Minimal contribution of marrow-derived endothelial precursors to tumor vasculature

During embryogenesis, vascular and hemopoietic cells originate from a common precursor, the hemangioblast. Recent evidence suggests the existence of endothelial precursors in adult bone marrow cells, but it is unclear whether those precursors have a role in tumor neovascularization. In this report, we demonstrate that murine bone marrow contains endothelial progenitors, which arise from a cell with self-renewing capacity, and can integrate into tumor microvasculature, albeit at a very low frequency. A transgenic double-reporter strategy allowed us to demonstrate definitively that tumor bone marrow-derived endothelial cells arise by transdifferentiation of marrow progenitors rather than by cell fusion. Single cell transplants showed that a common precursor contributes to both the hemopoietic and endothelial lineages, thus demonstrating the presence of an adult hemangioblast. Furthermore, we demonstrate that increased vascular endothelial growth factor (VEGF)-A secretion by tumor cells, as well as activation of VEGF receptor-2 in bone marrow cells does not alter the mobilization and incorporation of marrow-derived endothelial progenitors into tumor vasculature. Finally, in human umbilical cord blood cells, we show that endothelial precursors make up only approximately 1 in 10(7) mononuclear cells but are highly enriched in the CD133+ cell population. By ruling out cell fusion, we clearly demonstrate the existence of an adult hemangioblast, but the differentiation of marrow stem cells toward the endothelial lineage is an extremely rare event. Furthermore, we show that VEGF-A stimulation of hemopoietic cells does not significantly alter this process.     

Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol

Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k(inact) = 0.24 versus 0.39 min(-1)). Additionally, the overall inhibition constant (K(i)) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nm). Univalent electron flux (O(2)(.) formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving approximately 50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.     

Heavy metals resistant plasmid-mediated utilization of solar by Pseudomonas aeruginosa AA301

Solar-degrading bacteria, Pseudomonas aeruginosa strains, were isolated from Egyptian soil by Mineral Salt Medium (MSM) supplemented with Solar (motor fuel) from different oil-contaminated sites in Sohag province. The strain AA301 of Pseudomonas aeruginosa showed appreciable growth in MSM medium containing high concentrations of Solar ranging from 0.5 to 3% (v/v), with optimum concentration at 1.5%. Solar was used as a sole carbon source and a source of energy by the bacterium. The ability to degrade Solar was found to be associated with a single 60-kb plasmid designated pSOL15. The plasmid-cured variant, which was obtained by culturing in LB broth with kanamycin, lost the plasmid indicative the ability to degrade Solar must depend on this plasmid. The wild type isolate, Pseudomonas aeruginosa AA301 and transformant strain, have maximum growth (OD600 = approximately 2) on Solar, however the plasmid-cured variant did not have any significant growth on Solar. Moreover, resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ was also 60-kb plasmid-mediated. Therefore, the strain AA301 could be good candidate for remediation of some heavy metals and oil hydrocarbons in heavily polluted sites.     

A Simple and Efficient DNA Isolation Method for Salvia officinalis

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4?M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6?μg DNA g^?1 of dry leaf tissue, and the absorbance ratios 260/280 and 260/230?nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.     

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.     

Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target

Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells. We have constructed a test system to investigate HR between a plasmid and a chromosome in carbon-starved populations of the soil bacterium Pseudomonas putida restoring the expression of phenol monooxygenase gene pheA. Our results show that prolonged starvation of P. putida in the presence of phenol stimulates HR. The emergence of recombinants on selective plates containing phenol as an only carbon source for the growth of recombinants is facilitated by reactive oxygen species and suppressed by DNA mismatch repair enzymes. Importantly, the chromosomal location of the HR target influences the frequency and dynamics of HR events. In silico analysis of binding sites of nucleoid-associated proteins (NAPs) revealed that chromosomal DNA regions which flank the test system in bacteria exhibiting a lower HR frequency are enriched in binding sites for a subset of NAPs compared to those which express a higher frequency of HR. We hypothesize that the binding of these proteins imposes differences in local structural organization of the genome that could affect the accessibility of the chromosomal DNA to HR processes and thereby the frequency of HR.     

Synthesis of some quinolinyl chalcone analogues and investigation of their anticancer and synergistic anticancer effect with doxorubicin

Two derivatives of 2-(4-acetylanilino)quinolines (IIIa, b) were synthesized as scaffolds for synthesis of open chalcone analogues (Va-f) through Claisen-Schmidt condensation with a set of aromatic aldehydes (IVa-d). Derivatives (Va, b) were further manipulated into cyclic ??,??-unsaturated ketones by Michael-addition of acetylacetone and ethylacetoacetate affording derivatives (VI?CVII). Deethoxycarboxylation of derivatives (VIIa, b) afforded cyclohexenons (VIIIa, b) allowing formation of a mini library of ??,??-unsaturated ketones for screening their anticancer and synergistic anticancer effect with doxorubicin using colon cancer cell line (Caco-2). Two open enones, (Vb) and (Ve), showed significant anticancer activity with IC₅₀ of 5.0 and 2.5 ??M respectively. Only one cyclic enone, (VIa) showed synergistic anticancer activity with doxorubicin at 10 ??M.     

Cerebrolysin administration reduces oxidative stress‐induced apoptosis in limphocytes from healthy individuals

Cerebrolysin is the only drug available for clinical use containing active fragments of some important neurotrophic factors obtained from purified porcine brain proteins, which has long been used for the treatment of dementia and stroke sequels. Cerebrolysin has growth factor‐like activities and promotes neuronal survival and sprouting, however, its molecular mechanism still needs to be determined. It has been shown that Cerebrolysin may interact with proteolytic pathways linked to apoptosis. Administration of Cerebrolysin significantly reduces the number of apoptotic neurons after glutamate exposure. Furthermore, it has been reported that Cerebrolysin inhibits free radicals formation and lipid peroxidation. In vitro we evaluated the protective effects of Cerebrolysin towards spontaneous and induced apoptotic death in cells from healthy individuals. Peripheral blood lymphocytes (PBLs) from 10 individuals were used as cell model; 2‐deoxy‐D‐ribose (dRib), a highly reducing sugar, was used as paradigm pro‐apoptotic stimulus. Apoptosis was analysed using flow cytometry and fluorescence microscopy. Our results showed that Cerebrolysin significantly reduced the number of apoptotic PBLs after dRib treatment, although it had no significative effects on cells cultured in standard conditions. Our work showed a protective effect of Cerebrolysin on oxidative stress‐induced apoptosis and suggested that PBLs can be used as an easy obtainable and handy cell model to verify Cerebrolysin effects in neurodegenerative pathologies.     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.