Loss-of-function mutations in the EGF-CFC gene CFC1 are associated with human left-right laterality defects.

All vertebrates display a characteristic asymmetry of internal organs with the cardiac apex, stomach and spleen towards the left, and the liver and gall bladder on the right. Left-right (L-R) axis abnormalities or laterality defects are common in humans (1 in 8,500 live births). Several genes (such as Nodal, Ebaf and Pitx2) have been implicated in L-R organ positioning in model organisms. In humans, relatively few genes have been associated with a small percentage of human situs defects. These include ZIC3 (ref. 5), LEFTB (formerly LEFTY2; ref. 6) and ACVR2B (encoding activin receptor IIB; ref. 7). The EGF-CFC genes, mouse Cfc1 (encoding the Cryptic protein; ref. 9) and zebrafish one-eyed pinhead (oep; refs 10, 11) are essential for the establishment of the L-R axis. EGF-CFC proteins act as co-factors for Nodal-related signals, which have also been implicated in L-R axis development. Here we identify loss-of-function mutations in human CFC1 (encoding the CRYPTIC protein) in patients with heterotaxic phenotypes (randomized organ positioning). The mutant proteins have aberrant cellular localization in transfected cells and are functionally defective in a zebrafish oep-mutant rescue assay. Our findings indicate that the essential role of EGF-CFC genes and Nodal signalling in left-right axis formation is conserved from fish to humans. Moreover, our results support a role for environmental and/or genetic modifiers in determining the ultimate phenotype in humans.

Sister chromatid exchanges in human lymphocytes treated in vitro with cadmium in G(o) and S phase of their cell cycles.

Sister chromatid exchanges (SCEs) were analyzed in human phytohemagglutinin-activated peripheral lymphocyte cultures exposed to varying concentrations (10(-7)-10(-3) M) of cadmium chloride in vitro at two different stages of the cell cycle, G(o) and early S phase. When cadmium chloride was administered at the G(o) phase, no increase in the SCEs were observed for the doses 10(-6) and 10(-5) M. Concentrations equal to or larger than 10(4) M cadmium chloride were lethal to human lymphocytes in our experimental conditions. A highly statistically significant increase was observed in the SCE frequency with increasing cadmium chloride concentration (10(-7)-10(-4)) when cadmium was administered at the early S phase, which was 24 h after culture initiation. The increase in SCE frequency was higher when the cultures were terminated at 54 h, compared to termination at 72 h. In order to examine the effects of cadmium administered at the S phase on SCE frequency in different individuals, 10(-5) M concentration was used and the cultures were terminated at 54 h after culture initiation. A 2- to 3-fold increase in the SCE frequency was observed in all six individuals examined. A progressive decrease in the proliferative index was also observed by increasing cadmium chloride concentration. These results demonstrate that the genotoxicity of cadmium chloride may be changed depending on the stage of the cell cycle in human lymphocytes. This may be one of the reasons of contradictory findings in the literature.

DNA single-strand breakage in rat lung, liver and kidney after single and combined treatments of nickel and cadmium.

Single-strand breaks were observed in rat lung and kidney after acute treatment of animals with CdCl2 (4 mg/kg body weight) injected intraperitoneally and NiCl2, (44.4 mg/kg body weight) injected subcutaneously. In the rat liver, no single-strand breakage was evident with those doses in single and combined metal treatments. The most susceptible tissue in rats to cadmium or nickel chloride treatment was the lung tissue. The single-strand breaks were higher in cadmium treatment than in nickel treatment in the rat lung. Also the response to cadmium treatment was obtained earlier than nickel. Rat kidney was also responsive to cadmium treatment. However, the response, although statistically significant, was much lower than the one obtained in rat lung. The combined treatment, which was done by administrating cadmium prior to nickel administration, reduced the number of single-strand breaks significantly and reversed them to control values in rat lung and kidney. This study confirms that cadmium and nickel create single-strand breaks when administered alone in the rat lung. This effect, which was seen in the single metal treatments, was reduced in the combined treatments.

Purification and characterization of two forms of soluble NADH cytochrome b5 reductases from human erythrocytes.

1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography. 2. The final preparations were purified 9070- and 4808-fold, respectively, over hemolysate. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and apparent monomer Mr of each reductase was determined to be 32,000 +/- 1300. 3. Vmax values of reductase II for the various electron acceptors, namely, 2,6-dichlorophenolindophenol, ferricyanide and cytochrome c through cytochrome b5 were found to be 1.9, 1.8 and 2 times higher than those of reductase I. 4. Some differences were noted for reductase I and reductase II fractions. Their elution profiles from a second DEAE-cellulose column were quite different and that suggested that reductase II is more acidic than reductase I. Reductase II was found to be more sensitive to heat treatment than reductase I.