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Biomedical subjects

C L Thomas

Publications and source records attributed to C L Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Oral or topical nasal steroids for hearing loss associated with otitis media with effusion in children.

BACKGROUND: Otitis media with effusion (OME) is common and may cause hearing loss with associated developmental delay. Treatment remains controversial. OBJECTIVES: To examine evidence for or against treating children with hearing loss associated with OME with systemic or topical intranasal steroids. SEARCH STRATEGY: We searched the Cochrane Ear, Nose and Throat Disorders Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library Issue 4 2005, MEDLINE (1966 to 2006), EMBASE (1974 to 2006), and the CINAHL, LILACS, Zetoc, IndMED, SAMED, KoreaMed, MEDCARIB and Cambridge Scientific Abstracts databases in January 2006. SELECTION CRITERIA: Randomised controlled trials of oral and topical intranasal steroids, either alone or in combination with another agent such as an antibiotic, were included. EXCLUSIONS: publications in abstract form only; uncontrolled, non-randomised or retrospective studies; studies reporting outcomes with ears (rather than children). DATA COLLECTION AND ANALYSIS: Data were extracted from the published reports by the authors independently using standardised data extraction forms and methods. The methodological quality of the included studies was independently assessed by the authors as described in the Cochrane Handbook. Dichotomous results were expressed as an odds ratio using a fixed-effect model together with the 95% confidence intervals. Continuous data were analysed using the weighted mean difference in a fixed-effect model. Tests for heterogeneity between studies were performed using a Mantel-Haenszel approach. In trials with a cross-over design, post cross-over treatment data were not used. MAIN RESULTS: No study prospectively documented hearing loss associated with OME prior to randomisation. Follow up was mainly short term. The odds ratio for OME persisting after short-term follow up in children treated with oral steroids compared to control was 0.22 (95% CI 0.08 to 0.63). The odds ratio for OME persisting after short-term follow up for children treated with oral steroids plus antibiotic compared to control plus antibiotic was 0.37 (95% CI 0.25 to 0.56). However, there was significant heterogeneity between studies included in the latter comparison (P < 0.01). Trends favoured steroids for most other comparisons, but confidence intervals included unity. There was no evidence of benefit for steroid treatment for OME or hearing loss associated with OME in the longer term. AUTHORS' CONCLUSIONS: Both oral and topical intranasal steroids alone or in combination with an antibiotic lead to a quicker resolution of OME in the short term, however, there is no evidence of longer term benefit.

Administration, Intranasal↗
PubMed2004

Plasmodesmata in Arabidopsis thaliana suspension cells.

A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.

Arabidopsis↗
PubMed2004

Nuclear phosphoinositides and their functions.

Phosphoinositides are minor components of biological membranes, which have emerged as essential regulators of a variety of cellular processes, both on the plasma membrane and on several intracellular organelles. The versatility of these lipids stems from their ability to function either as substrates for the generation of second messengers, as membrane-anchoring sites for cytosolic proteins or as regulators of the actin cytoskeleton. Despite a vast literature demonstrating the presence of phosphoinositides in the nucleus, only recently has the function(s) of the nuclear pool of these lipids and their soluble analogues, inositol polyphosphates, started to emerge. These compounds have been shown to serve as essential co-factors for several nuclear processes, including DNA repair, transcription regulation and RNA dynamics. In this light, phosphoinositides and inositol polyphosphates might represent high turnover activity switches for nuclear complexes responsible for these processes. The regulation of these large machineries would be linked to the phosphorylation state of the inositol ring and limited temporally and spatially based on the synthesis and degradation of these molecules.

Active Transport, Cell Nucleus↗
PubMed2003

Pea seed-borne mosaic virus seed transmission exploits novel symplastic pathways to infect the pea embryo and is, in part, dependent upon chance.

