An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase,a key enzyme in artemisinin biosynthesis

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg²⁺ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519–532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg²⁺. Such detailed understanding will probably help to design an improved enzyme.     

Determination of Trace Element Level in Different Tissues of the Leaping Mullet (Liza saliens, Mugilidae) Collected from Caspian Sea

The concentrations of Cr, Cu, Fe, Mn, Ni, Pb, Cd, and Zn were determined in the brain, heart, liver, gill, gonad, spleen, kidney, and red and white muscles of Liza saliens (leaping mullet). Trace element levels in fish samples were analyzed by flame atomic absorption spectrometry. Among the non-essential metals, the levels of Ni and Pb in the tissues were higher than limits for fish proposed by FAO/WHO, EU, and TFC. Generally, the levels of the non-essential metals were much higher than those of manganese in the red and white muscles. Fe distribution pattern in tissues was in order of spleen?>?liver?>?heart?>?gill?>?brain?>?kidney?>?gonad?>?red muscle?>?white muscle. Red muscle was not within the safe limits for human consumption because non-essential metal (Ni, Pb) contents were higher than standard limits.     

Genetic variation in RPOIILS gene encoding RNA polymerase II largest subunit from Leishmania major

Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis, cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, Leishmania tropica, and Leishmania aethiopica complex which 90 % of cases occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru. Recently, some reports showed that some strains of L. major have internal transcribed space (ITS-1) with differential size exhibiting homology with the related gene in a divergent genus of kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. After obtaining samples from 100 patients, DNA extraction was performed and ITS-1 was analyzed using PCR–RFLP. These samples were sequenced for verifying their homology. Then, RPOIILS gene was analyzed in the samples that their ITS-1 gene exhibiting homology with the related gene in Crithidia. Results showed that 10 % of the isolates have ITS-1 exhibiting different size with the routine ones. Sequencing of them showed their similarity to the one from Crithidia fasciculata. RPOIILS gene encoding RNA polymerase II largest subunit analysis showed genetic diversity. This study might also help in solving the problems concerning Leishmaniasis outbreak currently facing in Iran and some other endemic regions of the world.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Effects of temperature, aurintricarboxylic acid and cibacron blue on 2',5'-oligoadenylate binding protein (RNase L) activity in rabbit reticulocyte lysates

The binding of p3A4,3'-32P [pCp] to rabbit reticulocyte RNase L can be displaced by the trimer and tetramer triphosphates of 2',5'-oligoadenylates (2-5A). Using assay conditions of protein synthesis, 2-5A trimer or tetramer triphosphates are shown to be equally effective when the displacement is done at 4 degrees C (on ice). In contrast, at 30 degrees C, the tetramer triphosphates still displace whereas the trimer triphosphates become ineffective. When lysates are preincubated at temperature ranging from 4 degrees-37 degrees C, the same results are obtained even when the subsequent displacement is done on ice. Incubation temperature also significantly affects the ability of metabolically stable dyes cibacron blue and aurintricarboxylic acid to inhibit RNase L binding activity. Taken together, these results suggest that rabbit reticulocyte RNase L may assume multiple conformations which are differentially affected by various forms of 2-5A or other compounds.