Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of β-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.     

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.     

Kinetic studies of laccase enzyme of Coriolus versicolor MTCC 138 in an inexpensive culture medium

An attempt was made to use cyanobacterial biomass of water bloom, groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme production by Coriolus versicolor. Laccase production was found to be 10.15 ± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial pH 5.0 and 28 ± 2 °C temperature. Half life of enzyme was found to be 74 min at 60 °C. Kinetic analysis of laccase when made with substrate ABTS, K_m and V_max were found to be 0.29 mM and 9.49 μmol/min respectively. Azide and hydroxylamine were found to exert significant inhibition on thermostable laccase. Inhibitor constant (k_i) for azide and hydroxylamine were 1.33 and 0.18 mM respectively. This study forms the first report on the potential application of waste water cyanobacterial bloom and dyeing effluent as a medium for laccase production by C. versicolor MTCC138.     

Endonuclease G promotes cell death of non-invasive human breast cancer cells

The invasiveness of breast cancer cells was shown to be associated with the suppressed ability to develop apoptosis. The role of cell death DNases/endonucleases has not been previously examined in relation with the invasiveness of breast cancer cells. We have compared the activity of the endonucleases in seven human breast cancer cell lines different in the level of invasiveness and differentiation. The invasiveness of cell lines was confirmed by an in vitro Matrigel-based assay. The total endonuclease activity in the differentiated non-invasive (WDNI) cell lines was higher than that in the poorly differentiated invasive (PDI) cells. The expression of EndoG strongly correlated with the degree of estrogen receptor expression and showed an inverse correlation with vimentin and matrix metalloproteinase-13. The EndoG-positive WDNI cells were more sensitive to etoposide- or camptothecin-induced cell death than EndoG-negative PDI cells. Silencing of EndoG caused inhibited of SK-BR-3 WDNI cell death induced by etoposide. Human ductal carcinomas in situ expressed high levels of EndoG, while invasive medullar and ductal carcinomas had significantly decreased expression of EndoG. This correlated with decreased apoptosis as measured by TUNEL assay. Our findings suggest that the presence of EndoG in non-invasive breast cancer cells determines their sensitivity to apoptosis, which may be taken into consideration for developing the chemotherapeutic strategy for cancer treatment.     

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants. The results of this paper have been granted US Patent No. 6,791,009.     

KINETIC STUDIES OF L-ASPARAGINASE FROM Penicillium digitatum

L-Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and other related malignancies. Its further use includes reduction of asparagine concentration in food products, which may lead to formation of acrylamide. Currently bacterial asparaginase is produced at industrial scale, but the enzyme isolated from bacterial origin is often associated with adverse reactions. These side effects require development of asparaginase from alternative sources. In the present study, Penicillium digitatum was explored for the production of extracellular L-asparaginase using modified Czapek–Dox media. The enzyme was purified about 60.95-fold and then kinetic study showed that the Km value of the enzyme was 1 × 10^?5 M. The optimum pH and temperature for the enzyme were 7.0 and 30°C, respectively. The optimum incubation period for L-asparaginase was 15 min. This work concludes that this enzyme can be a suitable candidate due to its strong kinetic properties, and further research can usher into development of asparaginase formulation from fungal origin with less adverse effects.     

A Dual Tracer 18F-FCH/18F-FDG PET Imaging of an Orthotopic Brain Tumor Xenograft Model

Early diagnosis of low grade glioma has been a challenge to clinicians. Positron Emission Tomography (PET) using ¹⁸F-FDG as a radio-tracer has limited utility in this area because of the high background in normal brain tissue. Other radiotracers such as ¹⁸F-Fluorocholine (¹⁸F-FCH) could provide better contrast between tumor and normal brain tissue but with high incidence of false positives. In this study, the potential application of a dual tracer ¹⁸F-FCH/¹⁸F-FDG-PET is investigated in order to improve the sensitivity of PET imaging for low grade glioma diagnosis based on a mouse orthotopic xenograft model. BALB/c nude mice with and without orthotopic glioma xenografts from U87 MG-luc2 glioma cell line are used for the study. The animals are subjected to ¹⁸F-FCH and ¹⁸F-FDG PET imaging, and images acquired from two separate scans are superimposed for analysis. The ¹⁸F-FCH counts are subtracted from the merged images to identify the tumor. Micro-CT, bioluminescence imaging (BLI), histology and measurement of the tumor diameter are also conducted for comparison. Results show that there is a significant contrast in ¹⁸F-FCH uptake between tumor and normal brain tissue (2.65 ± 0.98), but with a high false positive rate of 28.6%. The difficulty of identifying the tumor by ¹⁸F-FDG only is also proved in this study. All the tumors can be detected based on the dual tracer technique of ¹⁸F-FCH/ ¹⁸F-FDG-PET imaging in this study, while the false-positive caused by ¹⁸F-FCH can be eliminated. Dual tracer ¹⁸F-FCH/¹⁸F-FDG PET imaging has the potential to improve the visualization of low grade glioma. ¹⁸F-FCH delineates tumor areas and the tumor can be identified by subtracting the ¹⁸F-FCH counts. The sensitivity was over 95%. Further studies are required to evaluate the possibility of applying this technique in clinical trials.     

Protein stabilization by introduction of cross-strand disulfides

Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.     

Assessment of Polycyclic Aromatic Hydrocarbons (PAH) and Heavy Metals in the Vicinity of an Oil Refinery in India

Petroleum products are one of the major sources of energy for industry and daily life. Growth of the petroleum industry and shipping of petroleum products has resulted in the pollution. Populations living in the vicinity of oil refinery waste sites may be at greater risk of potential exposure to polycyclic aromatic hydrocarbons (PAH) through inhalation, ingestion, and direct contact with contaminated media. PAH have often been found to coexist with environmental pollutants including heavy metals due to similar pollution sources. The levels and distribution patterns of Σ16 PAH (sum of the 16 PAH) and heavy metals (lead, copper, nickel, cobalt, and chromium) were determined in soil and sediment in the vicinity (5 km radius) of an oil refinery in India. Concentrations of Σ16 PAH in the soils and sediments were found to be 60.36 and 241.23 ppm, respectively. Higher amount of PAH in sediments as compared to soil is due to low water solubility of PAH, settled in the bottom of aquatic bodies. The levels of lead, copper, nickel, cobalt, and chromium (total) in soil were 12.52, 13.52, 18.78, 4.84, and 8.29 ppm, while the concentrations of these metals in sediments were 16.38, 47.88, 50.15, 7.07, and 13.25 ppm, respectively. Molecular diagnostics indices of PAH (Ratio of Phenanthrene/Anthracene, Fluranthene/Pyrene) calculated for soil and sediment samples indicate that the oil refinery environment is contaminated with PAH from petrogenic as well as pyrolytic origin and heavy vehicular traffic on the Agra- Delhi National highway. Sixteen PAH priority pollutants were detected in the United States in entire samples collected near oil refinery areas and concentrations of Σ16 PAH in soil was found to be 1.20 times higher than the threshold value for PAH in soil by ICRCL (Inter-Departmental Committee on the Redevelopment of Contaminated Land). This concentration could lead to disastrous consequences for the biotic and abiotic components of the ecosystem and may affect the soil quality, thus impairing plant growth and its bioaccumulation in food chain.