HLA haplotypes in primary sclerosing cholangitis patients of admixed and non-European ancestry.

Primary sclerosing cholangitis (PSC) is strongly associated with several human leukocyte antigen (HLA) haplotypes. Due to extensive linkage disequilibrium and multiple polymorphic candidate genes in the HLA complex, identifying the alleles responsible for these associations has proven difficult. We aimed to evaluate whether studying populations of admixed or non-European descent could help in defining the causative HLA alleles. When assessing haplotypes carrying HLA-DRB1*13:01 (hypothesized to specifically increase the susceptibility to chronic cholangitis), we observed that every haplotype in the Scandinavian PSC population carried HLA-DQB1*06:03. In contrast, only 65% of HLA-DRB1*13:01 haplotypes in an admixed/non-European PSC population carried this allele, suggesting that further assessments of the PSC-associated haplotype HLA-DRB1*13:01-DQA1*01:03-DQB1*06:03 in admixed or multi-ethnic populations could aid in identifying the causative allele.

Quantifying realized inbreeding in wild and captive animal populations.

Most molecular measures of inbreeding do not measure inbreeding at the scale that is most relevant for understanding inbreeding depression-namely the proportion of the genome that is identical-by-descent (IBD). The inbreeding coefficient FPed obtained from pedigrees is a valuable estimator of IBD, but pedigrees are not always available, and cannot capture inbreeding loops that reach back in time further than the pedigree. We here propose a molecular approach to quantify the realized proportion of the genome that is IBD (propIBD), and we apply this method to a wild and a captive population of zebra finches (Taeniopygia guttata). In each of 948 wild and 1057 captive individuals we analyzed available single-nucleotide polymorphism (SNP) data (260 SNPs) spread over four different genomic regions in each population. This allowed us to determine whether any of these four regions was completely homozygous within an individual, which indicates IBD with high confidence. In the highly nomadic wild population, we did not find a single case of IBD, implying that inbreeding must be extremely rare (propIBD=0-0.00094, 95% CI). In the captive population, a five-generation pedigree strongly underestimated the average amount of realized inbreeding (FPed=0.013

Pyrrolizidine alkaloids in food: downstream contamination in the food chain caused by honey and pollen.

In recent years, there has been a steadily growing number of published data on pyrrolizidine alkaloids (PAs) in honey and pollen. This raises the question whether honey and/or pollen used as ingredients in food processing might provoke a downstream contamination in the food chain. Here we addressed two different facets in connection with PAs in honey and pollen. First, we analysed the PA content of several food types such as mead (n = 19), candy (n = 10), fennel honey (n = 9), soft drinks (n = 5), power bars and cereals (n = 7), jelly babies (n = 3), baby food (n = 3), supplements (n = 3) and fruit sauce (n = 1) that contained honey as an ingredient in the range of 5% to approximately 37%. Eight out of 60 retail samples were tested as being PA-positive, corresponding to 13%. Positive samples were found in mead, candy and fennel honey, and the average PA content was calculated to be 0.10 µg g(-1) retronecine equivalents (ranging from 0.010 to 0.484 µg g(-1)). Furthermore, we investigated the question whether and how PAs from PA pollen are transferred from pollen into honey. We conducted model experiments with floral pollen of Senecio vernalis and PA free honey and tested the influence of the quantity of PA pollen, contact time and a simulated honey filtration on the final PA content of honey. It could clearly be demonstrated that the PA content of honey was directly proportional to the amount of PA pollen in honey and that the transfer of PAs from pollen to honey was a rather quick process. Consequently, PA pollen represents a major source for the observed PA content in honey. On the other hand, a good portion remains in the pollen. This fraction is not detected by the common analytical methods, but will be ingested, and it represents an unknown amount of 'hidden' PAs. In addition, the results showed that a technically and legally possible honey filtration (including the removal of all pollen) would not be an option to reduce the PA level of the final product significantly.

Pyrrolizidine alkaloids in honey: comparison of analytical methods.

