Molecular implications of the human glutathione transferase A-4 gene (hGSTA4) polymorphisms in neurodegenerative diseases

Several lines of evidence, including an increased level of lipid peroxidation and the depletion of antioxidant molecules like as glutathione (GSH), indicate that oxidative stress plays an important role in the pathogenesis of several neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD). We previously observed a significant increased level of DNA oxidative damage in peripheral blood cells of PD patients, with respect to controls, moreover, the activity of glutathione transferases (GSTs) measured in circulating plasma was higher in controls than in PD patients, suggesting a lower enzymatic protection in PD individuals. Among human GSTs, glutathione transferase A4-4 displays a high catalitic activity towards 4-hydroxy-2-nonenal (HNE), a marker of lipid peroxidation whose levels have been found significantly increased in the substantia nigra of Parkinson's disease patients, in respect to controls. We performed this study to determine the presence of allelic variants of functional interest in the coding region of the hGSTA4 gene on 60 PD patients and 60 healthy controls. By the combined effort of polymerase chain reaction/single-strand conformation polymorphisms (PCR/SSCP) techniques, we observed a single nucleotide polymorphism (SNP) G351A leading to the silent mutation Gln117Gln. No significant difference was observed in the distribution of this polymorphism between PD individuals and controls, moreover, we did not observe any other polymorphism in the hGSTA4 gene in our population. Further studies are required to test the role played by both factors regulating the level of the expression of the hGSTA4 gene and any possible post-translational modification of the protein, in the protection against oxidative damage in neuronal cells.     

Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon

Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.     

Drazepinone, a trisubstituted tetrahydronaphthofuroazepinone with herbicidal activity produced by Drechslera siccans

When grown in a minimal-defined medium, a strain of Drechslera siccans, a pathogenic fungus isolated from seeds of Lolium perenne, produced phytotoxic metabolites. This strain is one of the best toxin producers among several grass pathogenic fungal strains collected and tested to find phytotoxins to be used as natural herbicides of monocot weeds. From the culture filtrates of D. siccans, we isolated a new phytotoxic trisubstituted naphthofuroazepinone, named drazepinone, and characterised it as a 3,5,12a-trimethyl-2,5,5a,12a-tetrahydro-1H-naphtho[2',3':4,5]furo[2,3-b]azepin-2-one. Assayed at 2 microg microl(-1) solution the novel metabolite proved to have broad-spectrum herbicidal properties, without antibacterial and antifungal activities, and low zootoxic activity. Its original chemical structure and the interesting biological properties make drazepinone a potential natural herbicide.     

miR-15a and miR-16-1 down-regulation in pituitary adenomas

Micro RNAs (miRs) are small noncoding RNAs, functioning as antisense regulators of other RNAs. miR-15a and miR-16-1 genes are located at chromosome 13q14, a region which is frequently deleted in pituitary tumors. An inverse correlation has been shown in B cell chronic lymphocytic leukemia (B-CLL) between miR-15a and miR-16-1 expression and the expression levels of arginyl-tRNA synthetase (RARS), an enzyme which associates with the cofactor p43 in the aminoacyl-tRNA synthetase complex. When secreted, p43 regulates local inflammatory response and macrophage chemotaxis, and seems to have anti-neoplastic properties in mice. We explored miR-15a and miR-16-1 expression in 10 GH-secreting and in 10 PRL-secreting pituitary macroadenomas by Northern blot, and investigated the possible correlation with in vivo and in vitro characteristics. We found that miR-15a and miR-16-1 are expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Moreover, their expression inversely correlates with tumor diameter and with RARS expression (P < 0.05), but directly correlates with p43 secretion (P < 0.02). Therefore, miR15 and miR16 down-regulation in pituitary adenomas correlates with a greater tumor diameter and a lower p43 secretion, suggesting that these genes may, at least in part, influence tumor growth.     

Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency

Background

The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors.

Methods

We measured three different types of biomarkers, including desmosines, elastase-formed fibrinogen fragments and heparan sulfate epitope JM403, in plasma and urine for a period of 7 weeks in a group of 12 patients who participated in a placebo-controlled study to assess the safety of a single inhalation of hyaluronic acid.

Results

Effect of study medication on any of the biomarkers was not seen. Baseline desmosines in plasma and urine correlated with baseline CO diffusion capacity (R = 0.81, p = 0.01 and R = 0.65, p = 0.05). Mean coefficient of variation within patients (CVi) for plasma and urine desmosines was 18.7 to 13.5%, respectively. Change in urinary desmosine levels correlated significantly with change in plasma desmosine levels (R = 0.84, p < 0.01). Mean CVi for fibrinogen fragments in plasma was 20.5% and for JM403 in urine was 27.8%. No correlations were found between fibrinogen fragments or JM403 epitope and desmosines.

Conclusion

We found acceptable variability in our study parameters, indicating the feasibility of their use in an evaluation of biochemical efficacy of alpha-1-antitrypsin augmentation therapy in Pi Z subjects.     

Liquid chromatography/mass spectrometry analysis of exhaled leukotriene B4 in asthmatic children

Background

The role of leukotriene (LT) B₄, a potent inflammatory mediator, in atopic asthmatic and atopic nonasthmatic children is largely unknown. The lack of a gold standard technique for measuring LTB₄ in exhaled breath condensate (EBC) has hampered its quantitative assessment in this biological fluid. We sought to measure LTB₄ in EBC in atopic asthmatic children and atopic nonasthmatic children. Exhaled nitric oxide (NO) was measured as an independent marker of airway inflammation.

Methods

Fifteen healthy children, 20 atopic nonasthmatic children, 25 steroid-naïve atopic asthmatic children, and 22 atopic asthmatic children receiving inhaled corticosteroids were studied. The study design was of cross-sectional type. Exhaled LTB₄ concentrations were measured using liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS) with a triple quadrupole mass spectrometer. Exhaled NO was measured by chemiluminescence with a single breath on-line method. LTB₄ values were expressed as the total amount (in pg) of eicosanoid expired in the 15-minute breath test. Kruskal-Wallis test was used to compare groups.

Results

Compared with healthy children [87.5 (82.5–102.5) pg, median and interquartile range], exhaled LTB₄ was increased in steroid-naïve atopic asthmatic [255.1 (175.0–314.7) pg, p < 0.001], but not in atopic nonasthmatic children [96.5 (87.3–102.5) pg, p = 0.59)]. Asthmatic children who were receiving inhaled corticosteroids had lower concentrations of exhaled LTB₄ than steroid-naïve asthmatics [125.0 (25.0–245.0) pg vs 255.1 (175.0–314.7) pg, p < 0.01, respectively]. Exhaled NO was higher in atopic nonasthmatic children [16.2 (13.5–22.4) ppb, p < 0.05] and, to a greater extent, in atopic steroid-naïve asthmatic children [37.0 (31.7–57.6) ppb, p < 0.001] than in healthy children [8.3 (6.1–9.9) ppb]. Compared with steroid-naïve asthmatic children, exhaled NO levels were reduced in asthmatic children who were receiving inhaled corticosteroids [15.9 (11.5–31.7) ppb, p < 0.01].

Conclusion

In contrast to exhaled NO concentrations, exhaled LTB₄ values are selectively elevated in steroid-naïve atopic asthmatic children, but not in atopic nonasthmatic children. Although placebo control studies are warranted, inhaled corticosteroids seem to reduce exhaled LTB₄ in asthmatic children. LC/MS/MS analysis of exhaled LTB₄ might provide a non-invasive, sensitive, and quantitative method for airway inflammation assessment in asthmatic children.