On the origin of affinity--specificity negative correlation in DNA--probe interactions

Affinity (the fastness) and specificity (the correctness) are the two important factors that decide the efficiency of a nucleic acid probe to target its specific site on a DNA lattice. DNA-probe interactions differ from protein-ligand interactions in a way that here the specificity and the affinity of the interactions correlate negatively with each other. We present a simple phenomenological theory to explain the negative correlation between the specificity and the affinity of the probe towards its target site on the template DNA under solution conditions. We show that a simple random jump model can explain this fact and we also predict that the negative correlation between the affinity and specificity diminishes as the temperature increases or the viscosity of the medium decreases. Moreover, the length of target DNA and the distance between the initial position of the probe on the template DNA lattice and the target site increases the magnitude of affinity-specificity negative correlation. These results are consistent with experimental observations. Finally we propose practical strategies to coherently improve the specificity and the affinity with respect to important molecular biological techniques such as PCR and Southern blotting.     

Crystal structure of a novel regulatory 40-kDa mammary gland protein (MGP-40) secreted during involution

We have determined the crystal structure of a novel regulatory protein (MGP-40) from the mammary gland. This protein is implicated as a protective signaling factor that determines which cells are to survive the drastic tissue remodeling that occurs during involution. It has been indicated that certain cancers could surreptitiously utilize the proposed normal protective signaling by proteins of this family to extend their own survival and thereby allow them to invade the organ and metastasize. In view of this, MGP-40 could form an important target for rational structure-based drug design against breast cancer. It is a single chain, glycosylated protein with a molecular mass of 40 kDa. It was isolated from goat dry secretions and has been cloned and sequenced. It was crystallized by microdialysis from 20 mg ml(-1) solution in 0.1 m Tris-HCl, pH 8.0, and equilibrated against the same solution containing 19% ethanol. Its x-ray structure has been determined by molecular replacement and refined to a 2.9 A resolution. The protein adopts a beta/alpha domain structure with a triose-phosphate isomerase barrel conformation in the core and a small alpha+beta folding domain. A single glycosylation site containing two N-acetylglucosamine units has been observed in the structure. Compared with chitinases and chitinase-like proteins the most important mutation in this protein pertains to a change from Glu to Leu at position 119, which is part of the so-called active site sequence in the form of Asp(115), Leu(119), and Asp(186) and in this case resulting in the loss of chitinase activity. The orientations of two Trp residues Trp(78) and Trp(331) in the beta barrel reduces the free space, drastically impairing the binding of saccharides/polysaccharides. However, the site and mode of binding of this protein to cell surface receptors are not yet known.     

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0–2.8 M in the presence of camphor and 0–1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20–60 °C in the presence of the substrate and 20–47 °C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.     

Expression, purification and characterization of fourth FAS1 domain of TGFβIp-associated corneal dystrophic mutants

Corneal dystrophies (CDs) are a group of inherited bilateral disorders affecting the corneal tissue of the eye. Most of these CDs in the stromal layer of the cornea have been associated with mutations found on the TGFBI gene that codes for a 683-amino acid transforming growth factor induced protein (TGFβIp). These mutations have been found to induce atypical aggregation and progressive accumulation of protein aggregates in the cornea that leads to loss of corneal transparency and hence blindness. At present, 65 distinct pathogenic mutations have been identified in TGFBI that are associated with different clinical phenotypes. More than 80% of these missense mutations occur in the 4th FAS (fasciclin-like) 1 domain. Current treatment includes surgical intervention, which often involves high recurrence rates. Hence, it is imperative to examine the properties of the TGFβIp and the pathogenic mutant proteins to understand the pathology of the disease mechanism and to develop potent therapeutics. Here, we report the recombinant expression, purification, characterization and the effect of four clinically significant pathogenic TGFβIp mutants - R555W, H572R, A620D, and H626R on the biophysical properties of the wild type (WT) 4th FAS1 domain of TGFβIp. While a higher proportion of the R555W, H572R and H626R mutants of the 4th FAS1 domains remained stable, the A620D mutant largely existed as inclusion bodies in native state and aggregates under physiological conditions. These mutants present a unique platform to examine protein aggregation-prone diseases wherein single amino-acid mutations present distinct pathogenic phenotypes. Though pathogenically and phenotypically diverse, these mutants do not exhibit variations in secondary structure and stability, except for the A620D mutant, when examined by CD and UV spectroscopy.     

Opiate antagonist prevents μ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth

In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.     

Characterization of synthesized silver nanoparticles and assessment of its genotoxicity potentials using the alkaline comet assay

Nano-silver (Nano-Ag) particles were synthesized and then characterized using transmission electron microscopy (TEM) and X-ray diffractometry. TEM showed that Nano-Ag were spherical in shape and their size ranged from 40 to 60nm. X-ray diffractometry indicated that the sample was crystalline and had a face centered cubic structure of pure silver. Genotoxicity of this Nano-Ag was evaluated in human peripheral blood cells using the alkaline comet assay. Results indicated that Nano-Ag (50 and 100μg/mL) caused DNA damage following a 3h treatment. Subsequently, a short treatment of 5min also showed DNA damage. In conclusion, we have shown that the synthesized Nano-Ag induced DNA damage in human peripheral blood cells as detected by the alkaline comet assay. Results further indicated that treatment of cells with Nano-Ag in the presence of hydrogen peroxide did not induce any DNA damage.     

Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Changes in Soil Microbial Biomass and Residual Indices as Ecological Indicators of Land Use Change in Temperate Permanent Grassland

The relationship between microbial biomass, residues and their contribution to microbial turnover is important to understand ecosystem C storage. The effects of permanent grassland (100 % ryegrass—PG), conversion to modified grassland (mixture of grass and clover—MG) or maize monoculture (MM) on the dynamics of soil organic C (SOC), microbial biomass, fungal ergosterol and microbial residues (bacterial muramic acid and fungal glucosamine) were investigated. Cattle slurry was applied to quantify the effects of fertilisation on microbial residues and functional diversity of microbial community across land use types. Slurry application significantly increased the stocks of microbial biomass C and S and especially led to a shift in microbial residues towards bacterial tissue. The MM treatment decreased the stocks of SOC, microbial biomass C, N and S and microbial residues compared with the PG and MG treatments at 0–40 cm depth. The MM treatment led to a greater accumulation of saprotrophic fungi, as indicated by the higher ergosterol-to-microbial biomass C ratio and lower microbial biomass C/S ratio compared with the grassland treatments. The absence of a white clover population in the PG treatment caused a greater accumulation of fungal residues (presumably arbuscular mycorrhizal fungi (AMF), which do not contain ergosterol but glucosamine), as indicated by the significantly higher fungal C-to-bacterial C ratio and lower ergosterol-to-microbial biomass C ratio compared with the MG treatment. In addition to these microbial biomass and residual indices, the community level physiological profiles (CLPP) demonstrated distinct differences between the PG and MG treatments, suggesting the potential of these measurements to act as an integrative indicator of soil functioning.