Effect of DNA sequence and nucleotide cofactors on hRad51 binding to ssDNA: role of hRad52 in recruitment

hRad51 binding to ssDNA is significantly lowered in the presence of a nucleotide cofactor ATP/ADP/ATPgammaS. In these conditions, presence of trace amounts of hRad52 protein restores hRad51 binding to DNA. In the absence of any nucleotide cofactor where intrinsic binding of hRad51 to ssDNA is higher, hRad52 brings about no improved binding. hRad51 binding to ssDNA is strongly influenced by the DNA sequence. The protein binding to repeat sequences is poor compared to that of mixed DNA sequence. Interestingly, presence of hRad52 restores the ability of hRad51 binding to such DNA targets as well. Moreover, all the cooperative effects of hRad52 on hRad51 binding are highly specific to the latter's binding to ssDNA and not to dsDNA. These results help us to model important mechanistic steps of hRad51 presynapsis on ssDNA templates.     

Interaction of hRad51 and hRad52 with MCM complex: a cross-talk between recombination and replication proteins

Human Rad51 and Rad52 are implicated in DNA repair during replication. Here we show, by pull-down assays, that purified hRad51 and hRad52 interact with each other as well as with Mini chromosome maintenance (MCM) proteins in HeLa cell extracts. Furthermore, immunoprecipitation experiments corroborate the same where hRad51 and hRad52 proteins not only cross-talk with each other but also pull down MCM3 and MCM2/3 proteins, respectively. The interaction scoring assays, performed reciprocally, demonstrate the same specificity, based on which, we speculate that MCM complex exhibits strong propensity to get physically recruited to the sites where hRad51 and hRad52-mediated homologously aligned ends need to be replicationally repaired.     

Physical exercise, body mass index, subcutaneous adiposity and body composition among Bengalee boys aged 10-17 years of Kolkata, India

A comparative study of 215 sedentary (no regular physical exercise undertaken) and 313 physically active (regular physical exercise undertaken) Bengalee boys aged 10-17 years was undertaken to investigate the differences in overall adiposity (body mass index), subcutaneous adiposity (skinfolds) and body composition (percent body fat, fat mass and fat mass index). Both groups had a similar age. The results revealed that boys who did not undertake regular physical exercise (NPE) had a significantly greater mean body mass index (BMI) compared with those who undertook regular physical exercise (PE); p < 0.001. The means for all the skinfolds as well as percent body fat (PBF), fat mass (FM) and fat mass index (FMI) were significantly higher among the NPE group. The percentile distributions of all these variables and indices were consistently higher among the NPE group. The results of ANOVA of physical exercise (PE = yes, NPE = no) and PBF, FM and FMI, with age as covariate, revealed that PE had a significant negative effect on all these measures of body composition even after controlling for the impact of age. The means in each case were greater among the NPE group. In conclusion, this study provided evidence that Bengalee boys, who undertook regular physical exercise, had significantly less adiposity compared with those who did not undertake regular physical exercise.     

NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light activation

Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.     

Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the beta-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca(2+)) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca(2+)-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca(2+) ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca(2+) influx seen in the rd photoreceptors. Ca(2+)-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca(2+) influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.     

Clustering of protein structural fragments reveals modular building block approach of nature

Structures of peptide fragments drawn from a protein can potentially occupy a vast conformational continuum. We co-ordinatize this conformational space with the help of geometric invariants and demonstrate that the peptide conformations of the currently available protein structures are heavily biased in favor of a finite number of conformational types or structural building blocks. This is achieved by representing a peptides' backbone structure with geometric invariants and then clustering peptides based on closeness of the geometric invariants. This results in 12,903 clusters, of which 2207 are made up of peptides drawn from functionally and/or structurally related proteins. These are termed "functional" clusters and provide clues about potential functional sites. The rest of the clusters, including the largest few, are made up of peptides drawn from unrelated proteins and are termed "structural" clusters. The largest clusters are of regular secondary structures such as helices and beta strands as well as of beta hairpins. Several categories of helices and strands are discovered based on geometric differences. In addition to the known classes of loops, we discover several new classes, which will be useful in protein structure modeling. Our algorithm does not require assignment of secondary structure and, therefore, overcomes the limitations in loop classification due to ambiguity in secondary structure assignment at loop boundaries.     

Effect of forced convection on the skin thermal expression of breast cancer

A bioheat-transfer-based numerical model was utilized to study the energy balance in healthy and malignant breasts subjected to forced convection in a wind tunnel. Steady-state temperature distributions on the skin surface of the breasts were obtained by numerically solving the conjugate heat transfer problem. Parametric studies on the influences of the airflow on the skin thermal expression of tumors were performed. It was found that the presence of tumor may not be clearly shown due to the irregularities of the skin temperature distribution induced by the airflow field. Nevertheless, image subtraction techniques could be employed to eliminate the effects of the flow field and thermal noise and significantly improve the thermal signature of the tumor on the skin surface. Inclusion of the possible skin vascular response to cold stress caused by the airflow further enhances the signal, especially for deeply embedded tumors that otherwise may not be detectable.     

Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43-Tyr44 pair and specificity of staphylokinase-plasmin activator complex

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with plasmin (Pm) and changes its substrate specificity to create a plasminogen (Pg) activator complex. The His(43)-Tyr(44) pair of SAK resides within the active site cleft of the partner Pm and generates intermolecular contacts to confer Pg activator ability to the SAK-Pm bimolecular complex. Site-directed mutagenesis and molecular modeling studies unravelled that mutation at 42nd or 45th positions of SAK specifically disrupts cation-pi interaction of His(43) with Trp(215) of partner Pm within the active site, whereas pi-pi interaction of Tyr(44) with Trp(215) remain energetically favoured.     

Low modulus biomimetic microgel particles with high loading of hemoglobin

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.