Min-1 Mg-1 Protein Research Articles

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76–93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min−1 mg−1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

Response of selected scion and rootstock grape (<i>Vitis </i>spp.) genotypes to induced drought stress

Climate change is expected to elevate drought frequency, straining agricultural freshwater resources. Developing drought-tolerant grapevine varieties is crucial. This study examined grape scion and rootstock genotypes under well-watered (WW) and induced-drought (ID) conditions. ID treatment reduced vine length by 11.34-35.15%, with Vitis parviflora, 110R, and Male Hybrid rootstocks showing superior growth. Root length increased under ID, indicating an adaptive moisture-seeking response. The ID treatment led to substantial reduction in leaf count and average leaf area, especially in Flame Seedless (27.71 and 19.07 cm2, respectively). Drought stress elevated chlorophyll a:b ratio, affecting chlorophyll degradation in different genotypes. Significant variations were observed in leaf and root iron (Fe) and zinc (Zn) contents. Enzyme activities particularly peroxidase and polyphenol oxidase especially rose under drought, particularly in V. parviflora (3.39 μM guaiacol min-1 mg-1 protein and 1.33EU/ml/min respectively) likely to be contributing in drought tolerance mechanism. Principal component analysis (PCA) highlighted impact of traits on genotypes, emphasizing V. parviflora, Male Hybrid and Pusa Navrang as superior drought stress tolerant genotypes. Genotype clustering confirmed distinct groupings, while, correlation analysis unveiled intricate trait interactions.

Plant growth-promoting and biocontrol traits of endophytic Bacillus licheniformis against soft rot causing Pythium myriotylum in ginger plant.

Bacterial endophytes from plants harbor diverse metabolites that play major roles in biocontrol and improve plant growth. In this study, a total of 12 endophytic bacteria were isolated from the ginger rhizome. The strain K3 was highly effective in preventing mycelia growth of Pythium myriotylum (78.5 ± 1.5% inhibition) in dual culture. The cell-free extract (2.5%) of endophyte K3 inhibited 76.3 ± 4.8% mycelia growth, and 92.4 ± 4.2% inhibition was observed at a 5% sample concentration. The secondary metabolites produced by Bacillus licheniformis K3 showed maximum activity against Pseudomonas syringae (24 ± 1 mm zone of inhibition) and Xanthomonas campestris (28 ± 3 mm zone of inhibition). The strain K3 produced 28.3 ± 1.7 IU mL-1 protease, 28.3 ± 1.7 IU mL-1 cellulase, and 2.04 ± 0.13 IU mL-1 chitinase, respectively. The ginger rhizome treated with K3 in the greenhouse registered 53.8 ± 1.4% soft rot incidence, and the streptomycin-treated pot registered 78.3 ± 1.7% disease incidence. The selected endophyte K3 improved ascorbate peroxidase (1.37 ± 0.009 µmole ASC min-1 mg-1 protein), catalase (8.7 ± 0.28 µmole min-1 mg-1 protein), and phenylalanine ammonia-lyase (26.2 ± 0.99 Umg-1) in the greenhouse. In addition, K3 treatment in the field trial improved rhizome yield (730 ± 18.4 g) after 180 days (p < 0.01). The shoot length was 46 ± 8.3 cm in K3-treated plants, and it was about 31% higher than the control treatment (p < 0.01). The lytic enzyme-producing and growth-promoting endophyte is useful in sustainable crop production through the management of biotic stress.

Effect of Seed Priming with Plant Growth Regulators on Growth Parameters, Biochemical Changes of Rice (Oryza sativa L.)

The investigation entitled “Effect of seed priming with plant growth regulators on growth parameters, biochemical changes of rice (Oryza sativa L.)” was conducted during kharif season, 2018 at Student Instructional Farm (SIF) of Acharya Narendra Deva University of Agriculture &amp; Technology, Kumarganj, Ayodhya-224 229 (U.P.) in randomized block design with eight treatments and three replications on rice variety Sarju-52.The treatments were comprised of priming with plant growth regulators of different concentrations viz salicylic acid (50 ppm, 100 ppm and 150 ppm), KNO3 (1%, 1.5% and 2%) along with unprimed control and hydro-priming. Seed priming was done for 12 hours in different treatments and nursery was raised. Twenty one days old seedlings were transplanted. Observations were recorded on various growth parameters at 30, 60, 90 DAT such as leaf area index (LAI), relative water content (RWC), biochemical parameters like chlorophyll content (SPAD Value), catalase activity (EU mg-1 protein min-1), total soluble sugar (mg g-1 dry weight), at harvest stages. All the plant growth regulators viz salicylic acid (50 ppm, 100 ppm and 150 ppm) and KNO3 (1%, 1.5% and 2%) influenced positively on the growth characters, chlorophyll content, &amp; yield content in rice crop. Seed priming of KNO3 @ 2% was found superior followed by salicylic acid 150 ppm over rest of the treatments.

