The value of routine screening for peripheral arterial disease in stable outpatients with a history of coronary artery or cerebrovascular disease.

BACKGROUND: Frequently unrecognised, PAD is associated with reduced quality of life and an increased mortality rate because of a greater propensity for fatal ischaemic events. PAD commonly coexists with coronary and cerebrovascular disease and is associated with poorer outcomes in such patients. The Edinburgh Claudication Questionnaire (ECQ) and the ankle-brachial index (ABI) are screening methods to identify the presence of PAD. This study used these methods to estimate the rate of previously undiagnosed PAD and to validate the ECQ against ABI in a Canadian outpatient population with manifest cerebrovascular or coronary disease. METHODS: At a regular office visit, patients completed the ECQ and were categorised as ECQ(+) or ECQ(-). All ECQ(+) and a randomly selected 25% of ECQ(-) patients were referred for ABI measurement. An ABI < 0.9 was considered positive. The prevalence of PAD in the patient population and the sensitivity and specificity of the ECQ score against the ABI were assessed. RESULTS: Of 2235 patients enrolled, 815 were selected for an ABI [ECQ(-), n = 478; ECQ(+), n = 337]. Extrapolated PAD prevalence in the total population was 12.3% (highest arterial pressure [HAP] method) and 20.8% (lowest arterial pressure [LAP] method), with a significantly higher prevalence found in diabetic patients than non-diabetic patients (p < 0.0001). Because ECQ is only a measure of symptomatic disease, it had poor sensitivity (35.3% and 25.8%), but high specificity (88.2% and 88.3%) using the HAP and LAP methods of ABI measurement, respectively. CONCLUSIONS: Undiagnosed PAD is common in stable outpatients with a prior history of manifest cardiovascular disease, particularly in those with diabetes. The ECQ does not possess the diagnostic value of the ABI in detecting PAD in this high-risk population, but may be useful to raise suspicion of PAD to be confirmed by ABI assessment.

Immunospecificity of nuclear nonhistone protein-DNA complexes in colon adenocarcinoma.

Tumor-specific antisera against dehistonized chromatin isolated from transplantable colon adenocarcinoma (from male noninbred Sprague-Dawley rats) were produced. The specificities of these antisera were determined by complement fixation. In the presence of these antisera, only chromatin from colon adenocarcinoma significantly fixed complement, whereas chromatins isolated from normal rat colon epithelia were inactive. Administration of 1,2-dimethylhydrazine to rats produced an early change in the immunospecificity of colon epithelial chromatin similar to that for colon adenocarcinoma. Several lines of experimental evidence indicated that nuclear antigen was not a carcinoembryonic antigen-like substance. Common antigens were also present in human colon adenocarcinomas.

Tissue-specific antibodies against human lung and breast carcinoma dehistonized chromatins.

Tissue-specific antisera against human lung and breast carcinoma dehistonized chromatins were obtained. The specificity of these antisera was determined by complement fixation. In the presence of antiserum against human lung carcinoma, only chromatins from lung carcinoma fixed complement significantly, whereas chromatins isolated from human breast carcinoma, HeLa cells, normal lung tissue, breast tissue, or term placenta were negative (i.e., inactive). In a similar assay with the use of antiserum against dehistonized breast carcinoma chromatins, only breast carcinoma chromatins fixed complement. Immunohistochemical localization of the antigens by the horseradish peroxidase bridge method demonstrated their presence in the nuclei.

Cellular binding proteins for vitamin A in colorectal adenocarcinoma of rat.

Rat colorectal mucosa was examined during the course of carcinogenesis, induced by chronic administration of 1,2-dimethylhydrazine (DMH), for the presence and amount of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein. These two binding proteins are implicated in the action of vitamin A in normal and neoplastic tissue. Induced adenocarcinomas were found to contain low levels of cellular retinoic acid-binding protein (10 pmol/g), similar to the levels found in adjacent mucosa of the same animal and also in colorectal mucosa from normal rats or rats chronically treated with DMH. However, the adenocarcinomas had high levels of CRBP (300 to 500 pmol/g), and these levels were dramatically higher than levels of CRBP in adjacent mucosa of the same animal (40 to 100 pmol/g), colorectal mucosa from normal rats (20 pmol/g), or colorectal mucosa from rats chronically treated with DMH (22 to 25 pmol/g). Consequently, the increase in CRBP occurred only with tumor appearance and not with the general hyperplasia of the crypts caused by DMH administration. The CRBP of the tumor was associated with endogenous retinol (77 to 100% saturation) and was similar to, if not identical with, CRBP of normal tissue, as judged by fluorescence spectra, sedimentation behavior, and elution position on Sephadex G-75.

Isolation of rat alpha1-fetoprotein messenger RNA from Morris hepatoma 7777.

