Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial alpha-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.     

Mesenchymal precursor cells in the blood of normal individuals

Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).     

31P Nuclear Magnetic Resonance Studies on the Phosphorus-Containing Metabolites of the Developing Embryos of a Freshwater Catfish, Clarias batrachus (L.)

Changes in the phosphorus-containing metabolites were monitored by ³¹P nuclear magnetic resonance in the developing embryos of Clarias batrachus. Phosphomonoester, yolk phosphoprotein, phosphocreatine, ATP, and inorganic phosphate (Pi) were consistently observed in all the developmental stages of C. batrachus. None of these phosphometabolites exhibited any significant change in their concentration up to the blastula stage, whereas distinct decrease in all except inorganic phosphate was observed in the fry stage. Concomitantly an increase in the concentration of inorganic phosphate was observed. Further, from the resonance positions of α, β, and γ phosphate groups of ATP, it was evident that the ATP molecules in vivo were liganded either to Ca²⁺ or Mg²⁺. This study also revealed that the intracellular pH of the developing embryos was approximately 7.05 up to the gastrula stage, after which it decreased in the fry stage to 6.98 units. Received August 10, 1998; accepted November 3, 1998.     

Cuprizone demyelination of the corpus callosum in mice correlates with altered social interaction and impaired bilateral sensorimotor coordination

For studies of remyelination in demyelinating diseases, the cuprizone model of CC (corpus callosum) demyelination has experimental advantages that include overall size, proximity to neural stem cells of the subventricular zone, and correlation with a lesion predilection site in multiple sclerosis. In addition, cuprizone treatment can be ended to allow more direct analysis of remyelination than with viral or autoimmune models. However, CC demyelination lacks a useful functional correlate in rodents for longitudinal analysis throughout the course of demyelination and remyelination. In the present study, we tested two distinct behavioural measurements in mice fed 0.2% cuprizone. Running on a ‘complex'' wheel with varied rung intervals requires integration between cerebral hemispheres for rapid bilateral sensorimotor coordination. Maximum running velocity on the ‘complex'' wheel decreased during acute (6 week) and chronic (12 week) cuprizone demyelination. Running velocity on the complex wheel distinguished treated (for 6 weeks) from non-treated mice, even after a 6-week recovery period for spontaneous remyelination. A second behavioural assessment was a resident–intruder test of social interaction. The frequency of interactive behaviours increased among resident mice after acute or chronic demyelination. Differences in both sensorimotor coordination and social interaction correlated with demonstrated CC demyelination. The wheel assay is applicable for longitudinal studies. The resident–intruder assay provides a complementary assessment of a distinct modality at a specific time point. These behavioural measurements are sufficiently robust for small cohorts as a non-invasive assessment of demyelination to facilitate analysis of subsequent remyelination. These measurements may also identify CC involvement in other mouse models of central nervous system injuries and disorders.     

SEMG signal analysis at acupressure points for elbow movement

Surface electromyography signals (SEMG) are the most common form of non-invasive-measurement of muscle activities. To acquire proper SEMG for particular limb function, the placement of electrodes on the skin over respective active group of muscles becomes very important. Measurement of SEMG depends on a number of factors/parameters like amplitude, time and frequency domain properties. In the present investigation, analysis was carried firstly; to study the grip force vs. SEMG parameters at acupressure points on arm, using single channel approach. At all the selected acupressure points a linear increment of SEMG was observed. Secondly; to discriminate four elbow movements from different locations on arm using two channel approach with single parameter. The parameter for the analysis chosen was the root of mean of square (RMS) value of SEMG. Further; principal component analysis was used to verify the elbow movement discrimination. Extension and supination were the two operations which were observed to be easy to realize by prosthetic devices. The selection of these locations was done on the basis of acupressure points and anatomy of elbow. Matlab-softscope was used for acquiring the SEMG from line-in input port of PC-sound card. This study will also be helpful for the researchers in understanding the behavior of SEMG for elbow movement in development of prosthetic or exoskeleton devices.     

Phytotoxicity of decomposing below-ground residues of Ageratum conyzoides: nature and dynamics of release of phytotoxins

We investigated the dynamics of phytotoxicity and the quantitative changes in phenolics in decomposing root residues of Ageratum conyzoides over a 60-day period. A set of four treatments, viz. residues alone (R), residues mixed into soils (R + S, R + 3S), and soil alone (S), were maintained and the quantitative changes in phytotoxicity were monitored on 1, 5, 10, 15, 20, 25, 30, 45, and 60 day after decomposition (DAD). The phytotoxicity during the decomposition process was evaluated in a laboratory bioassay against radish (Raphanus sativus). The phytotoxicity of R, R + S, and R + 3S treatments increased during initial period of decomposition (up to 20-DAD), and declined afterwards (i.e., at >20-DAD). In general, the phytotoxicity was in the order R > R + S > R + 3S treatments. It was paralleled by a similar trend of changes in the amounts of water-soluble phenolics that increased up to 20-DAD and thereafter declined. The amount of phenolics was in the order R > R + S > R + 3S. At 1-DAD, the amount of water soluble phenolics in R, R + S, and R + 3S treatments was 765.3, 594.5, and 251.3 μg/ml, respectively. It enhanced to 1,266.76, 845.5, and 416.0 μg/ml at 20-DAD. However, at 60-DAD, the amounts of phenolics in R, R + S, and R + 3S treatments was 149.93, 142.6, and 100.0 μg/ml, respectively. The study concludes that the phytotoxicity of below-ground residues of A. conyzoides changes during decomposition and was reduced upon the addition of soil to the residues.     