Potential of Y. lipolytica epoxide hydrolase for efficient production of enantiopure (R)-1,2-octanediol

The recombinant Yleh from a tropical marine yeast Yarrowia lipolytica NCIM 3589 exhibited a high epoxide hydrolase activity of 9.34 ± 1.80 µmol min-1 mg-1 protein towards 1,2-epoxyoctane (EO), at pH 8.0 and 30 °C. The reaction product was identified as 1,2-Octanediol (OD) by GC-MS using EO and H2O18 as substrate, affirming the functionality of Yleh as an epoxide hydrolase. For EO, the Km, Vmax, and kcat/Km values were 0.43 ± 0.017 mM, 0.042 ± 0.003 mM min-1, and 467.17 ± 39.43 mM-1 min-1, respectively. To optimize the reaction conditions for conversion of racemic EO by Yleh catalyst to enantiopure (R)-1,2-octanediol, initially, Response Surface Methodology was employed. Under optimized reaction conditions of 15 mM EO, 150 µg purified Yleh at 30 °C a maximal diol production of 7.11 mM was attained in a short span of 65 min with a yield of 47.4%. Green technology using deep eutectic solvents for the hydrophobic substrate (EO) were tested as co-solvents in Yleh catalyzed EO hydrolysis. Choline chloride-Glycerol, produced 9.08 mM OD with an increased OD yield of 60.5%. Thus, results showed that deep eutectic solvents could be a promising solvent for Yleh-catalyzed reactions making Yleh a potential biocatalyst for the biosynthesis of enantiopure synthons.

Molecular cloning, expression analysis and functional characterization of chicken cytochrome P450 27A1: A novel mitochondrial vitamin D3 25-hydroxylase

Vitamin D3 is hydroxylated by cytochrome P450 (CYP) before exerting biological effects. The chicken CYP involved in vitamin D3 25-hydroxylation has yet to be cloned, and little is known about its functional characteristics, tissue distribution, and cellular expression. We identified a novel, full-length CYP27A1 gene cloned from chicken hepatocyte cDNA that encodes a putative protein of 518 amino acids. Swiss modeling revealed that chicken CYP27A1 has a classic open-fold form. Multisequence homology alignment determined that CYP27A1 contains conserved motifs for substrate recognition and binding. Quantitative real-time PCR analysis in 2-mo-old Partridge Shank broilers demonstrated that CYP27A1 mRNA levels were highest in the liver, followed by the thigh muscles, the breast muscles, and kidneys. The transcripts of CYP27A1 in breast muscles were significantly higher in males than in females. A subcellular localization analysis demonstrated that CYP27A1 was mainly expressed in the mitochondria. In vitro enzyme assays suggested that recombinant CYP27A1 hydroxylates vitamin D3 at the C-25 position to form 25-hydroxyvitamin D3 (25(OH)D3). The Km and Vmax values for CYP27A1-dependent vitamin D3 25-hydroxylation were estimated to be 4.929 μM and 0.389 mol min-1 mg-1 protein, respectively. In summary, these results suggest that CYP27A1 encodes a mitochondrial CYP that plays an important physiologic role in the 25-hydroxylation of vitamin D3 in chickens, providing novel insights into vitamin D3 metabolism in this species.

