Flow-cytometric analysis of the DNA-content in paraffin-embedded tissue from colorectal carcinomas and its prognostic significance

The nuclear DNA content of 163 colorectal carcinomas was determined by flow-cytometry (FCM) on formalin-fixed, paraffin-embedded tissue. DNA-aneuploidy was found in 97 cases (59.5%), in which no statistically significant correlations with sex, mean age, tumour stage (Dukes and pTNM) and tumour grade were noted. The frequency of aneuploidy was significantly higher in patients less than 70 years of age (p < 0.01) and in tumours localized in the left colon and rectum (p< 0.002), irrespective of their stage. The tumours in which different areas could be analysed (n = 80) showed a heterogeneous DNA-ploidy pattern in 18%. Comparison of the DNA content in primary tumours and in lymph node metastases (n = 49) showed a difference in DNA-ploidy in 38% of the DNA-aneuploid tumours, but in only 6% of the DNA-diploid carcinomas (p<0.02). DNA-aneuploid carcinomas tended to show a higher rate of local recurrence and were associated with an unfavourable prognosis (p = 0.04) in those patients in which complete resection of their tumours was possible (n = 72). The significantly higher mortality of patients with DNA-aneuploid carcinomas of stage pT3, as well as those with Dukes stage A and B tumours indicates that DNA-aneuploidy may be a stage-independent additional risk factor in colorectal cancer.     

Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O2 levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O2/1% CO2, an O2 tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O2/1% CO2/79% N2) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold (p less than 0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a approximately 1.9-fold (p less than 0.001) increase in number and a 1.2-fold (p less than 0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, X 2.5; Mn-superoxide dismutase, X 2.1; catalase, X 4.0; glutathione peroxidase, X 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity.     

Symmetric enzyme distribution in asymmetric UF polysulfone membranes

Invertase as well as as amyloglucosidase were immobilized within asymmetyric ultrafiltration membranes that were prepared from polysulfone or homogeneously modified polysulfone. The chemical modification was carried out by sulfonation and halomethylation. This additional change of the surface properties of the capillaries within the membrane offers the possibilities for various types of enzyme fixation, namely adsorption, charge interactions, or covalent bonding. By variation of the immobilization conditions the distribution of the enzyme could be adjusted over the membrane's cross section. At a distinct enzyme concentration in the loading solution a homogeneous enzyme distribution within the membrane could be verified. This was shown by diffusion experiments. Under ultrafiltration conditions using a solution that contains membrane-impermeable macromolecules as well as a membrane-permeable solute like saccharose the residence time within the membrane was increased due to gel formation atop the membrane yet the kinetic was no affected. The nonpermeable soluble starch was not reacted by the amyloglucosidase membrane, indicating that the skin layer was free of enzymes. (c) 1994 John Wiley & Sons, Inc.     

Effect of hydrogen peroxide on sugar transport inSchizosaccharomyces pombe. Absence of membrane lipid peroxidation

Stationary unaerated cells ofS. pombe containing endogenous substrates but not energized by any exogenous ones take up 2-deoxy-d-glucose, 6-deoxy-d-glucose,d-xylose andd-arabinose actively over diffusion equilibrium. The active uptake is inhibited by 20–100 mmol/L H₂O₂ which causes an increase inK _T but has no effect onJ _max. This “competitive inhibition” indicates that H₂O₂ affects directly the sugar binding sites of the transporters. The ATP-binding site of the plasma membrane H⁺-ATPase is also affected by 100 mmol/L H₂O₂; theK _T decreases 7-fold,J _max about 2.5-fold. These effects are not likely to be mediated by membrane lipid peroxidation which appears to be lacking inS. pombe, and this lack may be one of the reasons for the high resistance of this yeast to H₂O₂. Because of thisS. pombe represents a suitable system for studying direct effects of oxidants on membrane proteins.