Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

The essential requirement of an animal heme peroxidase protein during the wing maturation process in Drosophila

Background

Thus far, a handful of genes have been shown to be related to the wing maturation process in insects. A novel heme peroxidase enzyme known as curly suppressor (Cysu)(formerly CG5873), have been characterized in this report because it is involved in wing morphogenesis. Using bioinformatics tools we found that Cysu is remarkably conserved in the genus Drosophila (>95%) as well as in invertebrates (>70%), although its vertebrate orthologs show poor homology. Time-lapse imaging and histochemical analyses have confirmed that the defective wing phenotype of Cysu is not a result of any underlying cellular alterations; instead, its wings fail to expand in mature adults.

Results

The precise requirement of Cysu in wings was established by identifying a bona fide mutant of Cysu from the Bloomington Drosophila Stock Centre collection. Its requirement in the wing has also been shown by RNA knockdown of the gene. Subsequent transgenic rescue of the mutant wing phenotype with the wild-type gene confirmed the phenotype resulting from Cysu mutant. With appropriate GAL4 driver like engrailed-GAL4, the Cysu phenotype was compartmentalized, which raises a strong possibility that Cysu is not localized in the extracellular matrix (ECM); hence, Cysu is not engaged in bonding the dorsal and ventral cuticular layers. Finally, shortened lifespan of the Cysu mutant suggests it is functionally essential for other biological processes as well.

Conclusion

Cysu, a peroxinectin-like gene, is required during the wing maturation process in Drosophila because as a heme peroxidase, Cysu is capable of utilizing H₂O₂, which plays an essential role in post-eclosion wing morphogenesis.

     

Neuroadaptations in the Striatal Proteome of the Rat Following Prolonged Excessive Sucrose Intake

Obesity is a contemporary health problem of rapidly increasing prevalence. One possible cause of obesity is loss of control over consumption of highly palatable foodstuffs, perhaps mirroring the processes involved in drug addiction. Accordingly, the striatum may be a key neural substrate involved in both food and drug craving. We hypothesised here that prolonged exposure to 10 % sucrose solution might cause neuroadaptations in the striatum that are analogous to those previously reported following prolonged exposure to alcohol or recreational drugs. Male Wistar rats were given constant access to 10 % sucrose solution (in addition to normal lab chow and tap water) for 8 months and were compared with control rats receiving no sucrose access. Rats in the sucrose group typically drank more than 100 ml of sucrose solution per day and showed 13 % greater body weight than controls at the end of the 8 months. Striatal dopamine (DA) concentrations were decreased in the sucrose group rats relative to controls. Differential expression of 18 proteins was identified in the striatum of the sucrose group rats relative to controls. Down regulated proteins included pyridoxal phosphate phosphatase, involved in DA synthesis, and glutathione transferase, involved in free radical scavenging. Up regulated proteins included prolactin (which is under negative regulation by DA) and adipose differentiation-related protein, involved in fat synthesis. We hypothesise that DA-related neuroadaptations in the striatum caused by prolonged sucrose intake may partly drive compulsive intake and seeking of high palatability foodstuffs, in a similar way to that observed with drug and alcohol addictions.     

Bandwidth Enhancement of Planar Terahertz Metasurfaces via Overlapping of Dipolar Modes

Plasmonics - We present enhancement of operational bandwidths of planar terahertz metasurfaces by incorporating a complex unit cell that consists of a pair of concentric ring resonators. The inner...     

Significance of the 19-kDa hemolymph protein HP19 for the development of the rice moth Corcyra cephalonica: morphological and biochemical effects caused by antibody application

The hemolymph protein HP19 of the rice moth, Corcyra cephalonica, mediates the 20-hydroxyecdysone (20E)-dependent acid phosphatase (ACP) activity at a nongenomic level. Affinity-purified polyclonal antibody against HP19 (alphaHP19-IgG) was used in the present study to understand the role of HP19 during the postembryonic development of Corcyra. In the in vitro studies, HP19 action was blocked either by immuno-precipitation using alphaHP19-IgG, prior to its addition to the fat body culture or by the addition of the antibody directly to the culture, along with 20E and hemolymph containing HP19. The alphaHP19-IgG blocked the HP19-mediated 20E-dependent ACP activation. In the in vivo studies, the alphaHP19-IgG was injected into the fully developed last (final/Vth) instar larvae of Corcyra, to complex the HP19 in vivo, in order to block the action of HP19. The injection of alphaHP19-IgG resulted in defective development of larvae, which grew either into non-viable larvae or larval-pupal/pupal-adult intermediates relative to the effect of pre-immune IgG injected controls. The present study shows that HP19 plays an important role in controlling the metamorphosis of Corcyra by regulating the 20E-dependent ACP activity. Coupled with the earlier findings, the ecdysteroid hormone regulates this action at a nongenomic level.     

Child Centred Approach to Climate Change and Health Adaptation through Schools in Bangladesh: A Cluster Randomised Intervention Trial

Background

Bangladesh is one of the most vulnerable countries to climate change. People are getting educated at different levels on how to deal with potential impacts. One such educational mode was the preparation of a school manual, for high school students on climate change and health protection endorsed by the National Curriculum and Textbook Board, which is based on a 2008 World Health Organization manual. The objective of this study was to test the effectiveness of the manual in increasing the knowledge level of the school children about climate change and health adaptation.

Methods

This cluster randomized intervention trial involved 60 schools throughout Bangladesh, with 3293 secondary school students participating. School upazilas (sub-districts) were randomised into intervention and control groups, and two schools from each upazila were randomly selected. All year seven students from both groups of schools sat for a pre-test of 30 short questions of binary response. A total of 1515 students from 30 intervention schools received the intervention through classroom training based on the school manual and 1778 students of the 30 control schools did not get the manual but a leaflet on climate change and health issues. Six months later, a post-intervention test of the same questionnaire used in the pre-test was performed at both intervention and control schools. The pre and post test scores were analysed along with the demographic data by using random effects model.

Results

None of the various school level and student level variables were significantly different between the control and intervention group. However, the intervention group had a 17.42% (95% CI: 14.45 to 20.38, P = <0.001) higher score in the post-test after adjusting for pre-test score and other covariates in a multi-level linear regression model.

Conclusions

These results suggest that school-based intervention for climate change and health adaptation is effective for increasing the knowledge level of school children on this topic.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.     

Myrip uses distinct domains in the cellular activation of myosin VA and myosin VIIA in melanosome transport

Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read‐outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip–Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non‐essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte‐specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip‐specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a‐associated myosin‐activating protein that mediates cellular activation of MyoVa and MyoVIIa via non‐overlapping domains.