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Biomedical subjects

D Jefferies

Publications and source records attributed to D Jefferies.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1991

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

Animals↗
PubMed1991

KVP meter errors induced by plastic wrap.

The purpose of this study was to determine whether erroneous kVp meter readings, induced by plastic wrap, affected the actual kVp (output) of a dental X-ray machine. To evaluate the effect of plastic wrap on dental X-ray machine kVp meters, a radiation output device was used to measure output in mR/ma.s. An intraoral dental X-ray unit (S.S. White Model #90W) was used to make the exposures. First, the kVp meter was not covered with plastic wrap and output readings were recorded at various kVp settings with the milliamperage and time held constant. Secondly, the same kVp settings were selected before the plastic wrap was placed. Milliamperage and time were again held to the same constant. The X-ray console was then covered with plastic wrap prior to measuring the output for each kVp. The wrap possessed a static charge. This charge induced erroneous kVp meter readings. Out-put readings at the various induced kVp settings were then recorded. A kVp of 50 with no wrap present resulted in the same output as a kVp of 50 induced to read 40 or 60 kVp by the presence of wrap. Similar results were obtained at other kVp settings. This indicates that the plastic wrap influences only the kVp meter needle and not the actual kilovoltage of the X-ray machine. Dental X-ray machine operators should select kVp meter readings prior to placing plastic wrap and should not adjust initial settings if the meter is deflected later by the presence of wrap. The use of such a procedure will result in proper exposures, fewer retakes, and less patient radiation. If plastic wrap leads to consistent exposure errors, clinicians may wish to use a 0.5% sodium hypochlorite disinfectant as an alternative to the barrier technique.

Communicable Disease Control↗
PubMed1990

Trypanosoma brucei: constitutive activity of the VSG and procyclin gene promoters.

The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.

Animals↗
PubMed1989

Intradermal hepatitis B vaccine in thalassaemia and sickle cell disease.

Thirty two patients with beta thalassaemia and sickle cell disease who were having regular blood transfusions were selected to test the efficacy and immunogenicity of low dose (2 micrograms or 0.1 ml) intradermal hepatitis B vaccine compared with the standard (20 micrograms or 1 ml) intramuscular dose. There was no significant difference in the rates of seroconversion, seroconversion had occurred in all patients by seven months. There were no significant differences in antibody titres between the intramuscular and intradermal groups at 1, 2, and 6 months. Although the titres were significantly higher in the intramuscular group at seven months and at 12-18 months, the antibody titre in the intradermal group did not fall below 10 IU/l. The results of this study suggest that low dose intradermal hepatitis B vaccination is an effective and economical way of stimulating an immune response in patients with beta thalassaemia and sickle cell disease.

Anemia, Sickle Cell↗
PubMed1987

Cibarial infections of Trypanosoma vivax and T. congolense in Glossina.

In a percentage of laboratory bred Glossina infected with Trypanosoma congolense or T. vivax, trypanosomes colonized the cibarium as well as the midgut and/or the proboscis. Field-caught tsetse flies also had T. congolense and T. vivax cibarial infections. T. congolense cibarial infections occurred in all species of Glossina examined: G. tachinoides, G. palpalis palpalis, G. morsitans morsitans and G. m. submorsitans. T. vivax was only found in the cibarium of G. tachinoides. Trypanosomes were firmly attached in the cibarium, as shown by scanning electron microscopy. Two field-caught G. tachinoides were also discovered to have only cibarial infections, presumably of T. vivax. More information is required on the occurrence of cibarial infections in Glossina, especially in the field. It is suggested that examination of the cibarium should be carried out as part of the routine dissection of tsetse flies in the field.

Animals↗
PubMed1987

The effect of trypanocidal drugs on the transmission of Trypanosoma brucei brucei by Glossina morsitans centralis.

The effects of trypanocidal drugs on Trypanosoma b. brucei infections in Glossina m. centralis have been investigated. Pentamidine and suramin exhibited no significant effects but both berenil and samorin reduced the number of salivary gland infections in comparison with controls. Berenil at concentrations of 10, 1.0 and 0.1 microgram/ml significantly reduced the number of mature infections when fed to flies throughout the whole period of trypanosome development. A similar result was obtained with samorin at 0.1 microgram/ml. Subsequent experiments showed that administration of both drugs at an early stage was more effective in preventing the maturation of infection than a later but more prolonged administration. Reported drug levels in the blood of different experimental host animals are of the same magnitude as those used here. It is suggested that repeated feeding of T. b. brucei infected Glossina on drug-treated hosts may reduce transmission, although alternative bloodmeal sources would reduce this effect. These influences are worthy of investigation in the field.

Animals↗