Biomedical subjects

R T Johnson

Publications and source records attributed to R T Johnson.

At least 91 records · Page 5Linked to original sources

PubMed1990

Molecular cloning of a mammalian gene involved in the fixation of UV-induced mutations.

A mammalian DNA damage tolerance gene has been isolated by DNA transfection and cosmid rescue. Following cotransfection of mouse genomic DNA and pSV2neo into SVM, the UV hypersensitive mutant Indian muntjac cell line, clones with a 1.7 to 2.0-fold greater D37 value for UV killing were isolated. This trait was carried through three rounds of transfection. A neo gene and flanking sequences from a tertiary transfectant were cloned by cosmid rescue. The cosmid clone confers UV resistance to SVM and improves the ability of the cell to replicate UV damaged DNA. This replication appears to be error-prone; UV-induced 6-thioguanine-resistant mutants occur four to fivefold more frequently than in SVM or a wild-type Indian muntjac line. Thus, the gene isolated is not homologous to that defective in SVM. We believe that this is the first mammalian gene to be isolated that is directly involved in mutation fixation.

Animals↗

PubMed1990

Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line.

DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.

Animals↗

PubMed1990

Spontaneous proliferation of peripheral mononuclear cells in natural measles virus infection: identification of dividing cells and correlation with mitogen responsiveness.

Spontaneous proliferation of peripheral mononuclear cells is pronounced following measles virus infection at a time when patients mount effective humoral and cell-mediated immune responses and manifest a range of poorly understood immunologic abnormalities. We found spontaneous activity (measles 8000 +/- 1200 cpm vs control 1900 +/- 350 cpm; P less than 0.05) to wax and wane abruptly during the first week after the rash in parallel with expression of the lymphocyte activation marker OKT10. At peak activity, approximately 10% of circulating mononuclear cells were actively synthesizing DNA. Double labeling of individual mononuclear cells with autoradiography and immunoperoxidase demonstrated that B and T lymphocytes as well as monocytes participate in the spontaneous activity. Proliferative activity was increased 3- to 20-fold over control levels in all PBMC subsets such that close to one-third of circulating B cells and monocytes and 5-10% of CD4- and CD8-positive T cells were preparing to divide. Mitogen responsiveness was generally decreased in measles patients (58,800 +/- 4600 cpm vs control 97,700 +/- 15,500 cpm; P less than 0.002). Neither spontaneous proliferation nor mitogen responsiveness was correlated with age, sex, or the presence of complications. Patients with the lowest mitogen responses, however, had the greatest increases in B cell (P less than 0.03) and CD8-positive T cell (P less than 0.05) proliferation. These data demonstrate that all major immunologic cell types proliferate in response to measles virus infection. Mechanisms by which spontaneous proliferative activity in individual mononuclear subsets could contribute to depressed mitogen responsiveness are discussed.

Antibody Formation↗

PubMed1990

Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis.

Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine-induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures.

Animals↗

PubMed1990

Immune activation during measles: interferon-gamma and neopterin in plasma and cerebrospinal fluid in complicated and uncomplicated disease.

To study T cell and macrophage activity during measles, levels of interferon-gamma (IFN-gamma) and neopterin in plasma and cerebrospinal fluid (CSF) were measured. Plasma levels of IFN-gamma were elevated in measles (1.17 +/- 0.27) compared with healthy adults (0.13 +/- 0.06, P less than .05) and children (0.14 +/- 0.06, P less than .01). Plasma levels of neopterin were elevated in measles (32.5 +/- 2.7) compared with healthy adults (5.3 +/- 2.9, P less than .0001), healthy children (12.1 +/- 4.0, P less than .001), and children with other infectious diseases (20.6 +/- 4.0, P less than .02). IFN-gamma was increased in measles primarily during rash; neopterin remained elevated for several weeks. Levels of neopterin showed a significant positive correlation with plasma levels of soluble interleukin-2 receptor and soluble CD8, two other parameters of T cell activation. Children with measles complicated by pneumonia had higher levels of neopterin in serum than those with uncomplicated disease. Children with measles complicated by autoimmune encephalomyelitis had higher levels of neopterin in CSF than those with noninflammatory neurologic disease but lower than those with central nervous system infections. Thus, IFN-gamma seems to be produced in vivo during acute measles virus infection; deficiency of this lymphokine does not appear to correlate with increased susceptibility to secondary infections.

Adolescent↗

PubMed1990

Natural killer cell activity during measles.

