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Biomedical subjects

R Kingston

Publications and source records attributed to R Kingston.

At least 37 records · Page 2Linked to original sources
PubMed1995

An improved method for clenbuterol screening using high resolution selected ion recording.

A method has been developed using reliable GC derivatization techniques interfaced with a high resolution mass spectrometer. The method has proved successful for the detection of low levels (less than 1 ppm) of clenbuterol in complex biological matrices. Selected ion recording of two characteristic isotopic fragment ions provides a specific mode of detection by verifying the GC retention time of these ions and also by comparing their relative abundance. Analysis of urine samples demands higher mass spectrometric resolution, and 40 000 (10% valley) was found to be a prerequisite for accurate integration of the drug-related chromatographic peaks. The method developed is suitable for adaptation to a completely unattended automated routine incorporating sample injection, storage and retrieval of source tuning parameters, and data processing.

Adrenergic beta-Agonists↗
PubMed1995

Monoclonal antibodies identifying chick gonadal cells.

1. The aim of the present study was to raise monoclonal antibodies (MAbs) which discriminate gonadal cell types in chickens. 2. A panel of 180 MAbs was generated, from which 14 were chosen for their specificity. 3. These MAbs were characterised immunohistologically on frozen sections of embryonic, newly-hatched and adult, male and female, left gonads. 4. Three of these MAbs are described: AGC5, AGC7, and AGC13 which recognise respectively germinal epithelial cells, supporting cells and germ cells. 5. These MAbs may be useful in following the developmental pathways in the chicken gonad and analysing the interactive role(s) taking place between gonadal stromal cells and primordial germ cells.

Aging↗
PubMed1992

Gastrointestinal decontamination. Which method is best?

Why has ipecac syrup become less popular in emergency management of poisoning and overdose? When should gastric lavage, activated charcoal, cathartics, or a combination of methods be used? Which patients are candidates for whole-bowel irrigation with polyethylene glycol-electrolyte solution? Drs Harris and Kingston answer these questions and present their recommendations for each of the available management options.

Cathartics↗
PubMed1991

An improved method for the detection of DNA fragmentation.

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.

Animals↗
PubMed1990

Cell growth and gene rearrangement signals during the development of T lymphocytes within the thymus.

The thymus provides signals that control the proliferation and differentiation of T lymphocytes and select the repertoire of T-cell specificities. Antibodies to CD3 molecules inhibit full rearrangement of T-cell receptor beta chain genes in organ cultures of early embryo mouse thymus. Whether this effect is mediated through gamma delta CD3 expressing cells, which are present in small numbers at this stage, or through low amounts of CD3 on alpha beta precursor cells is unclear. A requirement for special gene rearrangement signals within the thymus is supported also by the observations that growth factors such as IL-2 and IL-4, although stimulating proliferation of precursor cells removed from the thymus, do not induce full T-cell receptor gene rearrangements. Recent studies show that newly formed thymic lymphocytes expressing alpha beta CD3 receptors are targets for negative selection (deletion) as a means of removing autoreactive cells. Signalling to immature thymocytes via the alpha beta CD3 complex induces the activation of endogenous endonucleases that cleave DNA into oligonucleosomal fragments. We suggest that the activation of this mechanism is the means by which autoreactive cells are removed.

Animals↗
PubMed1990

Newly generated thymocytes are not refractory to deletion when the alpha/beta component of the T cell receptor is engaged by the superantigen staphylococcal enterotoxin B.

It has been reported that, following the initial expression of the T cell receptor (TcR) alpha/beta, newly generated thymocytes pass through a developmental window characterized by ineffective coupling between the alpha/beta and CD3 components resulting in resistance to deletion (negative selection). However, we now provide evidence that the TcR alpha/beta on developing thymocytes is capable of delivering deletional signals in response to the superantigen staphylococcal enterotoxin B (SEB) as soon as the receptor is expressed. We also show that if TcR+ thymocytes are allowed to mature in organ cultures of embryonic thymus before SEB is added, they respond by proliferation giving rise to blast cells of CD4-CD8-, CD4+CD8- or CD4-CD8+ phenotypes.

Animals↗
PubMed1990

Developmentally regulated fetal thymic and extrathymic T-cell receptor gamma delta gene expression.

The gamma delta T-cell receptor (TCR) is the first TCR to be expressed in ontogeny in all vertebrates in which it has been examined thoroughly. Murine gamma delta cell-surface protein is detected by the fourteenth day of gestation. In this work, the activation of gamma delta RNA has been studied. Data indicate that the first TCR protein to appear in the thymus is encoded by gamma genes that are activated after cells colonize the thymus. However, the sequential appearance of different gamma delta TCR proteins during thymic ontogeny cannot be readily explained by differential temporal activation of V gamma genes in the thymus. There are distinct patterns of gamma and delta gene expression during fetal liver development and in the fetal gut (or tissue associated with it). Cells apparent in the liver of mice at birth express gamma delta cell-surface protein, but they disappear from the liver very soon afterward. One V gamma gene is rearranged and expressed prethymically. In addition, gamma gene expression is detectable in the livers of newborn athymic mice. Together, these observations indicate a thymic-independent pathway of activation of TCR genes.

Animals↗
PubMed1990

Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a.

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator E1a. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the E1a region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by E1a domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of E1a with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.

Adenovirus Early Proteins↗
PubMed1989

Apoptosis.

