Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.     

Administration of r-VEGF-A prevents hepatic artery ligation-induced bile duct damage in bile duct ligated rats

The hepatic artery, through the peribiliary plexus, nourishes the intrahepatic biliary tree. During obstructive cholestasis, the nutritional demands of intrahepatic bile ducts are increased as a consequence of enhanced proliferation; in fact, the peribiliary plexus (PBP) displays adaptive expansion. The effects of hepatic artery ligation (HAL) on cholangiocyte functions during cholestasis are unknown, although ischemic lesions of the biliary tree complicate the course of transplanted livers and are encountered in cholangiopathies. We evaluated the effects of HAL on cholangiocyte functions in experimental cholestasis induced by bile duct ligation (BDL). By using BDL and BDL + HAL rats or BDL + HAL rats treated with recombinant-vascular endothelial growth factor-A (r-VEGF-A) for 1 wk, we evaluated liver morphology, the degree of portal inflammation and periductular fibrosis, microcirculation, cholangiocyte apoptosis, proliferation, and secretion. Microcirculation was evaluated using a scanning electron microscopy vascular corrosion cast technique. HAL induced in BDL rats 1) the disappearance of the PBP, 2) increased apoptosis and impaired cholangiocyte proliferation and secretin-stimulated ductal secretion, and 3) decreased cholangiocyte VEGF secretion. The effects of HAL on the PBP and cholangiocyte functions were prevented by r-VEGF-A, which, by maintaining the integrity of the PBP and cholangiocyte proliferation, prevents damage of bile ducts following ischemic injury.     

Sterically hindered complexes of platinum(II) with planar heterocyclic nitrogen donors. A novel complex with 1-methyl-cytosine has a spectrum of activity different from cisplatin and is able of overcoming acquired cisplatin resistance

A very interesting series of water soluble platinum compounds violating some of the classical structure-activity relationships, but still showing antitumor activity, was reported by Hollis and collaborators some 25 years ago [L.S. Hollis, A.R. Amundsenm, E.W. Stern. J. Med. Chem. 32 (1989) 128-136]. The compounds, having formula [PtClA(2)L](+) (A(2)=two monodentate or a bidentate amine, L=a secondary or tertiary amine or a N-donor heterocycle), were characterized by a positive charge and three non-labile N-donor ligands. We have extended the investigation to analogous compounds in which 2,9-dimethyl-1,10-phenanthroline has taken the place of the A(2) ligand(s) and L is 2-picoline (1), 6-amino-2-picoline (2), or 1-methyl-cytosine (3). The X-ray analysis of 2 has revealed a bow-like distortion of the phenanthroline plane, a sloping of the phenanthroline plane with respect to the coordination plane, and an overall shielding of the metallic core by the ortho substituents of the phenanthroline and pyridine ligands. In vitro grow inhibition assays have been performed on the most water soluble complex 3. The results indicate that this complex is characterized by a potent growth inhibitory activity with mean IC(50) value (in a panel of 11 human tumor cell lines) of 1.1 microM to be compared with a mean value of 3.8 microM for cisplatin. The same compound also appears to completely overcome the acquired cisplatin resistance stemming from reduced uptake or a multifocal mechanism, thus pointing to a mechanism of action distinctly different from that of cisplatin.     

FACS analysis of oxidative stress induced on tumour cells by SERMs

This study confirms previous reports of the antiproliferative effect of OH-ferrocifen on both hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell models. Contrastive analysis of the proliferative effects of classical agonists (including estradiol and ethynylestradiol) and selective estrogen receptor modulators (SERMs) such as tamoxifen and its active metabolite, 4-hydroxy-tamoxifen, was also performed. Previous studies have attributed the effects of OH-ferrocifen on hormone-independent cells to an underlying mechanism based on intracellular oxidation, production of hydroxyl radicals and oxidative damage of DNA. Here we report that the chemically generated OH-ferrocifen cation (as well as the decamethylferrocenium cation) is less cytotoxic to both cell lines than the neutral parent complex. Moreover, fluorescence activated cell sorting (FACS) analysis of 8-oxo-guanine production indicates that, even at relatively high concentrations, OH-ferrocifen produces negligible oxidative DNA damage as compared to other SERMs, namely tamoxifen and 4-hydroxy-tamoxifen. Alternative pathways to explain the remarkable activity of OH-ferrocifen must therefore be sought.     

