Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation

Background

Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.

Findings

We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH _2-46/ΔCT₁₀₄).

Conclusions

These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.     

Resistance to selected organochlorin, organophosphate, carbamate and pyrethroid, in Spodoptera litura (Lepidoptera: Noctuidae) from Pakistan

The toxicity of the most commonly used insecticides of organochlorine, organophosphate, pyrethroid, and carbamate groups were investigated against Spodoptera litura (F.) (Lepidoptera: Noctuidae) populations collected for three consecutive years (2004-2006). For a chlorocyclodiene and pyrethroids tested, the resistance ratios compared with Lab-PK were in the range of 10- to 92-fold for endosulfan, 5- to 111-fold for cypermethrin, 2- to 98-fold for deltamethrin, and 7- to 86-fold for beta-cyfluthrin. For organophosphates and carbamates, resistance ratios were in the range of 3- to 169-fold for profenofos, 18- to 421-fold for chlorpyrifos, 3- to 160-fold for quinalphos, 6- to 126-fold for phoxim, 7- to 463-fold for triazophos, and 10- to 389-fold for methomyl and 16- to 200-fold for thiodicarb. Resistance ratios were generally low to medium for deltamethrin and beta-cyfluthrin and high to very high for endosulfan, cypermethrin, profenofos, chlorpyrifos, quinalphos, phoxim, triazophos, methomyl, or thiodicarb. Pairwise comparisons of the log LC50 values of insecticides tested for all the populations showed correlations among several insecticides, suggesting a cross-resistance mechanism. Integration of timely judgment of pest problem, delimiting growing of alternate crops such as arum, rotation of insecticides with new chemicals, and insect growth regulators in relation to integrated pest management could help in manageable control of this important pest.     

Comparative study of the inhibitory effect of antidepressants on cholinesterase activity in Bungarus sindanus (krait) venom, human serum and rat striatum

Cholinesterases are divided into two classes based on differences in their substrate specificity and tissue distribution: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes may be inhibited by several compounds, such as antidepressants. The antidepressants paroxetine, imipramine, clomipramine and sertraline inhibited both venom AChE as well as human serum BChE in a concentration-dependent manner but had no effect on AChE in the rat brain striatum. The IC(50) of venom calculated for imipramine was 0.3 mM, paroxetine 0.38 mM, clomipramine 0.34 mM and sertraline 0.35 mM. Analysis of kinetic data indicated that the inhibition caused by sertraline and paroxetine was mixed, i.e. K(m) values increased and V(max) decreased in a concentration dependent manner. Imipramine and clomipramine exhibited competitive inhibition, i.e. K(m) values increased and V(max) remained constant. The present results suggest that these therapeutic agents used for depression can also be considered as inhibitors of snake venom and human serum cholinesterase.     

Consequences of copper treatment on pigeon pea photosynthesis,osmolytes and antioxidants defense

An attempt was made to explore the effect of copper sulphate treatment on growth, photosynthesis, osmolytes and antioxidants in 15 days old seedlings of C. cajan (Pigeonpea). C. cajan seedlings were grown in 0, 1, 5 and 10 mM concentrations of copper sulphate in petriplates lined with Whatman filter paper for 15 days. Root length and shoot length was decreased in a dose dependent manner with highest decrease of 82.80 and 45.92% in 10 mM Cu stress. Photosynthetic efficiency (qP, qN and Y) was decreased in a dose dependent manner whereas NPQ was increased in 1 and 5 mM and decreased in 10 mM Cu. Photosynthetic pigments viz total chlorophyll and carotenoids were increased in low concentrations and decreased in high concentrations of Cu. Osmolytes such as proline, glycine betaine and sugars were found to be increased in a dose dependent manner. Similarly antioxidants such as superoxide dismutase and catalase increased to 129.17 and 169.7%, respectively under Cu stress. Vitamin C and vitamin E was also increased in different concentrations of Cu to a significant level. It can be concluded from the present study that C. cajan can tolerate Cu stress up to 5 mM by adjusting the proportion of proline, glycine betaine, sugars and vitamins along with increasing the activity of some of the antioxidant enzymes.     

