The use of uracil-N-glycosylase in the preparation of PCR products for direct sequencing.
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Base Sequence↗
Biomedical subjects
J K Ball
Publications and source records attributed to J K Ball.
Incorporation of single-stranded DNA binding protein early in polymerase chain reaction product sequencing reactions prevents enzyme pausing.
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Autoradiography↗
A common integration locus in type B retrovirus-induced thymic lymphomas.
Type-B leukemogenic retrovirus (TBLV) is a replication-competent type-B thymotropic retrovirus which lacks a transforming gene and whose genome is > 98% homologous to that of type-B mouse mammary tumor virus (MMTV). In contrast to MMTV, which induces mammary adenocarcinomas, TBLV induces a high incidence of T-cell thymic lymphomas in mice after a very short latent period. To investigate the molecular mechanisms by which TBLV induces T-cell lymphomas, we screened TBLV-induced tumor DNA for the frequent disruption of a particular cellular locus by TBLV proviral copies. In approximately 20% of the 55 primary tumors screened, the presence of proviruses in a common integration site was detected. This locus spans at least 53 kb of genomic DNA and maps to the mouse X chromosome. The presence of a functional gene at this locus is suggested by the conservation of nucleotide sequences from this locus among diverse animal species and by the expression of these sequences as mRNA in normal mouse tissues and tumors. The majority (17/18) of TBLV-induced primary tumors examined have elevated levels of this expressed mRNA.
Animals↗
A modified alkaline phosphatase enzyme amplification system and its application in an HIV antigen ELISA.
Alkaline phosphatase amplification systems increase the sensitivity of enzyme-linked immunoassays (ELISAs). Here we describe a modified method by which levamisole is a component of the substrate buffer. This increases sensitivity by reducing the signal from non-specific alkaline phosphatases. The modified procedure was applied to an 'in house' HIV-1 p24 antigen capture assay and was shown to increase the sensitivity 4-fold as compared to the unmodified system. The performance of the modified amplification system is comparable to that of commercially available systems.
Alkaline Phosphatase↗
Variations in inpatient mortality for AIDS in a national sample of hospitals.
In this project, we examined the spectrum of AIDS-related conditions and variations in associated inpatient mortality for AIDS patients treated in a national sample of hospitals. We identified 10,538 adult discharges with a diagnosis indicating AIDS from 258 hospitals from a national sample of 438 acute-care hospitals with 6 million discharges in 1986-1987. Opportunistic and other infections occurred in 55.9 and 37.9%, respectively, of AIDS discharges, and inpatient fatality rates varied considerably depending on complication type(s) and comorbidities. Clinical conditions were more important predictors of inpatient death than demographic or treatment site characteristics. Among opportunistic infections, odds of inpatient death were significantly increased for progressive multifocal leukoencephalopathy (odds ratio [OR] = 2.8), Pneumocystis carinii pneumonia (OR = 2.4), cryptococcosis (OR = 1.6), atypical mycobacterial infections (OR = 1.6), and toxoplasmosis (OR = 1.3). Odds of inpatient death were also significantly increased by non-AIDS-defining infections causing septicemia (OR = 3.1) or CNS involvement (OR = 1.6) or pulmonary involvement (OR = 1.5). After controlling for clinical conditions, significant differences in odds of death persisted across regions, age, and ethnic groups. Increases in hospitals' AIDS treatment experience were associated with a significant decrease in odds of inpatient death. These analyses provide a national perspective on the diversity of AIDS-related clinical conditions and their relative effects on inpatient mortality.
AIDS-Related Opportunistic Infections↗
A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB.
ts1 is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB which causes hindlimb paralysis in mice. Previously, it had been shown that the temperature-sensitive defect resided in the env gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly into two cleavage products, gp70 and Prp15E. This inefficient processing of gPr80env is correlated with neurovirulence. In this study, it was shown that a single amino acid substitution, Val-25----Ile in gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env at the restrictive temperature, and neurovirulence of ts1. At the restrictive temperature, a steady-state level of nonprocessed, endoglycosidase H-sensitive gPr80env remained in the endoplasmic reticulum of cells infected by ts1, but no endoglycosidase H-resistant gPr80env and only trace amounts of gp70 were detected in the infected cells. Since the host cell-encoded processing protease resides in the cis cisternae of the Golgi apparatus, inefficient processing of gPr80env at the restrictive temperature is most likely due to inefficient transport of gPr80env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to misfolded gPr80env being a poor substrate for the processing protease at the restrictive temperature.
