Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP⁺ (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K _m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.     

Moderate/subclinical calcium deficiency attenuates trabecular mass,microarchitecture and bone growth in growing rats

Adequate dietary calcium (Ca) intake is essential for bone accretion, peak bone mass (PBM) attainment, bone quality and strength during the mammalian growth period. Severe Ca deficiency during growing age results in secondary hyperparathyroidism (SHPT) and poor bone quality and strength. However, the impact of moderate Ca deficiency during rats early growth period on bone health and the reversibility with supplementing calcium later in adult life remains unclear. Female Sprague-Dawley (SD) rats (postnatal 28th day, P28) were initiated either with a moderate calcium-deficient diet (MCD, 0.25% w/w Ca) or a control diet (0.8% w/w Ca, control group) till P70. Thereafter, MCD rats were continued either with MCD diet or supplemented with calcium diet (0.8% w/w Ca, calcium supplemented group, CaS) till P150. Another group (control rats) were fed 0.8% w/w Ca containing diet from P28 till P150.MCD group, as compared to the control group, had significantly reduced serum ionized Ca and procollagen type 1 N-terminal propeptide (P1NP) at P70 while no significant change was observed in serum corrected Ca, inorganic phosphate (P), alkaline phosphatase (ALP), 25-hydroxy vitamin D [25(OH)D], intact parathyroid hormone (iPTH), and urinary C-terminal telopeptide of collagen 1 (CTX-1), Ca, and P. Femoral and tibial metaphysis in MCD rats had significantly reduced linear growth, cortical and trabecular volumetric BMD (vBMD), trabecular microarchitecture (BV/TV%, trabecular thickness, separation and number, structural model index and connectivity density), cortical thickness, and bone stiffness despite the absence of secondary hyperparathyroidism (SHPT). Continued MCD at P70–P150 results in persistence of compromised bone strength while calcium supplementation (CaS group) improved all the parameters related to bone strength and microarchitecture. Our results indicate that uncorrected moderate/subclinical calcium deficiency in growing rats can result in poor bone quality and strength despite the absence of SHPT. This finding could have relevance in children with poor calcium intake in childhood and adolescence.     

Cloning,characterization, expression analysis and inhibition studies of a novel gene encoding Bowman–Birk type protease inhibitor from rice bean

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman–Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5 h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.     

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.     

Socioeconomic Deprivation and the Incidence of 12 Cardiovascular Diseases in 1.9 Million Women and Men: Implications for Risk Prediction and Prevention

Background

Recent experimental evidence suggests that socioeconomic characteristics of neighbourhoods influence cardiovascular health, but observational studies which examine deprivation across a wide range of cardiovascular diseases (CVDs) are lacking.

Methods

Record-linkage cohort study of 1.93 million people to examine the association between small-area socioeconomic deprivation and 12 CVDs. Health records covered primary care, hospital admissions, a myocardial infarction registry and cause-specific mortality in England (CALIBER). Patients were aged ≥30 years and were initially free of CVD. Cox proportional hazard models stratified by general practice were used.

Findings

During a median follow-up of 5.5 years 114,859 people had one of 12 initial CVD presentations. In women the hazards of all CVDs except abdominal aortic aneurysm increased linearly with higher small-area socioeconomic deprivation (adjusted HR for most vs. least deprived ranged from 1.05, 95%CI 0.83–1.32 for abdominal aortic aneurysm to 1.55, 95%CI 1.42–1.70 for heart failure; I² = 81.9%, τ² = 0.01). In men heterogeneity was higher (HR ranged from 0.89, 95%CI 0.75–1.06 for cardiac arrest to 1.85, 95%CI 1.67–2.04 for peripheral arterial disease; I² = 96.0%, τ² = 0.06) and no association was observed with stable angina, sudden cardiac death, subarachnoid haemorrhage, transient ischaemic attack and abdominal aortic aneurysm. Lifetime risk difference between least and most deprived quintiles was most marked for peripheral arterial disease in women (4.3% least deprived, 5.8% most deprived) and men (4.6% least deprived, 7.8% in most deprived); but it was small or negligible for sudden cardiac death, transient ischaemic attack, abdominal aortic aneurysm and ischaemic and intracerebral haemorrhage, in both women and men.

Conclusions

Associations of small-area socioeconomic deprivation with 12 types of CVDs were heterogeneous, and in men absent for several diseases. Findings suggest that policies to reduce deprivation may impact more strongly on heart failure and peripheral arterial disease, and might be more effective in women.     

Induction and establishment of somatic embryogenesis in elite Indian cotton cultivar (Gossypium hirsutumL. cv Khandwa-2)

Embryogenesis in cotton is a difficult task due its genome dependency. We used 3 cotton cultivars (Khandwa-2, G. Cot. 10, and BC-68–2) and Coker-312 as control for regeneration. Efficient somatic embryogenesis was induced in agronomically important Indian cotton cultivars, Khandwa-2 and G. Cot. 10. For callusing in all the cultivars, different media combinations were tried. Embryogenesis was initiated on a hormone-free MS medium (MSB). For embryo maturation and recovery excess of L-glutamine and l-asparagine were used. Khandwa-2 somatic embryos were successfully regenerated into plants. However, no plantlet was obtained in case of G. Cot. 10. Callus induction was also observed in BC-68–2 but there was no embryogenesis observed. The study indicated that the medium and genotype significantly effects embryogenesis. An efficient protocol is described here for regenerating plants via somatic embryogenesis in an elite Indian cotton cultivar Khandwa-2.     

Isolation and characterization of biosurfactant producing <Emphasis Type="Italic">Bacillus</Emphasis> sp. from diesel fuel-contaminated site

Among hydrocarbon pollutants, diesel oil is a complex mixture of alkanes and aromatic compounds which are often encountered as soil contaminants leaking from storage tanks and pipelines or as result of accidental spillage. One of the best ecofriendly approaches is to restore contaminated soil by using microorganisms able to degrade those toxic compounds in a bioremediation process. In the present study, nineteen bacteria were isolated by enrichment culture technique from diesel spilled soil collected from electric generator shed of NBAIM, Mau. All the isolates were subjected to screening for lipase production and twelve isolates were found to be positive for lipase. When the isolates were screened for biosurfactant production using CTAB-methylene blue agar plates, only one isolate viz. 2NBDSH3 was found positive which was found to be phylogenetically closely related with Bacillus flexus. Despite having low emulsification index, the bacterium could degrade 88.6% of diesel oil in soil. Biosurfactant from the isolate was extracted and characterized through infra-red spectroscopy which indicated its possible lipopeptide nature which was further supported by strong absorption in UV range in the UV-Vis spectrum. The results of the present study indicated that the isolate either does not produce any bioemulsifier or produces very low amount of emulsifier rather it produces a lipopeptide biosurfactant which helps in degradation of diesel oil by lowering the surface tension. The bacterium thus isolated and characterized can serve as a promising solution for ecofriendly remediation of bacterium diesel contaminated soils.     

The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.