A double-antibody procedure has been developed for the isolation of alpha1-fetoprotein (AFP)-synthesizing polysomes from Morris hepatoma 7777. The polyadenylic acid-containing RNA, subsequently purified by differential sedimentation on sucrose gradient and oligodeoxythymidylic acid-cellulose chromatography, migrates as a single 21S component in polyacrylamide gel electrophoresis; in a cell-free translation system, it yields a peptide product immunoprecipitable by anti-rat AFP antiserum, but not by anti-rat albumin, and which migrates slightly faster than serum AFP on sodium dodecyl sulfate-urea-polyacrylamide gels. This messenger RNA fraction was used for the synthesis of a radioactive complementary DNA. In hybridization assays, the complementary DNA reassociated with its purified template at a Cr0t1/2 [product of RNA concentration (mol of nucleotides per liter) X half-time (sec)] of 1.5 X 10(-2). By constitute 3,2, and less than 0.01% of total polyadenylic acid-containing polysomal RNA of Morris hepatoma 7777, 10-day-old-rat liver, and adult rat liver, respectively. The high specificity of the polysome immunoprecipitation system, the electrophoretic homogeneity of the isolated messenger RNA fraction, its selective translation into AFP, and the specificity of the hybridization probe indicate that the procedure described yields a highly purified rat AJP messenger RNA.

Changes in tumor-specific nuclear antigen activity in carcinogen-treated colon by tumor promoter and carcinogen inhibitors.

Changes in the immunospecificity of the nuclear antigens were demonstrated in the colon chromatin of rats treated with 1,2-dimethylhydrazine. Tumor-specific nuclear antigen appeared in the early stages of chemically induced colon carcinogenesis. Sodium barbiturate, in conjunction with the carcinogen, induced a higher level of nuclear antigen activity than that obtained with carcinogen alone. The rise of immunoactivity in carcinogen-treated colon chromatin can be abolished by simultaneous treatment with disulfiram or butylated hydroxytoluene.

Tissue-specific DNA-protein complexes during azo dye hepatocarcinogenesis.

The immunological tissue specificity could be transferred from one chromatin preparation to another by reconstituting this protein fraction to the DNA and the remaining chromatin components."The immunological tissue specificity could be transferred from one chromatin preparation to another by reconstituting this protein fraction this protein fraction to the DNA and the remaining chromatin components.

Effect of sodium butyrate on alpha-fetoprotein gene expression in rat hepatoma cells in vitro.

Sodium butyrate has been reported to induce cellular differentiation and reduce the tumorigenicity of certain tumor cells. We have examined the effects of butyrate on alpha-fetoprotein (AFP) gene expression in 7777 and McA-RH8994 rat hepatoma cells and have found that nontoxic concentrations of the drug decrease AFP mRNA levels in both cell lines. However, McA-RH8994 requires a 10-fold lower concentration (0.5 mM) of butyrate to affect a 50% reduction in AFP mRNA levels within 48 h. At 2 mM, sodium butyrate reduces AFP mRNA levels in McA-RH8994 cells by at least 90% after 48 h, while having little effect on the expression of either the 7S RNA or Harvey-ras genes. Time-course studies show that the effect of butyrate on McA-RH8994 AFP mRNA levels is immediate and is accompanied by an accumulation of cells in the G1/G0 phase of the cell cycle. Sodium butyrate was found to reduce AFP mRNA levels in both dexamethasone-treated 7777 and McA-RH8994 cells; dexamethasone decreases AFP mRNA levels in the former cell line and increases AFP mRNA levels in the latter. Therefore, it is unlikely that butyrate acts simply by reducing the dexamethasone receptor concentration in 7777 cells.

Dexamethasone inhibition of rat hepatoma growth and alpha-fetoprotein synthesis.

The effect of dexamethasone on hepatoma growth and differentiation, as well as the production of alpha-fetoprotein (AFP) and albumin, was investigated. Treatment of rats with dexamethasone strongly reduced (by 83 to 98%) the serum levels of AFP in rats bearing Morris hepatomas 7777, 8994, 7288c , and 9618A2 . Reduced AFP levels were due in part to a large reduction in tumor load in dexamethasone-treated rats. Hepatoma weights, on the average, were reduced by 64 to 90% relative to controls, while a large bowel transplantable tumor was affected only slightly. Lower serum AFP levels in rats with hepatomas 7777, 8994, and 9618A2 also resulted from reduced AFP synthesis, as indicated by lower cytoplasmic AFP levels. Cytoplasmic albumin levels were higher in dexamethasone-treated rats bearing hepatomas 7777, 8994, and 7288c than they were in rats which did not receive dexamethasone. RNA dot hybridization also indicated that dexamethasone reduced the amount of AFP mRNA in hepatoma 7777 while increasing albumin mRNA. Two-dimensional gel electrophoresis of tumor cytosol proteins showed that dexamethasone reduced synthesis of all AFP variants which could be detected by this technique. A number of abundant hepatoma-associated and liver-associated proteins were not significantly affected by dexamethasone.