Animals↗
Characterization of cell markers in type B retroviral-induced thymic lymphomas--I. Surface antigen phenotype and karyotype in developing and primary lymphomas.
CFW/D mice injected neonatally with a type B retrovirus, (DMBA-LV), rapidly develop thymic lymphomas. The present study examined simultaneously the karyotype and the expression of surface antigens on thymocytes from DMBA-LV treated mice at 28, 35 and 42 days of age during the development of lymphomas, and of cells from primary lymphomas. DMBA-LV treatment resulted in disturbances in the proportions of thymic Lyt 1+2-, Lyt 1-2+ and Lyt 1+2+ subpopulations. As a group, virus-treated mice showed a significant decrease in the size of the Lyt 1+2- subpopulation in thymuses during tumor development. However, developing tumors and primary tumors showed individual patterns of alteration in the proportions of thymocyte subpopulations rather than consistent trends for any one thymocyte subpopulation to increase or decrease. Cells with the abnormal chromosome complement, trisomy 15, were present early in the development of tumors and became the predominant karyotype in fully developed tumors. A correlation between the appearance of trisomy 15 and a particular thymocyte surface antigen phenotype was not detected. The results suggest that retrovirus infection disrupts, but does not eliminate the ability of developing thymocytes to form phenotypically distinct subpopulations.
9,10-Dimethyl-1,2-benzanthracene↗
Characterization of cell markers in type B retroviral-induced thymic lymphomas--II. Surface antigen phenotype, karyotype and proviral integration pattern in cultured lymphoma cells and cloned lines.
This study examined the surface antigen phenotype, karyotype and proviral integration patterns of cultured cells from murine thymic lymphomas induced by injecting neonatal mice with the type B leukemogenic retrovirus (DMBA-LV). Cells from the primary thymic lymphomas were established in mass culture and from these, clonal tumor cell lines were derived. During in vitro culture of lymphoma cells, Lyt 1-2+ cells predominated with an apparent selection against cells of the Lyt 1+2- and Lyt 1+2+ phenotypes. Of 21 cloned lines established, five had a diploid chromosome complement and expressed the Lyt 1-2+ phenotype. The other 16 clones lines were characterized by trisomy of chromosome 15 and expressed the Lyt 1+2+ or Lyt 1-2+ phenotype. Cells characterized by either a diploid or trisomy chromosome complement were capable of growth in vivo. Southern blot analyses showed that during growth in culture, cells from the mass cultures and cloned lines continued to acquire low numbers of new integrated DMBA-LV proviral copies while maintaining the basic proviral integration pattern present in the DNA from cells of the primary lymphomas. These findings support the notion that the acquisition of new genetic information in cells from DMBA-LV-induced thymic lymphomas may contribute to the continual generation of tumor heterogeneity.
9,10-Dimethyl-1,2-benzanthracene↗
A severity classification system for AIDS hospitalizations.
The authors describe a new model for classifying hospitalized patients with acquired immunodeficiency syndrome (AIDS) according to their severity of illness. In the first phase of the project, a clinical model indicating the relative severity of AIDS complications was formulated, based on the clinical literature and expert opinion. In the second phase, the model was tested empirically using data on in-patient mortality of over 6,000 adult AIDS hospitalizations in New York State during 1985. The ordering of AIDS-related complications in the clinical model was revised to reflect a continuum of increasing likelihood of death in the hospital. The final classification system for AIDS hospitalizations has 20 substages, grouped into three stages, with in-patient mortality rates increasing from 6% to 60%. The system is automated and can be applied to different AIDS populations to analyze resource use and outcomes of hospital care.
