Pyrene and benzo(a)pyrene Metabolism by an Aspergillus Terreus Strain Isolated from a Polycylic Aromatic Hydrocarbons Polluted Soil

A strain of Aspergillus terreus was isolated from a polycyclic aromatic hydrocarbons (PAHs) polluted soil. The metabolism of pyrene and benzo(a)pyrene by this fungus was investigated in liquid submerged culture added of 50 and 25 ppm respectively of each compound. Depletion of pyrene and Benzo(a)pyrene was evident during the first stages of growth and was 60% and 27.5% respectively of the added amount after nine days of culture. Solvent extracts of the fermentation broth and mycelium were analysed for presence of metabolites by HPLC-MS technique. Under the present cultural conditions pyrene was mainly metabolised to pyrenylsulfate similarly to benzo(a)pyrene that led to benzo(a)pyrenylsulfate. The structure of 1-pyrenilsulfate was determined after purification of extracts and H-NMR analysis. The result show that the isolated A. terreus strain metabolises PAHs by reaction similar to those previously reported for non lignolinolytic fungi with a mechanism that suggests the hydroxylation by a cytochrome P-450 monooxygenase followed by conjugation with sulfate ion.     

Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster

We have recently identified in Drosophila melanogaster a new gene encoding a nuclear protein, DIP1. Here we report the developmental expression and the finding that DIP1 subcellular localization is in the nucleus and at the nuclear periphery during interphase in embryos. Interestingly, in humans, DIP1 antibody identified signals in nuclei from cultured cells and reacted with a rough 30kDa protein in Western blotting experiments, demonstrating evolutionary conservation.     

Calcium/calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation

Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1.     

Pore formation by equinatoxin II,a eukaryotic protein toxin,occurs by induction of nonlamellar lipid structures

Pore formation in the target cell membranes is a common mechanism used by many toxins in order to kill cells. Among various described mechanisms, a toroidal pore concept was described recently in the course of action of small antimicrobial peptides. Here we provide evidence that such mechanism may be used also by larger toxins. Membrane-destabilizing effects of equinatoxin II, a sea anemone cytolysin, were studied by various biophysical techniques. 31P NMR showed an occurrence of an isotropic component when toxin was added to multilamellar vesicles and heated. This component was not observed with melittin, alpha-staphylococcal toxin, or myoglobin. It does not originate from isolated small lipid structures, since the size of the vesicles after the experiment was similar to the control without toxin. Electron microscopy shows occurrence of a honeycomb structure, previously observed only for some particular lipid mixtures. The analysis of FTIR spectra of the equinatoxin II-lipid complex showed lipid disordering that is consistent with isotropic component observed in NMR. Finally, the cation selectivity of the toxin-induced pores increased in the presence of negatively charged phosphatidic acid, indicating the presence of lipids in the conductive channel. The results are compatible with the toroidal pore concept that might be a general mechanism of pore formation for various membrane-interacting proteins or peptides.     

Cutting edge: increased NK cell activity in HIV-1-exposed but uninfected Vietnamese intravascular drug users

We addressed the role of innate immunity in the protection against HIV-1 infection by studying NK cell function in 37 Vietnamese intravascular drug users (IDUs), who appeared to remain HIV-1 uninfected despite many years of high-risk exposure (exposed uninfected, EU), 10 IDUs who underwent seroconversion and 28 unexposed blood donors. Main results were: NK cell lytic activities against both the NK-susceptible K562 cell line and the NK-resistant Daudi cell line were significantly augmented in EU IDUs compared with either controls or seroconverters before or after seroconversion; NK cells producing the cytokines IFN-gamma and TNF-alpha and the beta chemokines CCL3, CCL4, and CCL5 were also increased in the EU IDUs, either after in vitro activation or without stimulation. The finding of an enhanced NK cell function in EU IDUs, especially compared with IDUs who became HIV-1 infected, supports the hypothesis that NK cells contribute to the protection against HIV-1 infection.     

Affinity chromatography techniques based on the immobilisation of peptides exhibiting specific binding activity

Affinity chromatography is one of the powerful techniques in selective purification and isolation of a great number of compounds. New challenges in scientific research, such as high-throughput systems, isolation procedures that allow to obtain a single substance from a complex matrix in high degree of purity, low costs and wide availability, have led to the discovery of new tailor-made synthetic recognition systems. In this review the design, synthesis, purification and characterisation of peptides with recognition properties are discussed. Applications of peptide ligands are described and analytical tools mentioned.     

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA₂=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.     

Helicobacter pylori antigen-specific T-cell responses at gastric level in chronic gastritis,peptic ulcer,gastric cancer and low-grade mucosa-associated lymphoid tissue (MALT) lymphoma

Host T-cell response to Helicobacter pylori is important for the clinical outcome of the infection. A Th1-polarized response, preferentially against CagA, is associated with peptic ulcer, whereas mixed Th1 and Th0 responses are present in non-ulcer gastritis. A deregulated H. pylori-driven Th0 cell-dependent B-cell activation is found in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue.     

Regulation of 3' splice site selection in the 844ins68 polymorphism of the cystathionine Beta -synthase gene

844ins68 is a frequent polymorphism of the cystathionine beta-synthase gene (CBS) that consists of a 68-bp insertion duplicating the 3' splice site of intron 7 and the 5'-end of exon 8. The presence of two identical 3' splice sites spaced by 68 bp should lead to either a selection of the proximal site or to at least two alternatively spliced CBS mRNA variants. Instead, an accurate selection of the distal 3' splice site is observed in the 844ins68 carriers. The duplication has generated a gene re-arrangement at the 3' splice site where two GGGG runs have been brought close to each other. Using a minigene system, we have investigated the effect this peculiar configuration might have on the selection of the 3' splice site of intron 7 in the CBS gene. Minimal disruption of the G runs resulted in a dramatic shift toward the proximal 3' splice site selection with inclusion of the 68-bp insertion and a consequent change of the reading frame. The insertional event created this peculiar configuration of two G repeats close to each other that subsequently acquired the ability to strongly bind heterogeneous nuclear ribonucleoprotein (hnRNP) H1, a specific trans-acting factor. The interaction of hnRNP H1 with G runs within the 844ins68 context might interfere with the recruitment of splicing factors to the proximal 3' splice site thus favoring the selection of the distal 3' splice site. Our results therefore suggest the possibility that the insertion was an evolutionary event that allowed the rescue of the wild-type sequence, so preserving protein function.