Molecular modeling study of intercalation complexes of tricyclic carboxamides with d(CCGGCGCCGG)₂ and d(CGCGAATTCGCG)₂

Tricyclic dyes with different mesoatoms such as xanthenes (fluorescein, eosin) anthracenes and acridines (proflavine) approved by the Food and Drug Administration (FDA) for use in foods, pharmaceuticals and cosmetic preparations interact with DNA, and some of them do so through intercalation. Hyperchem 7.5, Spartan 04, Yasara 10.5.14 program packages and molecular modeling, molecular mechanics and dynamics techniques with the oligonucleotides d(CCGGCGCCGG)2 and d(CGCGAATTCGCG)2 were utilized in order to examine the mode of binding to DNA of a range of tricyclic carboxamides bearing N,N-dimethylaminoethyl side chain, i.e., 9-amino-DACA, anthracene, acridine-1-carboxamide, acridine-4-carboxamide (DACA), azacridine, phenazine, pyridoquinoxaline, oxopyridoquinoxaline, phenoxazine and xanthenone or N,N-dimethylaminobutyl moiety, i.e., phenazine and acridine. The bicyclic quinoline-8-carboxamide was also examined for comparison reasons. On the basis of our data, prerequisite for the interaction between protonated N,N-dimethylaminoethyl moiety and guanine is the formation of only one internal hydrogen bond between carboxamide and peri NH + in the case of 9-amino-DACA or peri N in the cases of DACA, azacridine, phenazine and pyridoquinoxaline. The presence of an additional internal hydrogen bond between oxygen carboxamide and protonated N,N-dimethylamino group in the cases of tricyclic systems bearing peri NH (phenoxazine) or O (xanthenone) group, prevents the interaction between side chain and guanine. Also, the formation of one internal hydrogen bond between oxygen carboxamide and protonated N,N-dimethylamino group inhibits the interaction between side chain and guanine in the case of acridine-1-carboxamide. Our findings are in accordance with previously reported results obtained from the kinetic studies of the binding of acridine and related tricyclic carboxamides to DNA.     

Kinetic study of cell proliferation of Saccharomyces cerevisiae strains by sedimentation/steric field flow fractionation in situ

The technique of Sedimentation/Steric Field Flow Fractionation (Sd/StFFF) is applied to the kinetic study of cells proliferation of Saccharomyces cerevisiae strains. The experimental parameter varied is the time from the preparation of the yeast sample dispersion in the culture medium. The determination of the size and mass distributions of the yeast cells is combined with the growth of the yeast cells and their life cycle. The experimental results are compared with those obtained by scanning electron microscopy (SEM) and those found in the literature. Useful conclusions concerning the budding and the fission of these yeast cells were extracted.     

Lipid rafts associate with intracellular B cell receptors and exhibit a B cell stage-specific protein composition

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits alpha-1 and beta-2, vimentin, gamma-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.     

Differentiation of myomas by means of biomagnetic and doppler findings

Aim

To elucidate the hemodynamics of the uterine artery myomas by use of Doppler ultrasound and biomagnetic measurements.

Method

Twenty-four women were included in the study. Sixteen of them were characterised with large myomas whereas 8 of them with small ones. Biomagnetic signals of uterine arteries myomas were recorded and analyzed with Fourier analysis. The biomagnetic signals were distributed according to spectral amplitudes as high (140–300 ft/√Hz), low (50–110 ft/√Hz) and borderline (111–139 ft/√Hz). Uterine artery waveform measurements were evaluated by use of Pulsatility Index (PI) (normal value PI < 1.45).

Results

There was a statistically significant difference between large and small myomas concerning the waveform amplitudes (P < 0.0005) and the PI index (P < 0.0005). Specifically, we noticed high biomagnetic amplitudes in most large myomas (93.75 %) and low biomagnetic amplitudes in most small ones (87.5 %).

Conclusion

It is suggested that the biomagnetic recordings of uterine artery myomas could be a valuable modality in the estimation of the circulation of blood cells justifying the findings of Doppler velocimetry examination.

     

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12?836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.     

Respiratory distress syndrome (RDS) in premature infants is underscored by the magnitude of Th1 cytokine polarization

Respiratory distress syndrome (RDS) is a common problem and the leading cause of death in premature infants (PI). The introduction of surfactant treatment for RDS management has lowered mortality and morbidity; nevertheless, some neonates do not improve and are at increased risk of pulmonary hemorrhage. Inflammation, not only local but also systemic, seems to play an important role in the pathogenesis of RDS. To determine whether cytokine patterns characterize RDS and its outcome, we measured type-1 (IL-2, TNF-α, IFN-γ, IL-6) and type-2 (IL-4, IL-5, IL-10, TGF-β1) serum cytokines of 47 PI with established RDS and a control group of 30 healthy, appropriate for gestational age, full-term neonates. Cord blood samples were obtained at the time of delivery from PI and controls. Venous blood samples were collected from PI who received surfactant treatment and/or developed pulmonary hemorrhage. Significantly elevated cord blood cytokine levels were observed in PI at time of delivery, compared to controls, except for IL-5 and TNF-α levels that were within control range. The type-1/type-2 cytokine ratio was significantly increased in PI vs controls. Neonates who developed pulmonary hemorrhage between 2 and 3 days of life and/or died, presented the strongest Th1 and type-1 cytokine polarization that was mainly due to increased IFN-γ and TNF-α, and decreased TGF-β1. The majority of these PI were female with very low gestational age. Overall, PI with RDS present a Th1/type-1 cytokine polarization, which persists irrespective of the treatment provided, and is amplified when complications appear. Th1 polarization is associated with poor prognosis.     