Acquired Immunodeficiency Syndrome↗
Duration and costs of AIDS hospitalizations in New York. Variations by patient severity of illness and hospital type.
This study uses the Severity Classification for AIDS Hospitalizations to examine and contrast the duration and costs of hospitalizations among 6,142 AIDS discharges from New York hospitals during 1985. Multivariate regression analyses of resource-use differences suggest that more severely ill AIDS patients have considerably longer stays and higher total charges, regardless of hospital type. Severity of illness alone accounts for most of the explained variation in hospital length of stay (LOS) and total charges. In contrast, other patient characteristics, such as gender, race, and reported drug use, are less important predictors of resource use. It was also found that public teaching hospitals serve somewhat more severely ill AIDS patients than those treated in private teaching hospitals. The study concludes that the AIDS severity classification facilitates interhospital comparisons of illness levels and variations in resource use.
Acquired Immunodeficiency Syndrome↗
Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus.
We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.
Base Sequence↗
Characterization of early molecular biological events associated with thymic lymphoma induction following infection with a thymotropic type-B retrovirus.
A highly leukemogenic virus (DMBA-LV) induces thymic lymphomas with a very short (40 days) latent period. All induced tumors contain low numbers of new integrated DMBA-LV type-B proviruses and tumorigenicity of DMBA-LV is completely abolished by a monoclonal antibody directed toward an envelope determinant present on a type-B mammary tumor-inducing viral isolate. While the DMBA-LV type-B genome is very highly related to mammary tumor-inducing isolates it does have unique gp52 and p28 proteins as well as unique restriction endonuclease sites. In the present study the target cell specificity of DMBA-LV was contrasted with that of the mammary tumor-inducing isolate MMTV (C3H). The results indicated that infection of CFW/D mice with DMBA-LV could be detected in the thymus only as early as 17 days postinfection and by 40 days postinfection all 40 thymuses examined contained new integrated proviral copies of DMBA-LV. In contrast, when mice were injected intrathymically with MMTV (C3H) virus infection was transiently detected in the thymus only at 28 days postinfection. By 35 and 42 days postinfection there was no indication that virus-infected cells were still present. Analysis of individual thymic lobes following DMBA-LV infection suggested that independent tumors may be initiated in each of the separate lobes. Furthermore, there appeared to be a correlation between the weight of the lobe and the number of new DMBA-LV proviral copies, the larger the lobe the greater the number of newly integrated proviral copies.
Animals↗
Induction of thymomas by N-methyl-N-nitrosourea in AKR mice: interaction between the chemical carcinogen and endogenous murine leukaemia viruses.
AKR mice develop thymomas spontaneously when greater than 6 months old but when young AKR mice are treated with N-methyl-N-nitrosourea (MNU) they develop thymomas at 3-6 months of age. In this study the potential role of oncogene activation in the development of both the spontaneous and MNU-induced thymomas in AKR mice has been examined by DNA transfection into NIH3T3 mouse fibroblasts and by Southern analysis of tumour DNA. The results show that a high proportion of MNU-induced thymomas contain activated cellular rasK while no activated cellular ras genes were detected in spontaneous thymomas. Southern analysis of tumour DNA revealed that 2/30 spontaneous tumours and 2/52 MNU-induced tumours contained alterations in the c-myc gene while 5/29 spontaneous tumours and 6/56 MNU-induced tumours contained alterations in the Pim-1 gene. A more detailed analysis of the Pim-1 gene demonstrated that the alterations observed in most MNU-induced and spontaneous tumours resulted from proviral integration at the 3' end of this gene. Our analyses also demonstrated that the majority of MNU-induced tumours, including those containing rearrangements in the Pim-1 gene, lacked the somatically acquired recombinant MCF proviruses that are present in most spontaneous AKR lymphomas. These results provide evidence that the mechanisms of development of MNU-induced and spontaneous tumours in AKR mice are distinct and the development of thymomas that contain proviral integrations at the Pim-1 locus in the MNU-treated AKR mice involve cooperation between the chemical carcinogen and endogenous murine leukaemia viruses.
