Poult enteritis and mortality syndrome in turkeys in Great Britain.
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Animals↗
Biomedical subjects
M P Stevens
Publications and source records attributed to M P Stevens.
Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants.
BACKGROUND: Salmonella enterica serovar typhimurium (S typhimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. AIMS: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). METHODS: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. RESULTS: S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.S typhimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading S typhimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. CONCLUSIONS: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis.
Animals↗
A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene.
Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.
Amino Acid Sequence↗
Phosphorylation of the M2 protein of influenza A virus is not essential for virus viability.
M2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel. Using an exogenous incorporation assay, we have shown that incorporation of M2 into virus particles is type-specific and does not require phosphorylation of the cytoplasmic tail. In addition, phosphorylation of the cytoplasmic tail is not required for the directional transport of M2 in polarized MDCK cells. Using a reverse genetics and reassortment procedure, we generated a virus (Ra) specifically mutated in segment 7 such that the M2 cytoplasmic tail could no longer be phosphorylated. The virus was found to grow as well as wild-type virus in tissue culture and in eggs, was stable on passage in these systems, and possessed no second-site mutations in the engineered RNA segment. In vivo Ra replicated in Balb/c mice at least as well as the parent strain A/WSN/33. These studies indicate that phosphorylation of the M2 cytoplasmic tail is not required for in vitro or in vivo replication of influenza A virus.
Amino Acid Sequence↗
The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accumulation or the expression and replication of a model RNA.
The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.
Amino Acid Sequence↗
Regulation of the Escherichia coli K5 capsule gene cluster by transcription antitermination.
The expression of the Escherichia coli K5 (group II) capsular polysaccharide requires the rfaH gene. By reverse transcriptase-polymerase chain reaction (RT-PCR) it was possible to demonstrate that RfaH increases the transcription of region 2 genes by readthrough transcription from the region 3 promoter. A mutation in the rfaH gene reduced this readthrough transcription from the region 3 promoter by 10-fold as measured by quantitative RT-PCR. The region 3 promoter was mapped to 741 bp 5' of the initiation codon of the kpsM gene. Deletion and insertion mutagenesis of the JUMPstart sequence, which is 28 bp 5' of kpsM and is conserved upstream of RfaH-regulated operons and other polysaccharide biosynthesis genes, confirmed that this sequence was required for expression of the K5 antigen and for the antitermination activity of RfaH. The JUMPstart sequence could cause RfaH-dependent antitermination at other Rho-dependent terminators, suggesting that the JUMPstart sequence may function in a manner analogous to a lambda nut site. On the basis of these results we propose a model by which RfaH regulates expression of E. coli group II capsule gene clusters by allowing readthrough transcription to proceed from region 3 into region 2 and that sequences within the JUMPstart sequence are essential for this process.
Antigens, Bacterial↗
Model of extreme hypoglycemia in dogs made ketotic with (R,S)-1,3-butanediol acetoacetate esters.
The rationale behind this study is that controlled starvation of poorly differentiated (anaplastic) fast-growing tumor cells, but not host cells, might be possible in vivo. The energy metabolism of anaplastic tumor cells, but not host cells, is largely dependent on carbohydrate metabolism at all times. Therefore depleting plasma of carbohydrate fuels could place these tumor cells at a significant metabolic disadvantage. Hence an animal model was developed in which all cells would be required to oxidize fatty acids, ketoacids, and/or 1,3-butanediol to satisfy their energy needs. To achieve this aim, one would need ketosis, severe hypoglycemia, and low lactatemia. Anesthetized normal dogs were infused with somatostatin and a mixture of (R,S)-1,3-butanediol monoacetoacetate and (R,S)-1,3-butanediol diacetoacetate; these latter compounds are nonionized precursors of ketoacids. They were infused at 90% of the dog's caloric requirement. After establishment of a moderate ketosis (2-3 mM) over < 100 min, a severe degree of hypoglycemia (close to 0.5 mM) without rebound and without hyperlactatemia was induced by infusing insulin and dichloroacetate. Tracer kinetic measurements showed 1) a 20% decrease in the rate of appearance of glucose, 2) 50 and 62% increases in glycerol and nonesterified fatty acid rates of appearance, reflecting stimulation of lipolysis, and 3) no change in the rate of glutamine appearance. We suggest that this model may prove useful for selectively starving those cancer cells that are unable to utilize fat-derived fuels while preserving nutrient supply to vital organs.
