association of genetic variation with AD.     

Paenibacillus nicotianae sp. nov., isolated from a tobacco sample

A Gram-stain positive, facultative anaerobic endospore-forming bacterium, designated strain YIM h-19^T, was isolated from a tobacco sample. Cells were observed to be motile rods by means of peritrichous flagella and colonies were observed to be convex, yellow, circular and showed catalase-positive and oxidase-negative reactions. Strain YIM h-19^T was able to grow at 4–45 °C, pH 6.0–8.0 and 0–3 % NaCl (w/v). The predominant respiratory quinone was identified as MK-7. Major fatty acids were identified as anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The polar lipids were found to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. The genomic DNA G+C content was determined to be 54 mol%. 16S rRNA gene sequence analysis showed the strain YIM h-19^T was most closely related to Paenibacillus hordei RH-N24^T and Paenibacillus hunanensis FeL05^T with similarities of 98.30 and 94.64 % respectively. However, DNA–DNA hybridization data indicated that the isolate represented a novel genomic species with the genus Paenibacillus. All data from genotypic and phenotypic analyses support the conclusion that strain YIM h-19^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nicotianae sp. nov. is proposed. The type strain is YIM h-19^T (=CGMCC1.12819^T = NRRL B-59112^T).     

Paenibacillus yonginensis sp. nov., a potential plant growth promoting bacterium isolated from humus soil of Yongin forest

Strain DCY84^T, a Gram-stain positive, rod-shaped, aerobic, spore-forming bacterium, motile by means of peritrichous flagella, was isolated from humus soil from Yongin forest in Gyeonggi province, South Korea. Strain DCY84^T shared the highest sequence similarity with Paenibacillus barengoltzii KACC 15270^T (96.86 %), followed by Paenibacillus timonensis KACC 11491^T (96.49 %) and Paenibacillus phoenicis NBRC 106274^T (95.77 %). Strain DCY84^T was found to able to grow best in TSA at temperature 30 °C, at pH 8 and at 0.5 % NaCl. MK-7 menaquinone was identified as the isoprenoid quinone. The major polar lipids were identified as phosphatidylethanolamine, an unidentified aminophospholipid, two unidentified aminolipids and an unidentified polar lipid. The peptidoglycan was found to contain the amino acids meso-diaminopimelic acid, alanine and d-glutamic acid. The major fatty acids of strain DCY84^T were identified as branched chain anteiso-C_15:0, saturated C_16:0 and branched chain anteiso-C_17:0. The cell wall sugars of strain DCY84^T were found to comprise of ribose, galactose and xylose. The major polyamine was identified as spermidine. The DNA G+C content was determined to be 62.6 mol%. After 6 days of incubation, strain DCY84^T produced 52.96 ± 1.85 and 72.83 ± 2.86 µg/ml l-indole-3-acetic acid, using media without l-tryptophan and supplemented with l-tryptophan, respectively. Strain DCY84^T was also found to be able to solubilize phosphate and produce siderophores. On the basis of the phenotypic characteristics, genotypic analysis and chemotaxonomic characteristics, strain DCY84^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus yonginensis sp. nov. is proposed. The type strain is DCY84^T (=KCTC 33428^T = JCM 19885^T).     

Properties of arginase from liver of Macaca fascicularis: Comparison of normals with red blood cell arginase deficient monkeys

