Helminths of brown bears (Ursus arctos) in the Kola Peninsula.

We present data on the species composition of helminths in brown bears (Ursus arctos) from the Murmansk Region, Russia. The absence of any information about helminths of brown bear in the region necessitated the conduct of these studies. Samples were collected in 2014 and 2015 in the southern part of the Kola Peninsula from the White Sea coastal habitats. Annually, in the study area, 1-3 bears are legally hunted and biological samples for examination are very difficult to obtain. Therefore, we used fecal samples. We studied 93 feces and identified parasite eggs identified in 43 of them by morphometric criteria. The surveys revealed eggs of the following helminths: Dicrocoelium sp., Diphyllobothrium sp., Anoplocephalidae, Capillariidae, Baylisascaris sp., Strongylida 1, and Strongylida 2. These results represent the first reconnaissance stage, which allowed characterizing the taxonomic diversity and prevalence of parasites of brown bears of the Kola Peninsula.

A forensic DNA profiling system for Northern European brown bears (Ursus arctos).

A set of 13 dinucleotide STR loci (G1A, G10B, G1D, G10L, MU05, MU09, MU10, MU15, MU23, MU26, MU50, MU51, MU59) were selected as candidate markers for a DNA forensic profiling system for Northern European brown bear (Ursus arctos). We present results from validation of the markers with respect to their sensitivity, species specificity and performance (precision, heterozygote balance and stutter ratios). All STRs were amplified with 0.6ng template input, and there were no false bear genotypes in the cross-species amplification tests. The validation experiments showed that stutter ratios and heterozygote balance was more pronounced than in the tetranucleotide loci used in human forensics. The elevated ratios of stutter and heterozygote balance at the loci validated indicate that these dinucleotide STRs are not well suited for interpretation of individual genotypes in mixtures. Based on the results from the experimental validations we discuss the challenges related to genotyping dinucleotide STRs in single source samples. Sequence studies of common alleles showed that, in general, the size variation of alleles corresponded with the variation in number of repeats. The samples characterized by sequence analysis may serve as standard DNA samples for inter laboratory calibration. A total of 479 individuals from eight Northern European brown bear populations were analyzed in the 13 candidate STRs. Locus MU26 was excluded as a putative forensic marker after revealing large deviations from expected heterozygosity likely to be caused by null-alleles at this locus. The remaining STRs did not reveal significant deviations from Hardy-Weinberg equilibrium expectations except for loci G10B and MU10 that showed significant deviations in one population each, respectively. There were 9 pairwise locus comparisons that showed significant deviation from linkage equilibrium in one or two out of the eight populations. Substantial genetic differentiation was detected in some of the pairwise population comparisons and the average estimate of population substructure (F(ST)) was 0.09. The average estimate of inbreeding (F(IS)) was 0.005. Accounting for population substructure and inbreeding the total average probability of identity in each of the eight populations was lower than 1.1×10(-9) and the total average probability of sibling identity was lower than 1.3×10(-4). The magnitude of these measurements indicates that if applying these twelve STRs in a DNA profiling system this would provide individual specific evidence.

Genetic association between COPD and polymorphisms in TNF, ADRB2 and EPHX1.

There is evidence of a hereditary component in chronic obstructive pulmonary disease (COPD). A number of genetic association studies have been performed to find susceptibility genes of COPD. The current authors performed a case-control, genetic-association study and a meta-analysis of 16 studies, involving seven polymorphisms in three well-studied genes: microsomal epoxide hydroxylase (EPHX1); tumour necrosis factor; and beta2-adrenoreceptor. A total of 492 Caucasian smokers and former smokers were recruited from hospital databases and population cohort studies. In the present study, a protective effect of the EPHX1 Tyr113His polymorphism was found (homozygous odds ratio (OR) 0.5). In the meta-analysis, homozygotes for this single nucleotide polymorphism (SNP) also had a pooled OR of 0.5. The same effect has been found in several lung cancer studies. Effects for other candidate SNPs were weak or statistically insignificant, and probable genotyping error was common. In conclusion, the present data and meta-analysis support a role for microsomal epoxide hydroxylase in the aetiology of chronic obstructive pulmonary disease.

CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells.

CXC-chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC-chemokines in the inflammatory interactions between oxidized (ox-) low-density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without 'traditional' risk factors and 15 carefully matched controls. Our main findings were: (a) ox-LDL stimulated the release of the CXC-chemokines interleukin (IL)-8, ENA-78 and GRO-alpha from PBMC, particularly in CAD. (b) In platelets, ox-LDL induced release of ENA-78 and, when combined with SFLLRN, also of GRO-alpha, with significantly higher response in CAD. (c) Platelet-rich plasma, especially when costimulated with ox-LDL, enhanced the release of IL-8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL-8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox-LDL, platelets and PBMC in CAD patients involving CXC-chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients.

Effects of interferon-alpha on gene expression of chemokines and members of the tumour necrosis factor superfamily in HIV-infected patients.

We examined the effect of interferon (IFN)-alpha on the expression of 375 genes relevant to inflammatory and immunological reactions in peripheral blood mononuclear cells (PBMC) from HIV-infected patients by cDNA expression array and real-time quantitative RT-PCR. Our main findings were: (i) IFN-alpha induced up-regulation of several genes in the tumour necrosis factor (TNF) superfamily including the ligands APRIL, FasL, TNF-alpha and TRAIL, with particularly enhancing effects on the latter in HIV-infected patients. (ii) While IFN-alpha markedly up-regulated the expression of anti-angionetic ELR- CXC-chemokines (e.g. MIG and IP-10), it suppressed the expression of angiogenic ELR+ CXC-chemokines (e.g. GRO-alpha, IL-8 and ENA-78), with similar patterns in both patients and controls. (iii) IFN-alpha induced a marked increase in gene expression of the HIV co-receptor CCR5 in both patients and controls. We suggest that these effects may contribute to both the therapeutic and toxic effects of IFN-alpha. Moreover, our findings underscore that the biological effects of IFN-alpha in HIV infection are complex and that the clinical net effects of IFN-alpha treatment may be difficult to predict. However, the potent enhancing effect of IFN-alpha on several pro-apoptotic genes in the TNF superfamily and the enhancing effect on CCR5 expression suggest a possible pathogenic role of IFN-alpha in the progression of HIV-related immunodeficiency and suggests caution in the therapeutic use of IFN-alpha in HIV-infected -individuals.

Monocyte chemoattractant protein-1 enhances and interleukin-10 suppresses the production of inflammatory cytokines in adult rat cardiomyocytes.

OBJECTIVE: Chemokines control the migration of leukocytes to inflamed tissue, and in particular monocyte chemoattractant protein (MCP)-1 has been implicated in the pathogenesis of several cardiovascular disorders such as chronic heart failure (CHF) and myocarditis. We hypothesised that MCP-1 may directly contribute to an inflammatory response in the cardiomyocytes, and in the present study we examined in adult rat cardiomyocytes: (i) the effect of tumour necrosis factor (TNF)alpha on MCP-1 production, (ii) the effect of MCP-1 on production of other inflammatory cytokines, and (iii) if the anti-inflammatory cytokine interleukin (IL)-10 could suppress any TNFalpha-induced MCP- 1 production. METHODS: We used enzyme immunoassays, RNase protection assays and slot blot analysis to measure protein and mRNA levels of various cytokines in adult rat cardiomyocyte cultures. RESULTS: (i) We found a approximately 6.4-fold increase of the MCP-1 level accompanied by an increase in MCP-1 mRNA accumulation in cardiomyocyte cultures after TNFalpha stimulation. (ii) In contrast, TNFalpha had no effect on IL-10 and only a modest effect on IL-1beta and IL-6 levels in these cells. (iii) Importantly, MCP-1 stimulated inflammatory response in cardiomyocytes by enhancing IL- 1beta and IL-6 levels in these cells as found at both the protein and mRNA level. (iv) Co-stim-ulation with IL-10 resulted in a approximately 55% reduction in TNFalpha-stimulated MCP-1 levels in cardiomyocyte culture supernatants. CONCLUSION: The present study demonstrates for the first time that MCP- 1 can directly affect cardiomyocytes, and we introduce MCP-1 as a potential enhancer and IL- 10 as a potential suppresser of inflammatory responses within the myocardium.

