Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems

Background: Phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) is a NAD+-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH. Methods: The purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results: A single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively. Conclusion: The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH.     

Herbicide risk assessment during the Wheat Self-sufficiency Project in Iran

BACKGROUND: The present study describes the quantitative changes in herbicide use during the specific observation periods in the Wheat Self-sufficiency Project in Iran from 1994 to 2004 and the associated changes in herbicide risk, area and yield in this context. A risk index, the environmental impact quotient (EIQ), was used to estimate the environmental impacts (EIs) of herbicides applied to wheat agroecosystems. RESULTS: The results suggest that, during this period, the overall risk posed by the herbicides applied to wheat agroecosystems increased substantially, as evidenced by a 71% increase in herbicide usage (weight of pesticide applied) and a 62.2% increase in EI rating, in spite of an 8.2% decrease in overall EIQ rating and an 89.2% decrease in mean application rate. Furthermore, a 0.57% increase in the area and a 23.6% and 22.7% increase in irrigated and rainfed yields, respectively, were observed. The results also indicate that the EIQ(FUR) of two herbicides, dichlorprop-P/mecoprop-P/MCPA and difenzoquat, is much higher than that of other herbicides, and they could be qualified as high-risk herbicides. CONCLUSION: As a whole, an increase in herbicide usage (weight of pesticide applied) was an important factor on herbicide environmental impact boost. However, this increase has not led to a similar increase in wheat yield, which could be attributed in part to the negative impact of high herbicide consumption in wheat fields and subsequent threat to the long-term sustainability of these agroecosystems.     

Path analysis of the relationships between seed yield and some morphological and phenological traits in safflower (Carthamus tinctorius L.)

Path analysis was performed on plant characters of six safflower genotypes grown in a two year field experiment to determine for seed yield, the direct and indirect effects of the following traits: total biomass, stem yield, number of primary branches/plant, number of secondary branches/plant, number of seeds/m², number of seeds/capitulum, number of capitula/plant, capitulum diameter, 1000-seed weight, seed weight/capitulum, distance between ground level and the first fertile branch, plant height, number of days to the beginning of branching, number of days to the beginning of flowering, number of days to 50% flowering, number of days to full flowering and flowering duration. Seed yield was significantly correlated with the following traits: total biomass, stem yield, capitulum diameter, 1000-seed weight, seed weight/capitulum, distance between ground level and the first fertile branch, number of days to the beginning of branching and flowering duration. Total biomass, seed weight/capitulum, distance between ground level and the first fertile branch, 1000-seed weight and flowering duration had substantial direct effects, in that order, on enhancement of seed yield. The significant positive correlation coefficient of capitulum diameter with seed yield resulted from positive indirect effects of total biomass, seed weight/capitulum and 1000-seed weight. Conversely, the significant negative correlations between number of days to the beginning of branching and distance between ground level and the first fertile branch and seed yield resulted from negative indirect effects of the same three traits. Stepwise multiple regression analysis showed that 94% of the total variation in seed yield could be explained by variation in total biomass and by number of days to the beginning of branching (84 and 10%, respectively). Results suggest that total biomass and number of days to the beginning of branching are primary selection criteria for improving seed yield in safflower.     

Alteration in the Expression of Proteins in Unexplained Recurrent Pregnancy Loss Compared with in the Normal Placenta

The placenta is a unique pregnancy-related tissue and plays a key role in occurrence of unexplained recurrent pregnancy loss (URPL). Abnormal placentation might play a key role in occurrence of URPL. Therefore, the purpose of this study was to compare the human placental proteome between URPL placentas and normal placental matched for gestational week. Total placental proteins were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was used for separation of the placental proteomes. Protein spots differentially expressed between URPL and normal placentas were selected and identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF) technique after being digested in the gel. Moreover, quantitative real-time PCR and Western blot techniques were used to confirm the differential expression mass results for some differentially expressed proteins. The results indicated that at least 19 protein spots were differentially expressed between URPL and normal placentas (P < 0.05), and twelve of them were successfully identified. While only two proteins were downregulated (calumenin and enolase 1), the remaining ten spots (actin gamma 1 propeptide, cathepsin D prepropeptide, heat shock protein gp96, tubulin beta, tubulin alpha 1, glutathione S-transferase, vitamin D binding protein, prohibitin, actin beta, apolipoprotein A-I) showed increased expression in URPL cases in comparison with normal placentas. Real-time PCR also confirmed the downregulation of calumenin and upregulation of prohibitin and apolipoprotein A-I at the mRNA levels. In conclusion, the results of the present study showed that alteration in the expression of proteins involved in proliferation and migration of endothelial cells as well as control of coagulation by these cells might play an important role in the pathogenesis of URPL.     

