Oncogenic comparison of human papillomavirus type 58 E7 variants

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony‐forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage‐independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7‐driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow‐up strategy.     

High-throughput Assays for Superoxide and Hydrogen Peroxide: DESIGN OF A SCREENING WORKFLOW TO IDENTIFY INHIBITORS OF NADPH OXIDASES*

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.     

Sequential colonization by river periphyton analysed by microscopy and molecular fingerprinting

1. An artificial glass substratum was incubated in the River Danube for a period of 28 days in order to detect the sequential colonization of microorganisms.
2. Light and fluorescent microscopy showed that microalgae and the picoalgal fraction on the slides increased rapidly over the first 2 weeks of colonization. Diatoms were numerically the most abundant component of the periphyton and their species richness and diversity increased rapidly in the early phase of colonization whereas diversity subsequently increased moderately.
3. Evenness of the diatom community was initially high, lower in the intermediate phase and again higher later on. Succession involving early, intermediate and late colonizer species was observed. Community composition during the first 5 days of colonization was very different from later stages whereas there were only minor changes subsequently.
4. Molecular community analysis by means of terminal restriction fragment length polymorphism analysis of PCR amplified 16S rRNA and 18S rRNA genes pointed to even larger differences between the composition of samples obtained early and late in the period.
5. The number of 18S rRNA and 16S rRNA terminal restriction fragments (T-RF-s) was variable over the colonization period and the fragment patterns of both the bacterial and eukaryotic portion of the microbial community were variable, with most T-RF-s unique to a single sample, suggesting a wide diversity and dynamic properties of periphytic organisms.     

Effect of ecdysterone and juvenoid on the developmental involution of flight muscles in Acheta domestica

Morphogenesis and degeneration of the flight muscles in Acheta domestica was studied. The dorso-longitudinal flight muscles (DLMs) degenerate during the fourth day after adult ecdysis and the dorso-ventral flight muscles (DVMs) on the fifteenth day. In the presence of an intact innervation the degeneration of the DLMs can be retarded for 2 days by the injection of ecdysterone into very young adults. This retardation may also result in hypertrophy of the muscle fibres. The injection of ecdysterone, even in high doses, did not affect the flight muscle remnants. No notable changes have been found in the degeneration of DLMs by ovarectomy. Thus, the degeneration of flight muscles and the development of ovaries appear to be independent processes.The DLMs are homogeneous in fibre pattern in respect to succinic dehydrogenase, an important oxidative enzyme, and to ATPase activity, but the muscle fibres do not show any phosphorylase activity.     

Regioselective synthesis of isoxazole–mercaptobenzimidazole hybrids and their in vivo analgesic and anti-inflammatory activity studies

Regioselective synthesis of isoxazole–mercaptobenzimidazole hybrids and their efficiency in in vivo analgesic and anti-inflammatory activity was described. A comparison of structure–activity relationship for there compounds was also emphasized.     

Slow clearance of Plasmodium vivax with chloroquine amongst children younger than six months of age in the Brazilian Amazon

Plasmodium vivax is the most widespread parasite causing malaria, being especially prevalent in the Americas and Southeast Asia. Children are one of the most affected populations, especially in highly endemic areas. However, there are few studies evaluating the therapeutic response of infants with vivax malaria. This study retrospectively evaluated the parasitaemia clearance in children diagnosed with vivax malaria during the first five days of exclusive treatment with chloroquine (CQ). Infants aged less than six months old had a significantly slower parasitaemia clearance time compared to the group of infants and children between six months and 12 years old (Kaplan-Meier survival analysis; Wilcoxon test; p = 0.004). The impaired clearance of parasitaemia in younger children with vivax malaria is shown for the first time in Latin America. It is speculated that CQ pharmacokinetics in young children with vivax malaria is distinct, but this specific population may also allow the detection of CQ-resistant parasites during follow-up, due to the lack of previous immunity.     

Preparative chromatography for obtaining pure samples of rosaniline,magenta II,and new fuchsine

A simple low pressure liquid chromatographic method is reported that can separate the basic fuchsine homologues, rosaniline, magenta II and new fuchsine from an impure commercial dye. The chromatographic purity of the separated dyes is > 90%. All homologues were obtained in multi-milligram amounts per chromatographic run; precise yields depend on the composition of the starting material and potentially may be greater. This is a useful preparative procedure for generating chromatographically pure samples of basic fuchsine homologues, especially those that cannot be obtained in pure form by direct synthesis.     

Adding a Lysine Mimic in the Design of Potent Inhibitors of Histone Lysine Methyltransferases

Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.     

Structural and thermodynamic insights into the binding mode of five novel inhibitors of lumazine synthase from Mycobacterium tuberculosis

Recently published genomic investigations of the human pathogen Mycobacterium tuberculosis have revealed that genes coding the proteins involved in riboflavin biosynthesis are essential for the growth of the organism. Because the enzymes involved in cofactor biosynthesis pathways are not present in humans, they appear to be promising candidates for the development of therapeutic drugs. The substituted purinetrione compounds have demonstrated high affinity and specificity to lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis in bacteria and plants. The structure of M. tuberculosis lumazine synthase in complex with five different inhibitor compounds is presented, together with studies of the binding reactions by isothermal titration calorimetry. The inhibitors showed the association constants in the micromolar range. The analysis of the structures demonstrated the specific features of the binding of different inhibitors. The comparison of the structures and binding modes of five different inhibitors allows us to propose the ribitylpurinetrione compounds with C4-C5 alkylphosphate chains as most promising leads for further development of therapeutic drugs against M. tuberculosis.     

Design, synthesis, and bioactivity of simplified paclitaxel analogs based on the T-Taxol bioactive conformation

A strategy for the design and synthesis of simplified paclitaxel analogs based on the T-Taxol conformation is presented. The resulting compounds have both cytotoxic and tubulin polymerization activities, although less so than those of paclitaxel itself.