Validation of spermidine synthase as a drug target in African trypanosomes

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.     

Ironing out Parkinson's disease: is therapeutic treatment with iron chelators a real possibility?

Levels of iron are increased in the brains of Parkinson's disease (PD) patients compared to age-matched controls. This has been postulated to contribute to progression of the disease via several mechanisms including exacerbation of oxidative stress, initiation of inflammatory responses and triggering of Lewy body formation. In this minireview, we examine the putative role of iron in PD and its pharmacological chelation as a prospective therapeutic for the disease.     

Out of Pocket Expenditure for Hospitalization among Below Poverty Line Households in District Solan,Himachal Pradesh,India, 2013

Introduction

Health insurance schemes, like Rashtriya Swasthya Bima Yojana (RSBY), should provide financial protection against catastrophic health costs by reducing out of pocket expenditure (OOPE) for hospitalizations. We estimated and compared the proportion and extent of OOPE among below poverty line (BPL) families beneficiaries and not beneficiaries by RSBY during hospitalizations in district Solan, H.P., India, 2013.

Methods

We conducted a cross sectional survey among hospitalized BPL families in the beneficiaries and non-beneficiaries groups. We compared proportion incurring OOPE and its extent during hospitalization, pre/post-hospitalization periods in different domains.

Results

Overall, proportion of non-beneficiaries who incurred OOPE was higher than the beneficiaries but it was not statistically significant (87.2% vs. 80.9%). The median overall OOPE was $39 (Rs 2567) in the non-beneficiaries group as compared to $ 11 (Rs 713) in the beneficiaries group (p<0.01). Median expenditure on in house and out house drugs and consumables was $ 23 (Rs 1500) in the non beneficiaries group as compared to nil in the beneficiaries group (p<0.01). Non-beneficiary status was significantly associated [OR: 2.4 (1.3–4.3)] with OOPE above median independently and also after adjusting for various covariates.

Conclusion

RSBY has decreased the extent of OOPE among the beneficiaries; however OOPE was incurred mainly due to purchase of drugs from outside the health facility. The treatment seeking behaviour in beneficiaries group has improved among comparatively older group with chronic conditions. RSBY has enabled beneficiaries to get more facilities such as drugs, consumables and diagnostics from the health facility.     

Role of Se(VI) in counteracting oxidative damage in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. under Cr(VI) stress

Chromium (Cr) is considered to be one of the major environmental hazards and poses a threat to both plant and animal health. Selenium (Se), however, has been recognized as an essential micronutrient in plants. To understand the role of Se(VI) in oxidative stress management and regulation of antioxidative defence mechanism against heavy metal stress, the seedlings of Brassica juncea L. were raised in Petri plates containing nutrient media supplemented with only with Se(VI) and Cr(VI), or their combination. It was observed that of Cr(VI) causes an increase in reactive oxygen species (ROS) in the seedlings leading to oxidative stress. Histological studies using confocal and visible microscopy confirmed the biochemical results. Supplementation of up to 4 µM of Se(VI) to media containing 300 µM of Cr(VI) reduced the contents of ROS and increased enzymatic and non-enzymatic antioxidants in the seedlings. At a concentration of 6 µM, however, Se(VI) was toxic. The results suggested that at appropriate concentrations, the exogenous application of Se(VI) enabled the B. juncea seedlings to counteract the effects of Cr(VI), thereby increasing the resistance of plants.     

First structural evidence of a specific inhibition of phospholipase A2 by alpha-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and alpha-tocopherol at 1.8 A resolution

This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.     

