A locus for autosomal dominant reflex epilepsy precipitated by hot water maps at chromosome 10q21.3-q22.3

Hot water epilepsy (HWE) is a form of reflex or sensory epilepsy wherein seizures are precipitated by an unusual stimulus, the contact of hot water over the head and body. Genome-wide linkage analysis of a large family with ten affected members, provided evidence of linkage (Z _max = 3.17 at θ = 0 for D10S412) to chromosome 10q21. Analysis of five additional HWE families, for markers on chromosome 10, further strengthened the evidence of linkage to the same chromosomal region with three out of five families showing concordance for the disease haplotype and providing a two-point LOD score of 4.86 at θ = 0 and 60% penetrance for D10S412. The centromere-proximal and -distal boundaries of the critical genetic interval of about 15 Mb at 10q21.3-q22.3 were defined by D10S581 and D10S201, respectively. Sequence analysis of a group of functional candidate genes, the ion channels KCNMA1, VDAC2 and solute carriers SLC25A16, SLC29A3 revealed no potentially pathogenic mutation. We propose to carry out further analysis of positional candidate genes from this region to identify the gene responsible for this unusual neurobehavioral phenotype.     

Biochemical responses to drought stress in mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> L.): evaluation of proline,glycine betaine and abscisic acid accumulation in five cultivars

Five popularly grown mulberry cultivars (K-2, MR-2, TR-10, BC2-59 and S-13) were subjected to drought stress by withholding irrigation, to obtain leaf water potentials (Ψ_w) ranging from −0.75, −1.50 and −2.25 MPa. Accumulation of proline, glycine betaine and abscisic acid (ABA) were quantified in control and water stressed mulberry leaves. The activities of enzymes involved in proline accumulation including glutamate dehydrogenase (EC1.4.1.2-4), pyrroline-5-carboxylate synthetase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC1.5.1.2), ornithine transaminase (EC 2.6.1.13) were significantly enhanced in the leaves of all the cultivars with decreasing leaf water potentials, while the activities of proline dehydrogenase (EC 1.5.1.2) were reduced with progressive increase in water stress. Accumulation of proline, glycine betaine and abscisic acid was relatively higher in S-13 and BC2-59 compared to K-2, MR-2 and TR-10 under water deficit conditions. Our results demonstrate that S-13 and BC2-59 have superior osmoprotectant mechanisms under water-limited growth regimes.     

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l⁻¹). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l⁻¹ representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.     

Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation

With the goal of identifying hitherto unknown surface exosites of streptokinase involved in substrate human plasminogen recognition and catalytic turnover, synthetic peptides encompassing the 170 loop (CQFTPLNPDDDFRPGLKDTKLLC) in the β-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinase-plasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-constrained variant with a single lysine to alanine mutation showed significantly reduced capabilities of inhibition. Alanine-scanning mutagenesis of the 170 loop of the β-domain of streptokinase was then performed to elucidate its importance in streptokinase-mediated plasminogen activation. Some of the 170 loop mutants showed a remarkable decline in k_cat without any alteration in apparent substrate affinity (K_m) as compared with wild-type streptokinase and identified the importance of Lys¹⁸⁰ as well as Pro¹⁷⁷ in the functioning of this loop. Remarkably, these mutants were able to generate amidolytic activity and non-proteolytic activation in “partner” plasminogen as wild-type streptokinase. Moreover, cofactor activities of the 170 loop mutants, pre-complexed with plasmin, against microplasminogen as the substrate showed a similar pattern of decline in k_cat as that observed in the case of full-length plasminogen, with no concomitant change in K_m. These results strongly suggest that the 170 loop of the β-domain of streptokinase is important for catalysis by the streptokinase-plasmin(ogen) activator complex, particularly in catalytic processing/turnover of substrate, although it does not seem to contribute significantly toward enzyme-substrate affinity per se.     

Gγ1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration

The isoprenoid biosynthetic pathway leading from the production of mevalonate by HMGCoA reductase (Hmgcr) to the geranylation of the G protein subunit, Gγ1, plays an important role in cardiac development in the fly. Hmgcr has also been implicated in the release of the signaling molecule Hedgehog (Hh) from hh expressing cells and in the production of an attractant that directs primordial germ cells to migrate to the somatic gonadal precursor cells (SGPs). The studies reported here indicate that this same hmgcr→Gγ1 pathway provides a novel post-translational mechanism for modulating the range and activity of the Hh signal produced by hh expressing cells. We show that, like hmgcr, gγ1 and quemao (which encodes the enzyme, geranylgeranyl diphosphate synthetase, that produces the substrate for geranylation of Gγ1) are components of the hh signaling pathway and are required for the efficient release of the Hh ligand from hh expressing cells. We also show that the hmgcr→Gγ1 pathway is linked to production of the germ cell attractant by the SGPs through its ability to enhance the potency of the Hh signal. We show that germ cell migration is disrupted by the loss or gain of gγ1 activity, by trans-heterozygous combinations between gγ1 and either hmgcr or hh mutations, and by ectopic expression of dominant negative Gγ1 proteins that cannot be geranylated.     

