Biomedical subjects
Publications and source records attributed to J Lyons.
In this review, I describe the polymerase chain reaction (PCR) and its application to cancer diagnostics. I present recent improvements to PCR methodology, and describe some state-of-the-art detection methods and approaches used to diagnose various cancer markers, including ras genes, BCR/abl translocations, and G-proteins.
The development of malignant tumors of the peripheral nervous system (schwannomas) within a defined intracranial section of the rat trigeminal nerve ("trigeminal box") was used as a model to identify genetic alterations typically associated with the process of cell-lineage-specific oncogenesis induced by exposure to N-ethyl-N-nitrosourea on postnatal day 1. All 47 trigeminal schwannomas (and 12 extracranial neurinomas) investigated carried a T.A----A.T transversion mutation at nucleotide 2012 of the neu (erbB-2) gene sequence encoding the transmembrane domain of pg185neu. This mutation was absent in all 18 tumors in the brain and spinal cord (central nervous system) isolated from the same animals. Identical observations were made in cell lines derived from N-ethyl-N-nitrosourea-induced rat schwannomas vs. brain tumors. By asymmetric PCR and mutant-specific Mnl I restriction fragment length analyses, cells carrying the mutant neu allele became detectable and could be localized within the trigeminal box as early as 7 days after the carcinogen pulse. The proliferation rate of the mutant cells strongly exceeded that of the wild-type cells up to the time of maturation of the trigeminal nerve around postnatal day 30 and thereafter to a lesser extent until the appearance of schwannomas. A specific mutation of the neu gene thus represents a very early, probably the first, step in the malignant conversion of immature rat Schwann cells exposed to N-ethyl-N-nitrosourea in vivo and is diagnostic for a subset of proliferative cells at high risk of progressing toward the expression of fully malignant phenotypes. Loss of heterozygosity for the mutant neu allele is a candidate event for a critical second step in the process.
In the present study methods are described for the analysis of the genomic structure of DNA isolated from tissues that have been stored up to several years as air-dried or stained bone marrow smears, bone marrow biopsies in "Histicon", chromosomal preparations in fixative or as formalin fixed tissues embedded in paraffin. By application of the new techniques clonal B- and T-cell disorders, Philadelphia chromosome translocations and ras oncogene mutations in pancreatic carcinomas could be detected. Thus, these DNA extraction procedures may open new avenues to pathological archives and enable the analysis of samples when fresh material is not available.
This 1990 study examines the relationship between the degree of use of patient care resources and the degree of supervision of residents by attending physicians (as perceived by residents) at a large midwestern teaching hospital. Ratings of the degree of clinical autonomy allowed residents by 65 attending physicians--each of whom had a general internal medicine practice with a significant hospital component--were provided by 23 former internal medicine chief residents and 17 internal medicine residents who were in their third year at the time of the study. A regression model was used to test the association between hospital resource use (as shown by total hospital charges to patients and their lengths of stay) and the residents' mean ratings of the degrees of autonomy the attending physicians permitted residents, for 7,169 of these physicians' patients discharged between 1986 and 1989 in 28 diagnosis-related groups. The analysis was controlled for patients' insurance status and chronic disease comorbidities. The patients whose attending physicians were rated as allowing substantial clinical autonomy had significantly lower total charges and lengths of stay (p less than .0001). These results suggest that internal medicine residents have an inherently conservative practice style that values low-intensity workups and rapid discharge of patients.
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Aristolochic acid I (AAI), a nitrophenanthrene derivative, is the major component of the carcinogenic plant extract aristolochic acid, which has been used as a medicine since antiquity. Long term oral administration of AAI to male Wistar rats induces multiple tumors, mainly in the forestomach, ear duct, and small intestine. The presence of activated transforming genes was investigated in various tumors of 18 AAI treated rats, namely in 14 squamous cell carcinomas of the forestomach, 7 squamous cell carcinomas of the ear duct, 8 tumors of the small intestine, 3 tumors of the pancreas, 1 adenocarcinoma of the kidney, 1 lymphoma, and 2 metastases in the lung and the pancreas. By utilizing the tumorigenicity assay and Southern blot analysis, we have detected an activated c-Ha-ras gene in the DNAs of 5 of 5 squamous cell carcinomas of the forestomach. Direct sequencing of amplified material revealed an AT----TA transversion mutation at the second position of codon 61 of the c-Ha-ras gene (CAA to CTA) in all transfectants as well as in the 5 original rat tumors. Enzymatic amplification of ras sequences followed by selective oligonucleotide hybridization detected identical mutations in 93% (13 of 14) of forestomach tumors, in 100% (7 of 7) of ear duct tumors, and in the lung metastasis. Among those tumors tested, we had 4 cases in which the forestomach tumors and the ear duct tumors originated from the same rat, showing the same mutation in both tissues. Moreover, similar mutations were demonstrated at c-Ki-ras codon 61 in 1 of 7 ear duct tumors (CAA to CAT) and in 1 of 8 tumors of the small intestine (CAA to CTA) as well as at c-N-ras 61 (CAA to CTA) in a pancreatic metastasis. Additional transfection experiments of some tumors scoring negative for ras gene mutations in dot blot analyses revealed a CAA to CTA transversion at codon 61 of the c-Ha-ras gene in 1 forestomach tumor as well as at codon 61 of the c-N-ras in 1 hyperplasia of the pancreas and in 1 lymphoma. The apparent selectivity for mutations at adenine residues in AAI induced tumors is consistent with the identification of an N6-deoxyadenosine-AAI adduct formed by reaction of AAI with DNA in vitro, suggesting that carcinogen-deoxyadenosine adducts are the critical lesions in the tumor initiation by aristolochic acid.
Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.
We have analysed, by Northern blots, the expression of 14 cellular oncogenes in nine cell lines established from human teratocarcinomas. All lines expressed considerable amounts of p53, c-Ki-ras2, c-Ha-ras1, c-raf1, N-myc, and c-fos. Low level expression of c-myc was detected in some lines. Southern blot experiments revealed no amplification or rearrangement of the c-Ki-ras2, N-myc or c-fos genes. Using a rapid dot-blot screening procedure, based on a combination of in-vitro amplification of ras-specific sequences and oligonucleotide hybridization, we could detect no activation of Ha-ras or Ki-ras or any unexpressed N-ras sequences secondary to a point mutation at codons 12, 13, or 61.
Eight Mycobacterium tuberculosis and M. leprae genes were inserted into the vaccinia virus genome by in vivo recombination. The resulting virus recombinants were shown to express five different M. tuberculosis proteins (71, 65, 35, 19, and 12 kDa) and three M. leprae proteins (65 and 18 kDa and a biotin-binding protein) by Western immunoblot analysis, radioimmunoprecipitation, or black-plaque assay. When injected into BALB/c mice, the recombinants expressing the M. tuberculosis 71-, 65-, or 35-kDa protein and the M. leprae 65-kDa protein or the biotin-binding protein elicited antibodies against the appropriate M. tuberculosis or M. leprae protein. These vaccinia virus recombinants are being tested for the ability to elicit immune protection against M. tuberculosis or M. leprae challenge in animal model systems. The recombinants are also useful in generating target cells for assays aimed at elucidating the cellular immune responses to mycobacterial proteins in leprosy and tuberculosis. Furthermore, the M. tuberculosis 65-kDa protein and four of the other mycobacterial proteins share homology with known eucaryotic and procaryotic stress proteins, some of which may play a role in autoimmunity.
Activating mutations in the gene for the alpha-chain of Gs, the stimulatory regulator of adenylyl cyclase, have been identified in human GH-secreting pituitary tumors. Using the polymerase chain reaction and allele-specific oligonucleotide hybridization, we screened 25 GH-secreting tumors for the presence of the activating mutations. We also reviewed the clinical charts of the patients from whom the tumors were removed. Of 25 tumors, 10 (40%) contained activating mutations. Patients in the mutation-positive group came to surgery with smaller tumors and had lower GH levels. The activating mutations identify a subgroup of GH-secreting pituitary tumors that probably arise from a shared oncogenic mechanism.
The frequency of Ki-ras gene mutations was studied in 100 paraffin-embedded sections obtained from 63 pancreatic adenocarcinomas by in vitro amplification of target sequences via polymerase chain reaction (PCR) and selective oligonucleotide hybridization. Forty-seven (75%) of the tumors contained a Ki-ras mutation at codon 12. No predominant amino acid substitution or nucleotide transition at this codon was observed. Two carcinomas exhibited 2 distinct Ki-ras mutations. No particular correlation could be established between the incidence of Ki-ras mutation and clinical parameters (sex, age, survival), tumor grade or tumor stage.
