Differential combination of cytokine and interferon- γ +874 T/A polymorphisms determines disease severity in pulmonary tuberculosis

Background

Mycobacterium tuberculosis infects nearly 1/3 of the world population and this reservoir forms the largest pool from which new cases arise. Among the cytokines, IFN-γ is a key determinant in protection against tuberculosis. Single nucleotide polymorphisms (SNPs) in IFN-γ gene (+874 T/A) which determine TT high (^hi), AA low (^lo) and TA intermediate (^int) responder phenotypes have shown variable associations with tuberculosis disease outcome in different ethnic populations. The objective of the current study was to analyze IFN-γ gene combinations with other IFN-γ regulating cytokine genes (IL-10, TNF –α, IL-6) to see the effect of gene- combinations on disease severity outcome in pulmonary tuberculosis.

Methods and Findings

Study groups comprised of pulmonary TB patients stratified according to lung tissue involvement into mild (Pmd = 74) or advance (Pad = 23) lung disease and compared with healthy controls (TBNA = 166). Genotype analysis was carried out using amplification refractory mutation system-PCR (ARMS-PCR). IFN-γ gene (+874 T/A) functional SNP combinations in TNFα (−308 G/A), IL-10 (−1082 A/G) and IL-6 (−174 G/C) were analyzed. Single gene analysis (Pearson χ²) showed a dominant association of IFN-γ TT ^hi genotype (p = 0.001) and T allele (p = 0.001) with mild disease. IFN-γ ^lo -IL-10 ^lo genotype combination was associated with advanced disease (p = 0.002). IFN-γ ^hi –IL-6 ^hi combination was associated with mild disease (p = 0.0005) while IFN-γ ^lo –IL-6 ^int was associated with protection against both forms of pulmonary disease (p = 0.002).

Conclusion

Our results show that a limited number of IFN-γ gene combinations with other cytokine functional SNPs determine the outcome of disease severity in tuberculosis.     

In Vitro Growth of Curcuma longa L. in Response to Five Mineral Elements and Plant Density in Fed-Batch Culture Systems

Plant density was varied with P, Ca, Mg, and KNO₃ in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO₃ concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO₃. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO₃ and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.     

Phosphorylation and kinetics of mammalian cytochrome c oxidase

The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.     

Isotopic composition of the delta-18O--delta-13C from the otoliths of reef fish from Taiaro (Tuamotu, French Polynesia): isotopic and biological implications

Nuclei (larval stage) and outer parts (adult stage) of fish otoliths from the Taiaro closed lagoon (French Polynesia) and adjacent ocean have been analysed for the C-O isotopic compositions. delta 18O values of the nuclei of both populations indicate that isotopic equilibrium is reached. This implies that the lagoonal fish population has done its complete biological cycle in the lagoon and represents an adaptation in a closed system. delta 18O values of the outer parts show a slight isotopic disequilibrium (< 0.2@1000) interpreted in term of vital effect. All the delta 13C values exhibit a strong isotopic disequilibrium related to metabolic activity.     

Bioinformatics of granzymes: sequence comparison and structural studies on granzyme family by homology modeling

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B(h)) and rat (B(r)), as well as that of pro-granzyme K (K(h)) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A(h), M(h), B(m), and C(m) from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (K(h)), and granzyme B (B(h)). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2(') binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of beta-sheet structure with a little alpha-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B(h). For example, in granzyme M(h), valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu-->Phe), S3 (Ser-->Gly), and S4 (Arg-->Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.     

