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141.
Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.  相似文献   
142.
Lipocalin-2 (Lcn2, NGAL) is a member of the lipocalin super family with diverse function such as the induction of apoptosis, the suppression of bacterial growth, and modulation of inflammatory response. Much interest has recently been focused on the physiological/pathological role of the lipocalin-2 that is considered to be a novel protective factor against oxidative stress. However, its precise biological roles in this protection are not fully understood. In this report we intended to test the effect of lipocalin-2 on the expression of heme oxygenase (1, 2) and superoxide dismutase (1, 2) which are two strong antioxidants. NGAL was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing NGAL and the expression of HO-1, 2 and SOD1, 2 were compared with appropriate controls by RT-PCR and western blot. On the other hand, expression of NGAL was suppressed by siRNA transfection in order to study the effect of lipocalin-2 on mentioned genes/proteins. The results showed that the expression of HO-1 and SOD1, 2 enzymes were higher in cells expressing recombinant lipocalin-2 compared with the control cells. Although the expression of HO-1 was lower in NGAL silencing cells, the expression of SOD1 and SOD2 were higher. Our data suggest that NGAL is a potent inducer of HO-1 and somewhat SOD1 and SOD2 and it appears that part of antioxidant property of NGAL could be attributed to the induction of HO-1and SOD1, 2.  相似文献   
143.
Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different contaminants analyzed are considered. The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.  相似文献   
144.
A petroleum ether extract of Kadsura longipedunculata enhanced the GABA-induced chloride current (IGABA) by 122.5 ± 0.3% (n = 2) when tested at 100 μg/ml in Xenopuslaevis oocytes expressing GABA A receptors (α1β2γ2S subtype) in two-microelectrode voltage clamp measurements. Thirteen compounds were subsequently identified by HPLC-based activity profiling as responsible for GABA A receptor activity and purified in preparative scale. 6-Cinnamoyl-6,7-dihydro-7-myrceneol and 5,6-dihydrocuparenic acid were thereby isolated for the first time. The determination of the absolute stereochemistry of these compounds was achieved by comparison of experimental and calculated ECD spectra. All but one of the 13 isolated compounds from K. longipedunculata potentiated IGABA through GABA A receptors composed of α1β2γ2S subunits in a concentration-dependent manner. Potencies ranged from 12.8 ± 3.1 to 135.6 ± 85.7 μM, and efficiencies ranged from 129.7 ± 36.8% to 885.8 ± 291.2%. The phytochemical profiles of petroleum ether extracts of Kadsura japonica fruits (114.1 ± 2.6% potentiation of IGABA at 100 μg/ml, n = 2), and Schisandra chinensis fruits (inactive at 100 μg/ml) were compared by HPLC-PDA-ESIMS with that of K. longipedunculata.  相似文献   
145.
The K121Q polymorphism of the ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1) gene has been variably associated with insulin resistance and type 2 diabetes (T2D) in several populations. However, this association has not been studied in Iranian subjects and we hypothesized that the K121Q variant might be associated with T2D and related metabolic traits in this population. The K121Q genotypes were determined by PCR-restriction fragment length polymorphism in 377 normoglycemic controls and 155 T2D patients. T2D patients had significantly higher values for systolic and diastolic blood pressure, BMI, glucose, cholesterol, triglyceride, LDL, apoB, insulin, and HOMA-IR, and lower levels of HDL than the normoglycemic subjects. The frequency of the Q allele did not differ between T2D and normoglycemic subjects (OR 0.96, 95% CI 0.90-2.00, P?=?0.70). The Q allele frequency was 16.5% in T2D and 15.2% in normoglycemic subjects. The ENPP1 genotype (KQ?+?QQ) was not associated with the systolic and diastolic blood pressure, glucose, triglyceride, cholesterol, LDL-C and HDL-C, apo B, BMI, HOMA-IR, and insulin levels in both normoglycemic and T2D groups. Our results suggest that the ENPP1 121Q allele might not be associated with T2D and related metabolic traits among Iranian subjects.  相似文献   
146.
The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAELB400) are largely unknown. BphAEp4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAELB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAELB400 and BphAEp4 and examine the biochemical properties of two BphAELB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.  相似文献   
147.
Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.  相似文献   
148.
The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.  相似文献   
149.
Scutellaria L. in Lamiaceae subfamily Scutellarioideae is a subcosmopolitan genus with about 360 species. According to the latest subgeneric classification, two subgenera are recognized: S. subgen. Scutellaria and S. subgen. Apeltanthus. We studied pollen grains from 29 species of Scutellaria belonging to both subgenera and from both the Old and the New World to evaluate their taxonomic importance for the infrageneric classification of the genus. Pollen grains were acetolysed and studied by scanning electron microscope (SEM) and light microscope (LM). All examined pollen grains are isopolar, tricolpate, suboblate, oblate spheroidal, prolate spheroidal to subprolate. The exine is mainly bireticulate perforate, but with variations in lumina shape and size as well as perforation size and uniformity. Based on the exine sculpturing, two subtypes are recognized within the bireticulate pollen type. In subtype I the primary reticulum of the exine consists of angular muri; the lumen has an irregular polygonal shape and the perforations of the secondary reticulum are more or less uniform and of the same size. This pollen subtype was observed in species of subgenus Scutellaria, sectt. Scutellaria p.p., Anaspis, Perilomia and Salazaria. In subtype II the primary reticulum of the exine consists of a more or less curved murus; the lumen is rounded or obtusely angular and the secondary reticulum consists of one or more large central perforations surrounded by some smaller ones. This pollen subtype was observed in species of subgenus Scutellaria sectt. Scutellaria p.p., Salviifoliae and in all members of subgenus Apeltanthus. The results of this study reveal that the exine ornamentation is a diagnostic character useful for the infrageneric classification of Scutellaria.  相似文献   
150.
To study the effect of an exogenous cytokinin application on safflower yield, an experiment was conducted in 2012–2013. Two cultivars of safflower (Goldasht and Zendehrood) and five concentrations of 6-benzylaminopurine (BAP) (0, 25, 50, 75, and 100 μM) were applied at the flowering stage. Results indicated that the application of 75 μM of BAP showed increased seed and oil yield by 17.54 and 18.29 % over the control, respectively. The increase in seed yield by application of BAP was attributed to the increase in characters like number of heads per plant, number of seeds per head, and 1,000 seed weight. Applying of BAP increased oil content compared with the control. To determine the concentration of cytokinin which has the highest performance for increasing seed yield, regression analysis were estimated showing that in the Zendehrood cultivar, the application of 43 μM of BAP produced the highest seed yield, and in the Goldasht cultivar the application of 73 μM of BAP during flowering produced the highest seed yield.  相似文献   
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