Quantification of gliadin levels to the picogram level by flow cytometry.

BACKGROUND: Celiac disease is a widely prevalent enteropathy caused by intolerance to gliadin, one of the gluten proteins. We developed two methods for the analysis of gliadin levels. Both methods use flow cytometry and rat antibodies against a 16-residue peptide of gliadin. The peptide is common to the alpha-, beta-, gamma-, and omega-gliadins. METHODS: In the one-site assay, the antigen (gliadin standard or food extract) was adsorbed on 3-mum latex particles. Sensitized particles were then incubated, in this order, with rat anti-gliadin peptide antibodies and anti-rat immunoglobulin G antibodies labeled with fluorescein isothiocyanate. In the two-site assay, the antigen was trapped on the latex particles by rat anti-gliadin antibodies and then measured by the same antibodies labeled with fluorescein. RESULTS: Detection limits were 1 ng/ml for the one-site assay and 10 pg/ml for the two-site assay. The two-site assay displayed gliadin at concentrations above the limit proposed by the Codex Alimentarius in 2 of 40 gluten-free products. CONCLUSION: There is a growing concern that gliadin, even when present in gluten-free foods within the limit fixed by the Codex Alimentarius, over the long term may become toxic to patients with celiac disease. The techniques described in this study provide an opportunity to further decrease the acceptable limit of gliadin in gluten-free foods.     

Lipase-catalyzed transformations for the synthesis of butyl lactate: a comparison between esterification and transesterification

The alpha-hydroxy esters are increasingly employed in cosmetic, food, and pharmaceutical formulations as they determine reduced skin-irritant effects in comparison with the respective acids, offering similar hygroscopic, emulsifying, and exfoliating properties. The enzymatic synthesis of lactate esters in nonaqueous systems was studied as regards the influence of the critical process parameters, to enable a comparison between the most commonly used synthetic routes, namely, esterification and transesterification. The experimental results showed that the direct esterification of lactic acid with butanol may be limited by the reduced lipase stability in the presence of the acid (substrate) and of the water (product), in particular when solvent-free media are used. The stability of the enzyme is further reduced as polar solvents are required as a result of the polarity of the lactic acid. Therefore, the use of the lactic acid as substrate is of practical interest only when the acid is significantly cheaper in comparison with its short-chain esters. If this is not the case, the transesterification of the ethyl lactate with butanol is to be preferred for the higher flexibility in the choice of the experimental conditions, the operability of solvent-free systems, and the simplicity of the product removal assembly.     

Transient maintenance in bioreactor improves health of neuronal cells

Summary To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, −9, and −3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-α (TNF-α) and inter-leukin-1β (IL-1β) is coupled with a level of activated nuclear factor-ϰB (NF-ϰB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-β/δ (PPARβ/δ), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.     

HPLC analysis of the second-generation antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma

A liquid chromatographic method with ultraviolet detection was developed for the analysis of the recent antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma. The analytes were separated on a C8 reversed phase column, using a mobile phase composed of acetonitrile and a 12.3 mM, pH 3.0 phosphate buffer containing 0.1% triethylamine (35:65, v/v). Clomipramine was used as the Internal Standard. Using a solid phase extraction procedure with C2 cartridges high extraction yields (>94%) and good purification from matrix interference were obtained. Good linearity was obtained in the 7.5-250.0 ng mL(-1) range for sertraline and in the 10-500 ng mL(-1) range for N-desmethylsertraline. The analytical method was validated in terms of precision, extraction yield and accuracy. These assays gave R.S.D.% values for precision always lower than 3.9% and mean accuracy higher than 90%. Thanks to its good selectivity, the method proved to be suitable for the analysis of plasma samples from patients treated with sertraline as either monotherapy or polypharmacy.     

Sea urchins, sea stars and brittle stars from Terra Nova Bay (Ross Sea, Antarctica)

Although echinoderms constitute some of the most conspicuous taxa of the Antarctic benthic communities, the echinoderm fauna of Terra Nova has not been described yet. The present study provides the first species list of echinoids, ophiuroids and asteroids from Terra Nova Bay (30–500 m depth) and describes the depth distribution of these species. Preliminary observations of the summer reproductive condition of some of the species are also included.