Molecular mechanism of apoptosis: prediction of three-dimensional structure of caspase-6 and its interactions by homology modeling

Caspases, the intracellular cysteine proteinases, play a central role in the process of programmed cell death. Caspases induce apoptosis through a highly integrated and regulated biological, biochemical, and genetic mechanism. Although proper execution of apoptosis is fundamental for cell growth artificial caspase inhibition can be considered in certain degenerative diseases. This realization has attracted attention towards caspases as likely targets for pharmaceutical intervention. Here we analyze the structure of caspase-6 and also predict the possible glycosylation, phosphorylation, and myristoylation sites as very little is known about the functional role of these post translational modifications in the caspase family. These studies are expected to improve our understanding of associations of caspases with other molecules and the possible role played in apoptosis. The predicted tertiary structure of caspase-6 as well as the enzyme complexed with its inhibitor (tetra-peptide aldehyde Ac-IETD-CHO) shows similar binding feature as seen in other caspases. Cys/His catalytic dyad for caspase-6 and -8 show possible involvement of a third component, i.e., Pro29 and Arg258 in caspase-6 and caspase-8, respectively. Changes in the length and nature of loop between alpha5 and beta9, involved in defining the S4 subsite, result in modification of P4 (Ile) site. These interactions provide detail of inhibitor binding on structural level and also help in designing mutants for structure-function studies of these enzymes.     

Effect of caffeic acid phenethyl ester on treatment of experimentally induced methicillin-resistant Staphylococcus epidermidis endophthalmitis in a rabbit model

This study investigated the anti-inflammatory effects of caffeic acid phenethyl ester (CAPE), a natural bee-produced compound, and compared it with corticosteroids in the treatment of experimentally induced methicillin-resistant Staphylococcus epidermidis (MRSE) endophthalmitis in addition to intravitreal antibiotics. An experimental endophthalmitis model was produced in 24 New Zealand albino rabbits by unilateral intravitreal injection of 0.1 ml of 4.7 x 10(4) colony-forming units (CFU) methicillin-resistant S. epidermidis. The animals were then divided randomly into three treatment groups and a control group, group 1 (six rabbits), received only intravitreal vancomycin (1.0 mg/0.1 ml); group 2 (six rabbits), received both intravitreal vancomycin (1.0 mg/0.1 ml) and intravitreal dexamethasone (400 microg/0.1 ml) and group 3 (six rabbits), received both intravitreal vancomycin (1.0 mg/0.1 ml) and subtenon CAPE (10 mg/0.3 ml) after 24 h post-infection. No treatment was given to the control group. Treatment efficacy was assessed by clinical examination, vitreous culture and histopathology. There were no statististically significant differences between clinical scores of all groups in examinations at 24 and 48 h post-infection (p = 0.915 and p = 0.067 respectively), but in examinations at 72 h post-infection and after 7 days post-infection, although the clinical scores of treatment groups were not significantly different from each other, they were significantly lower than the control group (p < 0.05). The culture results of all groups were sterile. As a result, CAPE was found to be as effective as dexamethasone in reducing inflammation in the treatment of experimental MRSE endophthalmitis when used with antibiotics. More studies are needed to determine the optimal administration route and effective dosage of this compound.     

Diagnosis and Recommendation Integrated System Assessment of the Nutrients Limiting and Nutritional Status of Tomato

Tomato is an important field crop, and nutritional imbalances frequently reduce its yield. Diagnosis and Recommendation Integrated System (DRIS), uses ratios for nutrient deficiency diagnosis instead of absolute concentration in plant tests. In this study, local DRIS norms for the field tomatoes were established and the nutrient(s) limiting tomatoes yield were determined. Tomato leaves were analyzed for nutrients, to identify nutritional status using the DRIS approach. One hundred tomatoes fields were selected from Chatter Plain Khyber Pakhtunkhwa and the Sheikupura Punjab Pakistan. The first fully matured leaf was sampled, rinsed, dried and ground for analyzing P, K, Ca, Mg, Cu, Fe, Mn and Zn using an Inductively Coupled Plasma Atomic Emission Spectrophotometer (ICP AES). Plant tissue N and S were measured by the combustion method. The tomatoes yields were recorded at each location. The data were divided into high-yielding (≥3.79 kg/10 plant) and low-yielding (<3.79 kg/10 plant) populations and norms were computed using standard DRIS procedures. High-yielding plant population had a statistically greater mean S and Fe than the low-yielding population. The average balance index, the sum of functions, for S and Fe were −11.04 and −5.17 which reflected deficiency of S and Fe. Plant nutrients norms established may optimize plant nutrition in field tomatoes for high yield.