Deficiency of arginase (E.C. 3.5.3.1), the fifth enzyme of the urea cycle, was found in the red blood cells (RBCs) of Macaca fascicularis monkeys (<0.2 µmol arginine cleaved/g Hb/min; normal =49.2). Liver biopsies were obtained from two of these monkeys and from one monkey with normal levels of RBC arginase activity. Arginase from both groups of animals required Mn²⁺ for maximal enzyme activity and demonstrated a pH optimum of 10.2 in vitro. The activity of arginase in the livers of all three monkeys was 1.1 mmol arginine cleaved/g protein/min. The apparent K _m for arginine of arginase in the livers of the RBC-deficient monkeys was 7.4 and 5.9mm and in the normal monkey was 6.9mm. Similar patterns of heat denaturation were seen at 69 C without Mn²⁺ present and 79 C in the presence of 20mm Mn²⁺. No difference in mobility on either acidic or basic polyacrylamide gels for liver arginase from either RBC-deficient or normal monkeys was found. In addition, liver arginase from all three monkeys reacted similarly with anti-human liver arginase antibody. Liver arginases in RBC-deficient and normal monkeys were identical by ten criteria. These studies do not distinguish among several hypotheses for the genetic determination of arginase in different organs of this species and of man.     

Calcium-independent uncoupling activity of palmitic acid in liver mitochondria is regulated by the ion fluxes causing the interconversion of Δψ and ΔpH across the inner membrane

The effect of ion fluxes across the inner membrane on calcium-independent uncoupling activity of palmitic acid was investigated in experiments on rat liver mitochondria energized by the oxidation of succinate. The following compounds were used as the inductors of ion fluxes: the K⁺/H⁺ antiporter nigericin causing transformation of ΔpH into electrical potential difference (Δψ) across the inner membrane; tetraphenylphosphonium (TPP⁺) that freely crosses phospholipid membranes; protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) that induces a flow of H⁺ from the intermembrane space into the matrix and reduces Δψ and ΔpH. It was found that nigericin at a concentration of 20 nM, which causes an increase in maximal Δψ, partially inhibits the ability of palmitic acid to reduce Δψ and stimulates mitochondrial respiration. A specific inhibitor of the ATP/ADP antiporter (carboxyatractylate) and a substrate of the aspartate/glutamate antiporter (glutamate) increase Δψ and partially inhibit mitochondrial respiration in the presence of palmitic acid. Under these conditions, 10 μM cyclosporin A also inhibits respiration but has no effect on Δψ. The specific uncoupling activity of palmitic acid (V _U) and its specific components that characterize participation of the ATP/ADP antiporter (V _Catr), aspartate/glutamate antiporter (V _Glu), and cyclosporin-A-sensitive system (V _CsA) in the palmitic acid-induced uncoupling were estimated. It was shown that nigericin substantially reduces V _U, V _Catr and V _Glu but increases V _CsA. TPP⁺ at a concentration of 20 μM increases V _U and V _Glu, does not affect V _Catr and reduces V _CsA. FCCP at concentrations of 20 and 40 nM reduces Δψ by not more than 17% but does not affect V _U, V _Catr, V _Glu and V _CsA. The results suggest that the calcium-independent uncoupling effect of palmitic acid in liver mitochondria is caused by the return of protons to the matrix with participation of ADP/ATP and aspartate/glutamate antiporters and owing to activation of cyclosporin A-sensitive electron transport along the respiratory chain without affecting Δψ. The induced ion fluxes across the inner mitochondrial membrane can be considered as a factor of the calcium-independent regulation of uncoupling activity of palmitic acid in liver mitochondria with participation of the ADP/ATP and aspartate/glutamate antiporters and of the cyclosporin A-sensitive electron transport system.     

Litoribrevibacter albus gen. nov. sp. nov., isolated from coastal seawater,Fujian province,China

