Development of 6-substituted indolylquinolinones as potent Chek1 kinase inhibitors

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.     

Prevalence of high-risk human papillomavirus type 16/18 infection among women with normal cytology: risk factor analysis and implications for screening and prophylaxis

Objective: To determine the prevalence of high‐risk human papillomavirus (HR‐HPV) 16/18 infection of uterine cervix among women in the reproductive age group, with cytologically normal cervical (Pap) smears; to analyse the risk factors for HR‐HPV acquisition and to address their implications for cervical cancer screening and prophylaxis in a low resource setting. Methods: Cervical samples from 769 cytologically negative women (age 18–45 years) attending a tertiary care centre in Delhi were subjected to HPV DNA testing and HR‐HPV 16/18 and low‐risk (LR)‐HPV 6/11 sub‐typing by polymerase chain reaction. Univariate risk factor analysis was carried out in HR‐HPV positive (n = 86) versus HR‐HPV negative women (n = 683) by chi‐square test. Results: The overall HPV prevalence among cytologically normal women was 16.6%. HR‐HPV16 was detected in 10.1%, whereas HPV18 was detected in 1% of women. HR‐HPV 16/18 comprised 67% of the total HPV positives. There was no decline in HR‐HPV positivity with age, and women aged 40–44 years were at significantly increased risk for HR‐HPV prevalence (P = 0.03). Statistically significant associations of HR‐HPV infection were found with risk factors such as high parity (P = 0.04), cervicitis/hypertrophic cervix (P = 0.01), unhealthy cervix (P = 0.04), rural residence (P = 0.03), low socioeconomic status (P = 0.01) and illiteracy (P = 0.07). Conclusions: Although the sample size was small, based on the observation that HR‐HPV 16 and 18 contributed significantly to the overall HPV prevalence in our setting, we speculate that testing/prophylaxis for these prevalent high‐risk types could perhaps make cervical cancer screening and preventive programmes cost‐effective. Larger community‐based studies on HPV prevalence and persistence are required to validate these findings before definitive recommendations can be made to the policy makers.     

CD148/DEP-1 association with areas of cytoskeletal organisation in macrophages

In macrophages, tyrosine phosphorylation regulates many signalling pathways leading to growth, differentiation, activation, phagocytosis and adhesion. Protein tyrosine phosphatases (PTPs) represent a biochemical counterbalance to the activity of protein tyrosine kinases, thus regulating the dynamic phosphorylation state of a cell. CD148 is a receptor PTP that is highly expressed in macrophages and is further regulated by pro-inflammatory stimuli. CD148 is normally localised to the plasma membrane of macrophages. Following stimulation with CSF-1 or LPS, there was a re-distribution and concentration of CD148 in areas of membrane ruffling. Treatment of macrophages with anti-CD148 monoclonal antibody inhibited CSF-1-induced macrophage spreading, cytoskeletal re-arrangements and chemotaxis, without affecting cell survival. There were no detectable effects on the CSF-1 receptor-akt signalling pathway. These results are consistent with the hypothesis that CD148 is a regulator of macrophage activity.     

Extracellular expression of keratinase Ker P from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from pH 4 to 12 for 1 h with a t_1/2 of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, d-Val-Leu-Lys-pNA     

Role of Endoplasmic Reticulum Stress in α-TEA Mediated TRAIL/DR5 Death Receptor Dependent Apoptosis

Background

α-TEA (RRR-α-tocopherol ether-linked acetic acid analog), a derivative of RRR-α-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in α-TEA induced apoptosis in human breast cancer cells.

Methodology/Principal Findings

α-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2α), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) α-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) α-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP''s inhibition of caspase-8; and (iii) α-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling.

Conclusion

Taken together, ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.     

Two internal ribosome entry sites mediate the translation of p53 isoforms

The p53 tumour suppressor protein has a crucial role in cell-cycle arrest and apoptosis. Previous reports show that the p53 messenger RNA is translated to produce an amino-terminal-deleted isoform (DeltaN-p53) from an internal initiation codon, which acts as a dominant-negative inhibitor of full-length p53. Here, we show that two internal ribosome entry sites (IRESs) mediate the translation of both full-length and DeltaN-p53 isoforms. The IRES directing the translation of full-length p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of DeltaN-p53 extends into the protein-coding region. The two IRESs show distinct cell-cycle phase-dependent activity, with the IRES for full-length p53 being active at the G2-M transition and the IRES for DeltaN-p53 showing highest activity at the G1-S transition. These results indicate a novel translational control of p53 gene expression and activity.     