Natural killer cells are postulated to play an important role in host anti-viral defences. We measured natural killer cell activity in 30 individuals with acute measles (73 +/- 21 lytic units (LU)/10(7) cells) and 16 individuals with other infectious diseases (149 +/- 95 LU) and found it reduced compared with values for adults (375 +/- 70 LU; P less than 0.001) or children (300 +/- 73 LU, P less than 0.01) without infection. Reduced natural killer cell activity was found in measles patients with (84 +/- 30 LU) and without (55 +/- 18 LU) complications and was present for at least 3 weeks after the onset of the rash. Activity was increased by in vitro exposure of cells to interleukin-2. Depressed natural killer cell activity parallels in time the suppression of other parameters of cell-mediated immunity that occurs during measles.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Intrathecal synthesis of anti-HIV IgG: correlation with increasing duration of HIV-1 infection.

We determined intrathecal synthesis (ITS) of anti-HIV-1 immunoglobulin in 62 CSF samples from 51 HIV-1 seropositive homosexual men using an ELISA technique with paired serum and CSF samples diluted to a concentration of IgG of 10 micrograms/ml. All subjects were neurologically normal and none was taking zidovudine. We estimated duration of HIV-1 infection from semiannual serologic testing during the 3-year period before CSF analysis and detected ITS of anti-HIV-1 immunoglobulin in 2 of 12 (17%) of those with less than 18 months of HIV-1 seropositivity, in 3 of 21 (14%) with 19 to 36 months, and in 13 of 29 (45%) with greater than 36 months of HIV-1 seropositivity (p = 0.037). There was a trend toward an inverse relationship between level of ITS and the peripheral blood T-helper lymphocyte count. This study demonstrates that increasing ITS of anti-HIV-1 IgG is related to duration of HIV-1 infection and suggests an inverse correlation with systemic immune status. The detection of ITS of anti-HIV-1 immunoglobulin is not necessarily a marker of clinically overt neurologic involvement.

Adult↗

PubMed1990

Steroid-responsive myeloneuropathy in a man dually infected with HIV-1 and HTLV-I.

Two human retroviruses, HIV-1 and HTLV-I, have been associated with myelopathies in addition to other neurologic disorders. We report an American dually infected with HIV-1 and HTLV-I who developed steroid-responsive myeloneuropathy. This 28-year-old bisexual man developed interstitial pneumonitis and a transient midthoracic sensory level followed by the evolution of a slowly progressive spastic paraparesis and sensorimotor neuropathy. Serologic studies demonstrated coinfection with both HIV-1 and HTLV-I. Peripheral blood absolute CD4 count was persistently within the normal range. Cranial MRI was normal and spinal MRI showed T3-T10 atrophy. Serial CSF analyses demonstrated marked intrathecal synthesis of anti-HTLV-I IgG, lymphocytic pleocytosis, elevated protein and immunoglobulin G, and oligoclonal bands. HIV-1 was isolated from CSF but not from peripheral nerve. Lymphoproliferative studies confirmed spontaneous proliferation in both blood and CSF. Soluble interleukin 2 receptor and soluble CD8 were greatly elevated in blood and CSF when compared with patients with HIV-related vacuolar myelopathy and seronegative patients with other causes of myelopathy. Nerve biopsy showed epi- and endoneurial CD8+ lymphocytic infiltration without vasculitis; muscle biopsy showed features of acute and chronic denervation. A 6-week course of prednisone produced sustained improvement in leg strength and walking times. We speculate that the myeloneuropathy was caused by HTLV-I in the setting of coinfection with HIV-1.

AIDS Dementia Complex↗

PubMed1990

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis.

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.

Animals↗

PubMed1989

Immune activation in measles.

Measles is associated with alterations in immune regulation that sometimes lead to secondary infections or autoimmune encephalomyelitis. Simultaneously, an effective measles virus-specific immune response develops. To relate immune activation to measles and its complications, we studied the spontaneous proliferation of blood mononuclear cells and circulating levels of soluble interleukin-2 receptor and CD8 T-cell antigens in 126 patients with complicated or uncomplicated measles at various stages of the disease. Spontaneous proliferation of mononuclear cells, which was present through the first week of the rash, was greater in cells from patients with measles (8787 +/- 1403 cpm) than in those from healthy children (1529 +/- 237 cpm, P less than 0.0001). Levels of soluble interleukin-2 receptor (3385 +/- 195 units per milliliter) and CD8 (4145 +/- 437 units per milliliter) were higher in patients with measles than in those with other infectious diseases (2377 +/- 440, P = 0.003; 2399 +/- 771, P = 0.0374) or in healthy children (865 +/- 138, P less than 0.0001; 1026 +/- 169, P less than 0.0001). Levels of soluble interleukin-2 receptor were elevated before the onset of the rash and remained elevated for several weeks. In contrast, levels of soluble CD8 increased only when the rash appeared, and subsided quickly. Spontaneous proliferation of mononuclear cells and levels of soluble CD8 were similar in patients with uncomplicated disease, pneumonia, or encephalomyelitis, but soluble interleukin-2 receptor levels were lower in patients with encephalomyelitis (2312 +/- 314 vs. 3455 +/- 247 units per milliliter in uncomplicated measles; P = 0.01). In patients with encephalomyelitis, cerebrospinal fluid levels of soluble CD8 (686 +/- 350 units per milliliter), but not interleukin-2 receptor (9 +/- 8.3 units per milliliter), were increased. We conclude that the proliferative phase of the immune response, as measured by the release of soluble interleukin-2 receptor, begins before the rash appears, continues for several weeks in those without complications, but does not occur within the nervous system. In contrast, the effector phase of the immune response, as measured by the release of soluble CD8, coincides with the appearance and disappearance of the rash and occurs within the nervous system during encephalomyelitis.

Adolescent↗

PubMed1989

Lack of complementation between xeroderma pigmentosum complementation groups D and H.

The construction of permanent hybrid cell lines between xeroderma pigmentosum (XP) cells from different complementation groups allows analysis not only of the degree of repair correction but also of the restoration of biological activity to the UV-irradiated cells. With use of an immortal human cell line (HD2) that expresses excision repair defects typical of XP group D, a series of permanent hybrid cells has been produced with XP cells from groups A to H. Excision repair, as measured by incision analysis and unscheduled DNA synthesis, is restored to normal or near normal levels in crosses involving HD2 and cells from XP groups A, B, C, E, F, G, and I. All these hybrids show complementation for the recovery of normal UV resistance. As expected, hybrids expressing poor incision and hypersensitivity to UV were produced in crosses between HD2 and XPD fibroblasts, but they were also produced without exception when XPH was the partner. In the permanent HD2 x XPD or XPH hybrids, analysis of incision capacity reveals abnormally low activity and therefore that there has been no complementation. The true hybrid nature of HD2 x XPH cells has been confirmed by HL-A and -B tissue typing; moreover, detailed kinetic analysis of incision in these cells shows that the XPH phenotype, rather than the XPD, is expressed, i.e. breaks accumulate at low UV fluence of 1 J/m2. To help confirm these findings, another immortal XPD cell line was used in fusions involving HD2, XPH, or XPI. Cells resistant to ultraviolet were produced only with XPI fibroblasts. These data are discussed in terms of whether XPD and H mutations are likely to be allelic with respect to incision.

DNA Damage↗

PubMed1989

Molecular cloning and characterization of a mammalian excision repair gene that partially restores UV resistance to xeroderma pigmentosum complementation group D cells.

A hamster DNA repair gene has been isolated by cosmid rescue after two rounds of transfection of an immortalized xeroderma pigmentosum (XP) complementation group D cell line with neomycin-resistance gene (neo)-tagged normal hamster DNA and selection with G418 and ultraviolet irradiation. The functional length of the sequence has been defined as 11.5 kilobase pairs by measurement of the region of overlap between two hamster DNA-containing cosmids, cloned by selection for the integrated neo gene, that are able to confer an increase in resistance to ultraviolet irradiation on two XP-D cell line but not on an XP-A line. Detailed molecular characterization of the hamster repair gene has revealed no obvious similarities to two human excision repair genes (ERCC1 and ERCC2) that correct repair-defective hamster cells but have no effect on XP cells. Hybridization analyses of normal human and XP cell genomic DNAs and mRNAs, using a cosmid-clone probe from which repeated sequences have been removed, show that homologues are present and expressed in all cases.

Animals↗

PubMed1989

Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines.

The responses of two Indian muntjac cell lines to two monofunctional alkylating agents were investigated. An SV40-transformed line (SVM) had an increased sensitivity to cell killing when compared to the other, euploid line (DM) after exposure both to methyl nitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibited higher frequencies of sister chromatid exchanges (SCEs) following alkylation. The hypersensitivity of SVM to DMS correlates with the defective repair of single-strand breaks that results in the generation of long-lived breaks in the DNA following exposure, leading eventually to the formation of chromosome aberrations. In contrast no difference is seen in the formation of long-lived breaks in the DNA of SVM and DM after treatment with biologically relevant doses of MNU; in this case hypersensitivity may be due to the loss of O6-alkylguanine-DNA-alkyltransferase activity. The conclusion that the hypersensitivites of SVM to MNU and to DMS have different molecular bases is supported by transfection of SVM with plasmids containing the protein coding region of the Escherichia coli ada+ gene; subsequent expression within the cell corrects its hypersensitivity to the cytotoxic and SCE-inducing effects of MNU but has very little influence upon the lethality, SCE induction or the repair of long-lived DNA strand breaks after exposure to DMS.

Alkylating Agents↗

PubMed1989

CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers.

