Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

High-yield expression,purification, characterization,and structure determination of tag-free <Emphasis Type="Italic">Candida utilis</Emphasis> uricase

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2₁2₁2₁ with unit cell parameters a?=?69.16 Å, b?=?139.31 Å, c?=?256.33 Å, and α?=?β?=?γ?=?90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Impact of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> inoculation on indigenous bacterial communities during agricultural waste composting

This research was conducted to distinguish between the separate effects of the Phanerochaete chrysosporium inoculation and sample property heterogeneity induced by different inoculation regimes on the indigenous bacterial communities during agricultural waste composting. P. chrysosporium was inoculated during different phases. The bacterial community abundance and structure were determined by quantitative PCR and denaturing gradient gel electrophoresis analysis, respectively. Results indicated a significant stimulatory effect of P. chrysosporium inoculation on the bacterial community abundance. The bacterial community abundance significantly coincided with pile temperature, ammonium, and nitrate (P?<?0.006). Variance partition analysis showed that the P. chrysosporium inoculation directly explained 20.5 % (P?=?0.048) of the variation in the bacterial communities, whereas the sample property changes induced by different inoculation regimes indirectly explained up to 35.1 % (P?=?0.002). The bacterial community structure was significantly related to pile temperature, water-soluble carbon (WSC), and C/N ratio when P. chrysosporium were inoculated. The C/N ratio solely explained 7.9 % (P?=?0.03) of the variation in community structure, whereas pile temperature and WSC explained 7.7 % (P?=?0.026) and 7.5 % (P?=?0.034) of the variation, respectively. P. chrysosporium inoculation affected the indigenous bacterial communities most probably indirectly through increasing pile temperature, enhancing the substrate utilizability, and changing other physico-chemical factors.     

Promotion of shoot regeneration of <Emphasis Type="Italic">Swertia chirata</Emphasis> by biosynthesized silver nanoparticles and their involvement in ethylene interceptions and activation of antioxidant activity

A complete protocol for the in vitro induction of Eclipta alba tetraploids has been optimized to enhance the wedelolactone content, an anti-cancerous compound. The effects of different concentrations of colchicine (0, 0.01, 0.05, 0.1, 0.2 and 0.3%; w/v) along with treatment durations (12, 24, 36 and 48 h) were investigated on shoot tip (ST) and nodal segment (NS). The treated explants were then incubated on Murashige and Skoog (MS) medium having 1.5 mg L^?1 N⁶-benzylaminopurine and 0.5 mg L^?1 α-napthalene acetic acid for shoot regeneration and afterward root was induced on 1.0 mg L^?1 indole-3-acetic acid enriched ½MS medium. The tetraploids of E. alba were proficiently induced by the treatment of 0.1% colchicine for 24 h. The highest tetraploid induction efficiency was obtained from ST (30.56%) in comparison to the NS (22.22%). Analysis by spectrophotometry and flow cytometry showed that colchicine treated plants contained higher quantity of DNA than diploid plants. Cytological studies demonstrated doubled the chromosome number in tetraploids (2n?=?4x?=?44) than diploids (2n?=?2x?=?22). The ploidy level enhancement lead to alteration of other traits, like increased plant height, stem diameter, leaf size, stomatal size and chlorophyll content. As determined through high performance thin-layer chromatography, the ultimate achievement of this technique is the higher accumulation of wedelolactone in tetraploid plants (300.32 µg g^?1 dry weight) in evaluation to in vitro diploid (131.31 µg g^?1 dry weight) and in vivo diploid mother plants (93.26 µg g^?1 dry weight), thus improving the pharmaceutical value of E. alba.     

Development and validation of a new real-time assay for the quantification of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope?=??3.405, r²?=?0.976, E?=?96.64 %) and the positive recombinant plasmid (y?=??3.36, r²?=?0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10² copies/μL (CV%?≈?1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g^?1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r²?=?0.944) and with the soil plating method (r²?=?0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.     

