Differential responses of UV-B irradiation on the viability and intracellular calcium influx in goat hepatocytes–in vitro effect

Ultraviolet-B (UV-B) irradiation in the range of 280-320nm has shown to be a promising immunomodulatory tool in xenogenic hepatocyte transplantation. Most of the studies documenting the effect(s) of UV-B irradiation on hepatic transplantation have been carried out in small model systems with very little information available in larger animals. The aim of the present investigation was to study in vitro the effect(s) of UV-B irradiation (302 nm) at 0, 250, 500, 1250 and 2500 J/m2 on the viability and cellular responses in the isolated goat hepatocytes. The results showed that the cells irradiated at 0, 250, 500, 1250 and 2500 J/m2 demonstrated a viability of 90-95%. However, intracellular [Ca2+]i influx as quantitated by Flu 3-acetete showed a significant increase with irradiation as observed in confocal microscope. The intracellular pH (quantitated by the flourescence of BCCEF) although tend to show an increase with UV-B irradiation was not statistically significant. The present observations suggest that there is a modulation in the intracellular [Ca2+]i concentration within the hepatocytes at higher dose of UV-B irradiation without altering the viability of hepatocytes. These observations are significant for the xenotransplantation of cells.     

Stereochemical criteria for prediction of the effects of proline mutations on protein stability

When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.     

Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning

Formation of normal olfactory memory requires the expression of the wild-type amnesiac gene in the dorsal paired medial (DPM) neurons. Imaging the activity in the processes of DPM neurons revealed that the neurons respond when the fly is stimulated with electric shock or with any odor that was tested. Pairing odor and electric-shock stimulation increases odor-evoked calcium signals and synaptic release from DPM neurons. These memory traces form in only one of the two branches of the DPM neuron process. Moreover, trace formation requires the expression of the wild-type amnesiac gene in the DPM neurons. The cellular memory traces first appear at 30 min after conditioning and persist for at least 1 hr, a time window during which DPM neuron synaptic transmission is required for normal memory. DPM neurons are therefore "odor generalists" and form a delayed, branch-specific, and amnesiac-dependent memory trace that may guide behavior after acquisition.     

Management of filariasis using prediction rules derived from data mining

The present paper demonstrates the application of CART (classification and regression trees) to control a mosquito vector (Culex quinquefasciatus) for bancroftian filariasis in India. The database on filariasis and a commercially available software CART (Salford systems Inc. USA) were used in this study. Baseline entomological data related to bancroftian filariasis was utilized for deriving prediction rules. The data was categorized into three different aspects, namely (1) mosquito abundance, (2) meteorological and (3) socio-economic details. This data was taken from a database developed for a project entitled "Database management system for the control of bancroftian filariasis" sponsored by Ministry of Communication and Information Technology (MC&IT), Government of India, New Delhi. Predictor variables (maximum temperature, minimum temperature, rain fall, relative humidity, wind speed, house type) were ranked by CART according to their influence on the target variable (month). The approach is useful for forecasting vector (mosquito) densities in forthcoming seasons.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity

Optimization of adenosine analog inhibitors of bacterial NAD⁺-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3′- and 5′-substituents on the ribose. Compounds with log D values 1.5-2.5 maximized potency and maintained drug-like physical properties.     

Comparison of the dynamics of bovine and human angiogenin: a molecular dynamics study

Molecular dynamics simulations have been carried out for 1 ns on human and bovine angiogenin systems in an effort to compare and contrast their dynamics. An analysis of their dynamics is done by examining the rms deviations, following hydrogen-bonding interactions and looking at the role of water in and around the protein. The C-terminus of bovine angiogenin moves appreciably during dynamics suggesting a better structure for ligand binding. However, we do not find any evidence of a conformation where the glutamate residue that obstructs the active site takes on a different conformation. We observe a differential hydrogen-bonding pattern in the active site regions of bovine and human angiogenins, which could have a bearing on the different catalytic activities of the proteins. We also propose that the differential binding of the monoclonal antibody toward the two proteins might be due sequential and not conformational differences. Water molecules might play an important functional role in both proteins given their subtle functional differences. A simple computation on the molecular dynamics data has been carried out to identify locations in and around the protein that are invariably occupied by water. The locations of nearly half the waters we have identified from the simulation as being invariant in bovine angiogenin occupy similar locations in the bovine angiogenin crystal structure. The positions of the waters identified in human angiogenin differ considerably from that of bovine angiogenin.     

Tools for comparative protein structure modeling and analysis

The following resources for comparative protein structure modeling and analysis are described (http://salilab.org): MODELLER, a program for comparative modeling by satisfaction of spatial restraints; MODWEB, a web server for automated comparative modeling that relies on PSI-BLAST, IMPALA and MODELLER; MODLOOP, a web server for automated loop modeling that relies on MODELLER; MOULDER, a CPU intensive protocol of MODWEB for building comparative models based on distant known structures; MODBASE, a comprehensive database of annotated comparative models for all sequences detectably related to a known structure; MODVIEW, a Netscape plugin for Linux that integrates viewing of multiple sequences and structures; and SNPWEB, a web server for structure-based prediction of the functional impact of a single amino acid substitution.