Biochemical and Biological Evaluation of an L-Asparaginase from Isolated Escherichia coli MF-107 as an Anti-Tumor Enzyme on MCF7 Cell Line

Background: One of the most widely used anticancer agents is microbial L-ASNase. Herein, we assessed the biochemical and biological properties of an isolated L-ASNase from a Gram-negative bacteria strain, Escherichia coli MF-107. Methods:Using garden asparagus, we obtained several bacterial isolates. These strains were further screened for L-ASNase activity. A promising bacterial isolate was selected for L-ASNase production and subsequent purification. The molecular weight of purified L-ASNase was determined. The MTT assay was applied to assess the cytotoxic effect of the purified enzyme. Also, for caspase activity determination and the apoptotic effect of purified enzyme on in cells, we conducted a real-time PCR method. Results:The molecular weight of the enzyme was approximately 37 kDa. In the pH range of 7.5 to 8, the enzyme had considerable stability. At 35 °C, the purified L-ASNase optimum activity was recorded. The cytotoxic effect of the enzyme on treated cells was dose-dependent with an IC₅₀ value of 5.7 IU/ml. The Bax gene expression considerably raised by 5.75-fold (p < 0.001) upon L-ASNase treatment. On the other hand, the anti-apoptotic Bcl-2 gene expression showed a 2.63-fold increase compared to the control (p < 0.05). It was detected that the mRNA levels of caspase-3 and p53 were considerably upregulated (5.93 and 1.85-fold, respectively). We did not find any alternation in the caspase-8 activity of the treated cells compared to untreated cells. Conclusion:In this research, the proliferation of the breast cancer cells remarkably inhibited via the cytotoxic effect of isolated L-ASNase from microbial sources.Key Words: Apoptosis, Breast cancer, Escherichia coli, MCF7 cell line     

Interactive effects of zinc and boron on growth,photosynthesis, and water relations in pistachio

The effects of zinc (Zn) and boron (B) on some physiological and biochemical characteristics of pistachio (Pistacia vera L. cv. Badami) seedlings were evaluated in a greenhouse study. It was found that both high levels (10 and 20 mg B kg^?1 soil) and also lack of B account for the reduction in growth and photosynthesis parameters, especially at low Zn levels. Moreover, Zn deficiency resulted in a reduction in the net photosynthetic rate and stomatal conductance. However, the adverse effects of B deficiency and toxicity were alleviated by increasing Zn levels up to 10 mg kg^?1 soil. In addition, provided that B and Zn are adequate, synergism can be observed between the two nutrients. This is because sufficient amounts B accelerated the effects of Zn by raising the plant dry weight, photosynthesis parameters, carbonic anhydrase activity, and chlorophyll contents. This increase was reported higher in comparison with Zn alone application. It was concluded that Zn may act as a scavenger of reactive oxygen species (ROS) for mitigating the injury on biomembranes under B stress. Adequate concentration of Zn also prevents uptake and accumulation of excess B in leaf by increasing membrane integrity of root cells. Similarly, B application in adequate concentration plays an important structural role in the cell wall and influences a great variety of physiological processes in pistachio seedlings.     

Uptake and remobilization of selenium in Brassica napus L. plants supplied with selenate or selenium‐enriched plant residues

Selenium (Se) biofortification via crops is one of the best strategies to elevate the daily Se intake in areas where soil Se levels are low. However, Se fertilizer recovery (SeFR) is low and most of the Se taken up accumulates in non‐harvested plant parts and returns to the soil with plant residues. A pot experiment with soil was undertaken to study the efficiency of inorganic Se (Na₂SeO₄) and Se‐enriched plant residues for biofortification, as well as to identify the bottlenecks in Se utilization by Brassica napus L. The soil was fertilized with Na₂SeO₄ (0 and 7 µg Se kg^?1) or with Se in stem or leaf residues (0 and 7 µg Se kg^?1). A treatment with autoclaved soil was included (0 and 7 µg kg^?1 as Na₂SeO₄) to unravel the impact of microbial activity on Se uptake. The Se‐enriched plant residues produced a lower Se uptake efficiency (SeUPE) and SeFR than did inorganic Se, and soil autoclaving enhanced Se accumulation in the plants. The time required for decomposition seems to preclude crop residues as an alternative source of Se. Furthermore, B. napus had a limited capacity to accumulate Se in seeds. The study shows that the bottlenecks in Se biofortification appear to be its low bioavailability in soil and poor loading from the silique walls to seeds. Thus, improved Se translocation to seeds would be a useful breeding goal in B. napus to increase SeFR.     

Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

Response of industrial hemp to Salsola kali subsp. tragus densities under organic and conventional fertilisation

A 2-year field study was conducted to determine the effects of different nutrition systems and Salsola kali subsp. tragus densities on industrial hemp yield and its components. Experiments were conducted as a split plot in a randomised complete block design with three replicates. Factors were fertilisation (poultry manure, vermicompost and nitrogen fertiliser) as the main plot and weed density (0, 5, 10, 15 and 20 plants/m²) as the subplot. Results indicated that the maximum number of racemes per plant, seeds per raceme, 1000-seed weight, biomass, grain yield and harvest index (HI) of industrial hemp were obtained using vermicompost and were reduced with nitrogen fertiliser application. All measured properties in hemp plants decreased due to increasing S. kali subsp. tragus density, especially at 15 and 20 plants/m². The most significant reduction in hemp grain yield and its components was observed by increasing weed density with the application of nitrogen fertiliser. The application of vermicompost at high weed densities resulted in greater hemp grain yield and its components compared with nitrogen fertiliser. It is recommended to apply organic fertilisers, especially vermicompost at high S. kali subsp. tragus densities.     