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of toxicologically relevant secondary plant metabolites. Currently, two analytical methods are used to determine PA content in honey. To achieve reasonably high sensitivity and selectivity, mass spectrometry detection is demanded. One method is an HPLC-ESI-MS-MS approach, the other a sum parameter method utilising HRGC-EI-MS operated in the selected ion monitoring mode (SIM). To date, no fully validated or standardised method exists to measure the PA content in honey. To establish an LC-MS method, several hundred standard pollen analysis results of raw honey were analysed. Possible PA plants were identified and typical commercially available marker PA-N-oxides (PANOs). Three distinct honey sets were analysed with both methods. Set A consisted of pure Echium honey (61-80% Echium pollen). Echium is an attractive bee plant. It is quite common in all temperate zones worldwide and is one of the major reasons for PA contamination in honey. Although only echimidine/echimidine-N-oxide were available as reference for the LC-MS target approach, the results for both analytical techniques matched very well (n = 8; PA content ranging from 311 to 520 µg kg(-1)). The second batch (B) consisted of a set of randomly picked raw honeys, mostly originating from Eupatorium spp. (0-15%), another common PA plant, usually characterised by the occurrence of lycopsamine-type PA. Again, the results showed good consistency in terms of PA-positive samples and quantification results (n = 8; ranging from 0 to 625 µg kg(-1) retronecine equivalents). The last set (C) was obtained by consciously placing beehives in areas with a high abundance of Jacobaea vulgaris (ragwort) from the Veluwe region (the Netherlands). J. vulgaris increasingly invades countrysides in Central Europe, especially areas with reduced farming or sites with natural restorations. Honey from two seasons (2007 and 2008) was sampled. While only trace amounts of ragwort pollen were detected (0-6.3%), in some cases extremely high PA values were detected (n = 31; ranging from 0 to 13019 µg kg(-1), average = 1261 or 76 µg kg(-1) for GC-MS and LC-MS, respectively). Here the results showed significantly different quantification results. The GC-MS sum parameter showed in average higher values (on average differing by a factor 17). The main reason for the discrepancy is most likely the incomplete coverage of the J. vulgaris PA pattern. Major J. vulgaris PAs like jacobine-type PAs or erucifoline/acetylerucifoline were not available as reference compounds for the LC-MS target approach. Based on the direct comparison, both methods are considered from various perspectives and the respective individual strengths and weaknesses for each method are presented in detail.

[Glanders--a comprehensive review]. / Ein Ubersichtsreferat zur Rotzerkrankung.

Since 1990 the number of glanders outbreaks in race, military and pleasure horses in Asia and South America is steadily increasing. Glanders, which is eradicated in Western Europe, Australia and Northern America, is currently considered a re-emerging disease. Consequently, the disease may be introduced into glanders-free regions by subclinical carriers at any time. The causative agent of glanders, Burkholderia (B.) mallei, is highly contagious and leads to chronic disease in horses whereas in donkeys and mules the disease is acute and often fatal. Occurrence of the disease leads to international trading restrictions and infected animals immediately have to be culled and safely disposed off. In humans B. mallei infection results in a severe clinical course, and is fatal without appropriate therapy. Its pathogenicity makes B. mallei a potential biological agent that may be used in bioterroristic attacks. Due to the eradication of glanders in the second half of the last century, veterinarians in western European countries are no longer familiar with its clinical presentation in solipeds. Having these facts in mind, this review describes the epidemiology, clinical signs, pathology and the current eradication strategy of this interesting zoonosis. Pictures of imported endurance horses infected with glanders taken during an eradication campaign in Dubai, United Arab Emirates, in 2004 illustrate most typical clinical findings.

STR typing of human telogen hairs--a new approach.

A new approach to short tandem repeat (STR) typing of DNA extracted from telogen shed hairs is presented. Newly designed primer pairs with annealing positions close to the repeat units of the STR loci HUMFES, HUMTH01 and HUMTPOX were used for amplification. The typing results were compared to those obtained by the commonly used primer pairs by means of success rates. The primer pairs capable of producing very short amplicons (< 106 bp with HUMFES, < 86 bp with HUMTH01 and < 87 bp with HUMTPOX) described in this study significantly increased the success rates when typing telogen hairs.

Substance use among street children in Honduras.

The hypothesis that drug use among Honduran street children is a function of developmental social isolation from cultural and structural influences is examined. Data from 1,244 children working and/or living on the streets of Tegucigalpa are described, separating "market" children from "street" children. The latter group is then divided into those who sniff glue and those who do not to identify salient distinguishing factors. An OLS regression of drug usage on these variables results in a model that explains 75% of the variance, where family relations, length of time on the street, and delinquency are the most important factors.

[Short donation intervals in preoperative autologous blood donation in the concept of autologous transfusion]. / Kurze Abnahmeintervalle bei der präoperativen Eigenblutspende im Konzept der autologen Transfusion.

