Aberrant E-cadherin staining patterns in invasive mammary carcinoma

Background

Advances in our understanding of the molecular biology of colorectal cancer have fuelled the search for novel molecular prognostic markers to complement existing staging systems. Markers assessed in combination may perform better than those considered individually. Using high-throughput tissue microarray technology, we describe the prognostic value of combined p53 / Bcl-2 status in colorectal cancer.

Patients and methods

Tumour samples from 462 patients who underwent elective surgery to resect a primary colorectal cancer between 1994 and 2000 (mean follow-up of 75 months) were assembled in tissue microarray format. Clinico-pathological data including tumour grade, stage, vascular invasion status along with disease specific survival data has been collected prospectively. Immunohistochemical analysis of p53 and Bcl-2 expression was performed using antibodies DO-7 (p53) and 124 (Bcl-2), and results correlated with known clinico-pathological variables and outcomes.

Results

Abnormal nuclear p53 accumulation and Bcl-2 overexpression were detected in 221/445 (49.6%) and199/437 (45.5%) tumours respectively, with a significant inverse correlation between the two markers (p = 0.023). On univariate analysis no correlations were found between either marker and standard clinico-pathological variables, however nuclear p53 expression was associated with a significantly reduced survival (p = 0.024). Combined analysis of the two markers indicated that 112/432 (24.2%) cases displayed a p53(-)/Bcl-2(+) phenotype, this occurring more frequently in earlier stage tumours. Kaplan-Meier analysis revealed a significant survival advantage in these p53(-)/Bcl-2(+) tumours compared with the remaining cases (p = 0.0032). On multivariate analysis using the Cox proportional hazards model, neither p53 expression nor Bcl-2 expression alone were of independent prognostic significance, however the combined p53(-)/Bcl-2(+) phenotype was significantly associated with a good prognosis in this series (HR 0.659, 95%CI 0.452–0.959, p = 0.029).

Conclusion

Patient stratification by combined p53 / Bcl-2 phenotype provides stage-independent prognostic information in colorectal cancer. Specifically, that up to a quarter of patients display a good prognosis p53(-)/Bcl-2(+) phenotype. This may indicate a more clinically indolent phenotype and a subset of patients for whom less aggressive adjuvant treatment appropriate.     

Polarity and motility of large polymer-actin complexes

The polarity of polymer-actin complexes obtained by mixing F-actin with synthetic polymers carrying positive charges such as poly(L-lysine), x,y-ionene bromide polymers, and poly(N-[3-(dimethylamino)propyl]acrylamide) (PDMAPAA-Q) have been investigated. Actin complexes formed with poly(L-lysine) and PDMAPAA-Q, which carry charges on their side chains, show a higher polarity than those formed with x,y-ionene bromide polymers, which have charges on their chain backbones. All these polymer-actin complex gels show motility on the surfaces coated with myosin by coupling to adenosine 5'-triphosphate hydrolysis. A linear correlation between the polarity of polymer-actin complex gels and the motility is observed.     

Structure-activity relationship of 5'-substituted fluoro-neplanocin a analogues as potent inhibitors of S-adenosylhomocysteine hydrolase

Four 5'-substituted fluoro-neplanocin A analogues la-d were designed and synthesized, and the inhibitory activity against SAH was in the following order: NH2 > SH > F, N3, indicating a hydrogen bonding donor is essential for inhibitory activity.     

Hemifusion in SNARE-mediated membrane fusion

SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wild-type SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate.     

Variations in the behaviors to novel objects among five inbred strains of mice

Novelty stimuli cause various behavioral responses, such as exploration and avoidance, and contextual variables may contribute to the behavioral outputs. Here, we tried to compare the behavioral responses to novel objects of five inbred strains of mice (C57BL/6J, 129/svJae, C3H/HeJ, BALB/cJ and DBA/2J) by using a modified novel object test where a small light-weight cube wrapped with paper was presented to mice in a home cage without beddings. In response to these objects, the C57BL/6J, 129/svJae and C3H/HeJ mice showed mild exploratory behaviors, such as approaching, sniffing or brief contact. In striking contrast, the BALB/cJ and DBA/2J mice, which have been known to show high avoidance/low exploration in other behavioral paradigms, exhibited play-like secondary reactions toward the objects after initial primary exploratory behaviors. Specifically, DBA/2J mice would move the object around in the cage, holding it with their mouths, and BALB/cJ mice would gnaw the object, eventually stripping off the wrapping paper. Such behaviors decreased when similar objects were presented repeatedly. The present results suggest that active manipulations of novel objects may be a relevant parameter for measuring novelty-induced behaviors in mice and appear to be strongly influenced by genetic factors.     

Eutectic formation analysis of amino acid mixtures using molecular dynamics simulations

The mechanism of eutectic formation was investigated via computer-aided molecular dynamics techniques based on experimental results. The CBZ group mixtures CBZ-l-Asp/d-AlaNH2 x HCl/methanol, CBZ-l-Asp/l-PheOMe x HCl/methanol, and CBZ-l-Tyr/l-ArgNH2 x 2HCl/methanol formed transparent eutectic melts. The non-CBZ group mixtures l-Asp/d-AlaNH2 x HCl/methanol, l-Asp/l-PheOMe x HCl/methanol, and l-Tyr/l-ArgNH2 x 2HCl/methanol did not form eutectic melts. According to molecular dynamics simulation results, increase in the kinetic energy values of eutectic forming mixtures was much larger than the increase in potential energy over a temperature shift from 298 to 333 K. However, the results for non-eutectic forming mixtures were reversed. The Coulomb interaction energies of eutectic forming mixtures significantly decreased, because eutectic melting can increase the mobility of molecules in the mixtures. The enhancement of molecular mobility was confirmed by increased self-diffusion constant values, and the change of solid-to-liquid phase was detected by radial distribution function results. The periodic boundary conditions for calculation of molecular dynamics were found to be reliable.     

Construction and evaluation of drug-metabolizing cell line for bioartificial liver support system

Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.     

Typical three-domain cry proteins of Bacillus thuringiensis strain A1462 exhibit cytocidal activity on limited human cancer cells

Bacillus thuringiensis strain A1462 produced two parasporal inclusion proteins with a molecular mass of 88 kDa that were converted to 64-kDa toxins when activated by proteinase K digestion. Both toxins exhibited strong cytocidal activity against two human cancer cell lines, HL60 (myeloid leukemia cells) and HepG2 (liver cancer cells), while low or no toxicities were observed against 11 human and three mammalian cell lines, including four non-cancer cell lines. The cytotoxicity of both toxins on susceptible cells was characterized by rapid cell swelling. Gene cloning experiments provided two novel genes encoding 88-kDa Cry proteins, Cry41Aa and Cry41Ab. The amino acid sequences of the two proteins contain five block regions commonly conserved in B. thuringiensis insecticidal Cry proteins. This is the first report of the occurrence of typical three-domain Cry proteins with cytocidal activity preferential for cancer cells.     

Expression cloning of a variety of novel protein kinases in Lotus japonicus

To investigate protein kinases expressed in Lotus japonicus, a cDNA expression library of the root-nodule of L. japonicus was immunologically screened with monoclonal antibodies directed to a highly conserved region in protein serine/threonine kinases (Ser/Thr kinases). Among 178 positive clones obtained from the lambdaZAPII cDNA library, 164 clones were found to encode novel proteins possessing the subdomain VIB sequences characteristic of Ser/Thr kinases. By phylogenetic analysis on the basis of deduced amino acid sequences, the isolated clones could be classified into five different families of Ser/Thr kinases : the SnRK family, GSK-3 family, Ndr kinase family, Ark family, and receptor kinase family. These results suggest that this expression cloning using the kinase-specific antibodies will provide new clues for investigations of a wide variety of known and novel protein kinases in higher plants.