Comparison of three pyrethroid treatments of top-sheets for malaria control in emergencies: entomological and user acceptance studies in an Afghan refugee camp in Pakistan

Insecticide-treated bedding materials (sheets and blankets) could be protective against vectors of malaria and leishmaniasis--especially in complex emergencies, epidemics and natural disasters where people are more likely to sleep in exposed situations. Comparison of cotton top-sheets impregnated with different pyrethroids (permethrin 500 mg/m2, deltamethrin 25 mg/m2 or alphacypermethrin 25 mg/m2) for effectiveness against mosquitoes (Diptera: Culicidae) was undertaken in a refugee camp in Pakistan. Predominant species encountered were Anopheles stephensi Liston, An. pulcherrimus Theobald, An. nigerrimus Giles, Culex quinquefasciatus Say, Cx. tritaeniorhynchus Giles and other culicine mosquitoes. All three pyrethroid treatments performed significantly better than the untreated sheets in deterrence and killing of mosquitoes. No significant differences were found between the three insecticides tested in terms of entomological effect. Washing of the treated sheets greatly reduced their effectiveness. In a user acceptance study conducted among 88 families (divided into four groups), six families complained of irritation of the skin and mucous membranes. Of these reports, one was from the placebo group (using untreated sheets) and the other five (5/22=23%) from families using deltamethrin-treated sheets. All families allocated to permethrin and alphacypermethrin groups declared an appreciation for the intervention and reported no side-effects. Ten of the placebo group disliked the intervention, citing no prevention of mosquito biting as the reason. Side-effects associated with deltamethrin indicate that alphacypermethrin and permethrin are more appropriate first choice insecticides for treatment of sheets and blankets.     

The structure of the transition state of the heterodimeric topoisomerase I of Leishmania donovani as a vanadate complex with nicked DNA

Type IB topoisomerases are essential enzymes that are responsible for relaxing superhelical tension in DNA by forming a transient covalent nick in one strand of the DNA duplex. Topoisomerase I is a target for anti-cancer drugs such as camptothecin, and these drugs also target the topoisomerases I in pathogenic trypanosomes including Leishmania species and Trypanosoma brucei. Most eukaryotic enzymes, including human topoisomerase I, are monomeric. However, for Leishmania donovani, the DNA-binding activity and the majority of residues involved in catalysis are located in a large subunit, designated TOP1L, whereas the catalytic tyrosine residue responsible for covalent attachment to DNA is located in a smaller subunit, called TOP1S. Here, we present the 2.27A crystal structure of an active truncated L.donovani TOP1L/TOP1S heterodimer bound to nicked double-stranded DNA captured as a vanadate complex. The vanadate forms covalent linkages between the catalytic tyrosine residue of the small subunit and the nicked ends of the scissile DNA strand, mimicking the previously unseen transition state of the topoisomerase I catalytic cycle. This structure fills a critical gap in the existing ensemble of topoisomerase I structures and provides crucial insights into the catalytic mechanism.     

Relative Changes from Prior Reward Contingencies Can Constrain Brain Correlates of Outcome Monitoring

It is well-known that the affective value of an environment can be relative to whether it reflects an improvement or a worsening from a previous state. A potential explanation for this phenomenon suggests that relative changes from previous reward contingencies can constrain how brain monitoring systems form predictions about future events. In support of this idea, we found that changes per se relative to previous states of learned reward contingencies modulated the Feedback-Related Negativity (FRN), a human brain potential known to index discrepancies between predictions and affective outcomes. Specifically, we observed that environments with a 50% reward probability yielded different FRN patterns according to whether they reflected an improvement or a worsening from a previous environment. Further, we also found that this pattern of results was driven mainly by variations in the amplitude of ERPs to positive outcomes. Overall, these results suggest that relative changes in reward probability from previous learned environments can constrain how neural systems of outcome monitoring formulate predictions about the likelihood of future rewards and nonrewards.     

Previous treatment with ebselen and vitamin E alters adenine nucleotide hydrolysis in platelets from adult rats experimentally demyelinated with ethidium bromide

