Deletion of the aceE gene (encoding a component of pyruvate dehydrogenase) attenuates Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen. From a transposon insertion mutant library created previously using S. Enteritidis 10/02, one of the mutants was identified to have a 50% lethal dose (LD(50) ) at least 100 times that of the parental strain in young chicks, with an attenuation in a poorly studied gene encoding a component of pyruvate dehydrogenase, namely the aceE gene. Evaluation of the in vitro virulence characteristics of the ΔaceE∷kan mutant revealed that it was less able to invade epithelial cells, less resistant to reactive oxygen intermediate, less able to survive within a chicken macrophage cell line and had a retarded growth rate compared with the parental strain. Young chicks vaccinated with 2 × 10(9) CFU of the ΔaceE∷kan mutant were protected from the subsequent challenge of the parental strain, with the mutant colonized in the liver and spleen in a shorter time than the group infected with the parental strain. In addition, compared with the parental strain, the ΔaceE∷kan mutant did not cause persistent eggshell contamination of vaccinated hens.     

Application of long-range order to predict unfolding rates of two-state proteins

Predicting the experimental unfolding rates of two-state proteins and models describing the unfolding rates of these proteins is quite limited because of the complexity present in the unfolding mechanism and the lack of experimental unfolding data compared with folding data. In this work, 25 two-state proteins characterized by Maxwell et al. (Protein Sci 2005;14:602–616) using a consensus set of experimental conditions were taken, and the parameter long-range order (LRO) derived from their three-dimensional structures were related with their experimental unfolding rates ln(k(u)). From the total data set of 30 proteins used by Maxwell et al. (Protein Sci 2005;14:602–616), five slow-unfolding proteins with very low unfolding rates were considered to be outliers and were not included in our data set. Except all beta structural class, LRO of both the all-alpha and mixed-class proteins showed a strong inverse correlation of r = -0.99 and -0.88, respectively, with experimental ln(k(u)). LRO shows a correlation of -0.62 with experimental ln(k(u)) for all-beta proteins. For predicting the unfolding rates, a simple statistical method has been used and linear regression equations were developed for individual structural classes of proteins using LRO, and the results obtained showed a better agreement with experimental results.     

Discovery of new anti-depressants from structurally novel 5-HT3 receptor antagonists: design, synthesis and pharmacological evaluation of 3-ethoxyquinoxalin-2-carboxamides

A novel series of 3-ethoxyquinoxalin-2-carboxamides were designed as per the pharmacophoric requirements of 5-HT₃ receptor antagonist using ligand-based approach. The desired carboxamides were synthesized from the key intermediate, 3-ethoxyquinoxalin-2-carboxylic acid by coupling with appropriate amines in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt). The 5-HT₃ receptor antagonism was evaluated in longitudinal muscle myenteric plexus preparation from guinea pig ileum against 5-HT₃ agonist, 2-methy-5-HT, which was expressed in the form of pA₂ values. Compound 6h (3-ethoxyquinoxalin-2-yl)(4-methylpiperazin-1-yl)methanone was found to be the most active compound, which expressed a pA₂ value of 7.7. In forced swim test, the compounds with higher pA₂ value exhibited good anti-depressant-like activity and compounds with lower pA₂ value failed to show activity as compared to the vehicle-treated group.     

Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin αvβ₃ targeted PET tracer based on a cyclic RGD peptide

Labeling biomolecules with ¹⁸F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating ¹⁸F labeled probes based on the tetrazine-trans-cyclooctene ligation. Starting with only 30 μg (78 μM) of a tetrazine-RGD conjugate and 2 mCi (5 μM) of ¹⁸F-trans-cyclooctene, the ¹⁸F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The ¹⁸F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.     

Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-carnitine prevents carbon tetrachloride-induced oxidative stress in various tissues of Wistar rats

Acetyl-l-carnitine (ALCAR) has been shown to prevent experimental selenite cataractogenesis, a manifestation of oxidative stress, but little is known about its potential in other settings of oxidative stress. The present study was based on the hypothesis that ALCAR prevents carbon tetrachloride (CCl₄)-induced oxidative stress in vital tissues. Male albino Wistar rats were divided into three groups, each of six rats. Group I (control) rats received only vehicle (1 ml/kg b.w.) for 4 days; Group II (CCl₄-exposed, untreated) rats received CCl₄ (2 ml/kg b.w.) on the second and third days and vehicle on the first and fourth days; Group III (CCl₄-exposed, ALCAR-treated) rats received ALCAR (200 mg/kg b.w.) for 4 days and CCl₄ on the second and third days. All administrations were made intraperitoneally. After the experimental period, significantly (P < 0.05) elevated mean serum levels of aspartate transaminase, alanine transaminase, alkaline phosphatase, and lactate dehydrogenase were observed in Group II rats when compared to Group I and Group III rats. The mean levels of vitamin C, vitamin E, and reduced glutathione and the mean activities of superoxide dismutase, catalase, and glutathione peroxidase were significantly (P < 0.05) lower in samples of hemolysate and of liver, kidney, and brain tissues of Group II rats than those in Group I and Group III rats. The mean level of lipid peroxidation was significantly (P < 0.05) higher in Group II rats than that in Group I and Group III rats. Moreover, the CCl₄-induced upregulation of inducible nitric oxide synthase expression was prevented by ALCAR in the liver and brain tissues. These results suggest that ALCAR is able to prevent the CCl₄-induced oxidative stress.     