Homologous transfusion is associated with infectious and immunological risks. Preoperative autologous deposit reduces homologous transfusion requirements considerably. Usually donations are carried out at weekly intervals. In this study we investigated the effect of shorter donation intervals on erythropoiesis and perioperative transfusion requirements. METHODS. A total of 40 consecutive patients scheduled for hip arthroplasty and taking part in an autologous donation programme were randomly assigned to two groups: group I gave blood on days 0, 3, 7 (and 14), group II at weekly intervals. The aim was deposit of three blood units of 450 ml. A patient was deferred if hemoglobin concentration prior to donation fell below 11 g/dl, and in this case 100 mg Fe 2+ three times daily was prescribed. Blood was stored with CP-DA-1 anticoagulant. Surgery was performed between day 28 and 35. A perioperative hemoglobin concentration lower than 9 g/dl was considered a transfusion trigger. RESULTS. Group I was made up of 21 patients (10 women, 11 men, aged 39-69 years) who gave blood at short intervals, and group II of 19 patients (10 women, 9 men, aged 37-77 years) who gave blood at weekly intervals. General data, calculated blood volume and erythrocyte mass prior to donation were comparable. Each patient donated three units. Four patients had to be deferred once, one in group I, three in group II. The hemoglobin concentration in group I decreased from 13.9 +/- 1.2 g/dl (mean +/- SD) to 13.3 +/- 1.0 g/dl prior the operation, in group II from 13.5 +/- 1.3 g/dl to 12.5 +/- 1.1 g/dl. Preoperatively the hemoglobin concentrations differed significantly (P < 0.05), as did calculated erythrocyte mass (1633 versus 1474 ml, P < 0.05). Reticulocytes increased from 46 x 10(3)/microliters (median) to a maximum of 94 x 10(3)/microliters on day 7 in group I, and from 44 x 10(3)/microliters to 108 x 10(3)/microliters in group II on day 14. Serum ferritin decreased from 122 micrograms/l (median) to 82 micrograms/l in group I, and from 140 micrograms/l to 77 micrograms/l in group II. These parameters did not differ statistically between the two groups. Intra- and postoperative blood loss amounted to 2175 ml (median) in group I versus 1430 ml in group II (P < 0.05). The perioperative hemoglobin concentration was similar in the two groups. Homologous transfusion requirements were similar in the two groups (1 unit in group I, vs 3 units in one patient and 1 unit in two patients in group II). CONCLUSIONS. Short donation intervals resulted in a higher preoperative erythrocyte mass after similar preoperative deposit, and significantly higher blood loss was tolerated with similar homologous transfusion volume.

[Chronic aspecific respiratory tract diseases in workers of pig-breeding farms]. / Chronisch-unspezifische Atemwegserkrankungen bei Beschäftigten in der industriellen Schweineproduktion.

To study chronic nonspecific respiratory diseases of people working in industrial swine confinement buildings, a cross sectional study was initiated. Based on history of diseases, clinical and parameters of ventilatory screening in 238 swine producers a frequency of chronic respiratory symptoms were found in 25.6%. The possible dependence on age, sex, smoking history, working conditions and duration of activity was investigated. The influence of professional and non professional factors had different intensity. The results of investigations of working conditions did not exceed the permitted maximal working place concentrations. So they could not prove the large frequency of respiratory diseases.

Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity.

The inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [3H]dGTP in the absence of, and 1 molecule of [3H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 +/- 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 +/- 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity.

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of the exonuclease activities of DNA polymerase I by captan.

DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.

Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase.

Captan was used as an inhibitor of avian myeloblastosis virus reverse transcriptase to study the polymerase and RNase H catalytic activities. With purified enzyme, RNase H activity was 10-fold more sensitive to captan than was either the DNA-dependent or RNA-dependent DNA polymerase activity. Inhibition of the RNA-dependent polymerase activity could be prevented by dTTP. Conversely, inhibition of this polymerase activity was enhanced by template/primer. The calculated KdTTP of the uninhibited reaction was 5.6 microM. Kinetic studies allow for the proposition of a model for the interaction of captan with the polymerase active center. RNase H activity showed a sigmoidal relationship between activity and substrate concentration. Nuclease activity decreased in Vmax with no change in the Hill coefficient in the presence of captan. Addition of dithiothreitol to the incubation cocktail prevented inhibition by captan of both RNA-dependent polymerase and RNase H activities, suggesting that the (trichloromethyl)thio moiety of captan is involved in the inhibitory action. Captan inhibition suggests the presence of essential amino residues in both polymerase and RNase H active centers.

Modification of microsomal lipid peroxidation and drug metabolism by cytoplasmic copper.

NADPH-dependent microsomal lipid peroxidation (LPO) can be influenced by addition of cytoplasmic supernatant as was demonstrated by Gram and Fouts (Arch. Biochem. Biophys. 114, 331-335, 1966). Small quantities of hepatocellular supernatant stimulate LPO, higher concentrations are inhibitory. These opposed effects depend on the relative concentrations of copper and iron, either as ions or protein-bound. A mathematical model is given to predict the formation of malondialdehyde as indicator of LPO in dependence of these metals. The copper-containing superoxide dismutase was shown to be without effect on LPO, whereas coeruloplasmin was inhibitory. Inhibition of LPO by copper ions or by TRIS buffer increased the linearity of microsomal ethylmorphine demethylase.

[Epidemiology of chronic non-specific lung disease in persons employed in animal production]. / Untersuchungen zur Epidemiologie der CNSLD an Beschäftigten der Tierproduktion.

In 2,091 agricultural workers (animal production, plant production, agrochemistry) the parameters of ventilatory screening FVC and FEV 1.0 were investigated, completed by determination of PaO2 in 1,324 persons with the intention of making evident differences of pulmonary function for diagnosis between these fields of activity. The persons employed in animal production showed more obstructive disturbances of ventilation and highly significantly lower values of PaO2 than the persons in plant production. The regression-coefficient b1 for the decrease of PaO2 in dependence on the duration of activity was more than double as high in animal production than in plant production. The exposure to organic dust (of irritative, allergenic, infectious, toxic action) must be regarded as the essential etiologic factor in animal production.