Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

Genetic behavior of earliness and yield traits of some rice (Oryza sativa L.) genotypes

Rice (Oryza sativa L.) is a critical staple food crop that provides more than half of the world's population with its primary nutritional source. Breeders and growers of rice would profit from robust genotypes with improved morphological and yield-related characteristics. The aim of this work is to determine the nature and magnitude of gene action on yield quantity and quality, to define the best combinations of earliness and yield characters, develop hybrids that perform better on yield quantity and quality. Three replications were used in the experiment's randomized complete block design (RCBD). During the 2016 season, seven different parents, namely Sakha 101, Sakha 104, Sakha 105, Giza 177, Black rice 1, Black rice 2, and Black rice 3, were crossed using A 7 × 7 half-diallel set analysis without reciprocals to generate 21 F1 crosses. The results indicated that genotype-dependent mean squares were very significant for main characteristics. Significant combining ability SCA variance estimates were more considerable than general combining ability (GCA) variance for all characters except days to 50% flowering. It demonstrated that both additive and non-additive genetic variance played a role in expressing the attributes investigated. The Parents, Black rice, Sakha 105, and Sakha 101, were recognized as the best general combiner for most growth and yield attributes. Sakha105 × Black Rice 1, Sakha105 × Black Rice 2, Sakha101 × Sakha104, Sakha105 × Giza 177, and Sakha101 × Giza 177 all demonstrated non-additive gene activity for the majority of maturity and yield traits. Heterosis breeding would be most efficient for qualities where high performance was determined by dominance and dominance gene effects. The increased yield of crosses results from parents with a diverse genetic background and genetic diversity.     

The interaction between body position and vibration frequency on acute response to whole body vibration

PurposeThe present study was designed to investigate the electromyographic (EMG) response in leg muscles to whole-body vibration while using different body positions and vibration frequencies.MethodsTwenty male sport sciences students voluntarily participated in this single-group, repeated-measures study in which EMG data from the vastus lateralis (VL) and the lateral gastrocnemius (LG) were collected over a total of 36 trials for each subject (4 static positions × 9 frequencies).ResultsWe found that vibration frequency, body position and the muscle stimulated had a significant effect (P-values ranged from 0.001 to 0.031) on the EMG response. Similarly, the muscle × frequency and position × muscle interactions were significant (P < 0.001). Interestingly, the frequency × positions interactions were not significant (P > 0.05).ConclusionsOur results indicate that lower frequencies of vibration (25–35 Hz) result in maximal activation of LG, whereas higher frequencies (45–55 Hz) elicit the highest responses in the VL. In addition, the position P2 (half squat position with the heels raised) is beneficial both for VL and LG, independently of the vibration frequency.     

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression,purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V_max and K_m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.     

Investigations into specificity of azepinomycin for inhibition of guanase: Discrimination between the natural heterocyclic inhibitor and its synthetic nucleoside analogues

In our long and broad program to explore structure–activity relationships of the natural product azepinomycin and its analogues for inhibition of guanase, an important enzyme of purine salvage pathway of nucleic acid metabolism, it became necessary to investigate if the nucleoside analogues of the heterocycle azepinomycin, which are likely to be formed in vivo, would be more or less potent than the parent heterocycle. To this end, we have resynthesized both azepinomycin (1) and its two diastereomeric nucleoside analogues (2 and 3), employing a modified, more efficient procedure, and have biochemically screened all three compounds against a mammalian guanase. Our results indicate that the natural product is at least 200 times more potent toward inhibition of guanase as compared with its nucleoside analogues, with the observed K_i of azepinomycin (1) against the rabbit liver guanase = 2.5 (±0.6) × 10^?6 M, while K_i of Compound 2 = 1.19 (±0.02) × 10^?4 M and that of Compound 3 = 1.29 (±0.03) × 10^?4 M. It is also to be noted that while IC₅₀ value of azepinomycin against guanase in cell culture has long been reported, no inhibition studies nor K_i against a pure mammalian enzyme have ever been documented. In addition, we have, for the first time, determined the absolute stereochemistry of the 6-OH group of 2 and 3 using conformational analysis coupled with 2-D ¹H NMR NOESY     

Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates

Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (k_i) ranging from 0.3 × 10⁵ M^?1 min^?1 to 10.4 × 10⁵ M^?1 min^?1. When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE’s (? 1 × 10⁷ M^?1 min^?1) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling.     

Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.     

Diether derivatives of homo- or substituted piperidines as non-imidazole histamine H3 receptor ligands

Synthesis and biological activities of a series of homo- or substituted piperidine unsymmetrical diethers are described. The novel compounds were evaluated for histamine H₃ receptor binding affinities at recombinant human H₃ receptor stably expressed in HEK-293 cells. All diethers showed in vitro affinities in nanomolar concentration range. The most potent compounds are 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]-3-methylpiperidine 11 (K_i = 3.2 nM) and 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]azepane 13 (K_i = 3.5 nM).     

