Genetic Analysis of Resistance to Benzimidazoles in Physarum: Differential Expression of β-Tubulin Genes

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a β-tubulin with noval electrophoretic mobility, in addition to a β-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for β-tubulin, and that at least two β-tubulin genes are expressed in myxamoebae. Comparisons of the β-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) β-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.     

In vitro interactions of opioid peptides with phospholipids. IV. Methionine enkephalin versus leucine enkephalin

The interaction of methionine and leucine enkephalin with phosphatidylserine and phosphatidylcholine was studied by optical spectroscopy techniques. The data reported indicate that with both peptides the binding is controlled by ionic parameters. They also indicate that the differences in the binding behavior of the two peptides induced by changing these parameters are minor. Non-ionic interactions are also important in the binding phenomena, but the above observations hold in this case as well. Finally, the tridimensional structure of both enkephalins appears to be modified in the presence of phospholipids. Moreover, the changes induced by these lipids appear to differ from one peptide to the other.     

Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin

The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from "Streptomyces steffisburgensis" NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in L-rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host Streptomyces albus, allowing the isolation of a biosynthetic intermediate. In addition, the expression in S. albus of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of L-rhamnose, led to the production of steffimycin. Inactivation of the stfX gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the stfG gene, coding for a putative glycosyltransferase involved in the attachment of L-rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in S. albus upon expression of the entire cluster.     

Genetic susceptibility to obesity and diet intakes: association and interaction analyses in the Malmö Diet and Cancer Study

Gene–environment interactions need to be studied to better understand the obesity. We aimed at determining whether genetic susceptibility to obesity associates with diet intake levels and whether diet intakes modify the genetic susceptibility. In 29,480 subjects of the population-based Malmö Diet and Cancer Study (MDCS), we first assessed association between 16 genome-wide association studies identified obesity-related single-nucleotide polymorphisms (SNPs) with body mass index (BMI) and associated traits. We then conducted association analyses between a genetic risk score (GRS) comprising of 13 replicated SNPs and the individual SNPs, and relative dietary intakes of fat, carbohydrates, protein, fiber and total energy intake, as well as interaction analyses on BMI and associated traits among 26,107 nondiabetic MDCS participants. GRS associated strongly with increased BMI (P = 3.6 × 10^?34), fat mass (P = 6.3 × 10^?28) and fat-free mass (P = 1.3 × 10^?24). Higher GRS associated with lower total energy intake (P = 0.001) and higher intake of fiber (P = 2.3 × 10^?4). No significant interactions were observed between GRS and the studied dietary intakes on BMI or related traits. Of the individual SNPs, after correcting for multiple comparisons, NEGR1 rs2815752 associated with diet intakes and BDNF rs4923461 showed interaction with protein intake on BMI. In conclusion, our study does not provide evidence for a major role for macronutrient-, fiber- or total energy intake levels in modifying genetic susceptibility to obesity measured as GRS. However, our data suggest that the number of risk alleles as well as some of the individual obesity loci may have a role in regulation of food and energy intake and that some individual loci may interact with diet.     

ANTICANCER MEDICINES (DOXORUBICIN AND METHOTREXATE) CONJUGATED WITH MAGNETIC NANOPARTICLES FOR TARGETING DRUG DELIVERY THROUGH IRON

The uptake of iron is increased by cancer cells. Iron magnetic nanoparticles (MNP) can be used as a nanovehicle for immobilization of anticancer medicines and to integrate them at a target site. The anticancer medicines doxorubicin (DOX) and methotrexate (MTX) were immobilized separately and in combination onto MNP by a glutaraldehyde activation method and confirmed by magnetic nanoparticles linked immunosorbent assay (MagLISA) and Fourier-transform infrared (FTIR) spectroscopy. The phenol peaks of DOX and MTX at 2896.6 cm^?1 to 2912.5 cm^?1 in FTIR spectra of immobilized medicines indicated the conjugation. Affinity-purified anti-DOX and anti-MTX antibodies were used to evaluate the coupling of DOX and MTX onto MNP, and the binding was found 34.6% to 37.2% and 51.8% to 54.3% separately, respectively. The immobilization of DOX and MTX in combination onto MNP was 18% and 27%, respectively. HeLa and B cells were cultured with DOX-MNP, MTX-MNP, and DOX-MNP-MTX separately, and MagLISA indicated that the binding of DOX-MNP/MTX-MNP was 41.5% to 45% with HeLa cells and 20% to 26% with B cells. No significant difference was observed in binding of DOX-MNP-MTX with HeLa and B cells. Results also indicated that the release of medicines at pH 5.0 is more (39% to 44%) than at pH 7.4 (3.7% to 10.2%). Sixteen to 22% more killing effect was observed on HeLa cells than on B cells. In immunohistochemical staining, more deposition of brown color on HeLa cells than on B cells may be due to more expression of iron-binding sites on cancer cells. The dual property of MNP can be used for binding of medicines and for targeting drug delivery.     