Nuclear protein phosphokinases in normal and neoplastic tissues.

Protein phosphokinases were isolated from the nuclei of normal and fetal liver and neoplastic tissues. Chromatography on phosphocellulose columns resolved the normal and fetal liver kinases into five reproducible fractions. Each of the fractions differed in optimal divalent cation and substrate requirements. Hepatic proliferation was accompanied by quantitative changes in the kinase activity profiles (with endogenous phosphoprotein as natural substrate). An additional phosphoprotein kinase activity stimulated by Mn2+ was found in the nuclei of malignant cells. This tumor-specific kinase could not be detected either in tumor cytoplasm or in fetal or regenerating liver nuclei. Mn2+-dependent phosphoprotein kinase from Novikoff hepatoma phosphorylated only one major protein band detectable by polyacrylamide gel electrophoresis. This substrate could not be detected in chromatin of normal tissues.

Specificity of DNA-associated nuclear antigens in HeLa cells and distribution during the cell cycle.

Chromosomal nonhistone protein:DNA complexes prepared from synchronized HeLa cells were used to immunize white rabbits. The antisera reacted specifically in complement fixation tests with chromatins isolated from HeLa cells and not with those from a number of other human and animal tissues. Specificity to this cell type was also demonstrated by immunocytochemical reaction. Both immunochemical tests revealed that the specific antigens are continuously present throughout the cell cycle. The immunological activity was dependent upon chromosomal nonhistone protein(s) being bound to DNA. Our findings are consistent with these chromatin antigens being stable nuclear components [complexes of chromosomal non-histone protein(s) with DNA] characteristic of cellular differentiation.

The expression of c-myc and c-N-ras in human cirrhotic livers, hepatocellular carcinomas and liver tissue surrounding the tumors.

To study the possible role of proto-oncogenes in the multistep process of human liver hepatocarcinogenesis, we have examined the expression of c-N-ras and c-myc in human hepatocellular carcinomas and liver tissue surrounding the tumors as well as cirrhotic livers which are generally considered to precede the formation of human hepatocellular carcinoma. One to four-fold higher expression of the c-N-ras proto-oncogene was observed in twelve hepatoma patients as compared to normal liver. Increased expression of c-N-ras was also observed in liver tissue surrounding these tumors. Eight patients exhibited an apparent higher expression of the c-N-ras oncogene in adjacent liver tissue than in their corresponding tumor tissues. Six human liver cirrhosis patients also exhibited a slight increase in c-N-ras expression. Southern blot analysis demonstrated an amplified c-N-ras sequence in these tissues surrounding the tumors. In the study of the c-myc gene, variable degrees of highly enhanced expression were found in all twelve hepatoma patients as compared to normal liver. The c-myc gene was also expressed in the adjacent liver tissue and in some of the human cirrhotic livers. Our studies give further evidence that the expression of c-N-ras and c-myc proto-oncogenes are involved in the process of human hepatocarcinogenesis.

Testicular chromatin activation in hypophysectomized rats.

Incorporation of labeled thymidine into testicular DNA of hypophysectomized rats began to increase after the administration of testosterone propionate and choriogenic gonadotrophin. While the thymidine incorporation reached maximum in 4 days, the DNA polymerase activity did not culminate until 8 days after the initiation of hormone treatment. The high molecular weight (6--8 S), presumably cytoplasmic DNA polymerase accounted almost entirely for this increase. Administration of testosterone propionate and chorionic gonadotrophin to hypophysectomized rats results in an increase of testicular RNA polymerase and chromatin templating activity. Chain elongation and initiation studies revealed that the increased templating capacity of androgen-stimulated testicular chromatin was almost entirely caused by the increase in the number of initiation sites. While the nuclear polymerase I responded relatively rapidly to hormone stimulation and reached a prominent maximum in about three days, the activity of polymerase II was more sluggish and not as prominent. The in vivo incorporation of ortho[32P]phosphate into chromosomal phosphoproteins occurred early during the androgen treatment and reached a maximum in about 20 h. The protein phosphokinase activity peaked later, approx. 72 h after the first administration of hormones.

Hepatoma-associated nonhistone chromosomal proteins.