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Animals↗
PubMed1989

Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures.

The receptors found on most T lymphocytes bind to antigen presented on major histocompatibility complex proteins and consist of dimers of alpha- and beta-polypeptides associated with the invariant CD3 complex. A fully competent immune system requires a diverse array of T-cell antigen receptors (TCRs) with different specificities. This diversity is generated by rearrangement of TCR alpha- and beta-chain gene segments within the thymus where the receptors are first expressed. Any cells carrying self-reactive receptors must be eliminated, suppressed or inactivated so that destructive autoimmunity is avoided. Recently, compelling evidence has shown that one process involved in producing such self-tolerance is clonal deletion of autoreactive cells within the thymus by an as-yet-undefined mechanism. Here we show that engaging the CD3/TCR complex of immature mouse thymocytes with anti-CD3 antibodies produces DNA degradation and cell death through the endogenous pathway of apoptosis. Activation of this process in immature T cells by the binding of the TCR to self-antigens may therefore be the mechanism which produces clonal deletion and consequently self-tolerance.

Animals↗
PubMed1989

The effects of anti-CD2 antibodies on the differentiation of mouse thymocytes.

Using a rat monoclonal antibody against mouse CD2, we determined the expression of this marker on thymocytes during ontogeny. CD2 expression becomes detectable at day 15 and reaches adult levels (approximately 95% positivity) by day 19. Furthermore, the effect of anti-CD2 antibodies on T cell differentiation was analyzed by addition of antibodies to thymus organ cultures or repeated injection into newborn mice. Anti-CD2 antibodies inhibit CD2 expression in organ cultures and drastically reduce its expression on thymocytes and peripheral lymphocytes in vivo. In either situation, suppression of CD2 expression does not significantly alter the generation of T cells expressing CD3, CD4, CD8 and T cell receptor V beta 8. These results do not support a role for CD2 in early steps of thymocyte differentiation.

Animals↗
PubMed1989

Antigen-induced apoptosis in developing T cells: a mechanism for negative selection of the T cell receptor repertoire.

Herein we have investigated the ability of antigen to induce thymocyte death by apoptosis on the basis that this may be the mechanism for the deletion of autoreactive cells during T cell development. We show that the ability of the bacterial "superantigen" staphylococcal enterotoxin B to cause specific depletion of V beta 8+ cells when added to thymus organ cultures is accompanied by DNA degradation into oligonucleosomal fragments, indicating that depletion involves apoptosis. Our results provide the first direct evidence that antigen-induced apoptosis can be triggered in developing T cells.

Animals↗
PubMed1989

Developmental control of lymphokine gene expression in fetal thymocytes during T-cell ontogeny.

We have used the technique of in situ hybridization to investigate the expression of lymphokine genes by immature thymocytes during intrathymic development. In 13-day fetal thymocytes a population of cells constitutively produces low levels of interleukin 2 (IL-2) and interleukin 4 (IL-4) mRNAs. A second phase of lymphokine gene expression occurs in the majority of 15-day thymocytes, and a population of cells constitutively produces both IL-2 and IL-4 mRNAs. Thymocytes at 14 days of gestation and after 16 days up until birth do not express detectable lymphokine mRNA. By contrast, the population of IL-2 receptor mRNA-producing thymocytes increases progressively up to 15 days of gestation, and expression thereafter decreases up to birth. In addition, thymocytes expressing interferon gamma mRNA were not present until just prior to birth. Our findings indicate developmental control of lymphokine and lymphokine receptor gene expression in fetal thymocytes during ontogeny.

Animals↗
PubMed1988

Isolation of a functional human gene for brain creatine kinase.

There is evidence that the gene for the B isozyme of creatine kinase is regulated during cell differentiation, is under hormonal control, and is activated in a small cell lung carcinoma. In order to investigate further the mechanisms of these processes, the human gene was isolated and the structure of the promoter region was determined. A human DNA fragment of 8 kilobase pairs was shown to encompass the entire coding region and 850 base pairs (bp) of the 5'-flanking sequence. This fragment was transfected into three cell lines and shown to express functional enzyme. The 5'-end of the gene is split by a 230-bp intron that is located 12 bp upstream of the initiator ATG codon. Transcription initiation occurs at a site that is approximately 69 bp upstream of the 5'-end of this intron. The DNA sequence in the region upstream of the 5'-end of the mRNA is suggestive of two superimposed promoters that contain additional sequence elements that are known to regulate expression of other eukaryote genes. The 5'-region also has a remarkable homology to the overlapping promoters of the adenovirus EIIaE gene. These elements collectively form the basis for initial investigations of how this gene is controlled.

Amino Acid Sequence↗
PubMed1988

The effects of anti-CD3 antibodies on the development of T-cell receptor alpha beta + lymphocytes in embryonic thymus organ cultures.

Cells expressing the gamma delta T-cell receptor in association with CD3 molecules appear in the embryonic thymus before alpha beta CD3+ cells. We have added anti-CD3 antibodies to organ cultures of mouse thymus removed from embryos before any CD3+ cells have appeared, with the aim of blocking the function of the latter as they are generated in the cultures. Our results show that these antibodies affect the maturation of precursors of alpha beta + cells so that D-J but not V-D-J beta-chain arrangements predominate. In contrast, full length alpha gene transcripts are produced. These results are compatible with the view that the early population of gamma delta CD3+ cells plays a role in the maturation of the alpha beta + population.

Animals↗