Carum carvi mediated green synthesis of copper nanoparticles and its effect on Solanum lycopersicum seedlings

The present study aims to synthesis the copper nanoparticles (CuNPs) and their effect on the biochemical and physiological characteristics of Solanum lycopersicum. The results exhibited the color change in the Carum carvi aqueous extract from clear deep brown to a greenish color and this observation indicated the green-biosynthesis of CuNPs by reduction of Cu+ to Cu0. Also, the absorbance broadening band for the green-biosynthetic CuNPs appeared at the 340 nm wavelength using UV-Vis but the C. carvi aqueous extract not showed any peaks at this wavelength. SEM analysis exhibited the micrographic surface morphology and the shape of the green-biosynthetic CuNPs with a scan area of 50 μm and showed the spherical shape particles of CuNPs aggregation. The three-dimensional image and the surface morphology of green-biosynthesized CuNPs and C. carvi aqueous extract were examined using AFM analysis that showed the surface of C. carvi aqueous extract was 45.5 nm size with non-homologus and irregular form of distribution, but the green-biosynthesized CuNPs were 12.4 nm size in nanoscale with regular and homogenous distribution form. The results also showed that the effect of bio-synthesized copper nanoparticles was evident on the S. lycopersicum seedlings fresh and dry weight according to the different reading times after treatment with nanoparticles. Also, the concentration of 2.5 mg/mL (CuNPs) showed a significant increase in the chlorophyll content (58.51 μg/cm²) on the 21st day after treatment and a significant increase in the activity of peroxidase enzyme (35.12 U min-1 mg-1 protein) was obtained at the concentration of 2.5 mg/mL (CuNPs) at 21st day after germination.

Biochemical mechanisms of the energy-protective action of blockers of slow high-threshold L-type calcium channels

This review discusses information about the structure and function of calcium channels in the plasma membrane and mitochondria of the heart, and pharmacological methods for modulating their conductance. Experimental data are presented that characterize the change in the energy metabolism of cardiomyocytes against the background of the transformation of the conductivity of L-type calcium channels of the cell membrane in a non-invasive model of vibration-mediated (56 sessions of total vertical vibration, with a frequency of 44 Hz and an amplitude of 0.5 mm) hypoxia.&#x0D; It was shown that in animals treated with calcium channel blocker adalat (nifedipine INN) against the background of vibration, the rate of endogenous respiration (Ve), measured by the polarographic method using a closed Clark electrode in native homogenate of rabbit myocardial tissue, remained at the level of intact animals and amounted to 16.3 4.3 ng-O atom/ min mg of protein, amytal sensitivity increased by 39% (p 0.05) compared to the group of vibrated animals, low-natality decreased by 40% (p 0.05). The dynamics of the rate of substrate respiration (Vac and Vglu + mal) in the group with adalat returned to that of intact animals, which indicated the restoration of the physiological predominance of the activity of theNADH CoQ-reductase complex in redox reactions. It was found that the blockade of transport of Ca2+ ions at the level of high-threshold (HVA) voltage-dependent ion channels of the L-type of the cell membrane, normalizing the activity of the I enzyme-substrate complex of the respiratory chain and regulatoryly restraining the hyperactivity of succinate dehydrogenase in zone II of the enzyme-substrate complex, has an energy-protective effect. Adalat prevented a low-energy shift and the development of bioenergetic hypoxia in the myocardial tissue of experimental animals.

Seed priming with endophytes on physiological, biochemical and antioxidant activity of hybrid maize (Zea mays l.) COH (M) 8 seeds

Endophytes are important microorganisms that enhance the plant's stability through a symbiotic relationship, without any harmful effects and symptoms in the host plant. To study the effect of endophytes on overall performance of COH(M)8 hybrid maize seeds, the present study was conducted with different endophytic seed priming for 12 hrs duration with Beauveria bassiana @ 5% (T2), Metarhizium anisopliae @ 5% (T3) and Bacillus subtilis @ 8% (T4) along with hydro priming (T1) and untreated control (T0). The seed priming treatments with all the above three endophytes enhanced the seed quality parameters, among which M. anisopliae @ 5% (T3) registered maximum increase of germination (4.34%), shoot length (20.73%), root length (15.04%), dry matter production (15.22%) and vigour index (22.68%) over control. Similarly, the seeds primed with M. anisopliae @ 5% (T3) recorded the highest value of dehydrogenase activity (0.441 OD value), α- amylase activity (2.06 mg maltose min-1) and antioxidant activity viz., catalase (1.55 μmol H2O2 min-1 g-1 protein) and peroxidase (0.87 U mg-1protein min-1). Results of this study revealed that the endophytes can enhance overall the seed quality in maize.

