Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: purification and characterization

High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine residues at the N-terminal and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. Activity assay and SDS-PAGE demonstrate that the His-tagged xylanase was extracellularly expressed in P. pastoris and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). The purified PoXyn2 showed a single band on SDS-PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 50°C. The specific activity measured for Oat Spelt Xylan was 8549.85 U mg(-1). The apparent The K(M) and V(max) values were 8.33±0.7 mg ml(-1)and 58.82±0.9 μmol min(-1) ml(-1), respectively, as measured on Oat Spelt Xylan. This is the first report demonstrating the possibility of mass production of P. occitanis xylanase using P. pastoris.     

Correlation between the Chemical and Genetic Relationships among Thymus saturejoides Genotypes Cultured under in vitro and in vivo Environments

In this study, the in vitro and in vivo essential oil (EO) composition and genetic variability in six micropropagated genotypes of Thymus saturejoides Coss ., a Mediterranean medicinal and aromatic plant, were analyzed by GC/MS and randomly amplified polymorphic DNA (RAPD). Yield and composition of the EO varied between genotypes. Cluster analysis based on RAPD data and EO grouped the six genotypes in three groups in both culture conditions, thus showing considerable intraspecific genetic and chemical variations. Applying the Mantel test, the result showed a significant correlation between the two proximity matrices RAPD and EO obtained from in vitro genotypes, whereas this correlation was not observed when using the EO obtained from the in vivo genotypes.     

Human eosinophils exert TNF-α and granzyme A-mediated tumoricidal activity toward colon carcinoma cells

Peripheral blood and tissue eosinophilia is a prominent feature in allergic diseases and helminth infections. In cancer patients, tumor-associated tissue eosinophilia is frequently observed. Tumor-associated tissue eosinophilia can be associated with a favorable prognosis, notably in colorectal carcinoma. However, underlying mechanisms of eosinophil contribution to antitumor responses are poorly understood. We have in this study investigated the direct interactions of human eosinophils with Colo-205, a colorectal carcinoma cell line, and show that eosinophils induce apoptosis and directly kill tumor cells. Using blocking Abs, we found that CD11a/CD18 complex is involved in the tumoricidal activity. Coculture of eosinophils with Colo-205 led to the release of eosinophil cationic protein and eosinophil-derived neurotoxin as well as TNF-α secretion. Moreover, eosinophils expressed granzyme A, which was released upon interaction with Colo-205, whereas cytotoxicity was partially inhibited by FUT-175, an inhibitor of trypsin-like enzymatic activity. Our data present the first demonstration, to our knowledge, that granzyme A is a cytotoxic mediator of the eosinophil protein arsenal, exerting eosinophil tumoricidal activity toward Colo-205, and provide mechanistic evidence for innate responses of eosinophil against tumor cells.     

Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K⁺ efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K⁺ electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24–48 h of culture. High extracellular K⁺ (10–100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K⁺ efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (ΔΨ_m) that preceded development of apoptotic morphology. Changes in ΔΨ_m resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K⁺ efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K⁺ released locally from damaged cells may function as a survival signal for neutrophils.     

Calcaneal quantitative ultrasound measurements in young male and female professional dancers

The aim of this study was to assess the bone status of dancers using calcaneal quantitative ultrasound (QUS). Twenty-four male and 26 female dancers (aged 19-36 years) and 100 age- and sex-matched nonathletic controls were included in this study. QUS parameters (broadband ultrasound attenuation [BUA], speed of sound [SOS], quantitative ultrasound index [QUI], and estimated heel bone mineral density [eBMD]) were obtained for both heels in all subjects using a gel-coupled QUS device. Two-way analysis of variance, including the factors of dancing status and gender, revealed significant differences in all QUS parameters between the dancers and the controls (p < 0.001 for all), without a significant interaction with gender status. For each heel (right versus left), the mean QUI, BUA, SOS, and eBMD values of the male and female dancers were 22.0% vs. 20.9% and 16.6% vs. 16.0%; 21.6% vs. 24.1% and 13.2% vs. 14.3%; 2.3% vs. 2.0% and 1.7% vs. 1.7%; and 25.0% vs. 23.9% and 19.0% vs. 18.6% higher than those of the male and female controls, respectively. Analysis of covariance still revealed significant differences between the dancers and the controls after controlling for the influence of body mass index (p < 0.001). Furthermore, the finding that significant bilateral differences in BUA did exist in the controls but not in the dancers demonstrated site-specific effects of exercise on bone, indicating that it is the dance that improved bone properties. Calcaneal QUS, with a strong discriminative ability between those involved in professional dance and normally active individuals, emerges as an attractive technology for exploring the benefits of exercise on bone, which might be a challenge for those in the conditioning field, who need to identify those who need intervention in terms of bone status and promote participation in high-impact physical activity, such as dance, to enhance bone quality.     