A Gram-stain negative, short rod-shaped aerobic bacterium with flagella, designated strain Y32^T, was isolated from coastal seawater in Xiamen, Fujian Province of China. 16S rRNA gene sequence comparisons showed that strain Y32^T is a member of the family Oceanospirillaceae, forming a distinct lineage with species of the genus Litoribacillus. The 16S rRNA gene sequence similarities between strain Y32^T and other strains were all less than 94.0 %. Strain Y32^T was found to grow optimally at 28 °C, at pH 7.0–8.0 and in the presence of 4–5 % (w/v) NaCl. The major fatty acids were identified as Summed Feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c, 49.4 %), C_16:0 (17.7 %), C_14:0 (6.9 %) and C_18:1 ω9c (5.4 %). The major respiratory quinone was identified as ubiquinone-8 (Q-8). The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain Y32^T was determined to be 55.6 mol%. According to its morphology, physiology, fatty acid composition, polar lipids composition and 16S rRNA gene sequence data, strain Y32^T represents a novel species of a new genus in the family Oceanospirillaceae, for which the name Litoribrevibacter albus gen. nov. sp. nov. is proposed. The type strain of Litoribrevibacter albus is Y32^T (=MCCC 1F01211^T=NBRC 110071^T).     

Esterase-16 (Es-16): Characterization,polymorphism, and linkage to chromosome 3 of a kidney esterase locus of the house mouse

A polymorphism for an isozyme of a presumed arylesterase, esterase-16 (EC 3.1.1.2), has been detected in kidney, heart, and spleen of the house mouse, Mus musculus, by means of isoelectric focusing and by disc electrophoresis. Three phenotypes can be distinguished: the ES-16A phenotype (IEP 5.9) was found in C57BL/10Sn and many other laboratory inbred strains; the ES-16B phenotype (IEP 6.1) was found in M. m. molossinus; and the ES-16C phenotype (IEP 5.9; very weak activity) was found in Peru-Coppock. Esterase-16 is strongly inhibited by 10^?3 m p-chloromercuribenzoate, but not by 2·10^?4 m bis-p-nitrophenyl phosphate or by 10^?3 m Diamox. It stains well with indoxyl acetate and other indigogenic substrates but only weakly with α-naphthyl acetate. Esterase-16 is completely insoluble in water. It is apparently governed by a structural gene locus, Es-16, with three alleles, Es-16 ^a , Es-16^b, and Es-16 ^c, respectively. Es-16 is closely linked to Car-1 and Car-2 on chromosome 3. Typing of 94 animals of the backcross (C57BL/10Sn × M. m. mol.) F₁ × M. m. mol. revealed a recombination frequency of 8.51±2.9%.     

Affinity labeling of the active site of pig liver NADH-cytochrome b5 reductase by 5′-p-fluorosulfonylbenzoyladenosine

Lysosome-solubilized pig liver NADH-cytochrome b₅ reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K _obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K _d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK ₂=0.22 min^?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[¹⁴C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b₅ reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.     

Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability

In this study, the thermostability of an alkaline α-amylase from Alkalimonas amylolytica was significantly improved through structure-based rational and the introduction of multiple arginines (Arg) on the protein surface. Based on an analysis of the tertiary structure, seven residues (glutamine (Gln) 166, Gln 169, serine (Ser) 270, lysine (Lys) 315, Gln 327, asparagine (Asn) 346, and Asn 423) were selected as engineering targets and individually replaced with arginine. Five of the seven single-mutated enzymes—S270R, K315R, Q327R, N346R, and N423R—showed enhanced thermostability. Multiple arginines were subsequently introduced on the protein surface, and the quintuple-mutated enzyme S270R/K315R/Q327R/N346R/N423R showed a 6.4-fold improvement in half-life at 60 and a 5.4 °C increase in melting temperature (T _m) compared with those of wild-type enzyme. Concomitantly, the optimal temperature, optimal pH, and catalytic efficiency of this mutated enzyme also improved. The mutated enzyme displayed a large shift in optimal pH from 9.5 to 11.0. In addition, the optimum temperature increased from 50 to 55 °C, and the catalytic efficiency (k _cat/K _m) increased from 1.8?×?10⁴ to 3.6?×?10⁴ L/(g?·?min). The intramolecular interactions of mutated enzymes that contributed to increased thermostability were examined through comparative analysis of the model structures of wild-type and mutated enzymes. The thermostable mutated enzymes generated in this study have potential applications in the textile industry.