Metarhizium anisopliae and Isaria fumosorosea challenge the survival and immunity of the palm weevil, Rhynchophorus ferrugineus Olivier

Aim of study: Rhynchophorus ferrugineus Olivier is an invasive pest of palm worldwide. The use of insecticides by farmers for its management has been found insignificant. This study evaluated the potential use of entomopathogenic fungi for R. ferrugineus management with a particular focus on the fungal infection on the activities of different detoxification enzymes. Area of study: Grubs and adults of R. ferrugineus were collected from various infested date palm fields in the four provinces of Pakistan. Material and methods: Fungi Isaria fumosorosea (If-02) and Metarhizium anisopliae (Ma-M2) were evaluated against R. ferrugineus, and its immune responses were biochemically characterized. Main results: The highest mortality rate was recorded at concentration 3×108 spores mL-1 on the 7th day post infection in the populations treated with M. anisopliae from Punjab, Khyber Pakhtunkhwa (KPK), Sindh and Baluchistan (93.75, 90.0, 90.0 and 81.25% respectively). M. anisopliae with lowest LC50 (1.1×106 spores mL-1) from Sindh also proved to be the most lethal fungus against R. ferrugineus. Maximum acetylcholinesterase (AChE) and glutathione S-transferases (GSTs) activities were observed in Baluchistan (26.28 and 24.0 μmol min-1 mg-1 protein, respectively) and maximum esterases (EST) activity (35.4 μmol min-1 mg-1 protein) was observed in the KPK population on the 3rd-day post I. fumosorosea infection. Research highlights: Fungal infection by I. fumosorosea caused a significant increase in AChE, GST and EST activities which may hinder R. ferrugineus development. However, M. anisopliae, to some extent, also inhibited enzyme activities and yielded a sudden increase in mortality. Future bio-pesticides could be developed for integrated pest management (IPM) of palm weevil.

Salicylic acid mediated physiological and biochemical alterations to confer drought stress tolerance in Zinnia ( Z. elegans )

Background: Protective role of salicylic acid against drought has been widely reported, but its effects on Zinnia elegans remain unknown.&#x0D; Hypotheses: Foliar salicylic acid (SA) improves water status, pigments, and antioxidant systems of Zinnia plants under drought stress.&#x0D; Studied species: Z. elegans (cv. Dreamland) were purchased from Chanan Seeds Store Lahore, Pakistan.&#x0D; Study site and dates: MNS University of Agriculture, Multan, Punjab, Pakistan; March-May, 2018.&#x0D; Methods: After two weeks of transplanting, one set of seedlings was exposed to drought stress (60 % field capacity, FC) while other control set was maintained at 100 % FC. Foliar SA (100 mg L-1) was applied to plants at drought initiation.&#x0D; Results: Foliar SA treatment to water stressed plants (60 % FC) increased the leaf area (46.89 cm2), shoot and root lengths (25 cm and 26.67 cm, respectively), leaf relative water content (75.98 %); chlorophyll a (0.68 mg g-1), chlorophyll b (0.38 mg g-1) and carotenoid concentrations (1.10 mg g-1); and catalase (116.67 U min-1 mg-1 protein), guaiacol peroxide (72 U min-1 mg-1 protein) and superoxide dismutase (93 U min-1 mg-1 protein) activities.&#x0D; Conclusions: Foliar SA could be used to minimize impacts of water stress in Zinnia plants.

Effect of Plant Growth Regulators on Biochemical Studies of Mustard [Brassica juncea (L.) Czern &amp; Coss.] under Sodic Soil

A field study was conducted to evaluate the “Effect of plant growth regulators on biochemical parameters of mustard [Brassica juncea (L.) Czern &amp; Coss.] under sodic soil.” An experiment was laid out in randomized block design with seven treatments and three replications. The field experiment treatments consist of foliar spray with GA3 (50ppm, 100ppm, 150ppm) and salicylic acid (100ppm, 200ppm, 300ppm) were prepared on weight by volume basis as per desired concentration. Data confined to physiological changes like chlorophyll content, catalase activity were recorded at 40, 60 and 80 DAS. Oil content (%) in mature seeds was recorded at maturity. Significantly higher chlorophyll contents and catalase activity in leaves were estimated with the foliar spray of 100 ppm GA3 (2.51, 5.05, 2.24) chlorophyll content mg g-1 fresh weight and (69.17, 173.80, 171.30) catalase activity EU mg-1 protein min-1 and Significantly higher oil content (%) in mature seeds of mustard were estimated with the foliar application of 100 ppm GA3 (40.67 %) as compared to control. These biochemical parameters superior with foliar application of GA3 100ppm followed by Salicylic acid 200ppm.

