Labeling of skeletal myoblasts with a novel oxygen-sensing spin probe for noninvasive monitoring of in situ oxygenation and cell therapy in heart

We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen-sensing paramagnetic spin probe for noninvasive tracking and in situ monitoring of oxygenation in stem cell therapy using electron paramagnetic resonance (EPR) spectroscopy. SM cells were isolated from thigh muscle biopsies of mice and propagated in culture. Labeling of SM cells with the probe was achieved by coincubating the cells with submicron-sized (270 +/- 120 nm) particulates of the probe in culture for 48 h. The labeling had no significant effect on the viability or proliferation of the cells. The SM cells labeled with the probe were transplanted in the infarcted region of mouse hearts. The engraftment of the transplanted cells in the infarct region was verified by using MY-32 staining for skeletal myocytes. The in situ Po(2) in the heart was determined noninvasively and repeatedly for 4 wk after transplantation. The results showed significant enhancement of myocardial oxygenation at the site of cell transplant compared with untreated control. In conclusion, labeling of SM cells with the oxygen-sensing spin probe offers a unique opportunity for the noninvasive monitoring of transplanted cells as well as in situ tissue Po(2) in infarcted mouse hearts.     

Regulation of Caenorhabditis elegans and Pseudomonas aeruginosa machinery during interactions

The amenability of Caenorhabditis elegans against pathogen provides a valuable tool for studying host–pathogen interactions. Physiological experiments revealed that the P. aeruginosa was able to kill C. elegans efficiently. The effects of P. aeruginosa PA14, PAO1 and their isolated lipopolysaccharide (LPS) on the host system were analyzed. The LPS at higher concentrations (≥2 mg/ml) was toxic to the host animals. Kinetic studies using qPCR revealed the regulation of host-specific candidate antimicrobial genes during pathogen-mediated infections. In addition, the pathogen-specific virulent gene, exoT expression, was anlyzed and found to be varied during the interactions with the host system. Ability of the pathogens to modify their internal machinery in the presence of the host was analyzed by XRD, FTIR and PCA. LPS isolated from pathogens upon exposure to C. elegans showed modifications at their functional regions. LPS from PAO1 showed difference in d-spacing angle (Å) and °2Th position. FTIR spectra revealed alterations in polysaccharide (1,200–900 cm⁻¹) and fatty acid (3,000–2,800 cm⁻¹) regions of LPS from P. aeruginosa PAO1 exposed to the host system. These data provide additional insights on how the pathogens subvert its own and host machinery during interactions.     

Energy flow from spider eggs through dipteran parasite and hymenopteran hyperparasite populations

Summary The incidence of the parasites in the egg sacs of the spider Argiope pulchella was 100% for Sarcophaga banksi and 25% for Tachinobia repanda. In 1976, 237 spider eggs equivalent to 131 gcal/m² were present. Of these, 212 eggs/m² (=117 gcal) were consumed by S. banksi larvae, leaving 25 spiderlings/m² (=14 gcal) to emerge. The density of S. banksi larvae was l/m² (=87 gcal), of which 0.7/m² S. banksi (=41 gcal) successfully emerged; as few as 0.3 S. banksi/m ² (=25 gcal) were infected by T. repanda; only 14 T. repanda/m² (=8 gcal) successfully emerged. Exploitation efficiency was 89% for S. banksi and 29% for T. repanda. Ecological efficiency was 66% for S. banksi and only 9% for T. repanda. The egg sac area of A. pulchella holds a straight line relationship to the energy content of the eggs; the sacs were grouped into 8 different sizes and each one further into groups containing 1, 2, and 3 S. banksi larvae per sac. Analysis of the sacs at the appropriate time revealed that an S. banksi larva consumed a minimum of 114 eggs (=70 gcal), when present as one of a pair in the smallest sac (0.6 cm² area), and a maximum of 476 eggs (=234 gcal), when present alone in the largest sac (1.3 cm²). Despite this wide difference in food intake, all S. banksi (barring those infected by T. repanda) successfully emerged. The energy content of a pharate pupa, which was 125, 92, and 68 gcal in a sac with 1 cm² area containing 1, 2, or 3 S. banksi, depended on the size of the sac and the number of S. banksi per sac. The corresponding values for the imago were 82, 61, and 45 gcal. The efficiency of S. banksi ranged between 60 and 80% for food conversion and between 35 and 56% for pupation.This work was completed at our Palni Centre     

Extracting kinetic rate constants from surface plasmon resonance array systems

Surface plasmon resonance imaging systems, such as Flexchip from Biacore, are capable of monitoring hundreds of reaction spots simultaneously within a single flow cell. Interpreting the binding kinetics in a large-format flow cell presents a number of potential challenges, including accounting for mass transport effects and spot-to-spot sample depletion. We employed a combination of computer simulations and experimentation to characterize these effects across the spotted array and established that a simple two-compartment model may be used to accurately extract intrinsic rate constants from the array under mass transport-limited conditions. Using antibody systems, we demonstrate that the spot-to-spot variability in the binding kinetics was <9%. We also illustrate the advantage of globally fitting binding data from multiple spots within an array for a system that is mass transport limited.     

Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55

Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.     

Surfacing frequency as an index of bioenergetics components of air breathing fishes

Surfacing frequency (Sf) in obligatory air-breathing fishes is a behavioural index of O₂ uptake; as both are interrelated, there is a possibility of predicting one from the other. From the point of cause and effect relationship, feeding (C) is regarded as the cause and Sf and O₂ uptake as its effects. Hence, the last two are predictable from the cause. A pathway for predicting the bioenergetics components, feeding (C), metabolism (M) and growth (P) from a behavioural index of surfacing frequency has been proposed. In this model, C occupies the focal point connecting on the horizontal plane Sf on one side and P on the other side and M vertically. The developed multiple regression models predict the bioenergetics components of the fishes,Macropodus cupanus andChanna striatus of different weight classes ex-posed to different rations and temperatures; the percentage of variation accounted for by the equations is over 90. The pathway can also be followed for predicting bioenergetics components of gill-breathing fishes, if feeding rate alone is known. On application of the relevant part of the pathway toSalmo trutta, the percentage of variation accounted by the equations is 95.