Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.     

Neurotoxicity of fluoride: neurodegeneration in hippocampus of female mice

Light microscopic study of hippocampal sub-regions demonstrated significant number of degenerated nerve cell bodies in the CA3, CA4 and dentate gyrus(Dg) areas of sodium fluoride administered adult female mice. Ultrastructural studies revealed neurodegenrative characteristics like involution of cell membranes, swelling of mitochondria, clumping of chromatin material etc, can be observed in cell bodies of CA3, CA4 and dentate gyrus (Dg). Fluoride intoxicated animals also performed poorly in motor co-ordination tests and maze tests. Inability to perform well increased with higher fluoride concentration in drinking water.     

The Th1-specific costimulatory molecule,m150, is a posttranslational isoform of lysosome-associated membrane protein-1

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4(+) T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4(+) T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.     

Analytical method for monitoring concentrations of cyclosporin and lovastatin in vitro in an everted rat intestinal sac absorption model

Cyclosporin A (CSA) and lovastatin (LV) are lipophilic drugs, which show poor and erratic absorption when administered perorally. The permeability of these compounds can be increased transiently by altering the membrane characteristics of the absorptive epithelium by the use of sorption promoters (SPs). In the present work a simple validated HPLC method utilizing an isocratic mobile phase with short retention times for CSA and LV was developed in order to monitor their concentrations in Kreb's Ringer bicarbonate (KRB) solution in vitro in intestinal sac absorption model. The same method was utilized to determine the apparent permeability coefficients and absorption profiles of CSA and LV by a modified Wilson-Wiseman method. Drugs were analysed by a reversed-phase HPLC method using a Shim-pack C18 column. An isocratic mobile phase containing acetonitrile and water in the proportions 70:30 and 80:20 was used for the HPLC analysis of CSA and LV, respectively. The flow-rate was 2 ml/min and quantitative determinations were carried out at 215 nm at 70 degrees C for CSA. In the case of LV the flow-rate was 1 ml/min and detection was done at 238 nm at 25 degrees C. The method was found to be specific as none of the proposed SPs, components of KRB or intestinal sac artefacts interfered with the drug peaks. Recovery studies and intra- and inter-day variations were within statistical limits. The limits of detection were 250 and 10 ng/ml and the limits of quantitation were 400 and 30 ng/ml for CSA and LV, respectively. The calibration curve was found to be linear in concentration range of 0.5-6 microg/ml for CSA and 0.05-0.4 microg/ml for LV. The proposed method was found to be rapid and selective and hence can be applied for continuous monitoring of CSA and LV in vitro in intestinal sac absorption studies.     

Reversal of fluoride induced cell injury through elimination of fluoride and consumption of diet rich in essential nutrients and antioxidants

The objective of the present communication is to address the issues concerning reversal of fluoride induced cell injury and disease (i.e. fluorosis) through the elimination of fluoride and consumption of a diet containing essential nutrients and antioxidants. Humans afflicted with fluorosis, as a result of consuming fluoride contaminated water or food, have been investigated. Hospital based diagnostic procedure for early detection of fluorosis, through retrieval of history, clinical complaints, testing of blood, urine and drinking water for fluoride using ion selective electrode technology, along with X-ray of the forearm have been carried out. Confirmed cases of fluorosis were introduced to an intervention protocol consisting of (1) provision of safe drinking water with fluoride levels less than 1 mg/L and (2) counselling on nutritional supplementation with focus on adequate intake of calcium, vitamins C, E and antioxidants. The patients were monitored at frequent intervals up to one year and the results are reported. With a standardized early diagnosis, elimination of fluoride intake and supplementation of a diet rich in essential nutrients and antioxidants, we have shown that the fluorosis can be reversed.     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Laser-assisted microinjection into targeted animal cells

A pulsed (17 nanoseconds) Nd:YAG laser (1064 nm) was used to inject impermeable dyes (propidium iodide andiodide and merocyanine 540) and a plasmid (pEGFP-N1) encoding green fluorescent protein (GFP) into human breast adenocarcinoma cells (MCF-7). The cell membrane integrity and viability were fully preserved in this laser-assisted transfer.     

Controlled rotation of biological microscopic objects using optical line tweezers

Controlled, continuous rotation of cells or intracellular objects was achieved using optical tweezers with an elliptic beam profile (line tweezers), which was generated by placing a cylindrical lens in the path of the trapping beam. By rotating the cylindrical lens, rotation of the elliptic trapping beam and hence of the object trapped therein was achieved. Compared to previously reported techniques for rotation of microscopic objects, this approach is much simpler, gives better utilization of available laser power and also allows much easier control of the trap beam profile. We have used this approach for rotation of biological objects varying in size from 2 to 40 m. At 25 mW trapping beam power at the object plane E. coli bacteria could be rotated at speeds approaching 10 Hz and an intracellular object (presumably a calcium oxalate crystal) trapped inside Elodea densa plant cell could be rotated with speeds of up to 4 Hz. To our knowledge, this is the first report for rotation of an intracellular object.