3-Hydroxybutyric Acid↗
Regulation of Escherichia coli K5 capsular polysaccharide expression: evidence for involvement of RfaH in the expression of group II capsules.
Expression of the Escherichia coli K5 antigen was used as a model system to study the role of known regulators of gene expression on production of group II capsules in E. coli. Only mutations in the rfaH gene had an effect on production of the K5 antigen, abolishing the expression of any detectable capsule at 37 degrees C. None of the mutations studied induced capsule expression at 18 degrees C. A sequence, termed JUMPstart, found in group II capsule gene clusters and upstream of a number of polysaccharide biosynthesis genes in enteric bacteria is homologous to sequences found in RfaH regulated operons. This may indicate a common mode of regulation of these polysaccharide biosynthesis genes by RfaH.
Antigens, Bacterial↗
Osteoarthritis of the knee: comparison of radiography, CT, and MR imaging to assess extent and severity.
Although conventional radiography is the method most frequently used for monitoring progression of osteoarthritis, it may not show osteoarthritic changes of the knee until late in the disease, and it may show involvement of only one or two compartments in patients who have tricompartmental disease. We compared radiography, CT, and MR imaging for assessing the extent and severity of osteoarthritis of the knee in 20 patients. Radiography included posteroanterior weight-bearing, true lateral, and sunrise patellar projections. Axial CT scans were reformatted in sagittal and coronal planes. MR imaging consisted of spin-echo (600-800/20; 2000/60, 120 [TR/TE]), and gradient-echo (600/30, theta = 30 degrees) sequences. The severity of osteoarthritic changes was graded from 0 to 3. MR frequently showed tricompartmental cartilage loss when radiography and CT showed only bicompartmental involvement in the medial and patellofemoral compartments. In the lateral compartment, MR showed a higher prevalence of cartilage loss (60%) than radiography (35%) and CT (25%) did. In the medial compartment, CT and MR showed osteophytes in 100% of the knees, whereas radiography showed osteophytes in only 60%. Notably, radiography often failed to show osteophytes in the posterior medial femoral condyle. On MR images, meniscal degeneration or tears were found in all 20 knees studied. Partial and complete tears of the anterior cruciate ligament were found in three and seven patients, respectively. MR is more sensitive than radiography and CT for assessing the extent and severity of osteoarthritic changes and frequently shows tricompartmental disease in patients in whom radiography and CT show only bicompartmental involvement. MR imaging is unique for evaluating meniscal and ligamentous disease related to osteoarthritis.
Adult↗
Prepatellar calcifications from repeated kneeling.
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Calcinosis↗
Human papillomavirus genotype prevalence in cervical biopsies from women diagnosed with cervical intraepithelial neoplasia or cervical cancer in Melbourne, Australia.
Multicenter international phase III clinical trials using multivalent human papillomavirus (HPV) vaccines for cervical cancer (CC) prevention are underway. As HPV immunity is type specific, defining HPV genotype prevalence in different regions to ascertain whether predominant types differ geographically is considerably important prior to vaccine implementation. This study aimed to define HPV genotypes present in CC and high-grade dysplasia among women in Melbourne, Australia. HPV genotype analysis of a cross section of women in Melbourne with cervical dysplasia/cancer was performed. A total of 493 cervical biopsies from patients being treated for moderate (n= 122) or severe (n= 180) cervical intraepithelial neoplasia (CIN II/III) or CC (n= 191) were tested for HPV genotypes using the PGMY09/11 primer system and line blot assay. HPV detection rates were 63.9%, 72.8%, and 86.9% in CIN II, CIN III, and CC biopsies, respectively. The most prevalent HPV genotypes among CC biopsies were HPV-16 (52.9%), HPV-18 (18.3%), HPV-45 (6.3%), HPV-39 (3.1%), and HPV-73 (2.6%). Multiple HPV infections, comprising two to five types, were identified in 14.4% of biopsies, being significantly fewer (5.2%) among CC biopsies (P < 0.0001). These results indicate that the two most prevalent CC-associated HPV genotypes in Australia parallel those described internationally, with type variations thereafter.
Adult↗