Male- and female-specific variants of doublesex gene products have different roles to play towards regulation of Sex combs reduced expression and sex comb morphogenesis in Drosophila

Sexually dimorphic characters have two-fold complexities in pattern formation as they have to get input from both somatic sex determination as well as the positional determining regulators. Sex comb development in Drosophila requires functions of the somatic sex-determining gene doublesex and the homeotic gene Sex combs reduced. Attempts have not been made to decipher the role of dsx in imparting sexually dimorphic expression of SCR and the differential function of sex-specific variants of dsx products in sex comb development. Our results in this study indicate that male-like pattern of SCR expression is independent of dsx function, and dsx ^F must be responsible for bringing about dimorphism in SCR expression, whereas dsx ^M function is required with Scr for the morphogenesis of sex comb.     

Cholesterol depletion perturbs calcium handling by rat submandibular glands

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.     

Sleep fragmentation and motor restlessness in a Drosophila model of Restless Legs Syndrome

Restless Legs Syndrome (RLS), first chronicled by Willis in 1672 and described in more detail by Ekbom in 1945, is a prevalent sensorimotor neurological disorder (5%-10% in the population) with a circadian predilection for the evening and night. Characteristic clinical features also include a compelling urge to move during periods of rest, relief with movement, involuntary movements in sleep (viz., periodic leg movements of sleep), and fragmented sleep. Although the pathophysiology of RLS is unknown, dopaminergic neurotransmission and deficits in iron availability modulate expressivity. Genome-wide association studies have identified a polymorphism in an intronic region of the BTBD9 gene on chromosome 6 that confers substantial risk for RLS. Here, we report that loss of the Drosophila homolog CG1826 (dBTBD9) appreciably disrupts sleep with concomitant increases in waking and motor activity. We further show that BTBD9 regulates brain dopamine levels in flies and controls iron homeostasis through the iron regulatory protein-2 in human cell lines. To our knowledge, this represents the first reverse genetic analysis of a "novel" or heretofore poorly understood gene implicated in an exceedingly common and complex sleep disorder and the development of an RLS animal model that closely recapitulates all disease phenotypes.     

Compensatory secondary structure alterations in protein glycation

Glycation, a local covalent interaction, leads to alterations in secondary and tertiary structures of hemoglobin, the changes produced by fructose being more pronounced than those caused by glucose. The Stokes diameter of hemoglobin increases upon glycation from 7 to 14 nm and a concurrent inter-chain cross-linking and heme loss are also observed, particularly in the later stage of glycation. An initial increase of tryptophan (trp) fluorescence was observed in both glucation and fructation. In case of frucation however there was a decrease in tryptophan fluorescence that was accompanied by an increase in fluorescence of the advanced glycosylation end products (AGEs). This fluorescence behavior is indicative of energy transfer between tryptophan and the AGEs formed during the late stage of glycation. Emergence of an isosbestic point in the fluorescence spectra (taken at different time intervals) implies existence of two distinct glycation stages. The late glycation stage is also marked by an increase of beta structure and random coil at the expense of alpha helix. It is further observed that this compensatory loss of alpha helix (reported for the first time) and increase in beta sheet and random coil elements depend on the number of solvent-accessible glycation sites (rather than total number of such sites) and the subunit assembly of the protein.     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P _alsSD promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic–anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P _alsSD promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.     

A size dependent folding contour for cytochrome C

The paper describes an experimental construct of the folding route of the heme protein cytochrome-C. The construct highlights a slowing down near the nose of the folding funnel caused by the multiplicity of the energy traps near the native conformation created as a result of complex heme-peptide interaction. Interestingly the hydrodynamic size, the size heterogeneity and peroxidase activity serve as a triple measure of the distance of this near equilibrium departure from native conformation. Accordingly, the folding process is marked with a gradual and reversible reduction of mean hydrodynamic size, size heterogeneity and peroxidase activity (higher in unfolded state). The Dynamic Light Scattering based straightforward illustration of hydrodynamic size variation may serve as a model to slow folding observed in case of heme proteins, the heme itself serving as a natural facilitator for the native peptide conformation.     

Animal models of Parkinson's disease

Animal models are important tools in experimental medical science to better understand pathogenesis of human diseases. Once developed, these models can be exploited to test therapeutic approaches for treating functional disturbances observed in the disease of interest. On the basis of experimental and clinical findings, Parkinson's disease (PD) was the first neurological disease to be modeled and, subsequently, to be treated by neurotransmitter replacement therapy. Agents that selectively disrupt or destroy catecholaminergic systems, such as reserpine, methamphetamine, 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have been used to develop PD models. Recently, it has been found that agricultural chemicals, such as rotenone and paraquat, when administered systemically, can reproduce specific features of PD in rodents, apparently via oxidative damage. Transgenic animals that over-express alpha-synuclein are used to study the role of this protein in dopaminergic degeneration. This review critically discusses animal models of PD and compares them with characteristics of the human disease.