Seed transmission of pea seed-borne mosaic virus (PSbMV) depends upon symplastic transport of the virus from infected maternal cells to the embryo. Such transport pathways have not been identified in higher plants. To identify these pathways, we have studied the ultrastructure of the tissues and cells around the micropyle of young developing seeds and compared transmitted and nontransmitted virus isolates. A characteristic of PSbMV infection was the presence of cylindrical inclusions positioned over plasmodesmal openings. The presence of cylindrical inclusions on the testa-endosperm boundary wall, together with immunogold labelling for virus-specific products on the wall and in the endosperm, indicated that symplastic connections existed at this interface. Close examination of the endosperm-suspensor boundary at the base of the suspensor revealed discontinuities in the suspensor sheath wall as porelike structures, which the virus might pass through en route to the embryo. A nontransmitted PSbMV isolate was able to invade the maternal tissues of the developing seed but was excluded from the embryo, although it was detected at a low level in the endosperm. Since the endosperm did not support virus replication, it appeared that passive accumulation determined the amount, timing, and location of the virus relative to the base of the suspensor. Rarely, therefore, could the nontransmitted virus isolate reach the correct location in the endosperm at the correct time for embryo infection via the suspensor to occur.

Embryo, Mammalian↗
PubMed2001

Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector.

Using a recombinant potato virus X (PVX) vector, we investigated the relationship between the length of RNA sequence identity with a transgene and the ability to promote post-transcriptional gene silencing (PTGS) and transgene methylation. The lower size limit required for targeting reporter transgene mRNA de novo using PTGS was 23 nucleotides (nt) of complete identity, a size corresponding to that of small RNAs associated with PTGS in plants and RNA interference (RNAi) in animals. The size and sequence specificity were also explored for PTGS-associated transgene methylation and for the targeting of the vector RNA. The PTGS-competent short sequences resulted in similar patterns of methylation. In all cases, including specific sequences of 33 nt with or without symmetrical cytosine residues, the methylation was distributed throughout the transcribed region of the transgene. In contrast, short sequences lacking symmetrical cytosines were less efficient at promoting PTGS of the transgene mRNA. Short gfp sequences in the PVX vector provided as effective a target for the degradation of viral RNA as was found for PVX carrying the complete gfp cDNA. Short sequences were able to initiate PTGS of an endogenous gene, phyotene desaturase, although this occurred in the absence of DNA methylation. This experimental approach provides important insights into the relationship between short RNA sequences and PTGS.

DNA Methylation↗
PubMed2001

Optimizing pretransfusion antibody detection and identification: a parallel, blinded comparison of tube PEG, solid-phase, and automated methods.

BACKGROUND: The ideal pretransfusion testing strategy identifies maximal significant antibodies at minimal cost. Objectives of this study were to compare the characteristics of three testing methods and determine their optimal incorporation into the following generic testing sequence: 1) screen, for antibodies 2) if results are positive, use primary identification method, 3) if results are inconclusive, use secondary identification method. STUDY DESIGN AND METHODS: A total of 2000 consecutive, unselected, coded specimens were tested with three screening methods-PEG IAT, manual and automated solid phase red cell adherence assay (SPRCA). Of 202 positive results, 187 were of sufficient volume and were tested with both PEG and manual SPRCA identification panels, yielding 82 with significant antibodies, plus one that was negative by both methods found on retrospective review of nonstudy results. Calculations were made on the 1985 volume-sufficient specimens, simulating the possible testing permutations. RESULTS: Manual SPRCA was the most sensitive antibody screen (67/83 = 81%) and the least specific (1840/1902 = 97%); automated SPRCA was the least sensitive (54/83 = 65%) and most specific (1883/1902 = 99%); and PEG was intermediate in both sensitivity (64/83 = 77%) and specificity (1860/1902 = 98%). Of the identification panels, manual SPRCA identified more antibodies than PEG (67 versus 66) but had more inconclusive results (41 versus 20). Of overall strategies, manual SPRCA screening with either sequence of identification methods identified the most antibodies (66). The combination of PEG screen, PEG identification, and manual SPRCA identification identified slightly fewer antibodies (63) but had the lowest reagent cost, total (reagent plus labor) cost, and cost per antibody identified. The sequence of automated SPRCA screening with manual SPRCA identification, and PEG identification had the lowest hands-on time. CONCLUSIONS: The most cost-effective pretransfusion strategy is PEG screen with PEG identification, plus manual SPRCA identification when PEG identification is inconclusive.

Antibodies↗
PubMed2001

Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing.

Phosphoinositide turnover regulates multiple cellular processes. Compared with their well-known cytosolic roles, limited information is available on the functions of nuclear phosphoinositides. Here, we show that phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) stably associates with electron-dense particles within the nucleus that resemble interchromatin granule clusters. These PtdIns(4,5)P2-containing structures have a distribution which is cell-cycle dependent and contain components of both the transcriptional and pre-mRNA processing machinery, including RNA polymerase II and the splicing factor SC-35. Immunodepletion and add-back experiments demonstrate that PtdIns(4,5)P2 and associated factors are necessary but not sufficient for pre-mRNA splicing in vitro, indicating a crucial role for PtdIns(4,5)P2-containing complexes in nuclear pre-mRNA processing.

Antibodies, Monoclonal↗
PubMed2000

Pea embryonic tissues show common responses to the replication of a wide range of viruses.

The response of pea embryonic tissues to the replication of a range of different viruses was investigated using in situ hybridization to analyze changes in the expression of two host genes, heat shock protein 70 (hsp70) and lipoxygenase (lox1). Excised pea embryos were infected using microprojectile bombardment with a nonseed transmissible strain of Pea seed-borne mosaic potyvirus, or with Pea early browning tobravirus (PEBV), White Clover mosaic potexvirus, or Beet curly top geminivirus. Collectively, these examples represent families of viruses with differing genomic features, differing numbers of genomic components and differing replication strategies. In all cases, there was an induction of hsp70 associated with virus replication and, in most cases, a downregulation of lox1. Hence, either each virus has a direct inducer of these common responses or the induction is indirectly the result of a generic feature of virus infection. By exploiting the bipartite nature of the PEBV genome, the coat protein gene and genes involved in vector transmission were excluded as potential inducers.

Cotyledon↗
PubMed2000

Post-translational proteolytic processing of procollagen C-terminal proteinase enhancer releases a metalloproteinase inhibitor.

Activity of matrix metalloproteinases (MMP) is regulated by a family of proteins called tissue inhibitors of metalloproteinases (TIMP). Four TIMPs have been cloned, and their molecular weights range from 29,000 to 20,000. By reverse zymography, we have observed a metalloproteinase inhibitor with an apparent molecular weight of 16, 500 from medium conditioned by human brain tumor cells. Antibodies directed against TIMPs failed to react with the 16,500 molecular weight inhibitor, indicating that it was not a truncated form of a known TIMP. The inhibitor was isolated from conditioned medium using affinity and ion exchange chromatography. N-terminal sequences of the inhibitor matched amino acid sequences within the C-terminal domain of a protein known as procollagen C-terminal proteinase enhancer (PCPE). Thus, the inhibitor was named CT-PCPE. Comparison of the N-terminal domain of TIMP with CT-PCPE revealed that both contained six cysteine residues. As in the case of TIMP, reduction and alkylation abolished the inhibitory activity of CT-PCPE. Purified CT-PCPE inhibited MMP-2 with an IC(50) value much greater than that of TIMP-2. This implies that MMPs may not be the physiologic targets for CT-PCPE inhibition. However, these results suggest that CT-PCPE may constitute a new class of metalloproteinase inhibitor.

Amino Acid Sequence↗
PubMed2000

Phylogenetic analysis of two potyvirus pathogens of commercial cowpea lines: implications for obtaining pathogen-derived resistance.

As a prelude to developing engineered resistance to two important potyvirus pathogens of cowpea, a phylogenetic analysis of strains of Cowpea aphid-borne mosaic virus (CAbMV) and Bean common mosaic virus--blackeye cowpea strain (BCMV-B1C) was undertaken. Nucleotide sequences for the coat protein genes and 3'-untranslated regions of four CAbMV and one BCMV-B1C strains were determined and included in an analysis with published sequences. While all the newly sequenced viruses showed strong homology with the existing respective sequences in the database, the CAbMV group showed a divergence into two subgroups. These groups differed from each other by more than some CAbMV strains differed from the South African Passiflora virus (CAbMV-SAP), which has distinct biological characteristics. The implications of the sequence analyses are discussed with respect to a strategy for the generation of engineered resistance to both groups of viruses.