Parasitism Rate of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> to <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> Larvae: Residual Effect of Deltamethrin,Fenvalerate and Azadirachtin

Sublethal concentrations of chemical insecticides may cause changes in some behavioral characteristics of natural enemies such as functional responses. The residual effect of three synthetic insecticides including deltamethrin, fenvalerate and azadirachtin were studied on functional response of Habrobracon hebetor Say to Ephestia kuehniella Zeller larvae. Seven host densities (2, 4, 8, 16, 32, 64 and 96) were used during a 24 h period. The resulting data were appropriately fit to Type II functional response models in all treatments: (1) control (0.0916 h^?1; and T _h  = 0.2011 h); (2) deltamethrin (a = 0.0839 h^?1; and T _h  = 0.3560 h); (3) fenvalerate (a = 0.0808 h^?1 and T _h  = 0.3623 h); and (4) azadirachtin (a = 0.0900 h^?1 and T _h  = 0.2042 h). Maximum theoretical parasitism rate (T/T _h ) was 119.34 estimated for control wasps. There was no significant difference between the values of attack rates (a and a + D _a ) in all treatments while the handling time was statistically affected in female wasps treated with fenvalerate. Our findings will be useful in safe application of these insecticides in pest management programmes.     

Kinetic Analyses of the Substrate Inhibition of <Emphasis Type="Italic">Paramecium</Emphasis> Arginine Kinase

Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R², AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, V_max^SES?=?30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, V_max^ES?=?241.7. The dissociation constant for the SES complex (K_i^SES: 0.34 mM) was two times smaller than that of the ES complex (K_s^ES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower V_max^SES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the K_i^SES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, V_max^ES and V_max^SES values for the mutants decreased by one-third, except for the V_max^SES of the S79A mutant, which had a value that was comparable with the value for the wild-type.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

Cloning of <Emphasis Type="Italic">phaCAB</Emphasis> genes from thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA _Cma , 1179 bp), acetoacetyl-CoA reductase (phaB _Cma , 738 bp), and PHA synthase (phaC _Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD₆₀₀, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC _Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

High genetic diversity in an endangered medicinal plant, <Emphasis Type="Italic">Saussurea involucrata</Emphasis> (<Emphasis Type="Italic">Saussurea</Emphasis>, Asteraceae), in western Tianshan Mountains,China

Saussurea involucrata (Asteraceae) is a medicinal and second-degree national priority endangered plant that is mainly distributed in the high latitude region of the western Tianshan Mountains. The population is fragmented and isolated, and extensive human impact merits a suitable and specific conservation strategy, which can be compiled based on the genetic diversity, population structure, and demographic history. Phylogeographic studies were conducted on a total of five natural populations and 150 individuals were sampled. Data from three cpDNA intergenic spacer regions (trnL-F, matK, and ndhF-rpl32) and nrDNA ITS sequences showed that twelve haplotypes in cpDNA and five haplotypes in nrDNA indicated high genetic diversity among populations sampled (H _T?=?0.820 and 0.756) and within populations sampled (H _S?=?0.792 and 0.721). Additionally, the high genetic diversity did not mirror genetic structure in either cpDNA (F _ST?=?0.03153, G _ST?>?N _ST, p?<?0.05) or nrDNA (F _ST?=?0.03666, meaningless G _ST and N _ST). Two groups (north and south) were determined for a SAMOVA analysis. Based on this analysis, the demographic history was conducted with a Bayesian Skyline Plot and Isolation with Migration analysis, which showed sustainable and stable extension without a marked bottleneck. Divergence time was indicated at c. 6.25 Mya (90%HPD: 15.30–0.22 Mya) in the Miocene, which is consistent with the formation of the Kelasu section of Tianshan. The southern populations in the Bayanbulak and Gonglu regions require additional attention and transplanting would be an effective way to restore rare cpDNA haplotypes, increase effective population size, and migration rate. Our results suggested that in situ conservation of S. involucrata in western Tianshan should be the main strategy for protection and recovery of the species.     