Enhanced gene expression of chemokines and their corresponding receptors in mononuclear blood cells in chronic heart failure--modulatory effect of intravenous immunoglobulin.

OBJECTIVES: We sought to study the gene expression of chemokines and their corresponding receptors in mononuclear blood cells (MNCs) from patients with chronic heart failure (CHF), both of which were cross-sectional and longitudinal studies during therapy with intravenous immunoglobulin (IVIg). BACKGROUND: We have recently demonstrated that IVIg improves left ventricular ejection fraction (LVEF) in patients with CHF. Based on the potential pathogenic role of chemokines in CHF, we hypothesized that the beneficial effect of IVIg may be related to a modulatory, effect on the expression of chemokines and their receptors in MNCs. METHODS: We examined: 1) the gene expression of C, CC and CXC chemokines and their receptors in MNCs from 20 patients with CHF and 10 healthy blood donors; and 2) the expression of these genes in MNCs from 20 patients with CHF randomized in a double-blind fashion to therapy with IVIg or placebo for 26 weeks. RESULTS: Our main findings in CHF were: 1) markedly raised gene expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and interleukin (IL)-8; 2) enhanced gene expression of their corresponding receptors; 3) modulation in a normal direction of this abnormal chemokine and chemokine receptor gene expression during IVIg, but not during placebo therapy; 4) down-regulation of MIP-1alpha, MIP-1beta and IL-8 during IVIg at the protein level in plasma; and 5) a correlation between down-regulation of MIP-1alpha gene expression and improved LVEF during IVIg therapy. CONCLUSIONS: Our results further support a pathogenic role for chemokines in CHF and suggest that IVIg may represent a novel therapeutic approach, with the potential to improve LVEF in patients with CHF, possibly by modulatory effects on the chemokine network.

Myocardial gene expression of leukaemia inhibitory factor, interleukin-6 and glycoprotein 130 in end-stage human heart failure.

BACKGROUND: Studies in different animal models and plasma analyses in humans suggest that members of the interleukin-6 (IL-6) cytokine family may be involved in the pathogenesis of congestive heart failure (CHF). Accordingly, we have examined IL-6-related cytokines in chronic CHF in humans by analysing gene and protein expression in myocardium derived from patients with end-stage heart failure and donor hearts. METHODS: Gene expression of cytokines/receptors of the IL-6 family was documented in myocardial samples using cDNA array hybridization and RNase protection assays. Immunohistochemistry was used to detect leukaemia inhibitory factor (LIF), IL-6 and glycoprotein 130 (gp130) in myocardial tissues. RESULTS: Myocardial gene activity was documented for the majority of IL-6 family cytokines and their receptors. Immunohistochemical analysis localized IL-6, LIF and their common receptor subunit gp130 to myocytes and vascular smooth muscle cells. LIF mRNA levels were enhanced in the left ventricles of CHF patients relative to the left ventricles of donor hearts (patients 4.6 +/- 4.7 vs. donors 0.3 +/- 0.3, P < 0.005). Myocardial IL-6 and gp130 mRNA levels were not statistically different between patients and donors, but in contrast to LIF mRNA expression in heart explants, gp130 mRNA levels were significantly higher in left atrium compared with left ventricle in both patients and donors. CONCLUSIONS: Both mRNA and proteins of gp130 and its ligands IL-6 and LIF are expressed in both nonfailing and failing human myocardium. The elevated LIF mRNA levels in left ventricles from patients with end-stage heart failure suggest a role for LIF in the pathogenesis of CHF.