Animals↗
Spontaneous and induced primary oncogenesis in natural killer (NK)-cell-deficient beige mutant mice.
Spontaneous tumor development and primary oncogenesis were compared in a large number of NK4-deficient, homozygous C57Bl/6-bg/bg mice and their NK normal, heterozygous +/bg littermate controls. In a group of 167 retired breeders followed for spontaneous tumors, the probability of survival for mice eventually dying with a tumor was greater for the NK-competent, +/bg than for the NK-deficient, homozygous C57BL/6-bg/bg mice (p = 0.0019), although the higher overall incidence of tumors in the bg/bg group (48%) was not significantly different from that in the +/bg group (37%). In the bg/bg group the incidence of tumor death appeared to increase relatively sharply in the 25- to 29-month age bracket compared to the fairly regular increase in incidence observed in the +/bg group. All the spontaneous tumors except 2 (discovered in +/bg mice) were classified histologically as widely disseminated malignant lymphomas. The other two were one squamous-cell carcinoma and one sarcoma. A total of 73 bg/bg mice injected s.c. with benzo[alpha]pyrene (BP) had a higher overall incidence of tumors (81%) (rhabdomyosarcomas) than 138 +/bg mice (64%) and in the largest group (0.3 mg, n = 85) the bg/bg group developed tumors, at a higher incidence (p = 0.01) and with a shorter latency (p = 0.025) than the +/bg group. On the other hand, mice injected with dimethylbenzanthracene or given 4 weekly doses of 160 rads of gamma irradiation showed no difference in overall tumor incidence. In addition, mice injected with various doses of DMBA-induced murine leukemia virus (DMBA-LV) also showed no significant difference in tumor incidence. Others have reported that some of these treatments (DMBA, split-dose irradiation) cause profound NK suppression, thereby reducing NK differences between the two groups of mice. These results suggest that a partial NK impairment in beige mutant mice early in life may lead to significantly greater rates of death with spontaneous malignant tumors late in life. Some primary oncogenesis treatments (BP) but not others (DMBA, split-dose irradiation, leukemia viruses) cause tumors with a greater incidence and shorter latency in beige mice. The results suggest, but do not prove, that NK cells play a role in surveillance against spontaneously arising, and possibly some types of carcinogen-induced, tumors.
9,10-Dimethyl-1,2-benzanthracene↗
Thymotropic envelope gene recombinants of Moloney leukemia virus have highly conserved envelope structures.
Envelope gp70s were isolated from the thymotropic recombinant viruses related to Moloney murine leukemia virus (RM-M-MuLVs) which were generated by the inoculation of two strains of ecotropic M-MuLV (strain 1869 and temperature-sensitive mutant-1) into BALB/c or CFW/D mice. Chymotrypsin oligopeptide maps of parental ecotropic MuLV, RM-M-MuLV, and inducible xenotropic MuLV showed each of the above virus types had a distinctly characteristic peptide map. The majority of RM-M-MuLV gp70 molecules examined showed a high degree of peptide homology. Data from restriction endonuclease mapping demonstrated that the newly acquired sequences in each of the RM-M-MuLVs were very related and encompassed both the polymerase and the envelope genes. The source of the sequences acquired by the RM-M-MuLV was from endogenous nonecotropic and nonxenotropic proviruses. This suggested that the family of endogenous proviruses which combined with the parental ecotropic virus was either specifically selected or was much more available than other endogenous proviruses. Although slight variations of envelope-specific sequences and peptides existed among various RM-M-MuLV isolates; within a single thymoma, individual clones of tumor cells yielded RM-M-MuLV gp70s which were identical to each other. These findings are discussed within the context of the leukemogenic potential of RM-MuLVs.
Animals↗
The involvement of a type-B retrovirus in the induction of thymic lymphomas.