Synthesis of substituted diphenyl sulfones and their structure–activity relationship with the antagonism of 5-НТ6 receptors

Substituted diphenyl sulfones (10a–n) were synthesised, and the structures were confirmed by NMR, LC–MS and X-ray crystallography. Their antagonistic activities towards 5-HT₆ receptor were assessed in a cell-based functional assay. Diphenyl sulfone 10a, in spite of being the smallest and simplest known sulfonyl-containing 5-HT₆R antagonist, showed a strong potency (K_i = 1.6 μM). Its derivative with a methylamine substituent, 10g (N-methyl-2-(phenylsulfonyl)aniline), was ～66-times as active as diphenyl sulfone (K_i = 24.3 nM). Addition of a piperazinyl moiety in the para-position relative to the sulfonyl group in compound 10m (N-methyl-2-(phenylsulfonyl)-5-piperazin-1-ylaniline) led to a further 150-fold increase in potency (K_i = 0.16 nM) to block the serotonin-induced response of HEK-293 cells that were stably transfected with the human recombinant 5-HT₆ receptor.     

Internal conductance to CO2 transfer of adult Fagus sylvatica: Variation between sun and shade leaves and due to free-air ozone fumigation

Two separate objectives were considered in this study. We examined (1) internal conductance to CO₂ (g_i) and photosynthetic limitations in sun and shade leaves of 60-year-old Fagus sylvatica, and (2) whether free-air ozone fumigation affects g_i and photosynthetic limitations. g_i and photosynthetic limitations were estimated in situ from simultaneous measurements of gas exchange and chlorophyll fluorescence on attached sun and shade leaves of F. sylvatica. Trees were exposed to ambient air (1× O₃) and air with twice the ambient ozone concentration (2× O₃) in a free-air ozone canopy fumigation system in southern Germany (Kranzberg Forest). g_i varied between 0.12 and 0.24 mol m⁻² s⁻¹ and decreased CO₂ concentrations from intercellular spaces (C_i) to chloroplastic (C_c) by approximately 55 μmol mol⁻¹. The maximum rate of carboxylation (V_cmax) was 22–39% lower when calculated on a C_i basis compared with a C_c basis. g_i was approximately twice as large in sun leaves compared to shade leaves. Relationships among net photosynthesis, stomatal conductance and g_i were very similar in sun and shade leaves. This proportional scaling meant that neither C_i nor C_c varied between sun and shade leaves. Rates of net photosynthesis and stomatal conductance were about 25% lower in the 2× O₃ treatment compared with 1× O₃, while V_cmax was unaffected. There was no evidence that g_i was affected by ozone.     

Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.     

Synthesis and evaluation of novel azetidine analogs as potent inhibitors of vesicular [3H]dopamine uptake

Lobelane analogs that incorporate a central piperidine or pyrrolidine moiety have previously been reported by our group as potent inhibitors of VMAT2 function. Further central ring size reduction of the piperidine moiety in lobelane to a four-membered heterocyclic ring has been carried out in the current study to afford novel cis-and trans-azetidine analogs. These azetidine analogs (15a–15c and 22a–22c) potently inhibited [³H]dopamine (DA) uptake into isolated synaptic vesicles (K_i ? 66 nM). The cis-4-methoxy analog 22b was the most potent inhibitor (K_i = 24 nM), and was twofold more potent that either lobelane (2a, K_i = 45 nM) or norlobelane (2b, K_i = 43 nM). The trans-methylenedioxy analog, 15c (K_i = 31 nM), was equipotent with the cis-analog, 22b, in this assay. Thus, cis- and trans-azetidine analogs 22b and 15c represent potential leads in the discovery of new clinical candidates for the treatment of methamphetamine abuse.     

Synthesis and monoamine transporter affinity of 3α-arylmethoxy-3β-arylnortropanes

A series of 3-arylnortrop-2-enes and 3α-arylmethoxy-3β-arylnortropanes were synthesized and evaluated for binding affinity at monoamine transporters. The 3-(3,4-dichlorophenyl)nortrop-2-ene (6e) exhibited high affinity for the SERT (K_i = 0.3 nM). The 3α-arylmethoxy-3β-arylnortropanes were generally SERT selective with the 3α-(3.4-dichlorophenylmethoxy)-3βphenylnortrop-2-ene (7c) possessing subnanomolar potency (K_i = 0.061 nM). However, 3α-(3,4-dichlorophenylmethoxy)-3β-phenylnortrop-2-ene (7b) exhibited high affinity at all three transporters [(DAT K_i = 22 nM), (SERT K_i = 6 nM) and (NET K_i = 101 nM)].     