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure.

OBJECTIVES: Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS: We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS: Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION: The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.

Enhanced detection of mutations in BRCA1 exon 11 using restriction endonuclease fingerprinting-single-strand conformation polymorphism.

A novel approach to mutation screening in the large exon 11 (comprising 3427 bp) of the human BRCA1 gene is presented. Restriction endonuclease fingerprinting single-strand conformation polymorphism (REF-SSCP) is based on repeated detection of DNA sequence variants in different restriction endonuclease fragments, and we evaluated the method using blood samples from 25 Norwegian patients with hereditary breast/ovarian cancer. We compared REF-SSCP to constant denaturant gel electrophoresis (CDGE) and to the protein truncation test (PTT). REF-SSCP detected 12 different DNA variants. Four of these were not detected by CDGE, and only one variant detected by CDGE was missed by REF-SSCP. PTT detected 4 of these 13 variants. REF-SSCP was subsequently applied to a second patient series (Swedish, n=20). A total of 14 different DNA variants were detected by REF-SSCP, 6 of which were truncating mutations (PTT detected only 4). Nonsense and frameshift mutations that are putative breast/ovarian cancer mutations, were detected in 7 of the 25 Norwegian and 9 of the 20 Swedish patients.

Diverse PAH transcripts in lymphocytes of PKU patients with putative nonsense (G272X, Y356X) and missense (P281L, R408Q) mutations.

The majority of mutations in the human phenylalanine hydroxylase (PAH) gene that lead to the recessive disease phenylketonuria (PKU) are believed to affect the activity or stability of the PAH enzyme. In this study we have performed in vivo analyses of lymphocyte PAH mRNA from PKU patients homozygous for the PKU missense mutations P281L and R408Q as well as the nonsense mutations G272X and Y356X. The mutations G272X, P281L and R408Q, which are located outside the consensus splice site sequence, result in transcripts with one or more exons skipped in addition to full-length transcripts. The mutation Y356X results in transcripts with one or more exons skipped, but no full-length transcripts. Our findings question the value of functional and structural predictions of mutations at the protein level without analyses of the corresponding transcript.

[Molecular genetic diagnostics. Technology for detecting mutations in DNA]. / Molekylaergenetisk diagnostikk. Teknologi for påvisning av mutasjoner i DNA.

Analyses of the genetic material in both inherited and acquired diseases such as cancer are already part of the repertoire of many laboratories. Over the last ten to fifteen years research has revealed a very complex human DNA, and shown that the molecular basis for disease is both intricate and variable. Methods for detecting the various categories of genetic changes are needed. These methods should be simple, quick and easy to perform, and cost-effective, and at the same time be both specific and sensitive. No single technology can fulfil all these criteria, and a variety of methods has therefore been introduced. In this overview we describe the different methods and their strengths and limitations.

Constant denaturant gel electrophoresis (CDGE) in BRCA1 mutation screening.