Expression of recombinant goldfish glutamic acid decarboxylase 65 and evidence for differential pH and PLP responsiveness compared to the human enzyme

Variability of taurine (2-aminoethane sulfonic acid) was studied as a function of size in the mussel Mytilus galloprovincialis and tissue specificity. Isometric and/or allometric relationships were established with regard to total soft mass of the mussels between 20 and 60 mm shell length. Relative amounts of taurine dropped significantly with increasing mass of whole soft tissues with an allometric coefficient value of -0.15. The inverse relationship between taurine and increasing size of mussels was confirmed for gill epithelium and labial palp (allometric coefficient values of -0.16 and -0.10, respectively), tissues that, in turn, represented isometric functions with regard to total soft mass. Although relative amounts of taurine were significantly different in digestive gland, mantle and foot, relationships with increasing size of mussels remained unchanged in these tissues. Gill area of mussels was related to soft mass with an allometric coefficient of 0.70 by 2D Image Analysis, but increased to 0.85 when introducing a third dimension, i.e. gill thickness. Results are discussed according to gill structure analysis and taurine functionality.     

Efficacy of reconstituted and stored botulinum toxin type A: an electrophysiologic and visual study in the auricular muscle of the rabbit

Once botulinum toxin type A is reconstituted, the manufacturer recommends that it be used in approximately 4 hours. As a result, a significant amount of this costly drug is often discarded because it is not completely used in the recommended period. The purpose of the present study was to compare fresh versus stored reconstituted botulinum toxin type A for (1) initial potency, (2) duration of action, and (3) bacterial colonization.Using a rabbit model, 20 New Zealand White rabbits were divided into four groups (I to IV). All rabbits had an injection of 2.5 U of reconstituted botulinum toxin into the right anterior auricular muscle. The first group was injected with botulinum toxin type A that was freshly reconstituted and served as the control. The second, third, and fourth groups were injected with botulinum toxin type A that had been reconstituted and stored for 2, 6, and 12 weeks, respectively, in a conventional freezer. Each rabbit had daily visual evaluation of the ear, with the position of auricle being graded from I to III. In addition, each rabbit had a nerve conduction study performed on the right anterior auricular muscle before injection and every 2 weeks after injection. Amplitude was chosen as the principal variable in the data analysis because it is the best predictor of physiologic changes at the muscle motor unit level. The endpoint of the study was defined as the time at which the nerve conduction studies and the visual inspections returned to baseline, preinjection levels. Botulinum toxin type A was also cultured before injection into each group.Overall, the nerve conduction data revealed a trend with a faster recovery (return to baseline) with the stored botulinum toxin. Groups IV and III returned to baseline first, followed by groups II and I. However, there was no significant difference among the groups at 2 and 4 weeks after injection, indicating that initial potency was unchanged. The differences between the groups became significant (p < 0.05) at 6 weeks and onward, suggesting that the duration was affected. Group I (fresh botulinum toxin) and group II (toxin stored for 2 weeks) had comparable outcomes and were not significantly different at any time period. Under visual inspection, the mean recovery time for each group was as follows: group IV, 5.4 weeks; group III, 7.0 weeks; group II, 6.75 weeks; and group I, 7.80 weeks. The results showed significance (p < 0.05) beginning after 3 weeks among some groups. Again, there was an overall quicker trend to return to baseline with the longer storage of the botulinum toxin (groups III and IV). These results support the authors' conduction study data, which suggest that the initial potency is not affected but the duration of action is. Again, groups I and II had comparable results. Microbiology cultures showed no growth of either aerobic or anaerobic bacteria at 7 days.In conclusion, using the rabbit model, it seems that reconstituted and stored botulinum toxin type A has the same initial potency but the duration of action is affected sometime after 2 weeks of storage. No bacterial contamination was associated with storing unpreserved reconstituted botulinum toxin type A for up to 12 weeks.     

Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromium

The cells of the immune system form a strong line of defence against foreign substances. The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro. 5 x 10(6) cells were incubated with 10 or 25 microg ml(-1) of Cr (VI) in the form of K2Cr2O7 at 37 degrees C in the presence of 5% CO2 in air. At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated. Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages. The reduction by splenic and peritoneal macrophages was similar. Total thymocytes reduced 54% of the Cr (VI). Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated. Among the intestinal cells the maximum reduction of 100% (of 10 microg ml(-1)) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells. The reduction in the intestinal loop in situ was 100%. The time taken by each cell type for the peak reduction to Cr (VI) was markedly different. The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium. The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects.     

Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.