Ascorbate peroxidase, a scavenger of hydrogen peroxide in glyoxysomal membranes

Efficient destruction of hydrogen peroxide (H(2)O(2)) in peroxisomes requires the action of an anti-oxidant defense system, which consists of low molecular weight anti-oxidant compounds, such as ascorbic acid, along with protective enzymes, such as catalase and ascorbate peroxidase (APX). We investigated the contribution of the ascorbate enzyme system to the consumptions of H(2)O(2) and NADH within glyoxysomes of germinating castor beans (Ricinus communis). We solubilized the glyoxysomal membrane APX (gmAPX) using octyl-glucoside and purified its activity by gel filtration. The activity was associated with a 34kDa protein, as determined by SDS-gel electrophoresis and Western blotting. The enzymatic properties of gmAPX were studied and this enzyme was found to utilize ascorbic acid as its most effective natural electron donor but it would also use pyrogallol and guaiacol at a smaller extent. Cyanide and azide drastically inhibited gmAPX, as well as certain thiol-modifying reagents and some metal chelators. The inhibition by cyanide and azide of the enzyme combined with its absorption spectra confirmed that it is a hemoprotein. The apparent K(m) value of the enzyme for ascorbic acid was 300 microM while the K(m) for H(2)O(2) was 60 microM. APX in the glyoxysomal membrane can work in cooperation with monodehydroascorbate reductase to oxidize NADH, regenerate ascorbate, detoxify H(2)O(2), and protect the integrity of glyoxysomal proteins and membranes.     

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca²⁺-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using ??-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T_1/2 was 1 h at 100°C. The K _m of the partial purified enzyme was 1 mM and the V _max was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca²⁺ and Hg²⁺ stimulated lipase activity, whereas Zn²⁺, Co²⁺, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.     

Proteome of human perilymph

Current diagnostic tools limit a clinician's ability to discriminate between many possible causes of sensorineural hearing loss. This constraint leads to the frequent diagnosis of the idiopathic condition, leaving patients without a clear prognosis and only general treatment options. As a first step toward developing new diagnostic tools and improving patient care, we report the first use of liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to map the proteome of human perilymph. Using LC-MS/MS, we analyzed four samples, two collected from patients with vestibular schwannoma (VS) and two from patients undergoing cochlear implantation (CI). For each cohort, one sample contained pooled specimens collected from five patients and the second contained a specimen obtained from a single patient. Of the 271 proteins identified with high confidence among the samples, 71 proteins were common in every sample and used to conservatively define the proteome of human perilymph. Comparison to human cerebrospinal fluid and blood plasma, as well as murine perilymph, showed significant similarity in protein content across fluids; however, a quantitative comparison was not possible. Fifteen candidate biomarkers of VS were identified by comparing VS and CI samples. This list will be used in future investigations targeted at discriminating between VS tumors associated with good versus poor hearing.     

An improvement of the diffusion model for assessment of drought stress in plant tissues

The article discusses an improvement of a previously developed method for assessment of ion leakage from plant tissues as a gauge of membrane and cell wall performance under stressful environment. It employs conductometric measurements of the ion efflux from leaves and their quantitative interpretation by a theoretical model based on the laws of diffusion. Experimental data are readily fit with the model and results are in accordance with relative water content of dehydrated barley (Hordeum vulgare) seedlings of two distinct cultivars. Some new parameters obtained from fitting are proposed as reliable indicators of the leaf status. They appear to be helpful in further distinguishing the behavior of two separate cellular structures with respect to their electrolyte permeability. It is concluded that the established method based on the kinetics of ion leakage is adequate for evaluation of contrasting genotypes under normal and stress conditions. Furthermore, it could be used as a simple and powerful tool for routine analysis and screening for drought tolerance in crops.     

Psychosocial distress and functioning of Greek youth with cystic fibrosis: a cross-sectional study

Background

To assess psychosocial functioning and distress of children and adolescents with cystic fibrosis compared to healthy controls.

Methods

Thirty-six patients with cystic fibrosis aged 8–18 years (24 boys, mean age?±?SD: 11.5?±?2.6 years) and 31 sex- and age-matched healthy control subjects (18 boys, mean age?±?SD: 12?±?2.5 years) were enrolled in the study. In order to assess the self-esteem, social adjustment, and family functioning of these young people, the Culture-free Self-esteem Inventory, the Social Adjustment Scale–Self-Report, and the Family Assessment Device were administered. Emotional/ behavioral problems were assessed through the Youth Self Report and the Child Behavior Checklist given to both the subjects and their parents.