A highly leukemogenic virus (DMBA-LV) (in vivo leukemogenic titer 1-5 X 10(6) IU/ml, and 35-40 days to thymic lymphoma detection) is produced by a chemical carcinogen-induced transplanted thymic lymphoma. The virus preparation is a mixture of a type-B retrovirus highly related to exogenous type-B retroviral isolates and a biologically defective type-C retrovirus. The DNA of DMBA-LV-induced-tumors contains new type-B proviruses but no additional type-C proviruses could be detected. The leukemogenicity of DMBA-LV was completely neutralized by a monoclonal antibody against MMTV envelope glycoprotein, but was not affected by a broadly reacting Friend MuLV anti-gp70 serum which effectively neutralizes type-C ecotropic, xenotropic, and recombinant retroviruses and which completely abolishes the leukemogenic activity of Moloney leukemia virus. Three type-B mammary tumor-inducing retroviral isolates, while containing type-C retroviral sequences, were not leukemogenic. A further characterization of the type-C retroviral sequences present in DMBA-LV indicated that sequences characteristic of endogenous, nonxenotropic proviruses are present. In addition, using a variety of type-C-specific retroviral DNA probes, no evidence was obtained for the presence of a type-B-C-recombinant genome in DMBA-LV. Leukemogenesis was absolutely dependent upon the presence of a functional type-B retroviral envelope gp 52 and DMBA-LV does not appear to contain a leukemogenic retroviral type-C genome.
Animals↗
Karyotypic variation of cells from murine thymic lymphomas during short-term culture.
The present study examined the balance in the proportion of cells with either trisomy 15 or a diploid chromosome complement during short-term culture in vitro. Neonatal CFW/D mice were given an intrathymic injection of an endogenous virus (DMBA-LV) isolated from a DMBA-induced thymic lymphoma. The thymus was removed at the terminal leukemic stage, and the cells from each enlarged thymus were cultured in quintuplet. At 0, 6, 12, 24, and 48 hr, cultures were given colcemid for 2 hr prior to harvesting for chromosome examination. The analyzed lymphomas can be divided into three groups according to the characteristics of their stem lines and sidelines, as revealed by direct chromosome preparation (0 hr). Two groups consisted of lymphomas carrying a stem line with either trisomy 15 (group 1) or a diploid karyotype (group 2). A third group (group 3) consisted of lymphomas with a mixture of an abnormal stem line and sidelines. Chromosome analysis of cultures at subsequent times showed that there were fluctuations in the proportion of cells characterized as either stem line or sideline in all tumors at different times during culture. The initial sideline in group 1 tumors had a diploid complement. Although the proportion of the diploid cells increased in 5 of the 6 tumors following either 24 or 48 hr in culture, cells with a diploid karyotype remained as a sideline throughout the culture period. Similarly, the two tumors analyzed in group 2 showed that the initial sideline that contained trisomy 15 increased temporarily to become the stem line following 6 or 12 hr in culture. Group 3 tumors showed a slight variation in the proportion of cells with an abnormal stem line or sideline during culture. Considering all the data from the 10 tumors, 7 tumors had cells with a diploid karyotype, and, although the proportion of cells with this karyotype increased (4 tumors) or persisted (3 tumors) during 48 hr in culture, it remained as a sideline and was not overgrown by trisomy 15 stem line.
Animals↗
Molecular biological characterization of a highly leukaemogenic virus isolated from the mouse. IV. Viral proteins.
The proteins of a highly leukaemogenic murine virus (DMBA-LV) endogenous to the CFW/D mouse have been characterized. This virus does not contain ecotropic, xenotropic or polytropic type C retroviruses capable of replicating in established tissue culture cell lines. Analysis of the viral proteins indicated that proteins characteristic of both type C and type B retroviruses were present. All the proteins characteristic of a competent, mature type B retrovirus, such as the murine mammary tumour virus, were present in the virions and were expressed at high levels in a virus-producing thymic lymphoma cell line. Type C internal structural proteins were detected in the virus and cells producing virus. Type C envelope protein was detected at very low levels in both the virions and the virus-producing tumour cell lines. Chymotryptic polypeptide profiles of this gp70 indicated similarity with the envelope glycoproteins characteristic of xenotropic and recombinant type C retroviruses.
9,10-Dimethyl-1,2-benzanthracene↗