Responses of Gmelina arborea, a tropical deciduous tree species, to elevated atmospheric CO2: Growth, biomass productivity and carbon sequestration efficacy

The photosynthetic response of trees to rising CO₂ concentrations largely depends on source-sink relations, in addition to differences in responsiveness by species, genotype, and functional group. Previous studies on elevated CO₂ responses in trees have either doubled the gas concentration (>700 μmol mol⁻¹) or used single large addition of CO₂ (500-600 μmol mol⁻¹). In this study, Gmelina arborea, a fast growing tropical deciduous tree species, was selected to determine the photosynthetic efficiency, growth response and overall source-sink relations under near elevated atmospheric CO₂ concentration (460 μmol mol⁻¹). Net photosynthetic rate of Gmelina was ∼30% higher in plants grown in elevated CO₂ compared with ambient CO₂-grown plants. The elevated CO₂ concentration also had significant effect on photochemical and biochemical capacities evidenced by changes in F_V/F_M, ABS/CSm, ET₀/CSm and RuBPcase activity. The study also revealed that elevated CO₂ conditions significantly increased absolute growth rate, above ground biomass and carbon sequestration potential in Gmelina which sequestered ∼2100 g tree⁻¹ carbon after 120 days of treatment when compared to ambient CO₂-grown plants. Our data indicate that young Gmelina could accumulate significant biomass and escape acclimatory down-regulation of photosynthesis due to high source-sink capacity even with an increase of 100 μmol mol⁻¹ CO₂.     

Chronic hypoxia-induced alterations of key enzymes of glucose oxidative metabolism in developing mouse liver are mTOR dependent

Hypoxia is a potent regulator of gene expression and cellular energy metabolism and known to interfere with post-natal growth and development. Although hypoxia can induce adaptive changes in the developing liver, the mechanisms underlying these changes are poorly understood. To elucidate some of the adaptive changes chronic hypoxia induces in the developing liver, we studied the expression of the genes of mammalian target of rapamycin (mTOR) signaling and glucose metabolism, undertook proteomic examination with 2D gel-MS/MS of electron transport chain, and determined activities and protein expression of several key regulatory enzymes of glucose oxidative metabolism. To gain insight into the molecular mechanism underlying hypoxia-induced liver metabolic adaptation, we treated a subset of mice with rapamycin (0.5 mg/kg/day) to inhibit mTOR postnatally. Rapamycin-treated mice showed lower birth weight, lower body weight, and liver growth retardation in a pattern similar to that observed in the hypoxic mice at P30. Rapamycin treatment led to differential impact on the cytoplasmic and mitochondrial pathways of glucose metabolism. Our results suggest a decrease in mTOR activity as part of the mechanisms underlying hypoxia-induced changes in the activities of glycolytic and TCA cycle enzymes in liver. Chronic postnatal hypoxia induces mTOR-dependent differential effects on liver glycolytic and TCA cycle enzymes and as such should be studied further as they have pathophysiological implications in hepatic diseases and conditions in which hypoxia plays a role.     

Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese

Although, several studies have been reported on the effects of oxidants on the structure and function of other molecular chaperones, no reports have been made so far for the chaperonin GroEL. The ability of GroEL to function under oxidative stress was investigated in this report by monitoring the effects of hydrogen peroxide (H(2)O(2)) on the structure and refolding activity of this protein. Using fluorescence spectroscopy and light scattering, we observed that GroEL showed increases in exposed hydrophobic sites and changes in tertiary and quaternary structure. Differential sedimentation, gel electrophoresis, and circular dichroism showed that H(2)O(2) treated GroEL underwent irreversible dissociation into monomers with partial loss of secondary structure. Relative to other proteins, GroEL was found to be highly resistant to oxidative damage. Interestingly, GroEL monomers produced under these conditions can facilitate the reactivation of H(2)O(2)-inactivated rhodanese but not urea-denatured rhodanese. Recovery of approximately 84% active rhodanese was obtained with either native or oxidized GroEL in the absence of GroES or ATP. In comparison, urea-denatured GroEL, BSA and the refolding mixture in the absence of proteins resulted in the recovery of 72, 50, and 49% rhodanese activity, respectively. Previous studies have shown that GroEL monomers can reactivate rhodanese. Here, we show that oxidized monomeric GroEL can reactivate oxidized rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.     

Depletion of chondrocyte primary cilia reduces the compressive modulus of articular cartilage

Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88^fl/fl transgenic mice results in up-regulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88^fl/fl transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage.