Biomedical subjects
Publications and source records attributed to R T Johnson.
A patient with monocular polyopia underwent extensive neurologic investigation over a two-year period. Ocular examination disclosed a corneal degenerative change as the cause, and symptomatic relief was obtained with pilocarpine hydrochloride drops.
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In normal cells exposed to low u.v. doses the several enzymic steps of the excision repair process are closely coupled with the result that DNA gaps are transient and present at such low frequency that it is very difficult to detect them. Cells from a u.v.-sensitive human genetic disorder, Cockayne's Syndrome (CS) and from an immunodeficient individual 46BR, have been examined with respect to their incision capacity after u.v. in the presence and absence of inhibitors of DNA synthesis. We have measured the initial rates of DNA break accumulation in the presence of hydroxyurea and 1-beta-D arabinofuranosylcytosine and find that in both these groups the rate is only slightly lower than in normal cells. However, there is a marked difference between u.v. sensitive cells and normal in the accumulation of long-lived DNA breaks in the absence of inhibitors. While in normal cells practically no breaks could be detected, the u.v. sensitive cells accumulated significant numbers of DNA breaks within 15 min of incubation; 46BR cells showed almost the same level of DNA breaks without the inhibitors as with them. In CS break accumulation can be detected in the absence of inhibitors for only a short time after irradiation (approximately 30 min), but less so when deoxyribonucleosides are provided. The spontaneous break accumulation is related to the time elapsed since proteolytic detachment of the cells from monolayer; 24 h after replating CS breaks no longer accumulate in response to u.v. 46BR cells, on the other hand, accumulate breaks even 1 day after replating and express unligated gaps 2 h after irradiation with a relatively low u.v. dose such as 4 Jm-2. Provision of DNA precursors does not greatly reduce break accumulation. The extremely slow rate of gap sealing in 46BR cells is consistent with the hypothesis that a ligase defect is expressed in these cells. In the absence of inhibitors break accumulation in CS cells often fluctuates between experiments for unaccounted reasons; though 11961 fluctuates less than other CS strains this phenotypic trait helps to confirm its assignation as a Cockayne Syndrome. Spontaneous conditional break accumulation is not restricted to CS cells; fibroblasts from a normal individual also express similar behaviour though their ability to seal repair sites is considerably greater.
The effects of inhibitors of replicative DNA synthesis on repair DNA synthesis have been examined by autoradiography in several different cell types and in cells in different growth states. Hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), administered together, influence unscheduled DNA synthesis (UDS) in a manner which is independent of the status of the cell culture (normal or transformed) and of the species, but which is strongly affected by whether the cells are proliferating or quiescent. In proliferating human, Chinese hamster and Microtus cell cultures, UDS is not inhibited by HU and ara C, and may even appear to be stimulated. In quiescent cultures of these cells UDS is reduced by HU and ara C. In cells reseeded from a confluent culture and followed during proliferation and back to quiescence the effect of inhibitors parallels the growth pattern. The results are interpreted in terms of changes in the sizes of endogenous DNA precursor pools; they underline the potential problems associated with quantitating UDS in the presence of inhibitors.
Serum C-reactive protein levels become elevated coincident with the onset of the rash in patients with measles. Serum C-reactive protein elevations are prolonged in measles complicated by pneumonia and may show a second elevation in measles complicated by encephalitis.
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In this paper we examine DNA repair in the mouse. Using stringently applied inhibitors of DNA synthesis (hydroxyurea and 1-beta-D-arabinofuranosylcytosine) after ultraviolet irradiation we have quantitated an early step in the repair process - the frequency of single-strand DNA breaks, products of enzymic incision--in fibroblasts and epithelial cells grown in vitro from adults and embryos of different ages. We find that initial rates of incision are very similar in the earliest-passage fibroblasts, regardless of donor age, resembling the rates measured in other rodents while being considerably less than in diploid human fibroblasts. Consistently higher rates of incision are found in adult kidney epithelial cells. With increasing passage number the embryo cultures change with respect to growth rate and chromosome constitution, undergoing spontaneous transformation to established heteroploid cultures. This sequence is accompanied by characteristic changes in initial rates of inhibitor-sensitive incision. An early increase in incision rate, at a time when the cultures are enriched with tetraploid cells, is succeeded by progressive reduction in rate as the cultures become overtly transformed into permanent lines and heteroploidy is established. The lowest rate of incision occurs in a transformed line cloned from a late-passage culture. The decline in incision rate associated with transformation is accompanied by reduced unscheduled DNA synthesis (UDS). The sensitivity to inhibitors of UDS levels in the mouse cultures has been found to vary considerably; inhibitors are most effective in slowly proliferating cultures and apparently not at all when cultures are growing rapidly despite their ability to accumulate DNA breaks in these cells. Caution is needed, therefore, in the interpretation of repair data obtained in the presence of these commonly used inhibitors of DNA synthesis.
Fusion between mitotic and S-phase cells induces the formation of prematurely condensed chromosomes (PCC) in the interphase partner. Viewed in the light microscope, S-phase PCC derived from the Indian muntjac appear to be fragmented and heterogeneous. In scanning electron micrographs prepared by an osmium impregnation technique, which avoids the need to sputter-coat the specimen, the S-phase fragments derived from an individual cell are resolved into about 1000 fibre aggregates, together with more dispersed fibres. Aggregates are roughly spherical and vary in diameter between about 0.25 and 1.6 micron. The spatial distribution of the aggregates shows some order: chains of single aggregates and, less commonly, duplicated chains occur. Regions of the PCC where the fibres are more dispersed are considered to be likely candidates for sites of replication at the time of fusion. The relationship between the condensed aggregate structure of the S-phase PCC and replication clusters is discussed, and also the assembly of aggregates to form metaphase chromosomes.