3' Untranslated Regions↗
PubMed2000

Determining the best method for first-line assessment of neonatal blood glucose levels.

OBJECTIVE: To evaluate and compare the accuracy and performance of two electrochemical glucose meters. To determine the user acceptability of these glucose meters and the ABL 620 Blood Gas Analyser (Radiometer, Copenhagen, Denmark) with an electrochemical glucose oxidase electrode for use in a Level 2 special care baby unit. METHODOLOGY: A total of 108 blood samples were collected from 47 babies at risk for hypoglycaemia. The blood glucose level was measured with two glucose meters, the Advantage Glucose Meter (Roche Diagnostics, Castle Hill, Australia) and the Precision-G Blood Glucose Testing System (Medisense, Melbourne, Australia), and the true blood glucose (TBG) measured with the ABL 620 blood gas analyser. Results from the glucose meters were compared with the TBG (as a percentage of the TBG). RESULTS: The mean (SD) percentage difference between the Advantage Glucose Meter and TBG was 4.5% (12. 5), and Precision-G Glucose Meter and TBG was 15.4% (12.4). The sample haematocrit did not significantly influence the glucose meter/TBG differences. There was an overall preference by the nursing staff for the Advantage Glucose Meter. CONCLUSIONS: The Advantage Glucose Meter was significantly more accurate than the Precision-G with similar precision. It was the preferred method of screening for neonatal hypoglycaemia.

Blood Gas Analysis↗
PubMed2000

Limitations on the use of fused green fluorescent protein to investigate structure-function relationships for the cauliflower mosaic virus movement protein.

To investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP-MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.

Amino Acid Sequence↗
PubMed1999

Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis.

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.

Amino Acid Sequence↗
PubMed1999

Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.

It has previously been reported that complementary-sense gene products of wheat dwarf virus (WDV), a geminivirus of the genus Mastrevirus that infects monocotyledonous plants, bind to human and maize retinoblastoma (Rb) protein. Rb proteins control cell-cycle progression by sequestering transcription factors required for entry into S-phase, suggesting that the virus modifies the cellular environment to produce conditions suitable for viral DNA replication. Using a yeast two-hybrid assay, we have investigated whether the complementary-sense gene products of bean yellow dwarf virus, a mastrevirus that is adapted to dicotyledonous plants, also bind maize Rb protein. We demonstrate that whereas RepA binds to Rb protein, Rep does not, suggesting that RepA alone regulates host gene expression and progression of cells to S-phase. RepA mutants containing L --> I, C --> S, C --> G, and E --> Q mutations within the consensus Rb protein binding motif LXCXE retained the ability to bind to Rb, but with reduced efficiency. Most notably, the E --> Q mutation reduced binding by approximately 95%. Nonetheless, all LXCXE mutants were able to replicate in tobacco protoplasts and to systemically infect Nicotiana benthamiana and bean, in which they produced wild-type symptoms.

Binding Sites↗
PubMed1999

A heat shock transcription factor in pea is differentially controlled by heat and virus replication.

Since some heat-inducible genes [heat shock (hs) genes] can be induced by virus infection in pea [e.g. Hsp70; Aranda et al. 1996, Proc. Natl Acad. Sci. USA 93, 15289-15293], we have investigated the effect that heat and virus replication may have on the expression of a heat-shock transcription factor gene (Hsf). We have characterized what appears to be the only member of the Hsf family in pea, PsHsfA. Similar to Hsp70, PsHsfA is heat-inducible in vegetative and embryonic tissues, which is concordant with the presence of heat shock elements (HSEs) and stress responsive elements (STREs) on its promoter sequence. The expression of PsHsfA during virus replication was studied in pea cotyledons and leaves, and compared to that of Hsp70. In situ hybridization experiments showed that whereas Hsp70 is induced, there is no detectable increased accumulation of PsHsfA RNA associated with the replication of pea seed-borne mosaic potyvirus (PSbMV). These experiments indicate that there is a selective control of virus-induced hs gene expression, and suggest that different regulatory pathways control hs gene expression during heat shock and virus replication.

Amino Acid Sequence↗
PubMed1999

Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA.

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

Amino Acid Sequence↗