Results

No significant differences were found for self-esteem between the two study groups. Regarding social adjustment, children with cystic fibrosis reported significantly worse friendship and overall adjustment (P?<?0.05). Moreover, no difference was found in the levels of family functioning between the two groups. No significant differences between the groups were found in emotional/ behavioral problems from the self-reports. On the contrary, parents of children with cystic fibrosis reported significantly higher levels of withdrawal/ depression, thought problems, and delinquent behavior (P?≤?0.01) as compared to controls.

Conclusions

Children and adolescents with cystic fibrosis appear to be a psychosocially vulnerable group. A biopsychosocial approach should emphasize the assessment and treatment of the psychosocial distress of these patients alongside multiple somatic treatments.

     

FGF23 Deficiency Leads to Mixed Hearing Loss and Middle Ear Malformation in Mice

Fibroblast growth factor 23 (FGF23) is a circulating hormone important in phosphate homeostasis. Abnormal serum levels of FGF23 result in systemic pathologies in humans and mice, including renal phosphate wasting diseases and hyperphosphatemia. We sought to uncover the role FGF23 plays in the auditory system due to shared molecular mechanisms and genetic pathways between ear and kidney development, the critical roles multiple FGFs play in auditory development and the known hearing phenotype in mice deficient in klotho (KL), a critical co-factor for FGF23 signaling. Using functional assessments of hearing, we demonstrate that Fgf mice are profoundly deaf. Fgf mice have moderate hearing loss above 20 kHz, consistent with mixed conductive and sensorineural pathology of both middle and inner ear origin. Histology and high-voltage X-ray computed tomography of Fgf mice demonstrate dysplastic bulla and ossicles; Fgf mice have near-normal morphology. The cochleae of mutant mice appear nearly normal on gross and microscopic inspection. In wild type mice, FGF23 is ubiquitously expressed throughout the cochlea. Measurements from Fgf mice do not match the auditory phenotype of Kl ^−/− mice, suggesting that loss of FGF23 activity impacts the auditory system via mechanisms at least partially independent of KL. Given the extensive middle ear malformations and the overlap of initiation of FGF23 activity and Eustachian tube development, this work suggests a possible role for FGF23 in otitis media.     

SALL4 controls cell fate in response to DNA base composition

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A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants

Background

Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.

Results

The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 μmol/min/mg protein and the Km for methionine and ATP was 288 μM and 76 μM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 μM, while the MIC for 8-azaguanine was >1.0 mM.

Conclusion

The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.     

Crystal structure of the inclusion complex of the antibacterial agent triclosan with cyclomaltoheptaose and NMR study of its molecular encapsulation in positively and negatively charged cyclomaltoheptaose derivatives

The inclusion complexes of triclosan with native cyclomaltoheptaose (beta-cyclodextrin, betaCD) as well as with negatively and positively charged derivatives are studied. The structure of the inclusion complex betaCD/triclosan in the crystalline state [P1, a=15.189(5), b=15.230(6), c=16.293(6), alpha=91.07(4), beta=91.05(3) gamma=100.71(3)] comprises two crystallographically independent host macrocycles A and B. The packing results in betaCD dimers that align head-to-head and form infinite channels along the c-axis. Only one guest molecule statistically disordered over two positions, (the dichlorophenyl ring in the cavities of either A or B) corresponds to each dimer (a 2:1 host/guest complex). The enclosed dichlorophenyl ring enters the dimer through the primary side, whereas the hydrophilic chlorophenol ring extends in the space between dimers. Water molecules in five positions are also enclosed in the intradimer region, arranged on a plane perpendicular to the sevenfold axis of betaCD. The NMR spectroscopic studies in aqueous solution show the presence of both 1:1 and 2:1 betaCD/triclosan complexes. In the first case, two different 1:1 complexes are simultaneously present, each with either ring entering the narrow primary side of one betaCD molecule. In the 2:1 complex both rings of triclosan are included in two independent betaCD hosts, a precursor to the supramolecular arrangement found in the crystalline form. In the case of the negatively charged sodium heptakis[6-deoxy-6-(3-thiopropionate)]-betaCD, the NMR studies at pH 7.9 show a complete inclusion of triclosan inside the host in two orientations, one for the non-ionized (phenol) and reverse for the ionized (phenolate) form. Finally, for the positively charged heptakis(6-aminoethylamino-6-deoxy)-betaCD, inclusion of triclosan is possible only when the pH is raised to 10 and it is concluded that both aromatic rings are alternatively inside the cavity. However in that case also, inclusion of the entire guest in the elongated cavity is suggested.