Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.     

The chicken beta-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain

Imprinting of the mouse insulin-like growth factor 2 (Igf2) and H19 genes is regulated by an imprinting control region (ICR). The hypomethylated maternal copy functions as a chromatin insulator through the binding of CTCF and prevents Igf2 activation in cis, while hypermethylation of the paternal copy inactivates insulator function and leads to inactivation of H19 in cis. The specificity of the ICR sequence for mediating imprinting and chromatin insulation was investigated by substituting it for two copies of the chicken beta-globin insulator element, (Ch beta GI)(2), in mice. This introduced sequence resembles the ICR in size, and in containing CTCF-binding sites and CpGs, but otherwise lacks homology. On maternal inheritance, the (Ch beta GI)(2) was hypomethylated and displayed full chromatin insulator activity. Monoallelic expression of Igf2 and H19 was retained and mice were of normal size. These results suggest that the ICR sequence, aside from CTCF-binding sites, is not uniquely specialized for chromatin insulation at the Igf2/H19 region. On paternal inheritance, the (Ch beta GI)(2) was also hypomethylated and displayed strong insulator activity--fetuses possessed very low levels of Igf2 RNA and were greatly reduced in size, being as small as Igf2-null mutants. Furthermore, the paternal H19 allele was active. These results suggest that differential ICR methylation in the female and male germ lines is not acquired through differential binding of CTCF. Rather, it is likely to be acquired through a separate or downstream process.     

Linkage of prostate cancer susceptibility loci to chromosome 1

Three prostate cancer susceptibility genes have been reported to be linked to different regions on chromosome 1: HPC1 at 1q24-25, PCAP at 1q42-43, and CAPB at 1p36. Replication studies analyzing each of these regions have yielded inconsistent results. To evaluate linkage across this chromosome systematically, we performed multipoint linkage analyses with 50 microsatellite markers spanning chromosome 1 in 159 hereditary prostate cancer families (HPC), including 79 families analyzed in the original report describing HPC1 linkage. The highest lod scores for the complete dataset of 159 families were observed at 1q24-25 at which the parametric lod score assuming heterogeneity (hlod) was 2.54 (P=0.0006) with an allele sharing lod of 2.34 (P=0.001) at marker D1S413, although only weak evidence was observed in the 80 families not previously analyzed for this region (hlod=0.44, P=0.14, and allele sharing lod=0.67, P=0.08). In the complete data set, the evidence for linkage across this region was very broad, with allele sharing lod scores greater than 0.5 extending approximately 100 cM from 1p13 to 1q32, possibly indicating the presence of multiple susceptibility genes. Elsewhere on chromosome 1, some evidence of linkage was observed at 1q42-43, with a peak allele sharing lod of 0.56 (P=0.11) and hlod of 0.24 (P=0.25) at D1S235. For analysis of the CAPB locus at 1p36, we focused on six HPC families in our collection with a history of primary brain cancer; four of these families had positive linkage results at 1p36, with a peak allele sharing lod of 0.61 (P=0.09) and hlod of 0.39 (P=0.16) at D1S407 in all six families. These results are consistent with the heterogeneous nature of hereditary prostate cancer, and the existence of multiple loci on chromosome 1 for this disease.     

Sex-specific dynamics of global chromatin changes in fetal mouse germ cells

Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia.     

Spatial analysis of mortality caused by malignant tumors, especially by digestive system tumors, using empirical Bayes estimates in Fejér County, Hungary

General practitioners in the vicinity of Lake Velencei in Fejér County, Hungary, have reported an unusual pattern of recent cancer mortality. Our aim was to clarify whether the presumed mortality cluster is epidemiologically justified; whether it is restricted to the locations in question or also appears elsewhere in the county; and if it is associated with some particular disease group. County mortality from malignancies of the digestive system, other malignancies, and all other causes for the period 1994 to 1999 was analyzed in 15- to 64-year-old men and women, using conventional standardized mortality ratios and empirical Bayes estimates. A continuous surface was interpolated from settlement level data for mortality maps. A mortality cluster from men's digestive cancers is apparent north and east of Lake Velencei and also elsewhere in the county. Differences from the country average in the frequency of males' deaths from other malignancies are fairly limited. A number of problematic areas in men's mortality from other causes are identifiable, including some of the settlements under the primary focus. Women's digestive tract cancer mortality in the area of the men's cluster near Lake Velencei is below the national average. There are almost no differences from the country level in women's deaths from other malignancies. Female mortality from all other causes shows remarkable elevations in the south of the county. Our results suggest the possible role of geographically localized exposures. Men's high mortality from digestive tract cancers is a problem affecting a considerable area of the county, necessitating further investigation. Continued search for causes is also warranted by some estimates of exceptionally high death rates in women from causes other than malignancies.     

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.