In vitro and in vivo activities of Haplophyllum myrtifolium against Leishmania tropica

This study aimed to evaluate the in vitro and in vivo leishmanicidal activity of an endemic Turkish plant and compare its efficacy with a reference drug. In addition to the in vitro activities of the ethanol, acidified and alkaloid extracts and furoquinoline alkaloids skimmianine and gamma-fagarine, in vivo antileishmanial activitiy of the acidified extract of Haplophyllum myrtifolium Boiss. (Rutaceae) were investigated against Leishmania tropica (L. tropica), a causative agent of cutaneous leishmaniasis. All the extracts and pure compounds showed in vitro inhibitory activity against the promastigotes of. L. tropica. The in vitro 50% inhibitory concentrations of y-fagarine, acidified extract, ethanol extract, skimmianine and alkaloid extract against promastigotes were determined as 8.7, 9.4, 10.9, 25.7 and 25.8 microg/ml respectively. In vivo results of Haplophyllum myrtifolium acidified extract showed that this plant has a limited effect on decreasing the lesion size of experimental mice infected with Leishmania tropica. To the best of our knowledge, this is the first time both the in vitro and in vivo antileishmanial activity of Haplophyllum mrytifolium have been reported in the same research.     

Combined metabolic activators therapy ameliorates liver fat in nonalcoholic fatty liver disease patients

Nonalcoholic fatty liver disease (NAFLD) refers to excess fat accumulation in the liver. In animal experiments and human kinetic study, we found that administration of combined metabolic activators (CMAs) promotes the oxidation of fat, attenuates the resulting oxidative stress, activates mitochondria, and eventually removes excess fat from the liver. Here, we tested the safety and efficacy of CMA in NAFLD patients in a placebo‐controlled 10‐week study. We found that CMA significantly decreased hepatic steatosis and levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine, whereas found no differences on these variables in the placebo group after adjustment for weight loss. By integrating clinical data with plasma metabolomics and inflammatory proteomics as well as oral and gut metagenomic data, we revealed the underlying molecular mechanisms associated with the reduced hepatic fat and inflammation in NAFLD patients and identified the key players involved in the host–microbiome interactions. In conclusion, we showed that CMA can be used to develop a pharmacological treatment strategy in NAFLD patients.     

Oxidative cleavage of DNA by homo- and heteronuclear Cu(II)-Mn(II) complexes of an oxime-type ligand

Novel homodinuclear Cu(II) (K1), heterodinuclear Cu(II)-Mn(II) (K2) and homotrinuclear Cu(II) (K3) complexes with a novel oxime-type ligand have been prepared and their nucleolytic activities on pCYTEXP were established by neutral agarose gel electrophoresis. The analyses of the cleavage products obtained electrophoretically indicate that although the examined complexes induces very similar conformational changes on supercoiled DNA by converting supercoiled form to nicked form than linear form in a sequential manner as the complex concentration or reaction period is increased, K3 is less effective than the two others. The oxime complexes were nucleolytically active at physiological pH values but the activities of K1 or K2 were diminished by increasing the pH of the reaction mixture. In contrast, K3 makes dominantly single strand nicking by producing nicked circles on DNA at almost all the applied pH values. Metal complex induced DNA cleavage was also tested for inhibition by various radical scavengers as superoxide dismutase (SOD), azide, thiourea and potassium iodide. The antioxidants inhibited the nucleolytic acitivities of the oxime complexes but SOD afforded no protection indicating that the nucleolytic mechanism involves of copper and/or manganese complex-mediated reactive oxygen species such as hydroxyl radicals being responsible for the oxidative DNA cleavage.     

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host^1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors^4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process⁶. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes^7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance^9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.Download video file.^{(52M, mov)}     

The Correlation Between Smoking Status of Family Members and Concentrations of Toxic Trace Elements in the Hair of Children

Hair analysis is a promising tool for routine clinical screening and diagnosis of heavy metal exposure and essential trace element status in the human body. Systemic intoxications have been identified by anomalously high values of toxins in hair samples. The aim of the present study is to evaluate the relationship between smoking habit of the family members and the levels of toxic and nontoxic trace elements in hair samples of children. The randomized cross-sectional controlled study comprised of 95 children (41 girls and 54 boys) between the ages of 1 and 6 years. After written informed consent was obtained, a face-to-face interview was conducted with the families about educational background, total income of the family, and smoking habits of family members. The mineral elements considered in this study were Zn, Se, B, V, Co, Mo, Mn, iron (Fe), Be, aluminum (Al), As, cadmium (Cd), lead (Pb), Hg, chromium (Cr), Ag, Be, nickel (Ni), copper (Cu), Sn, and antimony (Sb). Hair mineral contents were measured by inductively coupled plasma-mass spectrometry. The results showed that the levels of Cd, Pb, Cr, Sb, Fe, and Al in hair samples of children whose parents smoked were significantly higher than those whose parents were nonsmokers. The number of smokers and the frequency of smoking at home were positively correlated with Pb, Cd, Cu, Ni, and Sb levels found. At the same time, it was found that there was no correlation between toxic element concentrations and family income or educational background excluding the levels of Cd. A correlation was observed between the smoking status of family members and levels of toxic trace elements in hair where this correlation was more significant with the levels of Pb and Cd. High socioeconomic status and the level of education of family members did not have any effect on toxic trace levels in hair samples of children.     

Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.     

Prostaglandin-mediated effects in early canine corpus luteum: In vivo effects on vascular and immune factors

Prostaglandins (PGs) are important regulators of the early corpus luteum (CL) in the dog. Whereas, initially, CL is gonadotropin independent, in the second half of its lifespan, hypophyseal support is required. The transition period is marked by decreased availability of PGs, in particular of PGE2. We previously reported that inhibition of COX2/PTGS2 in vivo suppressed luteal production of PGE2, lowered circulating progesterone and negatively affected luteal development. Therefore, bitches were treated with a COX2-specific blocker, firocoxib, for 5, 10, 20 and 30 days after ovulation, leading to suppression of the steroidogenic machinery. Control groups received a placebo for the same periods. Considering the wide range of possible modulatory roles of PGs shown in different organ systems, this follow-up project aimed to understand further possible PG-mediated effects in early canine CL. Thirty-four (34) factors related predominantly to vascularization and immune response were screened (mRNAs and proteins) on samples from the above described in vivo study. Most of the effects were observed during the transitional period (days 20 and 30). The inhibition of COX2 diminished the expression of angiopoietin family members ANGPT1, -2, Tie1 and -2 receptors. The expression of endothelin (ET)-1 was increased. Concerning the immune system, increased expression of the pro-inflammatory cytokines, IL1β, IL6 and IL12a, and elevated expression levels of CD4, was observed. Cumulatively, besides its involvement in regulating steroidogenesis, our results indicate a broader role of PGs in the canine CL, including modulation of angiogenesis, vascular stabilization and local immunomodulation. Possible cross-species translational effects are strongly implied.