Induced mutagenesis in Jatropha curcas L. using gamma rays and detection of DNA polymorphism through RAPD marker

The aim of this study is to examine the effect of different doses (control, 5, 10, 15, 20 and 25 Kr) of gamma irradiation on seed germination, flowering, fruit and seed traits of Jatropha curcas and to identify DNA polymorphism among the mutants through a Randomly Amplified Polymorphic DNA (RAPD) marker analysis. The improved agronomic traits such as flowering, fruits and seeds were recorded in 5 Kr dose and seed germination percentage in 10 Kr dose treated plants, while corresponding parameters were reduced significantly (P>0.05) in 25 Kr dose gamma rays treated plants when compared to that of control. All the twenty-three random primers used except six primers, namely OPAW16, OPAK07, OPAK15, OPS01, OPAK20 and OPAL09 were showed polymorphic bands. The primers: OPAW16, OPAK07, OPAK15, OPS01, OPAK20 and OPAL09 produced only one band each across the six mutants, while the primers: OPU13, OPAB 15, OPF01 and OPAB11 were produced with maximum number of bands (8). The number of amplicons varied from 1 to 8 with an average of 3.9 bands, of which 2.3 were polymorphic. The percentage of polymorphism per primer ranged from 0 to 100 with an average of 55.16%. The Jaccard's coefficients of dissimilarity varied from 0.324 to 0.397, indicative of the level of genetic variation among the mutants studied. The maximum dissimilarity value (0.397) was observed in 5 Kr mutant while the minimum value (0.250) was observed in 20 Kr mutant when compared to that of control. In a dendrogram constructed based on genetic similarity coefficients, the mutants were grouped into three main clusters; (a) control, 10, 15 and 20 Kr dose mutants clustered together, (b) 25 Kr dose grouped alone, (c) 5 Kr dose also grouped alone. The mutants showing the differences in morphological traits showed DNA polymorphism in PCR profile amplified by RAPD marker. It is concluded that DNA polymorphism detected by RAPD analysis offered a useful molecular marker for the identification of mutants in gamma radiation treated plants.     

BPR1K653, a novel Aurora kinase inhibitor, exhibits potent anti-proliferative activity in MDR1 (P-gp170)-mediated multidrug-resistant cancer cells

Background

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.

Principal Findings

BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.

Conclusions and Significance

BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.     

Different population histories of the Mundari- and Mon-Khmer-speaking Austro-Asiatic tribes inferred from the mtDNA 9-bp deletion/insertion polymorphism in Indian populations

Length variation in the human mtDNA intergenic region between the cytochrome oxidase II (COII) and tRNA lysine (tRNA^lys) genes has been widely studied in world populations. Specifically, Austronesian populations of the Pacific and Austro-Asiatic populations of southeast Asia most frequently carry the 9-bp deletion in that region implying their shared common ancestry in haplogroup B. Furthermore, multiple independent origins of the 9-bp deletion at the background of other mtDNA haplogroups has been shown in populations of Africa, Europe, Australia, and India. We have analyzed 3293 Indian individuals belonging to 58 populations, representing different caste, tribal, and religious groups, for the length variation in the 9-bp motif. The 9-bp deletion (one copy) and insertion (three copies) alleles were observed in 2.51% (2.15% deletion and 0.36% insertion) of the individuals. The maximum frequency of the deletion (45.8%) was observed in the Nicobarese in association with the haplogroup B5a D-loop motif that is common throughout southeast Asia. The low polymorphism in the D-loop sequence of the Nicobarese B5a samples suggests their recent origin and a founder effect, probably involving migration from southeast Asia. Interestingly, none of the 302 (except one Munda sample, which has 9-bp insertion) from Mundari-speaking Austro-Asiatic populations from the Indian mainland showed the length polymorphism of the 9-bp motif, pointing either to their independent origin from the Mon-Khmeric-speaking Nicobarese or to an extensive admixture with neighboring Indo-European-speaking populations. Consistent with previous reports, the Indo-European and Dravidic populations of India showed low frequency of the 9-bp deletion/insertion. More than 18 independent origins of the deletion or insertion mutation could be inferred in the phylogenetic analysis of the D-loop sequences.