Biomedical subjects

Karen Stevenson

Publications and source records attributed to Karen Stevenson.

11 recordsLinked to original sources

PubMed2007

Proteomic comparison of Mycobacterium avium subspecies paratuberculosis grown in vitro and isolated from clinical cases of ovine paratuberculosis.

Paratuberculosis (Johne's disease) poses a significant economic problem to beef, dairy and sheep industries worldwide, and is caused by Mycobacterium avium subspecies paratuberculosis. In this study, 2D PAGE was used as a tool to investigate the virulent state of M. avium subsp. paratuberculosis, incorporating the technique of beating the organism with zirconium/silica beads to provide a comprehensive representation of its proteome. A direct comparison of the proteomes of M. avium subsp. paratuberculosis scraped from the terminal ileum of ovine paratuberculosis cases, and the identical strain grown in vitro, is presented. These analyses identified a set of 10 proteins whose expression is upregulated during natural infection: 1-pyrroline-5-carboxylate dehydrogenase (RocA), a putative acyl-CoA dehydrogenase (FadE14), 2-methylcitrate dehydratase (2-mcd), arginosuccinate synthase (ArgG), universal stress protein (usp), 30S ribosomal protein S2 (RpsB), peptidyl-prolyl cis-trans isomerase (PpiA), luciferase-like monooxygenase (lmo), thiosulfate sulfurtransferase (SseA) and ATP-dependent Clp protease (ClpB). Most of the proteins identified do not have obviously related functions; however, ArgG and RocA function in the same pathway, and may have a concerted action for energy production in vivo.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed2006

Functional changes in bladder tissue from type III collagen-deficient mice.

OBJECTIVE: Collagen fibers impart tensile strength and transfer tension from bladder smooth muscle cells. We have previously shown that fibrotic bladders are characterized by an increased type III:type I collagen ratio. To determine the effect of decreased type III collagen on bladder function, type III collagen-deficient mice (COL3A1) were studied physiologically. METHODS: Bladders from wild-type (+/+) and heterozygous (+/-) COL3A1 mice were biochemically characterized to determine total collagen (hydroxyproline analysis) and collagen subtype concentration (cyanogen bromide digestion and ELISA). Alterations in collagen fiber diameter were assessed by electron microscopy. Bladder muscle strips were used to assess physiologic function. RESULTS: Hydroxyproline content decreased in heterozygous bladders, which had 50% less type III collagen. Wild-type bladders had a biphasic distribution of collagen fiber sizes, whereas heterozygous bladder collagen fibers spanned a broad range. Physiologically, there were no differences in contractile responses between wild-type and heterozygotes when stimulated with ATP, carbachol or KCl, indicating normal contraction via purinergic and muscarinic receptors, and in response to direct membrane depolarization. In contrast, tension generation in heterozygotes was decreased after field stimulation (FS), indicating decreased synaptic transmission. Length-tension studies showed that the heterozygote muscle strips generated less tension per unit length, indicating that they were more compliant than wild-type controls. CONCLUSIONS: Critical levels of type III collagen appear to be a requirement for normal bladder tension development and contraction. Our data show that a decrease in the type III:type I collagen ratio, and altered fiber size, results in a more compliant bladder with altered neurotransmitter function.

Adenosine Triphosphate↗

PubMed2006

The prevalence, nature, and importance of hematologic abnormalities in heart failure.

BACKGROUND: Anemia is an adverse prognostic marker in heart failure (HF), but its cause and relationships with other comorbidities are uncertain. METHODS: All index emergency HF admissions to one urban hospital during the year 2000 were studied. RESULTS: Of 528 consecutive admissions with HF, 45% of patients (51% men and 38% women) had anemia (Hb <13 g/dL in men and <11.5 in women), which was usually normocytic. Anemia was equally common in patients with reduced and preserved systolic function. Hematinic deficiencies were rare, but 162 (70%) of anemic patients were lymphopenic. Median (interquartile range) C-reactive protein was higher in anemic patients compared with nonanemic patients (21 [5-211] mg/L vs 13 [5-155] mg/L, P = .0086). Marked reductions in glomerular filtration rate (<30 mL/min per 1.73m(2)) were more common in anemic patients (24%) than nonanemic patients (9%) (P < .0001). Median follow-up was 814 days (range 632-978 days). One hundred twenty-five (54%) anemic patients died, compared with 93 (32%) nonanemic patients (P < .00001), and anemia was a predictor of death (in patients with reduced or preserved systolic function) and death or hospital readmission. CONCLUSIONS: Renal dysfunction is one probable cause of anemia in HF. The overlap of anemia and lymphopenia suggest that hematopoiesis may be more generally depressed. Anemia is a predictor of adverse outcome in HF, irrespective of systolic function.