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A survey completed by 280 nonvolunteer, urban emergency medicine technician (EMT)-paramedics revealed high levels of occupational stress. We used a four-component model of occupational stress in medical environments to show indications that much variation in the manifestation of stress was accounted for by the rank and job description of the EMT-paramedic, the district served by the EMT-paramedic, and the patient population served by the EMT-paramedic. Stress exhibited by field EMT-paramedics tended to manifest in more negative attitudes toward patients, whereas administrative-level paramedics exhibited more organizational stress. We noted that the age of the EMT-paramedic and the length of time employed as an EMT-paramedic correlated with the level of occupational stress (P less than .05). The recent occurrence of significant life events also was significantly related to the level of stress (P less than .05). An EMT-paramedic's gender, marital status, and number of calls per shift had no significant correlation to the level of occupational stress. Based on these results, we recommend tailoring occupational stress programs to meet the needs of individual EMT-paramedics. Special attempts should be made to identify and counsel EMT-paramedics who are undergoing stressful life events. Finally, we suggest that rotating EMT-paramedics through various districts on a regular basis may help alleviate the negative impact on patient care in areas that have been identified as particularly stressful. Further studies are needed to verify our hypothesis.
Phase and amplitude analysis was applied to intravenous digital left ventriculograms to avoid the artefacts associated with image subtraction. Eight controls and 40 patients with known coronary artery disease underwent digital left ventriculography before and after a symptom limited supine bicycle exercise test. The resultant images were subjected to phase and amplitude analyses. In the control group there was no deterioration in left ventricular wall motion after exercise. In 30 of the 40 patients there was a deterioration in wall motion on exercise. This group contained all eight patients with three vessel disease and 12 of the 17 patients with two vessel disease. Ten patients showed no change in wall motion--five with one vessel disease and five with two vessel disease. Phase and amplitude analysis of digital left ventriculograms is a method of detecting exercise induced myocardial ischaemia that may help in the assessment of patients with coronary artery disease.
Restriction fragment length polymorphisms (RFLPs) of the X-chromosome genes hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) were studied in 34 female patients with primary myelodysplastic syndromes (MDS). Twelve patients (35%) were heterozygous at the HPRT or PGK loci for BamHI or BglI RFLPs, respectively. In eight patients showing PGK polymorphisms, clonality was determined by X-chromosome inactivation analysis. These included patients from different morphologic subtypes: four with refractory anemia (RA), two with RA and ring sideroblasts (RARS), one patient with RA with excess of blasts (RAEB), and one with chronic myelomonocytic leukemia (CMML). A monoclonal pattern of X-chromosome inactivation was observed in seven cases. In a further case characterized by bone marrow hypoplasia, peripheral blood (PB) leukocytes were polyclonal in origin. Following low-dose cytarabine therapy, reversion to polyclonal hematopoiesis was observed in a case of RAEB indicating the presence of residual normal hematopoietic stem cells with the capacity for marrow reconstitution. The clonal relation of lymphoid and granulocyte/monocyte lineages was studied directly in two cases of CMML exhibiting somatic mutations of N-ras or Ki-ras oncogenes. By selective oligonucleotide hybridization to ras gene sequences amplified in vitro by the polymerase chain reaction, a mutated ras allele was demonstrated in PB granulocytes, monocytes, and B and T lymphocytes of both patients. We conclude that MDS arise from a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation.
In vitro DNA amplification and synthetic oligonucleotide hybridization was used to analyze 57 acute myelocytic leukemias (AML) for the presence of ras gene mutations. We demonstrated mutated alleles in 19% of primary AMLs (10/51) as well as in five of six secondary leukemias. Mutations occurred predominantly at N-ras codons 12, 13, or 61 (13 cases) and twice at Ki-ras codons 12 and 13. Ras gene mutations were preferentially associated with an M4 morphology according to the FAB (French-American-British) classification, but no particular correlation was observed with respect to clinical parameter (sex, age, course of disease) or immunophenotype and karyotype. Mutated ras alleles were absent in nine mutation-positive cases analyzed during remission. However, a more complex pattern emerged from the five patients analyzed in relapse exhibiting identical ras mutations in three cases, absence of a mutated allele in one patient, and acquisition of a N-ras mutation in yet another case, in which no mutation had been detected initially. Moreover, restriction fragment length polymorphisms (RFLP) of the X-chromosome genes hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) were studied in 19 of the AML patients. Nine cases (47%) were heterozygous for BglI or BamHI RFLPs at the PGK or HPRT loci, respectively, and therefore suitable for clonal analysis investigating X-chromosome inactivation. All of the patients exhibited a monoclonal leukemic cell population at presentation. In addition, five of seven cases studied in remission showed reemergence of a polyclonal pattern. However, two children exhibited persistence of monoclonal hematopoiesis despite complete clinical/hematological remission and a corresponding loss of a mutated ras allele in one of the patients. These data indicate the value of molecular genetic approaches for evaluation of the heterogeneous nature of remission and relapse in AML.
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