Screening for mutations in the breast and ovarian cancer susceptibility gene, BRCA1, is complicated by the wide spectrum of mutations found in this large gene. In the present study a constant denaturant gel electrophoresis (CDGE) mutation screening strategy was established for approximately 80% of the genomic coding sequence (exons 2, 11, 13-16, 20, 24). This strategy was applied to screen genomic DNA from 50 familial breast and/or ovarian cancer patients who had previously been examined for BRCA1 mutations by SSCP. A total of 14 carriers of 12 distinct disease-associated mutations and 7 carriers of 6 distinct rare substitutions leading to amino acid substitutions were identified. The SSCP failed to detect 40% of the different deletions/insertions (4/10) and 75% (6/8) of the different base substitutions leading to terminating codons or rare amino acid changes. SSCP did, however, identify one rare base substitution that could not be detected in the CDGE screening. To evaluate the CDGE mutation screening strategy further, 25 unrelated patients from Norwegian breast and/or ovarian cancer families were examined for BRCA1 mutations using a combined genomic DNA/cDNA approach covering the entire coding sequence of the gene. A total of six mutation carriers were detected, all of whom had cases of ovarian cancer in their families. Three patients from independent families carried an 1135insA mutation in exon 11, two others had a Gly484ter and an 1675delA mutation, respectively, and the sixth carried a splice mutation (5194-2 a-->c) causing deletion of exon 18. CDGE may become an efficient tool in diagnostic and population based screening for BRCA1 mutations.

Pituitary dwarfism in the R271W Pit-1 gene mutation.

UNLABELLED: The Pit-1 gene encodes the POU-domain transcription factor Pit-1 which is important for the differentiation of the anterior pituitary and regulation of the PRL, GH and TSH genes. As a member of the POU domain transcription factors, Pit-1 contains a DNA-binding region, consisting of a POU-specific domain and a POU homeodomain. Mutation of the Pit-1 gene causes hypoplasia of the pituitary gland and deficiencies of GH, PRL and TSH. In a DNA sample from a 3-month-old girl with severe growth deficiency from birth, single stranded conformational polymorphism analysis of the Pit-1 gene identified a gel shift in exon 6. DNA-sequencing disclosed a single base mutation in codon 271 (CGG to TGG) that changes arginine to tryptophan (R271W) in the POU homeodomain. The patient presented distinct facial features with prominent forehead, marked mid-facial hypoplasia with depressed nasal bridge, deep-set eyes and a short nose with anteverted nostrils. MRI examination showed a hypoplastic pituitary gland. Low serum GH did not respond to insulin-arginine provocation or GHRH tests. PRL levels below the detection limit did not increase in response to a TRH test. T4 and free T4 was below detection limit (< 20 nmol/l and < 4 pmol/l). TSH was 2.0 mU/l and showed a blunt response to 6.0 mU/l following TRH test. TBG was normal. In spite of inappropriately low TSH and very low T4, T3 was in the low normal range (1.4-1.6 nmol/l) and she was clinically euthyroid. The thyroid function tests are consistent with increased monodeiodination activity and increased conversion of T4 to T3, possibly related to the Pit-1 gene mutation. GH and T4 treatment resulted in catch-up growth continued during 5 years of therapy. CONCLUSION: Reports of nine other cases of R271W mutations of different populations as well as the present Norwegian patient suggest codon 271 of exon 6 to be a "hot spot" for Pit-1 mutations. To enable rapid and simple detection of this type of de novo mutation we have designed a specific amplification-created-restriction-site assay to check for the R271W mutation in patients suspected to have this rare form of genetic defect in growth hormone production.

Phenylketonuria and the peoples of Northern Ireland.

The comparison of regional patterns of recessive disease mutations is a new source of information for studies of population genetics. The analysis of phenylketonuria (PKU) mutations in Northern Ireland shows that most major episodes of immigration have left a record in the modern genepool. The mutation 165T can be traced to the Palaeolithic people of western Europe who, in the Mesolithic period, first colonised Ireland. R408W (on haplotype 1) in contrast, the most common Irish PKU mutation, may have been prevalent in the Neolithic farmers who settled in Ireland after 4500 BC. No mutation was identified that could represent European Celtic populations, supporting the view that the adoption of Celtic culture and language in Ireland did not involve major migration from the continent. Several less common mutations can be traced to the Norwegian Atlantic coast and were probably introduced into Ireland by Vikings. This indicates that PKU has not been brought to Norway from the British Isles, as was previously argued. The rarity in Northern Ireland of IVS12nt1, the most common mutation in Denmark and England, indicates that the English colonialization of Ireland did not alter the local genepool in a direction that could be described as Anglo-Saxon. Our results show that the culture and language of a population can be independent of its genetic heritage, and give some insight into the history of the peoples of Northern Ireland.

