The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

QTL for spot blotch resistance in bread wheat line Saar co-locate to the biotrophic disease resistance loci Lr34 and Lr46

Spot blotch caused by Bipolaris sorokiniana is a major disease of wheat in warm and humid wheat growing regions of the world including south Asian countries such as India, Nepal and Bangladesh. The CIMMYT bread wheat line Saar which carries the leaf tip necrosis (LTN)-associated rust resistance genes Lr34 and Lr46 has exhibited a low level of spot blotch disease in field trials conducted in Asia and South America. One hundred and fourteen recombinant inbred lines (RILs) of Avocet (Susceptible) × Saar, were evaluated along with parents in two dates of sowing in India for 3 years (2007–2008 to 2009–2010) to identify quantitative trait loci (QTL) associated with spot blotch resistance, and to determine the potential association of Lr34 and Lr46 with resistance to this disease. Lr34 was found to constitute the main locus for spot blotch resistance, and explained as much as 55 % of the phenotypic variation in the mean disease data across the six environments. Based on the large effect, the spot blotch resistance at this locus has been given the gene designation Sb1. Two further, minor QTL were detected in the sub-population of RILs not containing Lr34. The first of these was located about 40 cM distal to Lr34 on 7DS, and the other corresponded to Lr46 on 1BL. A major implication for wheat breeding is that Lr34 and Lr46, which are widely used in wheat breeding to improve resistance to rust diseases and powdery mildew, also have a beneficial effect on spot blotch.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.     

Design and synthesis of 2-substituted benzoxazoles as novel PTP1B inhibitors

A series of benzoxazole compounds containing oxamic acid were synthesized and screened for the PTP1B inhibition. Compound 31d showed best biochemical potency (K_i) of 6.7 μM. Structure–activity relationship were explained with the help of molecular modeling approach.     

Amino acids derived benzoxazepines: Design,synthesis and antitumor activity

Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride (LAH) reduction, debenzylation, bimolecular nucleophilic substitution (S_N2) reaction. The present study investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF-7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hallmarks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depolarization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could serve as a base for the development of a new group of effective breast cancer therapeutics.     

Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil

Dunaliella bardawil, a unicellular microalga, grows in relatively high concentrations of salt and has so far been refractory to Agrobacterium-mediated transformation. An inverse relationship between salt concentration and hygromycin resistance was observed. Co-cultivation at 0.2?M NaCl allowed growth of both D. bardawil and A. tumefaciens. Lowering salt concentrations also enabled the use of lower concentrations of hygromycin, the selection agent. Cells resistant to 100?mg?l^?1 hygromycin were selected and growth of Agrobacterium was completely eliminated in these cells using cefotaxime/potassium clavulanate. The concentration of sodium chloride was gradually increased to 1.0?M with simultaneous reduction of hygromycin concentration for better growth of D. bardawil. Agrobacterium was unable to survive in the growth medium used for Dunaliella. Expression of β-glucuronidase (uidA), green fluorescent protein (GFP) and hygromycin phosphotransferase (hpt) in the hygromycin-resistant culture was detected using X-gluc as substrate and Western blotting using GFP antibodies and RT-PCR respectively. Cells growing in 1.0?M NaCl (in the absence of hygromycin) retained their ability to grow in hygromycin even after 18 months of cultivation. These cells expressed GFP and PCR for hpt gene was positive. The stability of the integrated transgene and resistance to hygromycin in three different transformation events were ascertained periodically. Southern blotting of DNA extracted from hygromycin resistant cells (HRC) that were 15–18 months old established the presence of the integrated transgene in the DNA of D. bardawil. Results of the present study substantiate A. tumefaciens-mediated transformation of the unicellular marine alga D. bardawil. Agrobacterium tumefaciens-mediated transgene integration along with the massive outdoor cultivation methods used for D. bardawil may allow the commercial synthesis of secondary metabolites and heterologous proteins.     

Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies

Chitobiase (CHB) is an important enzyme for the production of N-acetyl-d-glucosamine from the chitin biopolymer in the series of chitinolytic enzymes. Majority of over-expressed CHB (58 %) in E. coli expression system led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-expression with GroEL/ES chaperonin, optimizing culture conditions and solubilization followed by refolding of remaining inactive chitobiase present in the form of inclusion bodies. The growth of recombinant E. coli produced 42 % CHB in soluble form and the rest (~58 %) as inclusion bodies. The percentage of active CHB was enhanced to 71 % by co-expression with GroEL/ES chaperonin system and optimizing culture conditions (37 °C, 200 rpm, IPTG—0.5 mM, l-arabinose—13.2 mM). Of the remaining inactive CHB present in inclusion bodies, 37 % could be recovered in active form using pulsatile dilution method involving denaturants (2 M urea, pH 12.5) and protein refolding studies (1.0 M l-arginine, 5 % glycerol). Using combinatorial approach, 80 % of the total CHB expressed, could be recovered from cells grown in one litre of LB medium is a step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.     

Effect of Footwear Modifications on Oscillations at the Achilles Tendon during Running on a Treadmill and Over Ground: A Cross-Sectional Study

Background

Achilles tendon injuries are known to commonly occur in runners. During running repeated impacts are transferred in axial direction along the lower leg, therefore possibly affecting the oscillation behavior of the Achilles tendon. The purpose of the present study was to explore the effects of different footwear modifications and different ground conditions (over ground versus treadmill) on oscillations at the Achilles tendon.

Methods

Oscillations were measured in 20 male runners using two tri-axial accelerometers. Participants ran in three different shoe types on a treadmill and over ground. Data analysis was limited to stance phase and performed in time and frequency space. Statistical comparison was conducted between oscillations in vertical and horizontal direction, between running shoes and between ground conditions (treadmill versus over ground running).

Results

Differences in the oscillation behavior could be detected between measurement directions with peak accelerations in the vertical being lower than those in the horizontal direction, p < 0.01. Peak accelerations occurred earlier at the distal accelerometer than at the proximal one, p < 0.01. Average normalized power differed between running shoes (p < 0.01) with harder damping material resulting in higher power values. Little to no power attenuation was found between the two accelerometers. Oscillation behavior of the Achilles tendon is not influenced by ground condition.

Conclusion

Differences in shoe configurations may lead to variations in running technique and impact forces and therefore result in alterations of the vibration behavior at the Achilles tendon. The absence of power attenuation may have been caused by either a short distance between the two accelerometers or high stiffness of the tendon. High stiffness of the tendon will lead to complete transmission of the signal along the Achilles tendon and therefore no attenuation occurs.