Effects of different carbon sources and dietary protein levels in a biofloc system on growth performance,immune response against white spot syndrome virus infection and cathepsin L gene expression of Litopenaeus vannamei

A 5‐week study was performed to evaluate the effect of spoilage date extract (SDE) as the biofloc carbon source on Litopenaeus vannamei (5.4 ± 0.3 g) performance. The two levels of dietary protein (15% and 25% crude protein) and two carbohydrate sources (molasses‐M and SDE‐P) were tested including: M15, M25, P15 and P25. The minimum (0.2 ± 0.0 mg/L) and the maximum (0.5 ± 0.0 mg/L) of total ammonia nitrogen were observed in the P15 and M25 groups respectively. The highest protein efficiency ratio (6.1 ± 0.3) and protein productive value (112.3 ± 5.8%) were found in the P15 group (p < 0.05). No significant difference was found between biofloc treatments in the expression of cathepsin L gene in hepatopancreas (p > 0.05). The number of total haemocyte count (THC), semigranular cells (SGC) and granular cells (GC) of shrimp in SDE‐based biofloc treatments was relatively higher than those in molasses‐based biofloc treatments. Following the white spot syndrome virus (WSSV) challenge, a significant decrease in THC, SGC, GC and hyaline cell values was observed in all treatments (p = 0.001). Plasma biochemical parameters were significantly influenced by dietary protein levels, biofloc carbon sources as well as WSSV challenge test. In conclusion, SDE successfully could be used as an alternative carbon source for establishing a biofloc system in L. vannamei production.     

Wide hybridization and introgression breeding in safflower: Effectiveness of different selection methods

Possibility of using interspecific hybridization to increase genetic diversity of drought tolerance in safflower (Carthamus tinctorius L.) and effectiveness of different selection methods is poorly understood. The main objectives of this study were to evaluate the effectiveness of (a) interspecific hybridization to expand genetic diversity in safflower for agronomic traits and drought tolerance in advance generations and (b) different selection methods (direct vs. indirect) for improving seed yield. Interspecific hybridizations were performed between species of C. tinctorius × C. palaestinus (TP), C. palaestinus × C. oxyacantha (PO) and C. tinctorius × C. oxyacantha (TO). Based on the field screening of genotypes in F3 generation, four groups of selected genotypes (derived from combination of direct and indirect selection under both drought and normal environments along with selection based on drought tolerance index (STI)) were evaluated in F4 and F5 generations. Indirect selection for seed yield was significantly more efficient than direct selection. Selection in stress environment resulted to more efficiency than selection in normal environment. No significant difference was observed between direct selection and selection based on STI. The result showed that TP was the best population to combine high seed yield and drought tolerance in safflower.     

Effect of Bacillus thuringiensis var. kurstaki on survival and mortality of immature and mature stages of Diadegma insulare parasitizing Plutella xylostella

Effects of Bacillus thuringiensis (Bt) on immature stages of Diadegma insulare (Cresson) (Hym., Ichneumonidae) within larvae of diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), and on the adult stage as a Bt-honey solution were evaluated at 25?±?1?°C, 65?±?10% r.h. and 16:8?h (L:D) photoperiod. P. xylostella and D. insulare pupae were collected from cabbage fields in Karaj, Iran. A commercial wettable powder formulation of Bt (var. kurstaki, serotype H-3a3b, strain Z-52) was used in these experiments. The value of LC₅₀ for third instar larvae of P. xylostella was 210?ppm. Parasitoid??s adult mortality at field rate of Bt was not significantly different from that of control. Formation of parasitoid??s pupae in Bt-parasitoid combined treatments was significantly lower than parasitoid alone. Formation of parasitoid??s pupae in larvae treated with LC₁₀, LC₃₀ and LC₅₀ of Bt and then exposed to parasitoids after 48?h was 5%, 3% and 2%, respectively, which was significantly lower than that of the parasitoid alone (41%). Mean percentage mortality of larvae of DBM in LC₃₀ and LC₅₀ concentrations of Bt combined with the parasitoid was approximately 100%. Our results showed that B. thuringiensis kills D. insulare larvae indirectly by killing susceptible hosts in which they are developing. Successful use of multiple biological control agents will depend on close monitoring of their compatibility in the field. Our results could be useful in this purpose.     

Sowing date effects on dry matter remobilization and yield of triticale (Triticosecale wittmack) under late season drought stress

A 2-year field experiment using a randomized complete block design with the treatments arranged as split-split-plot with three replicates was conducted to investigate the effects of different sowing dates. Different irrigation regimes and different triticale cultivars were tested during 2014 and 2015 growing seasons. Under cutting off irrigation at the milk development stage, Sanabad with 46.2% had higher relative water content on December 3 sowing date. Assimilate remobilization in cutting off irrigation at dough development was more than that at milk development and Sanabad had the highest assimilate remobilization on 3 December sowing date in both years. In both years, Sanabad had the highest remobilization efficiency. The highest contribution of pre-anthesis assimilates to grain was obtained on 3 December sowing date in both years in Sanabad. Lower grain yield in Juanillo cultivar under cutting off irrigation, appeared to be due to reduction in remobilization efficiency, especially by cutting off irrigation at milk development stage in late sowing date. Overall, Sanabad was more tolerant to cutting off irrigation than Juanillo.