The Ca(2+)-sensing receptor gene (PCAR1) mutation T151M in isolated autosomal dominant hypoparathyroidism.

Isolated autosomal dominant hypoparathyroidism is a heterogeneous disorder characterized by parathyroid hormone (PTH) deficiency, hypocalcemia and hyperphosphatemia. The candidate gene approach was used to study a large Norwegian family. The loci for the PTH gene, PTH receptor gene and RET protooncogene were excluded using dinucleotide markers and restriction fragment length polymorphism analysis. Complete cosegregation of this trait was found with the chromosomal region 3q13, using the short tandem repeat markers D3S1267, D3S1269, D3S1303, D3S1518, and RHO. This region contains the candidate locus for the Ca(2+)-sensing receptor (PCAR1). By single-strand conformation polymorphism (SSCP) analysis of all PCAR1 exons followed by automated sequencing, we identified a C to T transition in exon 2 (cDNA position 452) on the mutant allele in the family. The mutation predicts a substitution of Thr to Met in amino acid position 151 (T151M). A StyI restriction site created by the nucleotide substitution was used to confirm the mutation on all alleles, as well as to exclude it among 100 normal alleles (blood donors). SSCP analysis also identified a novel polymorphism of PCAR1 intron 4 (1609-88t --> c) on normal alleles. The T151M mutation is located in the extracellular N-terminal domain of PCAR1, which belongs to the superfamily of G protein-coupled receptors. We suggest that this is a gain-of-function mutation that increases the sensitivity of the receptor to [Ca2+], thereby decreasing the calcium set point.

Phenylketonuria genotypes correlated to metabolic phenotype groups in Norway.

UNLABELLED: In order to establish a genotype-phenotype relationship, we have identified both mutant phenylalanine hydroxylase (PAH) genes in 108 phenylketonuria (PKU) patients (27 different alleles, 54 different genotypes). One major group of patients with very high pretreatment phenylalanine values ("classical" PKU) exclusively comprised homozygotes of the PKU mutations I65T, G272X, F299C, Y356X, R408W, IVS12nt1, and compound heterozygotes of various combinations of these alleles with G46S, R261Q, R252W, A259T, R158Q, D143G, R243X, E280K, or Y204C. A second major group of patients with lower phenylalanine values ("mild" PKU) comprised mutations A300S, R408Q, Y414C in various compound heterozygous states, and R261Q, R408Q, Y414C in homozygotes. The phenylalanine values in these groups were non-overlapping. In addition, a smaller group of patients formed the transition between the two main groups. In sib pairs 4 of 15 had discordant pretreatment phenylalanine values. CONCLUSION: Our results are consistent with the view that allelic heterogeneity at the PAH locus dominates the biochemical phenotype in PKU and that genotype information is able to predict the metabolic phenotype in PKU patients.

Mutations in the iduronate-2-sulfatase gene in five Norwegians with Hunter syndrome.

We have identified the mutations in the iduronate-2-sulfatase (IDS) gene of five unrelated Norwegians with Hunter syndrome by reverse transcription-polymerase chain reaction (RT-PCR) analysis of IDS mRNA followed by single strand conformation polymorphism (SSCP) analysis and cDNA sequencing. One patient had a 5-bp deletion, located at the intron 5/exon 6 junction, that created a new alternative splice site. This expanded the deletion to 9 bp in mRNA, an in-frame deletion of the first 3 codons of exon 6 of the IDS gene. In two patients point mutations were identified, the S333L mutation, which has been reported previously, and A346D (a C-->A transversion at nucleotide 1161/exon 8), which is novel. Two patients had large 3' mRNA rearrangements. The A346D mutation was associated with the mild phenotype, all others with the severe form.