Many aspects of the relationship between the demyelinating pathology and platelet function need to be elucidated. Thus, the activity of NTPDase and 5'-nucleotidase enzymes was analyzed in platelets from rats demyelinated with ethidium bromide (EB) and previously treated with ebselen (Ebs) and vitamin E (Vit. E). The animals were divided into four groups: for ebselen, the groups were: I-control (saline), II-(saline and Ebs), III-(EB) and IV-(EB and Ebs); and for vitamin E, the groups were: I - control (saline), II-(saline and Vit. E), III-(EB) and IV-(EB and Vit. E). After 3 and 21 days, the blood was collected and the platelets were separated for enzymatic assays. For the treatment with Ebs, the NTPDase activity for ATP substrate was significantly lower in groups II, III and IV (p < 0.05) after 3 days, while after 21 days, a reduction was observed in group III (p < 0.05). ADP hydrolysis was reduced in group II (p < 0.05) and increased in group IV (p < 0.05) after 3 days, while after 21 days there was an increase in group IV (p < 0.05). In the treatment with Vit. E, ATP hydrolysis was lower in groups II, III and IV (p < 0.05) after 3 and 21 days. ADP hydrolysis was increased in group II (p < 0.05) after 3 days, and in group IV (p < 0.05) after 21 days. However, 5'-nucleotidase activity was not altered by the treatments. These findings demonstrate that NTPDase activity in platelets is diminished in demyelinating events and the treatments with Ebs and Vit. E modulated adenine nucleotide hydrolysis.     

Stereoselective formation and hydration of 12-methylbenz[a]anthracene 5,6-epoxide enantiomers by rat liver microsomal enzymes.

The K-region trans-5,6-dihydrodiols formed in the metabolism of 12-methylbenz[a]anthracene (12-MBA) by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats were found by chiral stationary-phase h.p.l.c. (c.s.p.-h.p.l.c.) analyses to contain (5S,6S)/(5R,6R) enantiomer ratios of 93:7, 88:12 and 97:3 respectively. The absolute stereochemistry of a 12-MBA trans-5,6-dihydrodiol enantiomer was elucidated by the exciton-chirality c.d. method. The 5,6-epoxides formed in the metabolism of 12-MBA by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide were isolated from a mixture of metabolites by normal-phase h.p.l.c., and their (5S,6R)/(5R,6S) enantiomer ratios were found by c.s.p.-h.p.l.c. analyses to be 73:27, 78:22 and 99:1 respectively. The absolute configurations of 12-MBA 5,6-epoxide enantiomers, resolved by c.s.p.-h.p.l.c., were determined via high-resolution (500 MHz) proton-n.m.r. and c.d. spectral analyses of the two isomeric methoxylation products derived from each of the 12-MBA 5,6-epoxide enantiomers. Enantiomeric pairs of the two methoxylation products were resolved by c.s.p.-h.p.l.c. The results indicate that enantiomeric 5S,6R-epoxide and 5S,6S-dihydrodiol were the major enantiomers preferentially formed in the metabolism at the K-region 5,6-double bond of 12-MBA by all three rat liver microsomal preparations. Optically pure 12-MBA 5S,6R-epoxide was hydrated predominantly at the C(6) position (R centre) to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 97:3. However, optically pure 12-MBA 5R,6S-epoxide was hydrated nearly equally at both C(5) and C(6) positions to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 57:43.     

A Pilot Study to Evaluate Haemostatic Function,following Shock Wave Lithotripsy (SWL) for the Treatment of Solitary Kidney Stones

PurposeThe number of patients undergoing shock wave lithotripsy (SWL) in the UK for solitary unilateral kidney stones is increasing annually. The development of postoperative complications such as haematuria and sepsis following SWL is likely to increase. Comparing a range of biological markers with the aim of monitoring or predicting postoperative complications following SWL has not been extensively researched. The main purpose of this pilot-study was to test the hypothesis that SWL results in changes to haemostatic function. Subsequently, this pilot-study would form a sound basis to undertake future investigations involving larger cohorts.MethodsTwelve patients undergoing SWL for solitary unilateral kidney stones were recruited. From patients (8 male and 4 females) aged between 31–72 years (median—43 years), venous blood samples were collected pre-operatively (baseline), at 30, 120 and 240 minutes postoperatively. Specific haemostatic biomarkers [platelet counts, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimer, von Willebrand Factor (vWF), sE-selectin and plasma viscosity (PV)] were measured.ResultsPlatelet counts and fibrinogen concentration were significantly decreased following SWL (p = 0.027 and p = 0.014 respectively), while D-dimer and vWF levels significantly increased following SWL (p = 0.019 and p = 0.001 respectively). PT, APTT, sE-selectin and PV parameters were not significantly changed following SWL (p>0.05).ConclusionsChanges to specific biomarkers such as plasma fibrinogen and vWF suggest that these represent a more clinically relevant assessment of the extent of haemostatic involvement following SWL. Analysis of such markers, in the future, may potentially provide valuable data on “normal” response after lithotripsy, and could be expanded to identify or predict those patients at risk of coagulopathy following SWL. The validation and reliability will be assessed through the assessment of larger cohorts.