Independent evolution of functional Pm3 resistance genes in wild tetraploid wheat and domesticated bread wheat

The Pm3 alleles of cultivated bread wheat confer gene for gene resistance to the powdery mildew fungus. They represent a particular case of plant disease resistance gene evolution, because of their recent origin and possible evolution after the formation of hexaploid wheat. The Pm3 locus is conserved in tetraploid wheat, thereby allowing the comparative evolutionary study of the same resistance locus in a domesticated species and in one of its wild ancestors. We have identified 61 Pm3 allelic sequences from wild and domesticated tetraploid wheat subspecies. The Pm3 sequences corresponded to 24 different haplotypes. They showed low sequence diversity, differing by only a few polymorphic sequence blocks that were further reshuffled between alleles by gene conversion and recombination. Polymorphic sequence blocks are different from the blocks found in functional Pm3 alleles of hexaploid wheat, indicating an independent evolution of the Pm3 loci in the two species. A new functional gene was identified in a wild wheat accession from Syria. This gene, Pm3k , conferred intermediate race-specific resistance to powdery mildew, and consists of a mosaic of gene segments derived from non-functional alleles. This demonstrates that Pm3 -based resistance is not very frequent in wild tetraploid wheat, and that the evolution of functional resistance genes occurred independently in wild tetraploid and bread wheat. The Pm3 sequence variability and geographic distribution indicated that diversity was higher in wild emmer wheat from the Levant area, compared with the accessions from Turkey. Further screens for Pm3 functional genes in wild wheat should therefore focus on accessions from the Levant region.     

Bacillus megaterium mediated mineralization of calcium carbonate as biogenic surface treatment of green building materials

Microbially induced calcium carbonate precipitation is a biomineralization process that has various applications in remediation and restoration of range of building materials. In the present study, calcifying bacteria, Bacillus megaterium SS3 isolated from calcareous soil was applied as biosealant to enhance the durability of low energy, green building materials (soil–cement blocks). This bacterial isolate produced high amounts of urease, carbonic anhydrase, extra polymeric substances and biofilm. The calcium carbonate polymorphs produced by B. megaterium SS3 were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and Fourier transmission infra red spectroscopy. These results suggested that calcite is the most predominant carbonate formed by this bacteria followed by vaterite. Application of B. megaterium SS3 as biogenic surface treatment led to 40 % decrease in water absorption, 31 % decrease in porosity and 18 % increase in compressive strength of low energy building materials. From the present investigation, it is clear that surface treatment of building materials by B. megaterium SS3 is very effective and eco friendly way of biodeposition of coherent carbonates that enhances the durability of building materials.     

Overexpression of hypoxia-inducible factor and metabolic pathways: possible targets of cancer

Cancer, the main cause of human deaths in the modern world is a group of diseases. Anticancer drug discovery is a challenge for scientists because of involvement of multiple survival pathways of cancer cells. An extensive study on the regulation of each step of these pathways may help find a potential cancer target. Up-regulated HIF-1 expression and altered metabolic pathways are two classical characteristics of cancer. Oxygen-dependent (through pVHL, PHDs, calcium-mediated) and independent (through growth factor signaling pathway, mdm2 pathway, HSP90) regulation of HIF-1α leads to angiogenesis, metastasis, and cell survival. The two subunits of HIF-1 regulates in the same fashion through different mechanisms. HIF-1α translation upregulates via mammalian target of rapamycin and mitogen-activated protein kinase signaling pathways, whereas HIF-1β through calmodulin kinase. Further, the stabilized interactions of these two subunits are important for proper functioning. Also, metabolic pathways crucial for the formation of building blocks (pentose phosphate pathway) and energy generation (glycolysis, TCA cycle and catabolism of glutamine) are altered in cancer cells to protect them from oxidative stress and to meet the reduced oxygen and nutrient supply. Up-regulated anaerobic metabolism occurs through enhanced expression of hexokinase, phosphofructokinase, triosephosphate isomerase, glucose 6-phosphate dehydrogenase and down-regulation of aerobic metabolism via pyruvate dehydrogenase kinase and lactate dehydrogenase which compensate energy requirements along with high glucose intake. Controlled expression of these two pathways through their common intermediate may serve as potent cancer target in future.