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Biomedical subjects

D Jefferies

Publications and source records attributed to D Jefferies.

At least 19 recordsLinked to original sources
PubMed2005

Inter-laboratory comparison of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in bleaching earth used in the refinement of edible oils.

Bleaching earth (dried, powdered, bentonite-montmorillonite clay) is commonly used as a processing aid in edible oil refinement. Used bleaching earth may be incorporated into animal feed indirectly, for example because it is included into seed meal, or directly (e.g., as a binding agent). Control must be demonstrated to ensure that the levels of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in feed ingredients do not infringe European Community regulations. The low legislative action level assigned is analytically challenging and may be at or below the limits of quantification achievable by many laboratories. A statistical comparison (following the IUPAC/ISO/AOAC protocol) was made of analyses of PCDDs and PCDFs in selected bleaching earth samples by laboratories from Europe and the USA to assess the comparability of data. Of 19 sets of results submitted by laboratories for replicate samples, 11 demonstrated acceptable agreement.

Aluminum Silicates↗
PubMed2005

Analysis of para-phenylenediamine allergic patients in relation to strength of patch test reaction.

BACKGROUND: Despite having a positive patch test reaction to para-phenylenediamine (PPD), some patients continue to dye their hair, while others are forced to give up or abandon this practice. This difference in patient behaviour could be due to the degree of sensitization. OBJECTIVES: To establish whether the ability to continue dyeing hair in PPD allergic patients is related to the strength of patch test reaction. To note differences in other clinical features in relation to the strength of patch test reaction. METHODS: We analysed retrospectively the patch test records of 400 sequential PPD-positive patients for the strength of patch test reaction (+, ++, +++) and different clinical features. Data were analysed using Cochran-Mantel-Haenszel chi2 tests. RESULTS: There was a strong linear relationship between the strength of patch test reaction and continuation with hair dyeing. Patients were more likely to report a history of hair dye reaction with increasing strength of patch test reaction. There was no difference in strength of patch test reaction in relation to age, site of rash, occupation (hairdressing) or history of atopic eczema. Overall concomitant reactivity with related aromatic amine allergens (benzocaine, N-isopropyl-N-phenyl-para-phenylenediamine, para-aminobenzoic acid) was infrequent. CONCLUSIONS: Patients with stronger patch test reactions (++, +++) are more likely to have a clear history of reacting to hair dye and are less likely to still be dyeing their hair.

Adolescent↗
PubMed2003

Expression of type X collagen, Indian hedgehog and parathyroid hormone related-protein in normal and tibial dyschondroplastic chick growth plates.

Tibial dyschondroplasia (TD) is a form of aberrant endochondral ossification in chickens, in that a plug of avascular cartilage (TD lesion) is formed within the growth plate. Histologically, the lesion is filled with apparently transitional chondrocytes that have been unable to differentiate to hypertrophic chondrocytes. We have examined the spatial expression of mRNAs for type X collagen, Indian hedgehog (Ihh) and Parathyroid Hormone-related protein (PTHrP) in the TD growth plate by in situ hybridization in order to ascertain at which stage chondrocyte differentiation is arrested in TD. In the normal growth plate, type X collagen mRNA was expressed by both prehypertrophic and hypertrophic chondrocytes. Indian Hedgehog mRNA was detected in a band of prehypertrophic chondrocytes and PTHrP expression was localized to a narrow band of prehypertrophic chondrocytes and in osteoblasts within the diaphysis. In TD sections, collagen X expression was seen within differentiating cells, within a small number of lesion cells, and within hypertrophic chondrocytes on the diaphyseal side of the lesion. Ihh expression was also seen within the differentiating cells and throughout the lesion. These data indicate that chondrocyte differentiation is arrested at the transitional stage just prior to hypertrophy. Contrary to the previously reported PTHrP expression patterns in TD chicks by immunohistochemistry, PTHrP mRNA was not detected in the TD lesion. This observation probably reflects the cessation of PTHrP gene expression by chondrocytes in the more severe TD lesions. The results from the present study also imply that the arrest of cell differentiation in TD is independent of PTHrP and that endochondral ossification in the post-hatch avian growth plate may involve additional regulatory pathways.

Animals↗
PubMed2001

Regulation of chondrocyte terminal differentiation in the postembryonic growth plate: the role of the PTHrP-Indian hedgehog axis.

