Positive regulation of TNFR1 signaling via SH3 recognition motif

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the “death domain”, it also activates IKKα/β, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A. Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.     

No genotoxic effect in exfoliated bladder cells of rat under the exposure of 1800 and 2100 MHz radio frequency radiation

In this study, we aimed to investigate the effects of 1800 and 2100?MHz Radio Frequency (RF) radiation on the number of micronucleus (MN) in exfoliated bladder cells of rat which shows the genotoxic damage. Exposure period was 30?min/day, 6 days/week for a month and two months exposure periods. Thirty male wistar albino rats were used for five groups: Group I (n?=?6): 1800?MHz RF exposed animals for one month, Group II (n?=?6): 2100?MHz RF exposed animals for one month, Group III (n?=?6): 2100?MHz RF exposed for two months, Group IV (n?=?6): control group for one month, Group V (n?=?6): control group for two months. Rats of the control groups were housed in their home cages during the entire experimental period without subjecting to any experimental manipulation. 1800 and 2100?MHz RF exposures did not result in any significant MN frequencies in rat bladder cells with respect to the control groups (p?>?0.05). There was no statistically significant difference between 2100?MHz RF exposed groups, either. Further studies are needed to demonstrate if there is any genotoxic effect, micronucleus formation in other tissues of rats.     

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three‐factor and three‐level Box–Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL‐CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL‐CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL‐CLEA were examined for the synthesis of (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96‐h reaction time, the yield–enantiomeric excess values (%) were 100?99, 100?21, 100?99, 83?91, 100?99, 100?72, and 100?14%, respectively, for (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile. The results show that PdHNL‐CLEA offers a promising potential for the preparation of enantiopure (R)‐mandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, and (R)‐4‐bromomandelonitrile with a high yield and enantiopurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog, 30:818–827, 2014     

Synthesis,characterization and carbonic anhydrase inhibitory activity of novel benzothiazole derivatives

N-protected amino acids were reacted with substituted benzothiazoles to give the corresponding N-protected amino acid-benzothiazole conjugates (60–89%). Their structures were confirmed by proton nuclear magnetic resonance (¹H NMR), carbon-13 nuclear magnetic resonance (¹³C NMR), IR and elemental analysis. Their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activities were determined against two cytosolic human isoforms (hCA I and hCA II), one membrane-associated (hCA IV) and one transmembrane (hCA XII) enzyme by a stopped-flow CO₂ hydrase assay method. The new compounds showed rather weak, micromolar inhibitory activity against most of these enzymes.     

Response of catalase activity to Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus

Catalase (CAT, EC 1.11.1.6) is an important enzyme in antioxidant defense system protecting animals from oxidative stress. Freshwater fish Oreochromis niloticus were exposed for 96 h to different concentrations of Ag(+), Cd(2+), Cr(6+), Cu(2+) and Zn(2+), known to cause oxidative stress, and subsequently CAT activities in liver, kidney, gill, intestine and brain were measured. In vivo, CAT was stimulated by all metals except Ag(+) in the liver and the highest increase in CAT activity (183%) resulted from 1.0 mg Cd(2+)/L exposure, whereas 0.5 mg Ag(+)/L exposure resulted in a sharp decrease (44%). In tilapia kidney, cadmium and zinc had no significant effects on CAT activity, whereas 0.1 mg Cr(6+)/L exposure caused a decrease (44%). Cadmium and zinc did not significantly affect the CAT activity in gill; however, 0.5 mg Ag(+)/L exposure caused an increase (66%) and 1.5 mg Cr(6+)/L exposure caused a decrease (97%) in CAT activity. All metals, except Cu(2+)(41% increase), caused significant decreases in CAT activity in the intestine. In brain, 1.0 mg Zn(2+)/L resulted in an increase in CAT activity (126%), while 1.5 mg Ag(+)/L exposure caused a 54% decrease. In vitro, all metals -- except Ag(+) and Cu(2+) in kidney -- significantly inhibited the CAT activity in all tissues. Results emphasized that CAT may be considered as a sensitive bioindicator of the antioxidant defense system.     

Production of paramylon,a β‐1,3‐glucan,by heterotrophic cultivation of Euglena gracilis on a synthetic medium

Euglena gracilis was cultivated on a synthetic medium of well‐defined components. Biomass and paramylon (β‐1,3‐glucan) concentrations were the most important variables monitored. Mass production in the bioreactor was carried out following studies of operating conditions in shaken flasks. E. gracilis grew best at about 30°C and at a low pH of 3. A pH control was not necessary, although the pH increased considerably at the end of the cultivation processes. Aeration could be performed at low stirrer frequency. Biomass concentrations of about 13–14 g/L were obtained with paramylon mass fractions of 50–60%, by starting with a synthetic medium containing 15 g/L of glucose as the main carbon source.     

N-butanoyl-l-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.