Y-SNP-genotyping - a new approach in forensic analysis.

Y-chromosomal DNA polymorphisms, especially Y-STRs are well established in forensic routine case work. The STRs are used for identification in paternity deficiency cases and stain analysis with complicate mixtures of male and female DNA. In contrast, Y-chromosomal SNPs are a new tool in forensic investigations. At present, Y-SNPs are mainly used in molecular anthropology for evolutionary studies. Nevertheless, these markers could also provide very useful information for the analysis of forensic cases. The aim of the presented study was to test Y-SNP-typing for stain analyses using different methods-SNaPshot and MALDI-TOF MS. Both methods are based on the principle of minisequencing. The selected Y-SNP markers are suited to define the most important European haplogroups.

Degradation of 2-fluorophenol by the brown-rot fungus Gloeophyllum striatum: evidence for the involvement of extracellular Fenton chemistry.

Iron-containing liquid cultures of the brown-rot basidiomycete Gloeophyllum striatum degraded 2-fluorophenol. Two simultaneously appearing degradation products, 3-fluorocatechol and catechol, were identified by gas chromatography and mass spectrometry (GC-MS). Concomitantly, fluoride was produced at approximately 50% of the amount that theoretically could be achieved upon complete dehalogenation. Defluorination was strongly inhibited in the presence of either the hydroxyl radical scavenger mannitol or superoxide dismutase, as well as in the absence of iron. The addition of the natural iron chelator oxalate caused a clear but less extensive inhibition, whereas supplementation with the artificial iron chelator nitrilotriacetic acid increased fluoride production. Extracellular 2-fluorophenol degradation was evidenced by defluorination, observed upon addition of 2-fluorophenol to cell-free culture supernatants derived from iron-containing fungal cultures. Ultrafiltered culture supernatants oxidized methanol to formaldehyde, known as a product of the reaction of methanol with hydroxyl radical. In addition, G. striatum was found to produce metabolites extractable with ethyl acetate that are capable of reducing Fe3+. GC-MS analysis of such extracts revealed the presence of several compounds. The mass spectrum of a prominent peak matched those previously reported for 2,5-dimethoxyhydroquinone and 4,5-dimethoxycatechol, fungal metabolites implicated to drive hydroxyl radical production in Gloeophyllum. Taken together, these findings further support an extracellular Fenton-type mechanism operative during halophenol degradation by G. striatum.

Genosnip: SNP genotyping by MALDI-TOF MS using photocleavable oligonucleotides.

A photocleavable o-nitrobenzyl CE phosphoramidite building-block was synthesised and incorporated within oligonucleotides. After allele-specific primer extension, desalting was performed using genostrep purification plates. Release of the SNP information containing part through photocleavage created shortened molecules that are easily accessible for MALDI-TOF analysis. Additionally, incorporation of mass modified nucleosides enables flexible design of multiplex genotyping.

Hydroxylated metabolites of 2,4-dichlorophenol imply a fenton-type reaction in Gloeophyllum striatum.

While degrading 2,4-dichlorophenol, two strains of Gloeophyllum striatum, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fenton's reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-(14)C]dichlorophenol into (14)CO(2) and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative in Gloeophyllum.

Degradation of 2,4-dichlorophenol and pentachlorophenol by two brown rot fungi.

Wheat straw cultures of the brown rot fungi Gloeophyllum striatum and G. trabeum degraded 2,4-dichlorophenol and pentachorophenol. Up to 54% and 27% 14CO2, respectively, were liberated from uniformly 14C-labeled substrates within 6 weeks. Under identical conditions Trametes versicolor, a typical white rot species employed as reference, evolved up to 42% and 43% 14CO2 and expressed high activities of laccase, manganese peroxidase, and manganese-independent peroxidase. No such activity could be detected in straw or liquid cultures of Gloeophyllum. Moreover, G. striatum degraded both chlorophenols most efficiently under non-cometabolic conditions, i.e. on a defined mineral medium lacking sources of carbon, nitrogen and phosphate.

Metabolic fate of free and peptide-linked phenylalanine during infusion into portal or femoral vein of rats.

