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.     

Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

Nutrient retention by the Kis-Balaton Water Protection System

Water quality of Lake Balaton, particularly that of its south-western basin (the Keszthely-Bay), has been deteriorating faster and faster. The greatest transporter of inorganic nutrients to the Bay is the Zala River.The lower valley of the river was part of the lake until about 200 years ago. Later, a large part of the area was turned to a wetland, while the smaller part dried up.To retain the nutrients from Keszthely-Bay, the former marshland is now being reconstructed: the so-called Kis-Balaton Water Protection System (KBWPS). The first part of the System, the Hidvégi-Pond, has been operating since the middle of 1985. The second stage, the Fenéki-Pond, to be finished by the end of this century, is now under construction. The main purpose of the KBWPS is to gain enough time to build up the technology necessary for an adequate protection of Lake Balaton.From the beginning of the operation of the Hidvégi-Pond a multidisciplinary research program has been carried out to monitor the hydrological, hydraulical, ecological, and biological processes in the reservoir. This program focused on nitrogen and phosphorus loadings and retention. Input and output loads were estimated from daily measurements. The mechanisms of retention were studied in special projects.With the exception of TN, nutrient retention increased from year to year. In 1990, 96% of PO₄-P, 87% NO₃-N, and 58% of TP were retained.This efficiency exceeds expectations. Retention of TN is the lowest (about 20%), due to N₂-fixation by blue-green algae. However, the second stage needs to be implemented, since there is an additional external load from the watershed of the lower valley, and nutrient release takes place during decomposition of the organic matter which leaves the Hidvégi-Pond.     

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.     

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.