Chondrocyte differentiation during embryonic bone growth is controlled by interactions between PTHrP and Indian hedgehog. We have now determined that the major components of this signaling pathway are present in the postembryonic growth plate. PTHrP was immunolocalized throughout the growth plate, and semiquantitative RT-PCR analysis of maturationally distinct chondrocyte fractions indicated that PTHrP, Indian hedgehog, and the PTH/PTHrP receptor were expressed at similar levels throughout the growth plate. However, patched, the hedgehog receptor, was more highly expressed in proliferating chondrocytes. Although all fractionated cells responded to PTHrP in culture by increasing thymidine incorporation and cAMP production and decreasing alkaline phosphatase activity, the magnitude of response was greatest in the proliferative chondrocytes. Bone morphogenetic proteins are considered likely intermediates in PTHrP signaling. Expression of bone morphogenetic protein-2 and 4--7 was detected within the growth plate, and PTHrP inhibited the expression of bone morphogenetic protein-4 and 6. Although organ culture studies indicated a possible paracrine role for epiphyseal chondrocyte-derived PTHrP in regulating growth plate chondrocyte differentiation, the presence within the postembryonic growth plate of functional components of the PTHrP-Indian hedgehog pathway suggests that local mechanisms intrinsic to the growth plate exist to control the rate of endochondral ossification.

Animals↗
PubMed2000

Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia.

Tibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought.

Animals↗
PubMed2000

Chondrocytes and longitudinal bone growth: the development of tibial dyschondroplasia.

Growth plate cartilage is central to the process of bone elongation. Chondrocytes originating within the resting zone of the growth plate proceed through a series of intermediate phenotypes: proliferating, prehypertrophic and hypertrophic, before reaching a terminally differentiated state. Disruption of this chondrocyte maturational sequence causes many skeletal abnormalities in poultry such as tibial dyschondroplasia (TD), which is a common cause of deformity and lameness in the broiler chicken. Cell and matrix components of the growth plate have been studied in order to determine the cause(s) of the premature arrest of chondrocyte differentiation and retention of prehypertrophic chondrocytes observed in TD. Chondrocyte proliferation proceeds normally in TD, but markers of the differentiated phenotype, local growth factors, and the vitamin D receptor are abnormally expressed within the prehypertrophic chondrocytes above, and within, the lesion. Tibial dyschondroplasia is also associated with a reduced incidence of apoptosis, suggesting that the lesion contains an accumulation of immature cells that have outlived their normal life span. Immunolocalization studies of matrix components suggest an abnormal distribution within the TD growth plate that is consistent with a failure of the chondrocytes to fully hypertrophy. In addition, the collagen matrix of the TD lesion is highly crosslinked, which may make the formed lesion more impervious to vascular invasion and osteoclastic resorption. Recent studies have applied the techniques of differential display and semiquantitative reverse transcriptase-polymerase chain reaction to RNA obtained from discrete populations of growth plate chondrocytes of different maturational phenotypes. This strategy has allowed us to compare phenotypically identical cell fractions from normal and TD growth plates in an attempt to identify possible candidate genes for TD.

Animals↗
PubMed1999

Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes.

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.

Amino Acid Sequence↗
PubMed1999

Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy.

Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100% identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

Animals↗
PubMed1999

A novel integral membrane protein is differentially expressed in the chick growth plate and maps to chromosome 1.

The growth plate is a specialised region of cartilage located at the growing ends of long bones in higher vertebrates. It is responsible for longitudinal bone growth and is under the control of many local and systemic factors. The growth plate consists of an orderly arrangement of small proliferative and larger mature hypertrophic chondrocytes. This paper describes the isolation by differential display of a 988-bp cDNA fragment derived from a transcript that is more highly expressed in proliferating rather than hypertrophic chondrocytes of the chick growth plate. Using 3' RACE, a further 939 bp of cDNA sequence was obtained. The 1.9 kb sequence contains a 924-bp open reading frame encoding an unknown 308 amino acid protein. This protein has a putative transmembrane domain near its N-terminus and three dileucine motifs at its carboxy tail. This gene was expressed in all other tissues examined. A polymorphism was identified by SSCP analysis and the gene was mapped to the centromeric region of the short arm of chicken chromosome 1, close to the locus for autosomal dwarfism.

5' Untranslated Regions↗
PubMed1998

Cloning differentially regulated genes from chondrocytes using agarose gel differential display.

The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.

Animals↗
PubMed1998

Selection of novel ligands from phage display libraries: an alternative approach to drug and vaccine discovery?

Phage display involves the production and screening of large numbers of random peptide sequences of a specific length expressed on the surface of phage particles. This approach provides a powerful tool to probe the molecular basis of many biological processes, including host-parasite interactions. Phage display libraries have been used to study the binding specificity of numerous peptides and protein domains. Practical applications include the identification of peptide sequences that bind with high affinity to antibodies, enzymes or receptors, and that may serve as diagnostics and vaccine or drug candidates. Here, David Jefferies outlines the concept of phage display and summarizes recent developments in the field, with emphasis on those that may be of interest to parasitologists.