The modalities of the sexual cycle of Octopus vulgaris in the southern Moroccan Atlantic (Tantan, Boujdour)

The reproduction of Octopus vulgaris between Tan Tan and Boujdour is studied. The samples used are sorted monthly from commercial catches of coastal trawlers operating in this area from December 2001 to May 2003. The study shows that sexual maturity is in advance for males than for females. Two spawning periods are made out by the follow-up of the RGS: a spring period from March to July, and an autumnal period, which is less intense, between September and October. The division of spermatogonia and spermatocytes reaches its maximal intensity in July and December, whereas the spermiogenesis is active until the time of mating. The vitellogenesis starts in mid-December and extends until the following spawning period. This reveals a later gonadic development for females than for males; approximately seven months for females and three months for males.     

Unusual complexation of Cu(I) by pyrimidine/pyridine-pyrazole derived ligands exploiting the molecular function of 2-mercapto-4,6-dimethylpyrimidine - Syntheses, crystal structures and electrochemistry

Reaction of 2 equiv. amount of copper(II) chloride dihydrate with 2 equiv. of methyl-5-methyl-1-(4,6-dimethyl-2-pyrimidyl)pyrazole-3-carboxylate (DpymPzC) in presence of 1 equiv. of 2-mercapto-4,6-dimethylpyrimidine (DpymtH) at pH ∼ 6 afforded the tricoordinated copper(I) complex [Cu(DpymPzC)Cl] (1). The same reaction with copper(II) perchlorate hexahydrate, as the metal salt under the same equivalent ratio at pH ∼ 6 formed the tetracoordinated copper(I) complex [Cu(DpymPzC)₂]ClO₄ (2). In both the cases, the role of DpymtH is nothing but only to reduce the copper(II) salt in situ finally forming the copper(I) complex. On the other hand, the direct reaction between the copper(I) thiocyanate and DpymPzC in 2:2 equiv. ratio produced a tricoordinated copper(I) complex [Cu(DpymPzC)SCN] (3). In a similar reaction of 2 equiv. amount of copper(II) chloride dihydrate with 2 equiv. amount of ethyl-5-methyl-1-(2-pyridyl)pyrazole-3-carboxylate (PyPzC) in presence of 1 equiv. of DpymtH at pH ∼ 6, an intense red coloured microcrystalline compound (4) was obtained. In contrast, 1 equiv. of PyPzC and 2 equiv. of DpymtH on reaction with 1 equiv. of copper(II) chloride dihydrate at pH ∼ 6 produced a novel tetranuclear mixed coordinated [Cu₄(DpymtH)₄Cl₄] complex (5). Here DpymtH plays dual role - a reducing agent for the copper(II) salt followed by a chelating ligand towards copper(I) so formed in situ. Among the above species, 1, 2 and 5 are crystallographically characterized. In 1, the central copper atom is in distorted triangular planar geometry with N₂Cl chromophore whereas in 2, the same is in distorted tetrahedral geometry with N₄ chromophore. Notably, the extent of distortion from the ideal geometry is more in 2. In 5, which is in chair conformation, out of four copper atoms, two being in S₂Cl chromophore are tricoordinated and the remaining two are tetracoordinated with NS₂Cl chromophore. The metal centers are bridged through DpymtH in its ‘thione’ form. Interestingly, the chelation (in part) results in formation of the highly stable four-membered two chelate rings around the two tetracoordinated copper atoms in 5. The two copper centers along the long arm of the chair are separated through a distance of 5.190 Å while those in the short arm are at a length of 3.629 Å. The electronic, IR spectra and electrochemistry of the complexes 1, 2 and 5 have also been investigated.