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Biomedical subjects

J V Watson

Publications and source records attributed to J V Watson.

At least 91 records · Page 5Linked to original sources
PubMed1985

How does flow cytometry express Gaussian distributed biological information?

The analogue-to-digital conversion step in flow cytometric analysis introduces a positive skew into the distribution of any measured parameter. In this communication we describe a semi-analytical method to determine if a distribution skewed by the analogue-to-digital conversion step is compatible with a Gaussian origin. The procedure involves determining the errors associated with calculating the coefficient of variation from the skewed distribution and applying systematic corrections. The method was tested with a number of experimental data sets and is illustrated with 3 distributions. One of these was not compatible with a Gaussian origin, but the other 2 were, in spite of considerable positive skewness in the data.

Blood Cell Count↗
PubMed1985

Flow cytometric fluorescence emission spectrum analysis of Hoechst-33342-stained DNA in chicken thymocytes.

Hoechst-33342-stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom-built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green-to-violet ratio, due to a change in the "shape" of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties.

Animals↗
PubMed1985

Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle.

The presence and synthesis of c-myc protein and mRNA in the cell cycle has been studied. We find that c-myc mRNA is present, at equivalent levels, at all times in the cell cycle with the possible exception of mitosis. Furthermore, we demonstrate that this mRNA is transcribed in both G1 and G2 phases. An analysis of the c-myc protein in vivo shows that de novo synthesis occurs in G1 and G2 and the protein turns over with a half-life of approximately 20-30 min in both phases. Furthermore, the level of c-myc protein rapidly increases in cell populations when they re-initiate the cell cycle, thereafter decreasing as the culture reaches quiescence. The results therefore suggest that expression of c-myc can be rapidly modulated and that it is activated during the G0 to G1 transition, but is expressed thereafter in the cell cycle.

Animals↗
PubMed1985

Effects of X-irradiation and sodium butyrate on cell-cycle traverse on normal and radiosensitive lymphoblastoid cells.

We have used a multi-parameter flow-cytometric technique to analyse changes in cell-cycle phase distribution (early and late G1, S and G2+M phases) for normal and X-ray-sensitive (ataxia-telangiectasia, A-T) lymphoblastoid cells exposed to X-irradiation and sodium butyrate (either alone or in combination). Sodium butyrate, an inhibitor of histone deacetylase, is a useful pharmacological tool for determining the proposed role of a histone acetylation-based chromatin surveillance system in controlling cell-cycle responses to DNA damage. We report that X-irradiated A-T cells (acute doses up to 1.5 Gy) demonstrate deficiencies in the capacity to traverse G1 and G2+M phases, although we can find no evidence of the specific involvement of a sodium butyrate-sensitive process in normal cells or abnormalities in the responses of A-T cells to the drug. We conclude that abnormal cellular control of G1 transition in A-T may be the basis of disturbed cellular differentiation in vivo, particularly in non-proliferating tissues under conditions of accumulated environmental or spontaneous DNA damage.

Ataxia Telangiectasia↗
PubMed1985

Cyclosporin-A binds lymphocyte surface receptors induced by activation.

Cyclosporin A (CyA) is a new immunosuppressive drug of very considerable and widespread interest not only for clinicians and transplant immunologists but also for cell biologists concerned with activation mechanisms, as it causes a selective blockade of lymphocyte proliferation. Clearly, the molecular biology of CyA action is of fundamental importance, and in attempting to understand this we have looked for a cell surface receptor. We used indirect staining with antibody (at 4 degrees C and in azide) to ensure that only those CyA molecules held at the cell surface would be seen. The drug molecules which partition into the cell membrane, due to the extreme lipophilicity of CyA, are not detected by extracellular antibody. This technique differs from other methods using a directly labelled drug, where it is not possible to discriminate between specific and non-specific binding. Using high sensitivity flow cytometric analysis we were unable to find CyA on resting lymphocytes, whereas lymphocytes activated by concanavalin A (Con A) or mixed lymphocyte reaction (MLR) showed a CyA-dependent increase in fluorescence.

Cyclosporins↗
PubMed1985

Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells.

A multiparameter flow cytometric technique has been used to detect changes in the emission spectrum of the DNA-specific fluorochrome Hoechst 33342 during uptake by intact, human tumour cells and during the in vitro titration of permeabilized cells. The spectral shift phenomenon was associated with changes in dye: DNA ratio revealing heterogeneity in dye-binding sites. The degree of spectral shift was sensitive to changes in pH within the physiological range. Surprisingly, chromatin structure, in terms of DNase accessibility, was not a major factor in the generation of the spectral shift. The technique of fluorescence emission analysis permits cells with similar DNA contents to be distinguished on the basis of changes in the microenvironment of chromatin for both fresh and freezer-stored biopsy or experimental preparations.