Journal Article↗
PubMed1996

A promotor directing alpha-amanitin-sensitive transcription of GARP, the major surface antigen of insect stage Trypanosoma congolense.

The major surface antigen of procyclic and epimastigote forms of Trypanosoma congolense in the tsetse fly is GARP (glutamic acid/alanine-rich protein), which is thought to be the analogue of procyclin/PARP in Trypanosoma brucei. We have studied two T.congolense GARP loci (the 4.3 and 4.4 loci) whose transcription is alpha-amanitin sensitive. Whilst a transcriptional gap 5' of the first GARP gene in the cloned region of the 4.4 locus could not be detected, such a gap was present in the 5' flank of the first GARP gene in the 4.3 locus. We have located a GARP transcription start site and, using reporter gene constructs containing a putative GARP promoter region in transient transfection studies, we have demonstrated promoter activity for the test region in T.congolense. There are species-specific differences in sequences regulating expression of the two major surface antigens, GARP and procyclin/PARP: the GARP promoter is inactive in T.brucei while the procyclin/PARP promoter is inactive in T.congolense. We have defined the splice acceptor site for the 4.3 GARP gene by sequencing and by 5' RT-PCR and demonstrated microheterogeneity in GARP polyadenylation by 3' RT-PCR. It appears that some GARP and procyclin/PARP RNA processing signals, although similar, are also species-specific.

Amanitins↗
PubMed1995

The 3'-terminal region of the mRNAs for VSG and procyclin can confer stage specificity to gene expression in Trypanosoma brucei.

The variant surface glycoprotein (VSG) and procyclin are the respective major surface antigens of the bloodstream and the procyclic forms of Trypanosoma brucei. These proteins and their mRNAs are both the most abundant and absolutely characteristic of their respective life cycle stages. We show that the 3'-terminal region of these mRNAs regulates expression of a reporter gene in an inverse manner, depending on the developmental form of the parasite. In the case of VSG mRNA, the 97 nt sequence upstream from the polyadenylation site is responsible for these effects. The regulation occurs through a variation of mRNA abundance which is not due to a change in primary transcription. In the bloodstream form this effect is manifested by an increase in RNA stability, whereas in the procyclic form it seems to be related to a reduction in the efficiency of mRNA maturation. The 3'-end of VSG mRNA can obviate the 5- to 10-fold stimulation of transcription driven by the procyclin promoter during differentiation from the bloodstream to the procyclic form. The predominance of posttranscriptional over transcriptional controls is probably linked to the organization of the trypanosome genome in polycistronic transcription units.

Animals↗
PubMed1993

The ble resistance gene as a new selectable marker for Trypanosoma brucei: fly transmission of stable procyclic transformants to produce antibiotic resistant bloodstream forms.

We describe here the stable transformation of Trypanosoma brucei using a new selectable marker for kinetoplastid protozoa, the Sh ble, or phleomycin, resistance gene. A plasmid containing this gene targeted to the tubulin gene locus by homologous sequences was introduced into procyclic trypanosomes by electroporation and cells selected for antibiotic resistance. Southern analysis of stable transformants showed that the plasmid had been integrated into the tubulin locus by homologous recombination. Analysis of bloodstream stage transformants, produced by transmission through the vector Glossina, showed that the resistance gene was conserved and expressed in these forms in the absence of selective drug pressure. In both procyclic and bloodstream forms, transcription of the ble gene appears to originate from the upstream tubulin promoter, despite the presence of a VSG promoter in the integrated construct. The generation of stable bloodstream transformants for the first time will facilitate the study of gene function and expression during the trypanosome life cycle, and aid in the investigation of genetic exchange in these organisms.

Animals↗
PubMed1991

The actin gene promoter of Trypanosoma brucei.

The actin genes of Trypanosoma brucei are transcribed constitutively during the parasite life-cycle, by a polymerase sensitive to alpha-amanitin. The start region of the actin gene transcription unit was mapped by virtue of the accumulation of promoter-proximal transcripts which occurs following moderate UV irradiation. This region, located about 4 kilobases upstream from the genes, was able to direct transient expression of the bacterial Chloramphenicol Acetyl Transferase (CAT) gene in both bloodstream and procyclic forms of the parasite. The essential region of the promoter was defined by deletion, and appeared to be within 600 bp upstream from the putative transcription start site. It does not share significant homology with the other trypanosome promoters described so far (VSG, procyclin, rDNA), which all direct alpha-amanitin resistant transcription.

Actins↗