Aged↗

PubMed2004

A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host.

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island.

ATP-Binding Cassette Transporters↗

PubMed2003

Characterisation of IS901 integration sites in the Mycobacterium avium genome.

Data are presented on the identification and characterisation of 17 chromosomal integration loci of the insertion element IS901 in the Mycobacterium avium (cervine strain JD88/118) genome. Thirteen of these integration loci have been mapped to their corresponding positions on the M. avium strain 104 (an IS901(-) strain) genome (The Institute for Genome Research (TIGR) unfinished genome-sequencing project). Sequence data for both upstream and downstream sequence flanking regions were obtained for 12 insertion loci, while upstream sequence was obtained for five others. A consensus IS901 insertion target sequence compiled from all 17 integration sites was in broad agreement with earlier reports that were based on only two such loci. Analysis of IS901 integration site flanking sequences revealed that, like IS900 in M. avium subspecies paratuberculosis, IS901 inserts preferentially between a putative ribosome-binding sequence (RBS) and the translational start codon of an open reading frame (ORF). In BLAST X and BLAST P searches of the GenBank database, these ORFs were shown to share significant homologies with a number of other prokaryotic genes.

3' Flanking Region↗

PubMed2003

Characterization of genetic differences between Mycobacterium avium subsp. paratuberculosis type I and type II isolates.

A combination of representational difference analysis and comparative DNA sequencing revealed that four type I (sheep) isolates of Mycobacterium avium subsp. paratuberculosis were differentiated from nine type II (bovine) isolates by the presence of an 11-bp insertion in a novel M. avium subsp. paratuberculosis-specific region of genomic DNA. Further, our studies show that M. avium subsp. paratuberculosis type I isolates contain three type-specific loci that are missing in M. avium subsp. paratuberculosis type II but are present in M. avium subsp. avium. Taken together, the results are consistent with the hypothesis that M. avium subsp. paratuberculosis type I strains are an evolutionary intermediate between M. avium subsp. avium and M. avium subsp. paratuberculosis type II isolates or share a subset of M. avium subsp. avium type-specific loci through horizontal transfer.

Animals↗

PubMed2003

Do non-ruminant wildlife pose a risk of paratuberculosis to domestic livestock and vice versa in Scotland?

Paratuberculosis (Johne's disease) was long considered only a disease of ruminants. Recently non-ruminant wildlife species have been shown to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis. We review the known non-ruminant wildlife host range of M. avium subsp. paratuberculosis and consider their role in the epidemiology of paratuberculosis in domestic ruminant livestock. Mycobacterium avium subsp. paratuberculosis has been isolated from lagomorph, canid, mustelid, corvid, and murid species. In agricultural environments domestic ruminants may contact wildlife and/or their excreta when grazing or feeding on farm-stored feed contaminated with wildlife feces, opening up the possibility of inter-species transmission. Of the wildlife species known to harbor M. avium subsp. paratuberculosis in Scotland, the rabbit is likely to pose the greatest risk to grazing livestock. Paratuberculosis in domestic ruminants is a notoriously difficult disease to control; the participation of non-ruminant wildlife in the epidemiology of the disease may partially account for this difficulty.

Animal Feed↗

PubMed2002

Persistent non-specific FDG uptake on PET imaging following hip arthroplasty.

Hip arthroplasty is a common surgical procedure, but the diagnosis of infection associated with hip arthroplasty remains challenging. Fluorine-18 fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) has been shown to be a promising imaging modality in settings where infection is suspected. However, inflammatory reaction to surgery can result in increased FDG uptake at various anatomic locations, which may erroneously be interpreted as sites of infection. The purpose of this study was to assess the patterns and time course of FDG accumulation following total hip replacement over an extended period of time. Firstly, in a prospective study nine patients with total hip replacement were investigated to determine the patterns of FDG uptake over time. Three FDG-PET scans were performed in each patient at about 3, 6 and 12 months post arthroplasty. Secondly, in a retrospective analysis, the medical and surgical history and FDG-PET imaging results of 710 patients who had undergone whole-body scans for the evaluation of possible malignant disorders were reviewed. The history of arthroplasty and FDG-PET findings in the hip region were reviewed for this study. Patients with symptomatic arthroplasties or related complaints during FDG-PET scanning were excluded from the analysis. During the entire study period, all nine patients enrolled in the prospective study were demonstrated to have increased FDG uptake around the femoral head or neck portion of the prosthesis that extended to the soft tissues surrounding the femur. Among the patients reviewed in the retrospective study, 18 patients with a history of 21 hip arthroplasties who were asymptomatic at the time of FDG-PET scan met the criteria for inclusion. The time interval between the hip arthroplasty and the FDG-PET study ranged from 3 months to 288 months (mean+/-SD: 80.4+/-86.2 months). In 81% (17 of 21) of these prostheses, increased FDG uptake could be noted around the femoral head or neck portion of the prosthesis. The average time interval between arthroplasty and FDG-PET scan in these patients was 71.3 months. In only four prostheses (19%, 4 of 21) was no abnormally increased FDG uptake seen around the prostheses or adjacent sites. The average time interval in these patients was 114.8 months. It is concluded that following hip arthroplasty, non-specifically increased FDG uptake around the head or neck of the prosthesis persists for many years, even in patients without any complications. Therefore, to minimize the number of false-positive results for infection with PET studies obtained to evaluate a painful hip prosthesis, caution should be exercised when interpreting FDG uptake around the head or neck portion of the prosthesis.