Using hydrothermal time concept to describe sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) seed germination response to temperature and water potential

To quantify both temperature (T) and water potential (ψ) effects on sesame (Sesamum indicum L.) seed germination (SG) and also to determine the cardinal T _s for this plant, a laboratory experiment was carried out using hydrothermal time model (HTT). For this purpose, four sesame cultivars (‘Asbomahalleh’, ‘Darab’, ‘Dashtestan’ and ‘Yellowhite’) were germinated at seven constant T _s (20, 25, 30, 35, 37, 39 and 43 °C) at each of the following ψ _s (0, ? 0.12, ? 0.24 and ? 0.36 MPa; provided by PEG 8000). Germination rate (GR) and germination percentage (GP) significantly influenced by ψ, T and their interactions in all cultivars (P ≤ 0.01). There was no significant difference, based on the confidence intervals of the model coefficients, between cultivars, so an average of cardinal T _s was 14.7, 35.4 and 47.2 °C for the minimum (T _b), optimum (T _o) and maximum (T _c) T _s, respectively, in the control condition (0 MPa). Hydrotime values in all cultivars decreased when T was increased to T _o and then remained constant at T _s > T _o (15 MPa h^?1). An average value of ψ _b(50) was estimated to be ? 1.23 MPa at T _s ≤ T _o and then increased linearly (0.1041 MPa°Ch^?1, the slope of the relationship between ψ _b(50) and supra-optimal T _s) with T when T _s increased above T _o and finally reached to zero at T _c. The T _b and T _o values were not influenced by ψ, but T _c value decreased (from 47.2 for zero to 43.5 °C for ? 0.36 MPa) at supra-optimal T _s as a result of the effect of ψ on GR. Based on our findings, this model (as a predictive tool) and or the estimated parameter values in this study can easily be used in sesame SG simulation models to quantitatively characterize the physiological status of sesame seed populations at different T _s and ψ _s.     

Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of <Emphasis Type="Italic">Fomitopsis pinicola</Emphasis>

An efficient ß-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50°C. The enzyme showed high substrate specificity and high catalytic efficiency (k _cat?=?2,990 s^?1, K _m?=?1.76 mM, k _cat/K _m?=?1,700 mM^?1 s^?1) for p-nitrophenyl-β-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.     

Associations of <Emphasis Type="Italic">CTLA4</Emphasis> +49 A/G Dimorphism and HLA-DRB1*/DQB1* Alleles With Type 1 Diabetes from South India

The aim of present study was to elucidate the association of CTLA4 +49 A/G and HLA-DRB1*/DQB1* gene polymorphism in south Indian T1DM patients. The patients and controls (n?=?196 each) were enrolled for CTLA4 and HLA-DRB1*/DQB1* genotyping by RFLP/PCR-SSP methods. The increased frequencies of CTLA4 ‘AG’ (OR?=?1.99; p?=?0.001), ‘GG’ (OR?=?3.94; p?=?0.001) genotypes, and ‘G’ allele (OR?=?2.42; p?=?9.26?×?10^?8) were observed in patients. Reduced frequencies of ‘AA’ (OR?=?0.35; p?=?7.19?×?10^?7) and ‘A’ (OR?=?0.41; p?=?9.26?×?10^?8) in patients revealed protective association. Among HLA-DRB1*/DQB1* alleles, DRB1*04 (OR?=?3.29; p?=?1.0?×?10^?5), DRB1*03 (OR?=?2.81; p?=?1.9?×?10^?6), DQB1*02:01 (OR?=?2.93; p?=?1.65?×?10^?5), DQB1*02:02 (OR?=?3.38; p?=?0.0003), and DQB1*03:02 (OR?=?7.72; p?=?0.0003) were in susceptible association. Decreased frequencies of alleles, DRB1*15 (OR?=?0.32; p?=?2.55?×?10^?7), DRB1*10 (OR?=?0.45; p?=?0.002), DQB1*06:01 (OR?=?0.43; p?=?0.0001), and DQB1*05:02 (OR?=?0.28; p?=?2.1?×?10^?4) in patients were suggested protective association. The combination of DRB1*03+AG (OR?=?5.21; p?=?1.4?×?10^?6), DRB1*04+AG (OR?=?2.14; p?=?0.053), DRB1*04+GG (OR?=?5.21; p?=?0.036), DQB1*02:01+AG (OR?=?4.44; p?=?3.6?×?10^?5), DQB1*02:02+AG (OR?=?20.9; p?=?9.5?×?10^?4), and DQB1*02:02+GG (OR?=?4.06; p?=?0.036) revealed susceptible association. However, the combination of DRB1*10+AA (OR?=?0.35; p?=?0.003), DRB1*15+AA (OR?=?0.22; p?=?5.3?×?10^?7), DQB1*05:01+AA (OR?=?0.45; p?=?0.007), DQB1*05:02+AA (OR?=?0.17; p?=?1.7?×?10^?4), DQB1*06:01+AA (OR?=?0.40; p?=?0.002), and DQB1*06:02+AG (OR?=?0.34; p?=?0.001) showed decreased frequency in patients, suggesting protective association. In conclusion, CTLA4/HLA-DR/DQ genotypic combinations revealed strong susceptible/protective association toward T1DM in south India. A female preponderance in disease associations was also documented.     