β-Pinene inhibited germination and early growth involves membrane peroxidation

β-Pinene, an oxygenated monoterpene, is abundantly found in the environment and widely occurring in plants as a constituent of essential oils. We investigated the phytotoxicity of β-pinene against two grassy (Phalaris minor, Echinochloa crus-galli) and one broad-leaved (Cassia occidentalis) weeds in terms of germination and root and shoot growth. β-Pinene (0.02–0.80 mg/ml) inhibited the germination, root length, and shoot length of test weeds in a dose–response manner. The inhibitory effect of β-pinene was greater in grassy weeds and on root growth than on shoot growth. β-Pinene (0.04–0.80 mg/ml) reduced the root length in P. minor, E. crus-galli, and C. occidentalis over that in the control by 58–60, 44–92, and 26–85 %, respectively. In contrast, shoot length was reduced over the control by 45–97 % in P. minor, 48–78 % in E. crus-galli, and 11–75 % in C. occidentalis at similar concentrations. Further, we examined the impact of β-pinene on membrane integrity in P. minor as one of the possible mechanisms of action. Membrane integrity was evaluated in terms of lipid peroxidation, conjugated diene content, electrolyte leakage, and the activity of lipoxygenases (LOX). β-Pinene (≥0.04 mg/ml) enhanced electrolyte leakage by 23–80 %, malondialdehyde content by 15–67 %, hydrogen peroxide content by 9–39 %, and lipoxygenases activity by 38–383 % over that in the control. It indicated membrane peroxidation and loss of membrane integrity that could be the primary target of β-pinene. Even the enhanced (9–62 %) activity of protecting enzymes, peroxidases (POX), was not able to protect the membranes from β-pinene (0.04-0.20 mg/ml)-induced toxicity. In conclusion, our results show that β-pinene inhibits root growth of the tested weed species through disruption of membrane integrity as indicated by enhanced peroxidation, electrolyte leakage, and LOX activity despite the upregulation of POX activity.     

Repeatability of Quantitative Sodium Magnetic Resonance Imaging for Estimating Pseudo-Intracellular Sodium Concentration and Pseudo-Extracellular Volume Fraction in Brain at 3 T

The purpose of this study is to assess the repeatability of the quantification of pseudo-intracellular sodium concentration (C₁) and pseudo-extracellular volume fraction (α) estimated in brain in vivo using sodium magnetic resonance (MRI) at 3 T. Eleven healthy subjects were scanned twice, with two sodium MRI acquisitions (with and without fluid suppression by inversion recovery), and two double inversion recovery (DIR) proton MRI. DIR MRIs were used to create masks of gray and white matter (GM, WM), that were subsequently applied to the C₁ and α maps calculated from sodium MRI and a tissue three-compartment model, in order to measure the distributions of these two parameters in GM, WM or full brain (GM+WM) separately. The mean, median, mode, standard deviation (std), skewness and kurtosis of the C₁ and α distributions in whole GM, WM and full brain were calculated for each subject, averaged over all data, and used as parameters for the repeatability assessment. The coefficient of variation (CV) was calculated as a measure of reliability for the detection of intra-subject changes in C₁ and αfor each parameter, while intraclass correlation (ICC) was used as a measure of repeatability. It was found that the CV of most of the parameters was around 10–20% (except for C₁ kurtosis which is about 40%) for C₁ and α measurements, and that ICC was moderate to very good (0.4 to 0.9) for C₁ parameters and for some of the α parameters (mainly skewness and kurtosis). In conclusion, the proposed method could allow to reliably detect changes of 50% and above of the different measurement parameters of C₁ and αin neuropathologies (multiple sclerosis, tumor, stroke, Alzheimer’s disease) compared to healthy subjects, and that skewness and kurtosis of the distributions of C₁ and αseem to be the more sensitive parameters to these changes.     

Manipulation Through Liquid Culture in the Contents of Sucrose and Glutamine and Their Transformation to Starch and Protein in Wheat Grain

Detached ears of wheat were cultured in liquid medium manipulated for sucrose and glutamine contents, and the accumulation of starch and protein in relation to the activities of sucrose cleaving—, ammonia assimilating—, and transaminating enzymes was studied in the grain. With an increase in the concentrations of sucrose from 44 to 176 mM and glutamine from 6.4 to 25.7 mM (keeping their ratio at a constant value of 7:1), the contents of starch and protein increased in the grains. However, when the grains were cultured in the medium containing 8.5 to 34 mM glutamine and a fixed concentration of 117 mM sucrose, there was a gradual increase in protein and decrease in starch content in the grain. By such manipulation in the liquid medium, the content of free amino acids also increased in the grain up to 12 days culturing. Amongst sucrose cleaving enzymes, the activities of sucrose-UDP glucosyl transferase and soluble alkaline invertase were much lower than the activity of soluble acid invertase. At high concentration (34 mM) of glutamine in the medium, containing 117 mM sucrose, there was drastic decrease in the activities of soluble acid invertase and UDPG pyrophosphorylase but the activities of ADPG pyrophosphorylase, alkaline inorganic pyrophosphatase, glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase increased in the grain with increase in glutamine concentration in the culture medium. Evidently, an increase in the level of amino nitrogen, coupled with an optimum sucrose concentration in the grain raised through liquid culturing enhances the conversion of sucrose to protein at the cost of starch accumulation in wheat.