Benzimidazoles↗
PubMed1985

Detection of the c-myc oncogene product in testicular cancer.

A set of monoclonal antibodies was constructed by immunising mice with peptide fragments of the c-myc oncogene product. One such antibody, Myc 1-6E10 was shown to bind to a 62,000 dalton protein identifiable with the c-myc product (p62c-myc). The antigen recognised was not destroyed by paraffin wax embedding. Myc 1-6E10 was used to characterise the distribution of p62c-myc in archival testicular tumour material. Normal testes expressed only small amounts of p62c-myc. Seminomas showed increased nuclear and cytoplasmic staining. Undifferentiated teratoma showed little activity, whereas p62c-myc was abundant in the nuclei of differentiating epithelial structures, yolk sacs and embryoid bodies. Only small amounts of p62c-myc were seen in the tumours of 5 patients who subsequently died from their disease.

Antibodies, Monoclonal↗
PubMed1985

Establishment and characterisation of cell lines from patients with lung cancer (predominantly small cell carcinoma).

Tissue samples from 59 patients with lung cancer have been used to establish cell lines in culture. The primary diagnosis was small cell carcinoma in all except four. Most of the samples were of bone marrow but pleural effusions, lymph node biopsies and skin metastases were also included. The samples were usually split between HITES serum-free medium and HITES plus 2.5% foetal calf serum. A total of 19 cell lines were established and characterised. One line is large cell anaplastic lung carcinoma, four are B-lymphoblastoid and fourteen are small cell lung cancer. Considerable heterogeneity in gross morphology, neuroendocrine differentiation (by electron microscopy) and content of the enzyme L-dopa decarboxylase was seen. The use of HITES plus 2.5% foetal calf serum resulted in better establishment of cultures than did serum-free HITES.

Bone Marrow↗
PubMed1985

Abnormal retention of X-irradiated ataxia-telangiectasia fibroblasts in G2 phase of the cell cycle: cellular RNA content, chromatin stability and the effects of 3-aminobenzamide.

We have addressed three aspects of the abnormal sensitivity of SV40 transformed ataxia-telangiectasia (A-T) fibroblasts to X-irradiation, namely: (a) the radiogenic perturbations in cell-cycle traverse analysed by flow cytometry; (b) the involvement of 3-aminobenzamide-sensitive processes in cellular recovery in terms of viability and release from G2 + M phase delay; and (c) the functional and structural integrity of cells delayed in G2 + M phase using acridine orange as a probe for cellular RNA content and chromatin structure. We report that A-T cells show a dose-dependent and survival-related abnormal retention in G2 + M phase due to the lack of a recovery process, despite the capacity of such cells to synthesize ribosomal RNA and maintain the structural integrity of chromatin. Evidence is presented that the recovery process is dependent upon poly(ADP ribosyl)ation activity in both normal and A-T cells except that in the latter cell type recovery potential is rapidly saturated in terms of X-ray dose.

Ataxia Telangiectasia↗
PubMed1985

Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1.

Three children from two unrelated families had a history of recurrent bacterial infections, and their neutrophils were shown to have deficient phagocytic and respiratory responses and possible deficiencies in chemotaxis or adherence. Their neutrophils were strikingly deficient in the ability to ingest or give a respiratory burst in response to unopsonized bakers' yeast or zymosan (Z). Tests for neutrophil and monocyte CR1 (C3b/iC3b receptor) and CR3 (iC3b receptor) demonstrated rosettes with both EC3b and EC3bi. However, EC3bi were bound only to CR1, and not to CR3, because EC3bi rosettes were inhibited completely by anti-CR1. Neutrophils, monocytes, and natural killer (NK) cells also did not fluorescence stain with monoclonal antibodies specific for the alpha-chain of CR3 (anti-Mac-1, anti-Mol, OKM1, and MN-41). Quantitation of C receptors with 125I monoclonal anti-CR1 and anti-CR3 indicated that neutrophils from each patient expressed normal amounts of CR1 per cell but less than 10% of the normal amount of CR3. Examination of neutrophils by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that a normal glycoprotein of approximately 165,000 daltons was missing. Immunoblotting of these gels indicated that the missing band was the alpha-chain of CR3. Subsequent analysis of all three patients' cells also demonstrated a deficiency of LFA-1 alpha-chain and the common beta-chain that is shared by the CR3/LFA-1/p150,95 membrane antigen family. The deficiency of LFA-1 probably explained the absent NK cell function, as normal NK cell activity is inhibited by anti-LFA-1 but not by anti-CR3. The reduced phagocytic and respiratory responses to Z were probably due to CR3 deficiency, because treatment of normal neutrophils with anti-CR3, but not anti-FLA-1, inhibits responses to Z by 80% to 90%. Ingestion of Staphylococcus epidermidis by normal neutrophils was shown to be partially inhibited by monoclonal antibodies to the alpha-chain of either CR3 or LFA-1, and monoclonal antibody to the common beta-chain inhibited ingestion by 75%. Thus, both CR3 and LFA-1 may have previously unrecognized functions as phagocyte receptors for bacteria. The absence of this type of nonimmune recognition of bacteria by these children's neutrophils may be one of the reasons for their increased susceptibility to bacterial infections.