During the eukaryote cell cycle the nuclear envelope displays a series of major morphogenetic changes, the most significant of which include its breakdown and reconstitution as cells move up to, pass through and emerge from division. The three polypeptides, lamins A, B and C, are major components of the nuclear pore complex-lamina fraction of the nuclear envelope and their association with the nuclear membrane or their dispersal in the cytoplasm reflects the existing balance between polymerization and depolymerization in the envelope. We have perturbed the lamina polymerization cycle by means of cell fusion between mitotic and interphase cells, following the redistribution of nuclear lamina protein by means of immunofluorescence techniques. In these heterophasic heterokaryons changes in the distribution of lamina occur as a function of (1) the time elapsed after fusion; (2) the ratio of mitotic to interphase elements in the cell, and (3) the stage in the cell cycle occupied by the interphase partner at the time of fusion. Depolymerization of nuclear lamina occurs most rapidly in cells with high ratios of mitotic to interphase elements, and especially in G1 rather than S-phase nuclei. While lamina depolymerization predominates early after fusion, at later times lamina is deposited around both the original metaphase and interphase nuclear masses and this is associated with the resumption of interphase activity in the form of limited DNA synthesis. These observations lead us to conclude that lamina depolymerization is under positive control mediated by diffusible factors in the cytoplasm of the metaphase partner. Repolymerization is likely to be associated with the inactivation of these factors as the heterokaryons age and, as a result, pass into an interphase-like state.
In measles encephalitis we: Confirmed the decrease in mitogen responses and have shown that it does not correlate with complications. Demonstrated that the 'immunosuppression' is not universal but may be an abnormality of immune regulation as shown by the response to measles virus and myelin basic protein and by an abnormality of suppressor cell activity in patients with measles. Have evidence that there is early demyelination, and a response to myelin basic protein in a large proportion of the patients, and a lack of evidence of direct virus invasion of the brain. These findings lead to our present hypothesis that measles virus infection, probably of lymphoid cells, leads to a breakdown of immune regulation. This lack of regulation may lead to dissemination and allow secondary infection. It may also lead to a break in tolerance leading to autoimmune demyelination, a regulation which as Patterson (1979) has said 'may effectively restrain our ever present capacity to react immunologically against our own nervous tissue'.
Polyomaviruses have been implicated in several chronic human neurological diseases. In progressive multifocal leukoencephalopathy there is an opportunistic and lytic infection of oligodendrocytes of the central nervous system leading to subacute demyelinating disease. Although aspects of the pathogenesis remain undefined, the causal role of the viruses seems firm. Nonpermissive infection of neural cells with resultant cerebral tumors has been suggested by epidemiological studies, virus isolations, and detection of viral antigens and nucleic acids. Nevertheless, it is uncertain whether papovaviruses are causally related to human tumors. Possible association of papovavirus infection with other neurological diseases needs further study.
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We have measured repair DNA synthesis in UV-irradiated normal human fibroblasts, grown to a defined state of quiescence in order to avoid the problem of discriminating repair from replicative DNA synthesis. We have assessed the effects of various DNA synthesis inhibitors on repair. Inhibition of repair synthesis by hydroxyurea, 1-beta-D-arabinofuranosylcytosine and aphidicolin is associated with the ability to accumulate DNA breaks due to enzymic incision at DNA damage sites; the inhibition by novobiocin is in accord with its known ability to block incision.
The incision step of excision repair of UV-damaged DNA, represented by the accumulation of DNA strand breaks in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, was measured in cultured cells of Microtus agrestis during quiescence and at different times after release from quiescence. The UV dose-dependent kinetics of enzymic incision change rapidly through the first cell cycle after release, with a maximum potential for enzymic incision and a minimum affinity of the incision system for damage sites occurring in late G1. The potential for incision falls to a lower level as cells approach a randomly proliferating state, whilst the affinity rises. Incision activity declines rapidly after UV irradiation, leaving many damage sites unrepaired; the enhanced survival seen in cells held in quiescence after irradiation is thus partly due to some other repair mode.
Following UV-irradiation and in the presence of inhibitors of DNA synthesis (hydroxyurea and 1-beta-D-arabinofuranosylcytosine) human cells accumulate strand breaks in their DNA--as a result of enzymic incision without subsequent rejoining. We have developed a sensitive procedure which makes stringent use of these inhibitors so as to maximize the frequency of breaks detected after low levels of UV (0.25-10 Jm-2) and to permit analysis of the kinetics of break accumulation over short intervals after irradiation (up to 90 min). Since the rate of accumulation of breaks declines quickly with time of incubation (not simply as a consequence of substrate depletion), we have calculated initial rate constants by extrapolating to zero time for a range of UV doses (i.e. different substrate concentrations). Using these constants as indices of enzymic incision, we have compared a wide range of human cell types, and have (in some cases) been able to estimate the enzymatic parameters KM and Vmax for the incision step. Assessed in this way the human cells tested fall into a number of distinct categories. Fibroblasts from normal embryos and from xeroderma pigmentosum (XP) variant and Bloom's syndrome show high and uniform levels of incision readily distinguishable from XP(A), in turn distinct from XP(D). Tumour-derived cells and SV40-transformed fibroblasts also fall into a group with similar incision capacity, significantly lower than that of normal diploid cells. We discuss possible reasons for this distinction, and evaluate the use of inhibitors in repair studies.