An expanded family of proteins with BPI/LBP/PLUNC-like domains in trypanosome parasites: an association with pathogenicity?

Trypanosomatids are protozoan parasites that cause human and animal disease. Trypanosoma brucei telomeric ESs (expression sites) contain genes that are critical for parasite survival in the bloodstream, including the VSG (variant surface glycoprotein) genes, used for antigenic variation, and the SRA (serum-resistance-associated) gene, which confers resistance to lysis by human serum. In addition, ESs contain ESAGs (expression-site-associated genes), whose functions, with few exceptions, have remained elusive. A bioinformatic analysis of the ESAG5 gene of T. brucei showed that it encodes a protein with two BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelium clone)-like domains and that it belongs to a multigene family termed (GR)ESAG5 (gene related to ESAG5). Members of this family are found with various copy number in different members of the Trypanosomatidae family. T. brucei has an expanded repertoire, with multiple ESAG5 copies and at least five GRESAG5 genes. In contrast, the parasites of the genus Leishmania, which are intracellular parasites, have only a single GRESAG5 gene. Although the amino acid sequence identity between the (GR)ESAG5 gene products between species is as low as 15-25%, the BPI/LBP/PLUNC-like domain organization and the length of the proteins are highly conserved, and the proteins are predicted to be membrane-anchored or secreted. Current work focuses on the elucidation of possible roles for this gene family in infection. This is likely to provide novel insights into the evolution of the BPI/LBP/PLUNC-like domains.     

A Convenient Method for the Synthesis of (Prop-2-Ynyloxy)Benzene Derivatives via Reaction with Propargyl Bromide,Their Optimization,Scope and Biological Evaluation

A highly convenient method has been developed for the synthesis of (prop-2-ynyloxy) benzene and its derivatives. Differently substituted phenol and aniline derivatives were allowed to react with propargyl bromide in the presence of K₂CO₃ base and acetone as solvent. The compounds were synthesized in good yields (53–85%). Low cost, high yields and easy availability of compounds helped in the synthesis. Electron withdrawing groups favor the formation of stable phenoxide ion thus in turn favors the formation of product while electron donating groups do not favor the reaction. Phenol derivatives gave good yields as compared to that of aniline. As aprotic polar solvents favor S_N2 type reactions so acetone provided best solvation for the reactions. K₂CO₃ was proved to be good for the synthesis. Antibacterial, Antiurease and NO scavenging activity of synthesized compounds were also examined. 4-bromo-2-chloro-1-(prop-2-ynyloxy)benzene 2a was found most active compound against urease enzyme with a percentage inhibition of 82.00±0.09 at 100 µg/mL with IC₅₀ value of 60.2. 2-bromo-4-methyl-1-(prop-2-ynyloxy)benzene 2d was found potent antibacterial against Bacillus subtillus showing excellent inhibitory action with percentage inhibition of 55.67±0.26 at 100 µg/ml wih IC₅₀ value of 79.9. Based on results, it can be concluded that some of the synthesized compounds may have potential antiurease and antibacterial effects against several harmful substances.     

Pharmacokinetics of intravenous theophylline in mutant Nagase analbuminemic rats

It was obtained from our laboratories that the expression of hepatic microsomal cytochrome P450 (CYP) 1A2 increased approximately 3.5 times in mutant Nagase analbuminemic rats (NARs, an animal model for human familial analbuminemia), and theophylline was reported to be metabolized to 1,3-dimethyluric acid (1,3-DMU) and 1-methylxanthine (which was further metabolized to 1-methyluric acid, 1-MU, via xanthine oxidase) via CYP1A2 in rats. Hence, the pharmacokinetic parameters of theophylline, 1,3-DMU and 1-MU were compared after intravenous administration of aminophylline, 5 mg/kg as theophylline, to control Sprague-Dawley rats and NARs. In NARs, the total area under the plasma concentration-time curve from time zero to time infinity (AUC) of theophylline was significantly smaller (1,040 versus 1,750 microg min/ml) than that in control rats and this could be due to significantly faster renal clearance (CL(R), 1.39 versus 0.571 ml/min/kg, due to inhibition of renal reabsorption of unchanged theophylline) and nonrenal clearance (CL(NR), 3.36 versus 2.25 ml/min/kg, due to 3.5-fold increase in CYP1A2) than those in control rats. Based on in vitro hepatic microsomal studies, the intrinsic 1,3-DMU formation clearance was significantly faster in NARs than that in control rats (267 versus 180 x 10(-6) ml/min). After intravenous administration of 1,3-DMU, the renal secretion of 1,3-DMU was inhibited in NARs. Inhibition of renal secretion or reabsorption of various compounds in NARs was also discussed.