Cord blood leptin levels of healthy neonates are associated with IFN-γ production by cord blood T-cells

Leptin is a hormone synthesized by adipocytes and other tissues, including the placenta, and it regulates food intake and energy expenditure, reproductive and immune functions. To investigate the role of leptin in neonatal immunity, we measured serum leptin and cytokine (IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-12) levels in the cord blood (cb) of 510 healthy neonates, 14 small for gestational age (SGA), 312 appropriately grown for gestational age (AGA) and 184 large for gestational age (LGA). Median serum leptin concentration in the whole sample was 11 ng/ml. In 11.2% neonates (1 SGA, 32 AGA, 24 LGA), leptin levels were >90th percentile (median 39 ng/ml). In 33.3% of those (3.72% of total sample) with the highest leptin levels (median 46 ng/ml), significantly elevated levels of serum IFN-γ were also found (mean 27.11 pg/ml, range 17.5-38.5 pg/ml). In neonates with leptin levels ～50th percentile (median 12 ng/ml) or <10th percentile (median 1 ng/ml), serum IFN-γ levels were negligible. All other cytokines measured, were < the assays' detection limits. To investigate whether leptin can independently influence cytokine gene expression by cb T-cells and monocytes (Mc), we cultured cb T-cells or Mc, isolated from randomly selected AGA neonates or adult peripheral blood, with leptin. This resulted in upregulation of IL-2, IFN-γ and IL-4 gene expression in cb and adult T-cells and IL-10 expression mainly in cb-Mc. Significantly higher expression of IFN-γ occurred in female cb-T-cells cultured with leptin, compared with male cb-T-cells. In conclusion, the concurrent presence of high concentrations in both leptin and IFN-γ in cb of healthy infants, and leptin's ability to directly upregulate cytokine gene expression in cb T and Mc cells, indicate that abnormally high leptin levels can independently influence the immune system of healthy newborns, and may mediate gender differences in the development of a Th1 polarized immune response.     

Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization.

Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [3H]arachidonic acid from [3H]anandamide, in a time- and concentration-dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K(m) of 3.7 microM and V(max) 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9-10, was abolished by phenylmethylsulfonyl fluoride and was Ca(2+)- and Mg(2+)-independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.     

Synthesis of all-trans anandamide: a substrate for fatty acid amide hydrolase with dual effects on rabbit platelet activation

Anandamide (AEA) presents the four double bonds in the cis configuration, deriving from the arachidonic acid moiety. In the context of an antisense strategy based on the double bond configuration, all-trans AEA (t-AEA) was synthesized in high yield starting from all-trans methyl arachidonate and ethanolamine in the presence of KCN. t-AEA was assayed on rabbit platelet aggregation, obtaining effect only at high concentrations (>10(-4) M) after an also concentration-dependent lag phase. At lower concentrations it inhibited PAF-induced rabbit platelet aggregation with an IC(50)=4.6 x 10(-6) M. In contrast to anandamide, the activation of platelets was not due to the conversion of t-AEA to trans arachidonic acid, as ascertained by negative results with FAAH inhibitors. However, t-AEA was found to be a substrate for fatty acid amide hydrolase (FAAH), the enzyme that cleaves anandamide and regulates in vivo the magnitude and duration of the signaling induced by this lipid messenger.     

Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: relationship with increased luteinizing hormone levels

The objective of the study was the comparison of anti-Müllerian hormone (AMH) levels among obese or overweight and normal-weight women with the four different polycystic ovary syndrome (PCOS) phenotypes and healthy control subjects. AMH levels were evaluated in four age- and body mass index (BMI)-matched groups of 25 normal-weight and 25 obese or overweight women each, belonging to the four main subsets of the syndrome resulting from combinations of the three diagnostic criteria [group 1: oligo- and/or anovulation (ANOV), hyperandrogenemia (HA), and polycystic ovaries (PCO) on ultrasonographic evaluation; group 2: ANOV and HA; group 3: HA and PCO, group 4: ANOV and PCO], and in 50 (25 obese or overweight and 25 normal weight) age- and BMI-matched healthy control subjects. Age, BMI, waist circumference, FSH, LH, prolactin, testosterone, Delta(4)-androstenedione, dehydroepiandrosterone-sulfate, 17alpha-OH-progesterone, fasting insulin, glucose, AMH, free androgen index, and homeostasis model assessment for insulin resistance index were analyzed. AMH levels were significantly higher in PCOS groups 1 and 2 compared with groups 3 and 4 and the control group and higher in PCOS groups 3 and 4 compared with the control group. AMH levels were significantly increased in normal-weight compared with obese and overweight women. AMH concentrations were independently predicted, in order of significance, by LH and testosterone levels, BMI (negatively), and the total number of follicles 2-9 mm in diameter. The differences in circulating AMH levels between the main phenotypic groups of PCOS women appear to reflect the severity of the syndrome, but are negatively affected by obesity. Increased LH levels might be the most significant independent link between PCOS-associated disorders of ovulation and the observed increase in circulating AMH concentration.