Weed control and wheat (Triticum aestivum L.) yield under application of 2,4-D plus carfentrazone-ethyl and florasulam plus flumetsulam: Evaluation of the efficacy

Three field experiments were conducted at the research fields of Plant Protection Research Institute, Iran, at different locations in 2004–2005 to study the efficacy of different broadleaved herbicides to control weeds in wheat. Treatments were the full-season hand weeded and weed-infested controls, and post-emergence applications of florasulam plus flumetsulam at 8.75, 10.50, and 12.25 g a.i./ha, 2,4-D plus carfentrazone-ethyl at 210, 245, 280, and 490 g a.i./ha, bromoxynil plus MCPA at 75, 100, and 150 g a.i./ha, 2,4-D at 560, 720, and 1120 g a.i./ha, tribenuron methyl, and 2,4-D plus MCPA. Herbicides were applied at wheat tillering stage. Naturally occurring broadleaved weed populations were used in experiments. Results indicated that bromoxynil plus MCPA at 150 g a.i./ha, 2,4-D plus MCPA, and 2,4-D plus carfentrazone-ethyl at 490 g a.i./ha were the best options to control weeds. Bromoxynil plus MCPA at 150 g a.i./ha and 2,4-D plus MCPA also resulted in the highest wheat yield. Overall, it could be concluded that rotational application of bromoxynil plus MCPA at 150 g a.i./ha, 2,4-D plus MCPA, and 2,4-D plus carfentrazone-ethyl at 490 g a.i./ha would be the best option to achieve satisfactory weed control, high grain yield and prevention of evolution of herbicide-resistant weeds.     

Chemical control of weeds in wheat (Triticum aestivum L.) in Iran

Experiments were conducted in 2006–2007 at six research stations of the Iranian Plant Protection Research Institute to study the efficacy of different herbicides to control weeds in wheat. Treatments included mesosulfuron-methyl plus iodosulfuron-methyl-sodium plus mefenpyr-diethyl (WG) at 45 + 45 + 135 g a.i./ha, respectively, sulfosulfuron at 21, 31.5, 42 and 51 g a.i./ha, chlorsulfuron at 15 g a.i./ha, bromoxynil plus MCPA at 600 g a.i./ha with clodinafop propargyl at 64 g a.i./ha, sulfosulfuron plus metsulfuron-methyl at 36 g a.i./ha, mesosulfuron-methyl plus iodosulfuron-methyl-sodium plus mefenpyr-diethyl (OD) at 15 + 3 + 45 g a.i./ha, respectively, and a full season weed-free control. Herbicides were applied at wheat tillering. Results showed that sulfosulfuron plus metsulfuron-methyl, and bromoxynil plus MCPA with clodinafop propargyl resulted in satisfactory weed control and wheat grain yield at most locations. Weed control efficacy of both formulations of mesosulfuron-methyl plus iodosulfuron-methyl-sodium was variable across locations. Efficacy of the OD formulation appears to depend upon location, so that application of this herbicide at Shiraz and Gorgan resulted in better weed control compared to use of the WG. Satisfactory performance of the OD formulation at Gorgan and Shiraz could be attributed in part to the even pattern of rainfall distribution during the growing season and wheat cultivar used, respectively. With respect to grain yield, however, the OD formulation was better than WG formulation at most of the locations.     

Cross-resistance patterns of winter wild oat (<Emphasis Type="Italic">Avena ludoviciana</Emphasis>) populations to ACCase inhibitor herbicides

The level of resistance and patterns of cross-resistance to clodinafop, sethoxydim, and pinoxaden were examined in 12 putative resistant and one susceptible populations of winter wild oat (Avena ludoviciana) collected from Fars Province, in the southwest of Iran. The responses of biomass and length of coleoptiles to the increasing dosages of the three herbicides were determined in both whole-plant and seed bioassays. In the whole-plant bioassay, all 12 putative resistant populations were found to be resistant to clodinafop with resistance ratios (R/S) ranging from 1.76 to >47.04. Most clodinafop-resistant populations exhibited low levels of cross-resistance to sethoxydim. Three highly sethoxydim-resistant populations, F2, S2, and ES4, were slightly resistant to clodinafop. Six populations (M1, M2, F2, S2, S4, and ES4) showed high cross-resistance to pinoxaden with R/S values as large as 10.73 to 40.29. A highly clodinafop-resistant population, M2, was more sensitive to pinoxaden than the susceptible population. The results of the seed bioassay resembled those obtained from the whole-plant experiment suggesting seed bioassay as an inexpensive, rapid method for screening-resistant genotypes.