Antigens, Surface↗
PubMed1985

Family studies of erythrocyte complement receptor type 1 levels: reduced levels in patients with SLE are acquired, not inherited.

It has been claimed that patients with systemic lupus erythematosus (SLE) have an inherited deficiency of erythrocyte complement receptor type 1 (CR1, with ligand binding specificity for C3b, iC3b and C4b). CR1 functions as the only cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg. The activity of this receptor on red cells may be an important mechanism for handling immune complexes which have bound C3b or iC3b. Radioligand binding studies were performed using a monoclonal antibody to CR1, E11, to enumerate these receptors accurately. The results confirmed that patients with SLE have a reduced number of CR1 molecules per red cell, but showed no reduction in CR1 levels amongst their consanguineous relatives. Study of 13 normal families suggested the presence of heritable factors controlling the numbers of erythrocyte CR1 molecules; in particular there was a correlation between mean parental CR1 numbers and CR1 numbers in their children. However, amongst 17 families of 19 patients with SLE, four families were identified in which genotypically 'high CR1' SLE patients had persistently low phenotypes. This is not compatible with the hypothesis that the reduction in erythrocyte CR1 numbers in these patients is inherited.

Antibodies, Monoclonal↗
PubMed1984

Effect of cell density on intracellular adriamycin concentration and cytotoxicity in exponential and plateau phase EMT6 cells.

The relationship between cell number and available Adriamycin (ADM) has been investigated in EMT6 cells. Results have shown that the ratio between cell number and total available ADM is important in determining in vitro ADM uptake and surviving fraction. Having established this effect, the sensitivity of exponentially growing and plateau phase EMT6 cells to ADM was investigated. ADM was assayed by extraction followed by spectrofluorimetry and also by flow cytometry (FCM); both methods were found to give the same ratio of intracellular ADM between exponentially growing and plateau phase cells. We found that for a given exposure dose plateau phase cells were more sensitive than exponentially growing cells. For the same dose per cell, plateau cells take up more ADM than exponentially growing cells. But for a given intracellular ADM concentration exponentially growing cells have a lower surviving fraction than plateau phase cells. We conclude that the surviving fraction is dependent on the proliferative state of the cells and in order to draw that conclusion it is important to relate the ADM effect on cells in vitro to the total ADM available to each cell.

Animals↗
PubMed1983

Subcutaneous culture chamber for continuous infusion of monoclonal antibodies.

Monoclonal antibodies allow the precise definition of molecular components present on tumour cell surfaces. Some human monoclonal antibodies prepared by fusing intratumoral lymphocytes from patients undergoing craniotomy for malignant glioma with cells from a specially derived human lymphoid line (LICR-LON-HMy2) bind to glioma surface components. To study the feasibility of continuous administration of human monoclonal antibodies directed against glioma we designed a chamber which enables hybridoma cells to be cultured in the subcutaneous tissue. This chamber allows antibodies to diffuse out, and nutrients and oxygen necessary for continued cell viability to diffuse in. Cells are unable to traverse the membrane pores of the device, so there is no risk that malignant cells put in the chamber can metastasise. The chamber has now been inserted in one patient with recurrent glioma, in whom the kinetics of internally labelled antibody release has been monitored.

Antibodies, Monoclonal↗
PubMed1982

Human hybridomas from malignant gliomas.

Intratumoral lymphocytes from 12 patients undergoing craniotomy for malignant glioma were fused with a specially derived human myeloma line LICR-LON-HMy2. 71 stable hybridomas were obtained from 5 of the 12 patients. Such hybridomas had a DNA content equal to the sum of that present in lymphocytes and the parent myeloma. The hybridoma supernatants contained human monoclonal immunoglobulins. Glioma-binding activity was detected in 7 out of the 71 supernatants. These results suggest that malignant gliomas contain a population of B lymphocytes which may be involved in host defence against the tumour.

Adolescent↗
PubMed1982

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry.

Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming synchronously growing cell populations and (i) measurement coefficients of variation (CV) from 2 to 16%; (ii) constant measurement CV or VC's increasing from G1 to G2 phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time. The fractions of cells in the G1, S, and (G2 + M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10-20%. However, most methods tended to overestimate G1 fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental. The errors were only slightly larger than for histograms from asynchronously growing cell populations.

Analysis of Variance↗