Typha × glauca dominance and extended hydroperiod constrain restoration of wetland diversity

Urban wetlands typically have few plant species. In wetlands designed to improve water quality, nutrient-rich water and highly variable water levels often favor aggressive, flood-tolerant plants, such as Typha × glauca (hybrid cattail). At Des Plaines River Wetlands Demonstration Site (Lake Co., IL), we assessed T. × glauca dominance and plant community composition under varying hydroperiods in a complex of eight constructed wetlands. Plots flooded for more than 5 weeks during the growing season tended to be dominated by T. × glauca, while plots flooded fewer days did not. Plots with high cover of T. × glauca had low species richness (negative correlation, R² = 0.72, p < 0.001). However, overall species richness of the wetland complex was high (94 species), indicating that wetlands in urbanizing landscapes can support many plant species where T. × glauca is not dominant. T. × glauca-dominated areas resisted the establishment of a native plant community. Removing T. × glauca and introducing native species increased diversity initially, but did not prevent re-invasion. Although 12 of the 24 species we seeded became established in our cleared plots, T. × glauca rapidly re-invaded. In year 1, T. × glauca regained an average of 11 ramets m⁻², and its density doubled in year 2. The likelihood of planted species surviving decreased as duration of inundation increased, and in both seeded and planted plots, graminoids had greater survivorship through year 2 than forbs across a range of water levels. Within 4 years, however, T. × glauca was the most common plant, present in 92% of the cleared plots. Simply removing T. × glauca and adding propagules to an urban wetland is not sufficient to increase diversity.     

2,5-Diaryloxadiazoles and their precursors as novel inhibitors of cathepsins B,H and L

High levels of cathepsins indicated in various pathological conditions like arthritis, cancer progressions, and atherosclerosis explains the need to explore potential inhibitors of these proteases which can be of great therapeutic significance. We, in the present work, report the synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L. Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their K_i values have been calculated as 11.38 × 10⁻⁸ M and 66.4 × 10⁻⁸ M for cathepsin B and 4.2 × 10⁻⁹ M and 47.31 × 10⁻⁹ M for cathepsin L, respectively. However, cathepsin H activity was maximally inhibited by compounds, 1e and 2c with K_i values of 4.4 × 10⁻⁷ M and 5.6 × 10⁻⁷ M, respectively. Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes. The results have been compared with docking results obtained using iGemDock.     

Alpha and quantum yield of aquatic plants derived from PAM fluorometry: Uses and misuses

The slope of the initial linear range of a photosynthesis–irradiance (P–I) curve, alpha (α), is frequently, but often incorrectly, used to denote the maximal quantum yield (or the “efficiency” of photosynthesis) of higher plants and macroalgae under the conditions for which the P–I curve was measured. When using the increasingly popular method of pulse amplitude modulated (PAM) fluorometry, the determination of α from so-called rapid light curves (RLC) may lead to misinterpretations when comparing photosynthetic efficiencies under different environmental conditions. Furthermore, since PAM fluorometry measures the quantum yield (Y) directly, there may be no need to estimate it from the initial slopes of RLCs.We compared photosynthetic parameters derived from RLCs of Ulva sp. measured during winter and summer, and show large differences in α when electron transport rates (ETR) were plotted against incident irradiance (I_i) [α = 0.26 ± 0.00 versus 0.08 ± 0.01 during the winter (November–December) and summer (July–August), respectively], as is usually done. On the other hand, no differences in the initial slopes of the RLCs were apparent when plotting ETR versus the absorbed irradiance (I_a) (initial slope = 0.75 ± 0.01 versus 0.62 ± 0.12 during the winter and summer, respectively); this is called for since also ETR is calculated using I_a. Using the I_a based RLCs, it was also found that the values of the initial slopes equalled those of the first Y-value measurements of the RLCs (Y₀) (t-test, p > 0.05, r² = 0.85). Therefore, when using PAM fluorometry, we suggest (a) to present the x-axis of RLCs as I_a (I_i × AF × 0.5), and ETR on the y-axis as Y × I_a, and (b) that Y₀ can be taken as a correct measure of the maximal quantum yield instead of estimating it from an RLC.     

NAD-based inhibitors with anticancer potential

Three classes of novel inhibitors of inosine monophosphate dehydrogenase have been prepared and their anti-proliferative properties were evaluated against several cancer cell lines.(1) Mycophenolic adenine dinucleotide analogues (8–13) containing a substituent at the C2 of adenine ring were found to be potent inhibitors of IMPDH (K_i’s in range of 0.6–82 nM) and sub-μM inhibitors of leukemic K562 cell proliferation. (2) Mycophenolic adenosine (d and l) esters (20 and 21) showed a potent inhibition of IMPDH2 (K_i = 102 and K_i = 231 nM, respectively) and inhibition of K562 cell growth (IC₅₀ = 0.5 and IC₅₀ = 1.6 μM). These compounds serve both as inhibitors of the enzyme and as a depot form of mycophenolic acid. The corresponding amide analogue 22, also a potent inhibitor of IMPDH (K_i = 84 nM), did not inhibit cancer cell proliferation. (3) Mycophenolic-(l)- and (d)-valine adenine di-amide derivatives 25 (K_i = 9 nM) and 28 (K_i = 3 nM) were found to be very potent enzymatically, but did not inhibit proliferation of cancer cells.     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.     

Synthesis and CB1 cannabinoid receptor affinity of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles

A series of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles were synthesized regioselectively using click chemistry and evaluated at CB1 cannabinoid receptors. The n-propyl ester 11 (K_i = 4.6 nM) and phenyl ester 14 (K_i = 11 nM) exhibited the most potent affinity of the series.