Induction of Biochemical Changes in Santa Teriza Lime Leaves by Chitosan Application Influence Citrus Leaf Miner Damage

The effect of foliar spray of young lemon trees with Chitosan (CH) (200 - 300 - 400 ppm), on antioxidant enzymes activities (peroxidase POD, polyphenol oxidase PPO) and total phenolic content, and their influence on leaf miner (Phyllocnistis citrella) activity (tunnel length and damage density) under field condition was investigated. The results showed that treatment with chitosan 300 ppm enhanced the total phenolic content (5.975 mg/g) and the POD activity (0.533 min-1 mg-1 protein), while PPO activity was increased by chitosan 200 ppm (1.394 ΔA/min/g FW). The results demonstrated as well that chitosan treatment has a beneficial effect in reducing leaf miner activity, by decreasing tunnel length and damage density.

Characteration of a novel arylesterase from probiotics Lacticaseibacillus rhamnosus GG with the preference for medium- and long-chain p-Nitrophenyl esters.

The online version contains supplementary material available at 10.1007/s13205-021-03053-7.

Bio-molecule (Curcumin) Seed invigoration for extending the shelf-life of blackgram (Vigna mungo) under accelerated ageing

A study on curcumin seed invigoration for extending the storability of blackgram seeds under accelerated ageing was undertaken during 2021. Genetically pure seeds of blackgram (VBN 8) were treated with curcumin at different concentrations viz., 20,40,60,80,100 and 120 mg per kg. The treated seeds were subjected to accelerated ageing for 10 days. Every day of accelerated ageing, required quantity (50-100g) of seed samples were drawn and tested for physiological and biochemical seed quality attributes. The results revealed that the seeds invigorated with curcumin at 80 mg per kg of seeds significantly registered higher germination (66%), root (11.3cm) and shoot length (16.3cm) seedling vigour index I (1813) and II (12), alpha amylase (0.97µmol starch hydrolysed min-1 mg-1 protein) and dehydrogenase (1.642 OD value) enzymes activity. Overall results of this study conclude that blackgram seeds invigorated with curcumin at 80 mg per kg excelled the other treatments, irrespective of the accelerated ageing period.

Enhanced hydrolysis of β-cypermethrin caused by deletions in the glycin-rich region of carboxylesterase 001G from Helicoverpa armigera.

Carboxylesterase (CarE) is a major class of enzyme involved in the detoxification of toxic xenobiotics in various insect species. Previous work has shown that the carboxylesterase gene CarE001G found in Helicoverpa armigera is more active and can metabolize synthesized pyrethroids, such as β-cypermethrin, one of the commonly used commercial insecticides for lepidopteran pest control. In addition, CarE001G is very special as it has a very specific glycine-rich region located adjacent to its C-terminal. But whether mutations in this unique sequence can change the biochemistry and function of CarE001G are unknown. In this study, four variants of CarE001G with different deletions in the glycine-rich region were obtained and functionally expressed in Escherichia coli. The recombinant proteins were purified and confirmed by Western blot and mass spectrometry analyses. These mutant enzymes showed high catalytic efficiency toward the model substrate α-naphthyl acetate. Inhibition study showed that β-cypermethrin had relatively strong inhibition on CarE activities. In vitro metabolism assay showed that the mutant enzymes significantly enhanced their metabolic activities toward β-cypermethrin with specific activities between 4.0 and 5.6 nmol L-1 min-1 mg-1 protein. Molecular docking analyses consistently demonstrated that deletion mutations in the glycine-rich region may facilitate the anchoring of the β-cypermethrin molecule in the active binding pocket of the mutant enzymes. The data show that deletion mutations can cause qualitative change in the capacity of CarEs in the detoxification of β-cypermethrin. This indicates that deletion mutations in the glycine-rich region may have the potential to cause synthesized pyrethroid (SP) resistance in H. armigera in the future. © 2020 Society of Chemical Industry.