The CHO mutant UV61 was previously assigned to complementation group 6 of UV-sensitive rodent cell mutants. UV61 is less sensitive to killing by UV radiation than mutants such as UV5, which is highly defective in the incision process that acts on UV-induced lesions. The D37 for cell survival is approximately 4 J/m2 for UV61, compared with 10 J/m2 for the parental AA8 line and approximately 2 J/m2 for UV5. Similarly, mutation induction at the hprt and aprt loci shows an intermediate response to UV61. In a post-replication recovery assay, the kinetics of maturation of pulse-labelled nascent DNA were normal after UV irradiation in UV61. Data from alkaline elution and alkaline unwinding assays showed that the rates of break accumulation and resealing, measured 0-120 min after irradiation, were also normal in the mutant. This repair incision correlated with the rapid, normal removal of pyrimidine(6-4)pyrimidone photoproducts in UV61 measured using a radioimmunoassay that is specific for this class of damage. In contrast, after exposure to 10 or 15 J/m2, no detectable removal of cyclobutane dimers from DNA was found in UV61 while AA8 cells removed 32% by 24 h. We suggest that the mutation in UV61 specifically lowers the affinity of a repair protein for cyclobutane dimers, which are also inefficiently removed from the bulk DNA of normal CHO cells. The resistance of UV61 to killing by the direct acting chemical 7-bromomethylbenz[a]anthracene was only slightly greater than that of UV5, indicating defective repair of bulky chemical adducts in addition to cyclobutane dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Collaborative skill instruction for promoting positive interactions between mentally handicapped and nonhandicapped children.

Structuring cooperative learning activities has been shown to be an effective technique for integrating handicapped and nonhandicapped students. Previous research in this area has focused on the relative effects of cooperative versus competitive and individualistic learning situations upon peer relations and academic achievement. Few investigations have examined the various elements within the cooperative learning model that appear to promote positive peer interactions among handicapped and nonhandicapped students. The present study evaluated the influence of collaborative skill instruction versus no collaborative skill instruction on the social interaction behaviors of moderately/severely handicapped and nonhandicapped students participating in group science activities. These data reveal that students receiving collaborative skill instruction interacted more positively with one another than those who did not receive the instruction.

Adolescent↗

PubMed1989

Identification of mononuclear cells in CSF of patients with HIV infection.

Using immunocytochemical methods, CSF lymphocyte subpopulations were examined in different neurologic disorders associated with human immunodeficiency virus (HIV) infection. CSF pleocytosis was observed in asymptomatic neurologically normal subjects, in patients with aseptic meningitis, and those with inflammatory demyelinating neuropathies, but infrequently in subjects with AIDS dementia complex. The distribution of CSF lymphocyte subpopulations in HIV-infected patients differed from control subjects showing decreases in percentages of T helper (CD4) cells and increases in T suppressor (CD8) cells. Peripheral blood and CSF CD4:CD8 ratios were inverted in all of the neurologic disorders studied. In all disorders, the changes in CSF composition of mononuclear cells paralleled alterations in peripheral blood and in patients with AIDS dementia complex, there was a relationship between the severity of dementia and blood and CSF CD4 lymphocyte proportions.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Cerebrospinal fluid abnormalities in homosexual men with and without neuropsychiatric findings.

We have studied cerebrospinal fluid obtained from 38 homosexual or bisexual men participating in a prospective study of the neuropsychological disorders associated with human immunodeficiency virus (HIV) infection. Twenty-two subjects had neuropsychiatric findings and seropositivity, 11 asymptomatic subjects had seroconverted within 6 to 24 months, and 5 subjects were seronegative controls. Only 1 had acquired immunodeficiency syndrome-related complex, and none had the acquired immunodeficiency syndrome when initially studied. There was a high rate of cerebrospinal fluid abnormalities in men with neuropsychiatric findings, including pleocytosis (41%), elevated IgG and IgG index (47%), and oligoclonal bands (18%). Even in the absence of neuropsychiatric findings, the asymptomatic HIV-seropositive subjects frequently had spinal fluid abnormalities. Of those with neuropsychiatric findings, HIV p24 antigen was detectable in cerebrospinal fluid in only 1 individual, yet HIV was isolated in 11 of 16 (69%) compared to 2 of 7 (29%) of those without neuropsychiatric findings. The identification of cerebrospinal fluid abnormalities in individuals known to have become infected within 6 to 24 months supports the hypothesis that the nervous system is an early target for HIV.

Adolescent↗

PubMed1988

Spastic paraparesis and HTLV-I infection in Peru.

Three of 6 patients with spastic paraparesis in Lima, Peru, were found to have antibodies to human T-lymphotropic virus type I (HTLV-I). Blood and cerebrospinal fluid antibodies were confirmed by Western blot analysis. Multilobulated lymphocytes in blood and cerebrospinal fluid of the index case stained with monoclonal antibodies for T-helper cells and for T10, an activation marker. Blood mononuclear cells from patients with HTLV-I-associated myelopathy showed spontaneous proliferation in culture, evidence of interleukin-2 receptors, and decreased natural killer cell activity.

Adult↗