Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Phylogeography of the Lacerta viridis complex: mitochondrial and nuclear markers provide taxonomic insights

Based on broad, nearly rangewide sampling, we reanalysed the phylogeography of the Lacerta viridis complex using the mitochondrial cytochrome b gene and the intron 7 of the nuclear β‐fibrinogen gene. Using the mitochondrial marker, we identified in phylogenetic analyses 10 terminal clades clustering in four deeply divergent main lineages whose relationships are weakly resolved. These lineages correspond to Lacerta bilineata, L. viridis, the previously identified Adriatic or West Balkan lineage and a newly discovered fourth lineage from the Anatolian Black Sea coast and the south‐eastern Balkan Peninsula. Except for the latter lineage, there is considerable phylogeographic structuring in each lineage, with higher diversity in the south of the distribution ranges. This pattern indicates the existence of two distinct microrefugia in the Italian Peninsula and Sicily and of up to seven microrefugia in the Balkan Peninsula, but of only one refugium along the Black Sea coast of Anatolia. We identified secondary contact zones of the main lineages and of terminal clades within these lineages. However, most of the formerly described putative contact zone of L. bilineata and L. viridis turned out to be a contact zone between the Adriatic lineage and L. viridis, but L. bilineata seems to be involved only marginally. Our nuclear marker could not unambiguously resolve whether there is gene flow in contact zones. Thus, further research is necessary to decide whether the four main lineages are conspecific or whether they represent distinct biological species. We restrict the name L. v. meridionalis to the newly identified genetic lineage from Turkey and south‐eastern Europe, synonymize some previously recognized taxa and suggest a tentative nomenclature for the L. viridis complex.     

Long-chain polyunsaturated fatty acids profile in plasma phospholipids of hyperphenylalaninemic children on unrestricted diet

IntroductionThe aim of the present study was to examine whether hyperphenylalaninemic children on unrestricted diet (MHP) may exhibit a different LCPUFA profile from PKU or healthy children in plasma phospholipids.Patients and methodsForty-five MHP children (age 9–14 years) were age and sex matched with 45 PKU and 45 healthy children. Fatty acids were determined and expressed as % of total fatty acids.ResultsMHP children showed docosahexaenoic acid (DHA) levels higher than PKU children (mean difference, 0.2%; 95% confidence interval, 0.02%–0.38%), although difference was not significant after correction for multiple comparisons, and lower levels than healthy children (?0.8%; ?1.01% to ?0.59%). Concentration of n-3 PUFA was higher in MHP than PKU children (0.6%; 0.4% to 0.8%),ConclusionsThe results suggest that low DHA levels in plasma phospholipids not only are evident in PKU but also may occur in MHP children, who are on unrestricted diet, as compared to healthy children.     

Fine structure of leydig and sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata

An ultrastructural investigation revealed the presence of true Leydig cells in the testis of sexually mature specimens of Torpedo marmorata. They showed the typical organization of steroid-hormone-producing cells, which, however, changed as spermatocysts approached maturity. In fact, they appeared as active cells among spermatocysts engaged in spermatogenesis, while in regions where spermiation occurred, they progressively regressed resuming the fibroblastic organization typically present in the testis of immature specimens. Such observations strongly suggest that these cells might be engaged in steroidogenesis and actively control spermatogenesis. Sertoli cells, too, appeared to play a role in spermatogenesis control, since, like Leydig cells, they showed the typical aspect of steroidogenic cells. In addition, the presence of gap junctions between Sertoli cells suggests that their activity might be coordinated. After sperm release, most Sertoli cells were modified and, finally, degenerated, but few of them changed into round cells (cytoplasts) or round cell remnants, which continued their steroidogenic activity within the spermatocyst and the genital duct lumen. From the present observations, it can be reasonably concluded that, in T. marmorata, spermatogenesis depends on both Leydig and Sertoli cells, and, as postulated by Callard (1991), in cartilaginous fish, the function of the Leydig cells as producers of steroids might be more recent and subsequent to that of Sertoli cells. In this regard, it is noteworthy that, in immature males, when Leydig cells showed a fibroblastic organization, Sertoli cells already displayed the typical organization of a steroidogenic cell.     

Neuroprotective activities of bacopa,lycopene, astaxanthin, and vitamin B12 combination on oxidative stress-dependent neuronal death

Oxidative stress is considered the common effector of the cascade of degenerative events in many neurological conditions. Thus, in this paper we tested different nutraceuticals in H₂O₂ in vitro model to understand if could represent an adjuvant treatment for neurological diseases. In this study, nutraceuticals bacopa, lycopene, astaxanthin, and vitamin B12 were used alone or in combination in human neuronal differentiated SH-SY5Y cells upon hydrogen peroxide-induced injury and neuroprotective, neuronal death pathways were analyzed. The nutraceuticals analyzed were able to protect H₂O₂ cytotoxic effects, through increasing cell viability and proteins involved in neuroprotection pathways and restoring proteins involved in cell death pathways. On this basis, it is possible to propose the use of these compounds as dietary supplement for the prevention or as adjuvant to the only symptomatic treatments so far available for neurodegenerative diseases.     

Perturbation of maize rhizosphere microflora following seed bacterization with Burkholderia cepacia MCI 7

Introduction of a large quantity of exogenous microorganisms may disrupt a local ecosystem and affect the natural microflora. In this work we investigated the effects of the introduction of a plant growth promoting strain of Burkholderia cepacia into the rhizosphere of maize on both indigenous B. cepacia populations and microbial community structure of total culturable bacteria using the concept of r/K strategy. Moreover we studied the distribution of bacterial populations in the root system at various soil depths. Seed bacterization was used as application method. Root colonization of the introduced strain occurred mainly on roots close to the plant stem, whereas indigenous B. cepacia was recovered at higher amounts from the lower parts of root systems of mature plants. As far as total culturable bacteria are concerned, an almost uniform distribution in the root system of mature plants was observed. The release of the exogenous bacterial strain affected mainly the microbial populations of young growing plants rather than mature plants. Indeed it caused only short-term perturbations in the microbial community of maize rhizosphere. Colonization of maize roots by indigenous B. cepacia was not significantly affected by the presence of the exogenous strain.     

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo

Background

Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.

Methodology/Principal Findings

The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.

Conclusion

Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.