English

Drought induced oxidative stress triggers anti-oxidative system in cell to minimize oxidative damage where catalase plays vital role to neutralize H2O2. In this work, catalase activity was evaluated to examine the role of catalase in barley (Hordeum vulgare L.) seedlings under drought stress. As compared to control, catalase activity increased with decreasing soil moisture where 219% higher activity were recorded at 10% soil moisture of field capacity (FC) compared to control (75% FC), but was reduced below 10% FC. Four different catalase isozymes that specifically accumulated in barley leaves in response to drought (10% FC) which nominated catalase, particularly CAT4 and CAT2, as key players for H2O2 scavenging were identified. However, for future study, one catalase was purified from barley leaves with an apparent molecular weight of 54 kDA and specific activity of 871.32 &micro;mol min-1 mg-1 protein. Therefore, in this study, it was found that four CAT isozymes in barley leaf under drought, and the purified catalase needs characterization at molecular level for further biotechnical use.&nbsp; &nbsp; Key words: Catalase, isozymes, barley, drought, purification. &nbsp

Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004mmol L-1) and high (VM = 153.4 ± 7.7nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5nmolPimin-1mg-1 protein, K0.5 = 0.16 ± 0.01mmol L-1), K+ (VM = 485.3 ± 24.3nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05mmolL-1), Na+ (VM = 425.0 ± 23.4nmol Pi min-1mg-1 protein, K0.5 = 5.1 ± 0.3mmol L-1) and NH4+ (VM = 497.9 ± 24.9nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5mmolL-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8µmolL-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Syzygium samarangense Volatile Oil Inhibited Bacteria Growth and Extracellular Protease of Salmonella typhimurium.

Medicinal plants are fast becoming essential pharmaceuticals for disease and infection management. The vast antimicrobial properties of these plants reside in the inhibitory properties of their endogenous secondary metabolites. Therefore, this study aimed to assess if the volatile oil of Syzygium samarangense inhibits enteric bacteria growth and its effect against the caseinolytic activity of the extracellular protease of Salmonella typhimurium. The volatile oil was extracted by hydrodistillation, while the antimicrobial assay was assessed with the microdilution method. The extracellular protease was partially purified by salting out, followed by size-exclusion chromatography. The mode of inhibition of this enzyme was deduced from the enzyme-substrate kinetics using a line-weaver burke plot. The antimicrobial properties of the oil were reported against ten enteric bacteria. Proteus vulgaris has the highest IC50 value of 0.75±0.004% v/v, while S. typhimurium, the most sensitive bacterium, showed the lowest IC50 value of 0.17±0.005% v/v. The extracellular protease of S. typhimurium was partially purified to achieve 3.73 purification fold and 314.2 μmol min-1 mg-1 protein. The optimal caseinolytic activity of this enzyme was found at pH 7.5 and 40 °C. The protease showed significantly higher activity in the presence of Zn2+ (9.3±0.33 U min-1) as compared to the control (7.0±0.58 U min-1) (p<0.05), however, K+, Ca2+, Co2+, Ba2+, Pb2+ and Hg2+ considerably reduced the enzyme activity. The activity of this enzyme was competitively inhibited by the volatile oil as an inhibitor. The volatile oil of S. samarangense inhibited a wide range of enteric bacteria and, therefore proposed as a potential antimicrobial agent. Inhibiting the extracellular protease of S. typhimurium may be one of its modes of action against these pathogens.

Abamectin resistance and resistance mechanisms in Tetranychus urticae populations from cut flowers greenhouses in Turkey

ABSTRACTTetranychus urticae Koch is a common pest of many crops in Turkey and the world. In this study, abamectin LC values, resistance rates, detoxification enzyme activities and the presence of G314D point mutation in the target site were investigated in T. urticae collected from cut flower production greenhouses in Antalya. The LC values of the susceptible and field populations were determined by spray tower-leaf disc method. The resistance rates of field populations ranged from 1.60 to 850.12-fold compared to the susceptible population. Activities of three detoxification enzymes were determined by a microplate reader. The activity of esterase and GST ranged from 4.04 to 8.21 and from 4.40 to 11.69 mOD min−1 mg−1 proteins, respectively, in different populations. On the other hand, the P450 enzyme activity of the field populations was 0.52-1.80 RFU 30 min-1 mg-1 protein. Screening the populations for the presence of G314D point mutation in glutamate-gated chloride channels (GluCls) by real-time PCR using the mutation-specific TaqMan probe revealed no mutations in any populations tested. The results showed that the resistance in the populations tested in this study is likely associated with activity of detoxifying enzymes rather than target site mutations in the GluCls channels.