The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Surrogate variable analysis using partial least squares (SVA-PLS) in gene expression studies

MOTIVATION: In a typical gene expression profiling study, our prime objective is to identify the genes that are differentially expressed between the samples from two different tissue types. Commonly, standard analysis of variance (ANOVA)/regression is implemented to identify the relative effects of these genes over the two types of samples from their respective arrays of expression levels. But, this technique becomes fundamentally flawed when there are unaccounted sources of variability in these arrays (latent variables attributable to different biological, environmental or other factors relevant in the context). These factors distort the true picture of differential gene expression between the two tissue types and introduce spurious signals of expression heterogeneity. As a result, many genes which are actually differentially expressed are not detected, whereas many others are falsely identified as positives. Moreover, these distortions can be different for different genes. Thus, it is also not possible to get rid of these variations by simple array normalizations. This both-way error can lead to a serious loss in sensitivity and specificity, thereby causing a severe inefficiency in the underlying multiple testing problem. In this work, we attempt to identify the hidden effects of the underlying latent factors in a gene expression profiling study by partial least squares (PLS) and apply ANCOVA technique with the PLS-identified signatures of these hidden effects as covariates, in order to identify the genes that are truly differentially expressed between the two concerned tissue types. RESULTS: We compare the performance of our method SVA-PLS with standard ANOVA and a relatively recent technique of surrogate variable analysis (SVA), on a wide variety of simulation settings (incorporating different effects of the hidden variable, under situations with varying signal intensities and gene groupings). In all settings, our method yields the highest sensitivity while maintaining relatively reasonable values for the specificity, false discovery rate and false non-discovery rate. Application of our method to gene expression profiling for acute megakaryoblastic leukemia shows that our method detects an additional six genes, that are missed by both the standard ANOVA method as well as SVA, but may be relevant to this disease, as can be seen from mining the existing literature.     

Isoniazid and thioacetazone may exhibit antitubercular activity by binding directly with the active site of mycolic acid cyclopropane synthase: Hypothesis based on computational analysis

Isoniazid and thioacetazone are the two important antitubercular drugs. In case of thioacetazone it is established that it inhibits mycolic acid cyclopropane synthase but the exact binding site accounting for such inhibition is presently unknown. In case of isoniazid its action on the said enzyme is unexplored. In this work we have analyzed the binding of isoniazid and thioacetazone with mycolic acid cyclopropane synthase (CmaA1 and CmaA2) using tools of computational biology. We have observed that thioacetazone fits well at the active site of CmaA1 and CmaA2 while isoniazid binds at the active site of CmaA1 only. We have recommended experimental validation of such results. If such results are proved to be fact it will explore the exact binding site of thioacetazone and discover a new mechanism of anti-tubercular action of isoniazid.     

The cholesterol turnover, synthesis, and absorption in two sisters with familial hypercholesterolemia (type IIa).

To explore the mechanisms of the profound plasma cholesterol elevations in familial homozygous hypercholesterolemia (type IIa), cholesterol turnover, sterol balance, cholesterol absorption, and low density lipoprotein studies were carried out under controlled dietary conditions in two sisters (aged 13 and 16). Cholesterol turnover was prolonged. The half-life of the first exponential of the plasma cholesterol specific activity decay curve was double that of normal adults. The rate constants for the removal of cholesterol from pool A (KAA = 0.0652) and for the excretion of cholesterol from the system (Kaa = 0.0197) were less than half of normal. The production rates of cholesterol were low, only 6.30 and 6.86 mg/kg per day as measured by cholesterol turnover and sterol balance techniques, respectively. Fecal neutral steroid and bile acid excretion were 5.22 and 1.64 mg/kg per day, which is remarkably low in comparison to those of normal and heterozygous children. Cholesterol absorption was within the upper limit of the values reported for normal adults. THE HDL cholesterol values were extremely low (27 mg/dl) in contrast to profoundly elevated LDL levels. The fractional catabolic rate of LDL (0.127 per day) and the rate of synthesis and catabolism of apo-LDL (15 mg/kg per day) were low in comparison to previously reported values in homozygotes. These composite data indicated that the definable metabolic defects of these two sisters with homozygous familial hypercholesterolemia were the sluggish clearance of cholesterol from the body coupled with low total body synthesis of cholesterol.     

Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles.

Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell.