-8p12-23 and +20q are predictors of subtypes and metastatic pathways in colorectal cancer: construction of tree models using comparative genomic hybridization data

A substantial body of evidence suggests the genetic heterogeneous pattern and multiple pathways in colorectal cancer initiation and progression. In this study, we construct a branching tree and multiple distance-based tree models to elucidate these genetic patterns and pathways in colorectal cancer by using a data set comprised of 244 cases of comparative genomic hybridization. We identify the six most common gains of chromosomal regions of 7p (37.0%), 7q11-32 (34.8%), 8q (48.3%), 13q (49.1%), 20p (36.1%), and 20q (50.4%), and the nine most common losses of 1p13-36 (30.9%), 4p15 (24.3%), 4q33-34 (24.3%), 8p12-23 (50.9%), 15q13-14 (23.5%), 15q24-25 (24.3%), 17p (34.8%), 18p (36.5%), and 18q (61.7%) in colorectal cancer. We classify colorectal cancer into two distinct groups: one preceding with -8p12-23, and the other with +20q. The sample-based classification tree also demonstrates that colorectal cancer can be classified into multiple subtypes marked by -8p12-23 and +20q. By comparing chromosomal abnormalities between primary and metastatic colorectal cancer, we identify five potential metastatic pathways: (-18q, -18p), (-8p12-23, -4p15, -4q33-34), (+20q, +20p), (+20q, +7p, +7q11-32), and +8q. -8p12-23 and +20q are inferred to be the two marker events of colorectal cancer metastasis. The current oncogenetic tree models may contribute to our understanding towards molecular genetics in colorectal cancer. Particularly, the metastatic pathways we describe may provide pivotal clues for metastatic candidate genes, and thus impact on the prediction and intervention of metastatic colorectal cancer.     

Comparison of the adhesion and proliferation characteristics of HUVEC and two endothelial cell lines (CRL 2922 and CRL 2873) on various substrata

Endothelial cell coverage of blood-contacting devices is crucial to their eventual success in the clinic. Two established human cell lines derived from HUVEC (human umbilical vascular endothelial cells), CRL 2922 and CRL 2873, have been widely utilized to study and model endothelial cell biology. However, it is not clear if these two cell lines would be useful for modeling primary endothelial cell interaction with newly-formulated biomaterials in tissue engineering applications. Hence, this study was conducted to compare the adhesion and proliferation characteristics of HUVEC grown on seven different substrata, tissue culture polystyrene (TCPS), gelatin, chitosan, poly-L-lysine, hyaluronan, poly-L-lactic acid (PLLA), and polylactic-co-glycolic acid (PLGA). The short-term adhesive behavior (2 h) of HUVEC on the various substrata was not closely-replicated by either CRL 2873 or CRL 2922. This was likely because the 2 h timeframe is too short for identification of differences in the interaction among the three cell types grown on various substrata. There was much faster proliferation of CRL 2922 on all seven substrata when compared to HUVEC and CRL 2873. Moreover, the proliferation rates of CRL 2922 on the various substrata showed little variation. In contrast, HUVEC and CRL 2873 displayed similar trends in proliferation rates, with gelatin and TCPS yielding the highest rates, and PLLA and PLGA yielding the lowest rates. Hence, CRL 2873 is better suited for modeling primary endothelial cell interaction with newly-formulated biomaterials than CRL 2922. The advantage of using CRL 2873 over HUVEC for biomaterial screening is that it is immortalized and displays much less inter-batch variability than primary culture.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

In vivo correction of complement regulatory protein deficiency with an inhibitor targeting the red blood cell membrane

Because of the complement system's involvement in many human diseases and potential complications associated with its systemic blockade, site-specific regulation of this effector system is an attractive concept. We report on further developments of such an approach using a single-chain Ab fragment as a vehicle to deliver complement regulatory proteins to a defined cell type. In a model system in which RBCs deficient in complement receptor 1-related gene/protein y (Crry) are rapidly cleared after injection into wild-type animals by a complement-dependent mechanism, we selectively reconstituted these cells with N- and C-terminally targeted recombinant forms of Crry. Transfusion of Crry-coated knockout RBCs into C57BL/6 mice extended their in vivo half-life from <5 min to approximately 2 days. Maintenance of protective levels of Crry (by a combined treatment of donor and recipient RBCs) led to nearly normal RBC survival. Uniform in vitro and in vivo coating of the RBCs and the more efficient complement inhibitory capacity of C-terminally tagged Crry were other interesting features of this experimental system. These results suggest the possibility of using the single-chain Ab fragment-mediated targeting concept of complement regulatory proteins to restrict complement inhibition to the site of its excessive activation.     

Chain conformation and bioactivity of water‐soluble polysaccharide extracted from Rhizoma Panacis Japonici

A water‐soluble α‐(1→4)‐D ‐glucan heteropolysaccharide with 37% degree of branch extracted by base from Rhizoma Panacis Japonici, coded as RPS3, was fractionated into six fractions by the method of nonsolvent addition. Their weight‐average molecular mass (M_w), polydispersity index (M_w/M_n), and radius of gyration (〈s²〉_z^1/2) were determined with laser light scattering (LLS) and size exclusion chromatography combined with LLS. The structure of the fraction was determined by methylation analyses and ¹³C NMR. The dependences of intrinsic viscosity ([η]) and 〈s²〉_z^1/2 on M_w were established as [η] = 0.71 M_w^{0.27 ± 0.01} (cm³/g) and 〈s²〉_z^1/2 = 1.53 M_w^{0.27 ± 0.02} (nm) in the M_w range from 5.62 × 10⁴ to 3.05 × 10⁶ (g/mol) for RPS3 in 0.15M NaCl aqueous solution at 25°C. On the basis of the current theory of the polymer solution, the fractal dimension (d_f), unperturbed chain dimension (A), and characteristic ratio (C_∞) were calculated to be 3.0, 1.48 Å, and 15.1, respectively. The results revealed that the RPS3 chains existed as spherical conformation in the aqueous solution. Transmission electron microscope further provided the evidence of the sphere shape of the RPS3 and its fractionated molecules in water. In vitro cytotoxicity assay indicated that the fractions could inhibit the tumor cells and showed no harm to normal cells at low dose. The bioactivity was relative with molecular mass of the samples. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 383–390, 2010. This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office atbiopolymers@wiley.com     

Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria

Mitochondrial cholesterol 27-hydroxylase (CYP27A1) plays an important role in the maintenance of intracellular cholesterol homeostasis. Cholesterol delivery to the mitochondrial inner membrane is believed to be a rate-limiting step for the "acidic" pathway of bile acid synthesis. This work reports that proteinase K treatment of mitochondria markedly increases CYP27A1 specific activity. With endogenous mitochondrial cholesterol, treatment with proteinase K increased CYP27A1 specific activity by 5-fold. Moreover, the addition of the exogenous cholesterol in beta-cyclodextrin plus proteinase K treatment increased the specific activity by 7-fold. Kinetic studies showed that the increased activity was time-, proteinase K-, and substrate concentration-dependent. Proteinase K treatment decreased the apparent K(m) of CYP27A1 for cholesterol from 400 to 150 microM. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity compared with mitochondria freshly isolated from rat liver tissue.