[14C]Phenylalanine, either as free amino acid or as glycyl-phenylalanine, was continuously infused over 3 h into the portal or femoral vein of anaesthetized Wistar rats. Radioactive phenylalanine and metabolites, as well as labelled proteins, were determined in plasma during infusion and at the end of the experiments in kidney, liver, brain, heart, and femoral muscle. No differences in plasma and tissue pools were observed, if the results after portal versus femoral infusion are compared. The infusion of peptide-linked phenylalanine results in a distribution of radioactivity similar to that occurring after infusion of the free amino acid. Peptidase activity toward glycyl-phenylalanine was found in plasma as well as tissues. The unhydrolyzed dipeptide could not be detected in plasma or in the tissues. These data suggest that administration of nutrients into the peripheral vein does not alter their metabolic fate.

Absorption of free and peptide-linked glycine and phenylalanine in children with active celiac disease.

Using a marker perfusing technique, the jejunal absorption rates of glycine and phenylalanine from test solutions containing either the free amino acids or glycyl-phenylalanine (30 mM each) were studied in five children with active celiac disease and in three controls. Changes in plasma amino acid levels during intestinal loading were also investigated. Mean values of intestinal uptake rates (micromoles/min/20 cm segment) in patients and controls were found to be as follows: free glycine, 19.3 +/- 21.8 and 74.5 +/- 10.3; peptide-linked glycine, 45.0 +/- 12.4 and 110.7 +/- 20,1; free phenylalanine, 33.6 +/- 19.6 and 106.1 +/- 10.6; peptide-linked phenylalanine, 44.5 +/- 14.3 and 113.6 +/- 11.1, respectively. Thus, amino acid absorption is higher from peptide-linked glycine and phenylalanine, and the advantage of individual amino acid absorption from dipeptide seems to be maintained in the disease state. Changes of amino acid levels in plasma do not reflect impaired intestinal absorption because the levels of phenylalanine increased to the same extent in patients and in controls, and the increment of glycine was found to be higher in the patients group.

[A new bile contrast in pediatrics (author's transl)]. / Vergleichende Studie über die Anwendung eines neuen Gallenkontrastmittels im Kindesalter.

Biligram was used for 42 cholegraphies in children aged 2 months to 15 years. A 35% concentration of Biligram was given as intravenous injection in 14 children, a 17% or 3,4% concentration as an intravenous infusion in 16 or 12 patients, respectively. The quality of x-ray films with both Biligram 35% and 17% was equally good whereas Biligram 3,4% gave a weak contrast only. The best time for taking films was 30 and 60 min after injection of Biligram. Allergic reactions or an effect on liver enzymes were not observed.

Sclerosis of the intestinal tract with severe malabsorption.

The clinical findings and pathological changes in a 14 year old boy who died from severe malabsorption are presented. Early signs of the illness were attacks of abdominal pain, diarrhoea, and intermittent periods of constipation. Later, severe malabsorption together with chronic ileus were apparent but no organic obstruction was found at several laparotomies. X-ray examination revealed extremly distended intestinal loops and a very slow transit time of barium. Malnutrition could not be improved by dietary, medical, or surgical therapy. Microscopic examination of the tissues obtained at autopsy showed changes in the small vessels of the lungs, heart, kidneys, and intestine. Increased fibrous tissue combined with atrophy of the muscular coat was observed in the wall of the entire intestine. Although cutaneous changes of scleroderma were not evident the deposition of collagenous material and the vascular changes seem typical of systemic sclerosis.

[Intravenous cholangio-cystography during childhood (author's transl)]. / Die intravenöse Cholangiozystographie im Kindesalter.

Twenty-five intravenous cholangio-cystograms were carried out in children aged 2 to 15 years (10 by injection, 15 by infusion). Total bilirubin, GOT, GPT, GLDH and alkaline phosphatase were determined before and after injection of the contrast medium. The contrast media used were "Biligram for infusion" (17%) and "biligram for injection" (35%). Contrast dose per kilo body weight depends on the age of the patient: a) For infusion: infants 1.6 ml/kg/KG, small children 1.2 ml/kgKG, older children 0.8 ml/kg/KG. b) for injection: infants 0.8 ml/kg/KG, small children 0.6 ml/kg/KG, older children 0.4 ml/kg/KG. Both methods, in the above doses, provided good demonstration of the biliary tree and gall bladder. Films were taken at 30 minutes, 60 minutes and 90 minutes after the end of the injection, and 40 minutes after a fatty meal. No allergic reactions were observed, nor any effect on the liver enzymes.