Arthroplasty, Replacement, Hip↗

PubMed2002

Development of a peptide-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk.

Based on phage display technology, a peptide-mediated magnetic separation technique was developed to facilitate selective isolation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from bulk milk of naturally infected dairy herds. Nine recombinant bacteriophages binding to M. paratuberculosis were isolated from a commercial phage-peptide library encoding random 12-mer peptides. Nucleotide sequencing revealed the deduced sequence of the binding peptides. One peptide with the sequence NYVIHDVPRHPA, designated aMP3, was chemically synthesized with an amino-terminal biotin residue attached via an amino-hexacarbonic acid spacer molecule. Paramagnetic beads coated with the phage or with peptide aMP3 enabled the capture of M. paratuberculosis from milk. Combining this peptide-mediated magnetic separation with an ISMav2-based PCR allowed the detection of M. paratuberculosis in artificially spiked milk down to a concentration of 10(1) ml(-1). Experiments using milk from naturally infected cows and bulk milk samples from infected herds demonstrated that the peptide-mediated capture PCR is sufficiently sensitive to detect single strong shedders in pooled milk samples. The method, for the first time, applies phage display technology to microbial diagnostics and has potential value as a completely standardizable tool for the routine M. paratuberculosis screening of bulk milk samples at acceptable costs.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of pigmented and nonpigmented isolates of Mycobacterium avium subsp. paratuberculosis.

Five pigmented isolates of Mycobacterium avium subsp. paratuberculosis were examined by pulsed-field gel electrophoresis (PFGE), IS900 restriction fragment length polymorphism (IS900-RFLP), and IS1311 polymorphism analysis using PCR. All of the pigmented isolates exhibited one of three distinct PFGE profiles with SnaBI, designated 9, 10, and 11, and with SpeI, designated 7, 8, and 9, which generated three multiplex profiles designated [9-7], [10-8], and [11-9]. All of the pigmented isolates had the same IS900-RFLP BstEII and PvuII profiles. The IS900-RFLP BstEII profile was new, but the IS900-RFLP PvuII profile corresponded to PvuII type 6 of a sheep strain described by Cousins and colleagues (D. V. Cousins, S. N. Williams, A. Hope, and G. J. Eamens, Aust. Vet. J. 78:184-190, 2000). IS1311-PCR analysis typed all of the pigmented isolates as sheep (S) strains. The genetic relationship between pigmented and nonpigmented isolates was investigated by using multiplex PFGE data from the analysis of both the 5 pigmented isolates and 88 nonpigmented isolates of M. avium subsp. paratuberculosis from a variety of host species and geographic locations. It was possible to classify the isolates into two distinct types designated type I, comprising the pigmented isolates, and type II, comprising the nonpigmented isolates, which exhibit a very broad host range.

Animals↗

PubMed2001

Unique expression of a highly conserved mycobacterial gene in IS901(+) Mycobacterium avium.

Expression of a gene encoding a novel protein antigen of 40 kDa (p40) was detected in IS901(+) strains of Mycobacterium avium, but not in any other species or subspecies of Mycobacterium tested, including IS901(-) M. avium and the other members of the M. avium complex. Although Southern hybridization revealed that the p40 gene is widely distributed within the genus, expression of the antigen could not be detected on Western blots of mycobacterial cell lysates. Nucleotide sequence analysis of the cloned p40 gene, and a database search, revealed high levels of sequence identity with a homologous gene in IS901(-) M. avium, M. avium subsp. paratuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium smegmatis and Mycobacterium tuberculosis. Further analysis of upstream sequences identified a putative promoter region. The p40 gene is the first example of a gene that is widely distributed within the genus Mycobacterium but expressed only in association with the presence of a genomic insertion element, in this case IS901, in strains of M. avium isolated from birds and domestic livestock.

Animals↗