Mixing rates in weakly differentiated stocks of greater amberjack (<Emphasis Type="Italic">Seriola dumerili</Emphasis>) in the Gulf of Mexico

The greater amberjack (Seriola dumerili) is a commercially and recreationally important marine fish species in the southeastern United States, where it has been historically managed as two non-mixing stocks (Gulf of Mexico and Atlantic). Mark-recapture studies and analysis of mitochondrial DNA have suggested the two stocks are demographically independent; however, little is currently known about when and where spawning occurs in Gulf of Mexico amberjack, and whether stock mixture occurs on breeding grounds. The primary objective of this study was to quantify stock mixture among breeding populations of amberjack collected from the Atlantic and Gulf of Mexico. Genetic data based on 11 loci identified very low, though statistically significant differentiation among Gulf of Mexico samples (G_ST = 0.007, \(G_{{{\text{ST}}}}^{\prime }\) = 0.009; all P?=?0.001) and between reproductive adults collected from two spawning areas (G_ST = 0.007, \(G_{{{\text{ST}}}}^{\prime }\) = 0.014; all P?=?0.001). Naïve Bayesian mixture analysis supported a single genetic cluster [p(S|data)?=?0.734] whereas trained clustering (using Atlantic and Gulf spawning fish) gave the highest support to a two-cluster model (p(S|data)?=?1.0). Our results support the argument that the genetic structuring of greater amberjack is more complex than the previously assumed two, non-mixing stock model. Although our data provide evidence of limited population structure, we argue in favour of non-panmixia among reproductive fish collected from the Gulf of Mexico and Florida Keys.     

Terpenoid bioactive compound from <Emphasis Type="Italic">Streptomyces rochei</Emphasis> (M32): taxonomy,fermentation and biological activities

The present study emphasized the production of biologically active terpenoid compound from Streptomyces rochei M32, which was isolated from Western Ghats ecosystem, South India. The presence of resistant genes like mecA, vanA of Staphylococcus aureus and bla _SHV, bla _TEM of Pseudomonas aeruginosa was confirmed by molecular studies. The isolated compound from Streptomyces rochei M32 inhibited wide range of standard and clinical drug resistant pathogens and enteric pathogens. The rice bran supplemented basal medium influenced the active compound production on 8th day of fermentation and yielded 1875 mg of crude extract from 10 g of rice bran substrate. Purification and characterization of crude ethyl acetate extract was achieved by preparative thin layer chromatography. The active fraction was identified as terpenoid class compound by chemical screening. Based on the results of spectral studies (NMR, LC–MS, FTIR, etc.), the active compound was tentatively identified as 1, 19-bis (3-hydroxyazetidin-1-yl) nonadeca-5, 14-diene-1, 8, 12, 19-tetraone with molecular weight 462.41 g/mol. Minimum inhibitory concentration value ranges between 7.6 and 31.2 µg/mL against test organisms was observed. The cytotoxicity results on cervical cancer (HeLa) cell line showed IC₅₀ value of 2.034 µg/mL. The corresponding compound is not previously reported from any microbial resources.     

Enhancement of medium-chain-length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis>

Objectives

To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHA_MCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways.

Results

Pseudomonas mendocina NK-01 produces PHA_MCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHA_MCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(?alg), NK-U-2 (?alg?galU), NK-U-3(alg?galU?rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHA_MCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHA_MCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHA_MCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHA_MCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %.

Conclusion

The PHA_MCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.