Effect of eluent pH on the HPLC-UV analysis of hyperforin from St. John's Wort (Hypericum perforatum L.)

The effect of the pH of the mobile phase in HPLC analysis of hyperforin was investigated. Working with an extract of St. John's Wort (Hypericum perforatum L.) that is rich in hyperforin, significant differences were observed in conventional chromatograms depending on whether the mobile phase was acidic or alkaline. Chromatogram changes were paralleled by changes in the UV spectrum of the hyperforin peak. The structural changes in hyperforin occur in the chromatographic column itself, as has been confirmed by UV spectroscopy performed on a sample of purified hyperforin, which showed that the UV spectrum is indeed dependent on the pH of its environment.     

Oxidative Stress in Skin Fibroblasts Cultures of Patients with Huntington’s Disease

Oxidative stress and mitochondrial dysfunction should play a role in the neurodegeneration in Huntington’s disease (HD). The most consistent finding is decreased activity of the mitochondrial complexes II/III and IV of the respiratory chain in the striatum. We assessed enzymatic activities of respiratory chain enzymes and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with HD. We studied respiratory chain enzyme activities, activities of total, Cu/Zn- and Mn-superoxide-dismutase, glutathione-peroxidase (GPx) and catalase, and coenzyme Q₁₀ (CoQ₁₀) levels in skin fibroblasts cultures from 13 HD patients and 13 age- and sex-matched healthy controls. When compared with controls, HD patients showed significantly lower specific activities for catalase corrected by protein concentrations (P < 0.01). Oxidized, reduced and total CoQ₁₀ levels (both corrected by citrate synthase (CS) and protein concentrations), and activities of total, Cu/Zn- and Mn-superoxide-dismutase, and gluthatione-peroxidase, did not differ significantly between HD-patients and control groups. Values for enzyme activities in the HD group did not correlate with age at onset and of the disease and with the CAG triplet repeats. The primary finding of this study was the decreased activity of catalase in HD patients, suggesting a possible contribution of catalase, but not of other enzymes related with oxidative stress, to the pathogenesis of this disease.     

Biochemical and molecular characterization of a lipase produced by Rhizopus oryzae

A novel strain of Rhizopus oryzae WPG secretes a noninduced lipase (ROLw) in the culture medium; purified ROLw is a protein of 29 kDa, the 45 N-terminal amino acid residues were sequenced, this sequence is very homologous to Rhizopus delemar lipase (RDL), Rhizopus niveus lipase (RNL) and R. oryzae lipase (ROL29) sequences; the cloning and sequencing of the part of the gene encoding the mature ROLw, shows two nucleotides differences with RDL, RNL and ROL29 sequences corresponding to the change of the residues 134 and 200; ROLw does not present the interfacial activation phenomenon when using tripropionin or vinyl propionate as substrates; the lipase activity is maximal at pH 8 and at 37 degrees C, specific activities of 3500 or 900 U mg(-1) were measured at 37 degrees C and at pH 8, using olive oil emulsion or tributyrin as substrates, respectively; ROLw is unable to hydrolyse triacylglycerols in the presence of high concentration of bile salts; it is a serine enzyme as it is inhibited by tetrahydrolipstatin and was stable between pH 5 and pH 8.     

Ceftriaxone-induced up-regulation of cortical and striatal GLT1 in the R6/2 model of Huntington's disease

Background  

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by cortico-striatal dysfunction and loss of glutamate uptake. At 7 weeks of age, R6/2 mice, which model an aggressive form of juvenile HD, show a glutamate-uptake deficit in striatum that can be reversed by treatment with ceftriaxone, a β-lactam antibiotic that increases GLT1 expression. Only at advanced ages (> 11 weeks), however, do R6/2 mice show an actual loss of striatal GLT1. Here, we tested whether ceftriaxone can reverse the decline in GLT1 expression that occurs in older R6/2s.     

Effects of the planting density on water relations and production of ‘Chemlali’ olive trees (Olea europaea L.)

Water relations are a key factor limiting olive production. In this study, effects of plating density on physiological aspects and productivity of ‘Chemlali’ olive trees were analyzed under rain-fed conditions in four planting densities (156, 100, 69 and 51 trees ha⁻¹), in an experimental olive orchard located in the center of Tunisia. Seasonal changes in leaf relative water content (RWC), leaf water potential, stomatal conductance (g _s), CO₂ assimilation rate and tree production were studied. Accompanying the changes in leaf water status, all the monitored trees reduced leaf stomatal conductance (g _s) and photosynthetic rate (A) throughout the summer drought, mirroring the increase in soil moisture deficit and vapor pressure deficit. However, the decrease in gas exchange was much more pronounced in high planting densities than in low ones. Our results confirm that the increase of tree-to-tree water competition with planting density was significant in the dry climate of Tunisia. Thus, planting density is critical when planting new olive orchards in arid regions.     

Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation

The plasma membrane Na+/H+ exchangers (NHE) require calcineurin B homologous protein (CHP) as an obligatory binding partner for ion transport. Here, we report the first crystal structure of CHP (CHP2 isoform) in complex with its binding domain in NHE1. We show that the cytoplasmic alpha-helix of NHE1 is inserted into the hydrophobic cleft formed by N- and C-lobes of CHP2 and that the size and shape of this crevice together with hydrogen bond formation at multiple positions assure a high degree of specificity for interaction with NHE members. Structure-based mutagenesis revealed the importance of hydrophobic interactions between CHP/NHE1 for the function of NHE1. Furthermore, the crystal structure shows the existence of a protruding CHP-unique region, and deletion of this region in CHP2 inhibited the NHE1 activity by inducing the acidic shift of intracellular pH dependence, while preserving interaction with NHE1. These findings suggest that CHP serves as an obligatory subunit that is required both for supporting the basic activity and regulating the pH-sensing of NHE1 via interactions between distinct parts of these proteins.     

Synthesis and DNA binding properties of a new benzo[f]imidazo[1,5b]-isoquinoline-butan-1,2-diol derivative

A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA, DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was used for the synthesis of a zipping duplex.     

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.     

Expression of Trophic Peptides and Their Receptors in Chromaffin Cells and Pheochromocytoma

Pheochromocytomas are catecholamine-producing tumors arising from chromaffin cells of the adrenal medulla or extra-adrenal location. Along with catecholamines, tumoral cells produce and secrete elevated quantities of trophic peptides which are normally released in a regulated manner by the normal adrenal medulla. Among these peptides, the amounts of pituitary adenylate cyclase-activating polypeptide (PACAP), adrenomedullin (AM), and neuropeptide Y (NPY) are particularly high. These peptides can exert endocrine, paracrine or autocrine effects in numerous cell types. In particular, they have been shown to be involved in cell proliferation and survival, catecholamine production and secretion, and angiogenesis. Some of these processes are exacerbated in pheochromocytomas, raising the possibility of the involvement of trophic peptides. Here, we review the expression levels of NPY, PACAP, and AM and theirs receptors in chromaffin cells and pheochromocytomas, and address their possible implication in the adrenal medulla tumorigenesis and malignant development of pheochromocytomas.