In order to compare nonhistone proteins in normal and neoplastic hepatocytes, we elicited antisera to Morris hepatoma 7777 dehistonized chromatin. By the enzyme-linked immunosorbent assay, the antisera demonstrated specificity for Morris hepatoma 7777 and little reactivity to normal rat liver chromatin. Morris hepatomas 7288c, 7800 and 5123tc shared some antigenic hepatoma nonhistone proteins. Neoplasia induced in rats fed 3'-methyl-4-dimethylaminoazobenzene changed the immunospecificity of the liver chromatin to a new type that was antigenically similar to Morris hepatoma 7777. Fetal rat liver chromatin and regenerating rat liver chromatin did not bind antibody. To further characterize the antigenic nonhistone proteins, we analyzed Morris hepatoma 7777 chromatin and normal rat liver chromatin by the immunoblot technique. Nonhistone proteins that demonstrated immunoreactivity were predominantly high molecular weight proteins.

Nuclear phosphoprotein kinase activities in normal and neoplastic tissues.

Nuclear phosphoprotein kinases from normal rat liver and transplantable neoplasms were fractionated and compared. A phosphoprotein kinase fraction activated by Mn2+ was found to be present only in the neoplasms. This nuclear protein kinase phosphorylated nuclear proteins represented by one major and several minor bands as determined by polyacrylamide gel electrophoresis (M approximately 50,000).

Effect of soil and a nonionic surfactant on BTE-oX and MTBE biodegradation kinetics.

The biodegradation kinetics of BTE-oX and MTBE, mixed all together, in the presence of 905 mg/L VSS of BTEX-acclimated biomass was evaluated. Effects of soil and Tergitol NP-10 in aqueous samples on substrate biodegradation rates were also evaluated. Biodegradation kinetics was evaluated for 36 hours, every 6 hours. MTBE biodegradation followed a first-order one-phase kinetic model in all samples, whereas benzene, toluene and ethylbenzene biodegradation followed a first-order two-phase kinetic model in all samples. O-xylene biodegradation followed a first-order two-phase kinetic model in the presence of biomass only. Interestingly, o-xylene biodegradation was able to switch to a first-order one-phase kinetic model when either soil or soil and Tergitol NP-10 were added. The presence of soil in aqueous samples retarded benzene, toluene and ethylbenzene removal rates. O-xylene and MTBE removal rates were enhanced by soil. The addition of Tergitol NP-10 to aqueous samples containing soil had a positive effect on substrate removal rate in all samples. Substrate percent removals ranged 77-99.8% for benzene, toluene and ethylbenzene. O-xylene and MTBE percent removals ranged 50.1-65.3% and 9.9-43.0%, respectively.

Regulation and activities of alpha-fetoprotein.

alpha-Fetoprotein (AFP) is one of the major serum proteins in the early life of mammals. The function of this protein is not yet fully understood. AFP is an oncodevelopmental gene product which is expressed at high levels in the embryonic yolk sac and fetal liver. The synthesis of AFP decreases dramatically after birth. Only trace amounts of AFP are synthesized in the adult liver. However, expression of the AFP gene is reactivated in the adult during liver regeneration and hepatocarcinogenesis. AFP is an excellent model system for studying the temporal and tissue-specific regulation of oncodevelopmental gene expression. Experiments with transgenic mice and DNA transfection studies have revealed several transcriptional control regions and cis-acting elements in the AFP gene. A large number of trans-acting protein factors interacting with these cis-acting elements have also been identified. Recent studies demonstrated that expression of AFP is regulated by at least two major signal transduction pathways in response to extracellular stimuli. The interactions between steroid hormone receptors and transcriptional factors which respond to separate signal transduction pathways result in transcriptional regulation of AFP gene expression. Trans-acting protein factors or steroid receptors complexed with given response elements can display different activities in different cell types due to cross-talk among both local protein-protein interactions within the DNA-binding domain, and distal protein-protein interactions. However, the detailed mechanisms of AFP gene expression are still not completely understood.

Colon tumor specific nuclear antigen with potential as a pre-tumor diagnostic probe.

Tumor specific nuclear antigen was demonstrated in early stages of chemically-induced colon carcinogenesis. At these early stages, there is no observable nuclear or cytoplasmic alteration in the colon mucosae. The rise in tumor specific nuclear antigen in carcinogen-treated animals can be abolished by simultaneous treatment with carcinogen inhibitor. The potential for tumor specific nuclear antigen to be used as a pretumor diagnostic probe is discussed.

Changes in hepatic levels of tyrosine aminotransferase messenger RNA during chemical hepatocarcinogenesis.

The activity of tyrosine aminotransferase (TAT) decreased biphasically in livers of rats fed 3'-methyl-4-dimethylaminoazobenzene (3'-MDAB). TAT activity decreased to an extremely low level at later stages of hepatocarcinogenesis. The activity of TAT is negatively correlated with alpha-fetoprotein (AFP) levels. The level of TAT enzyme activity in precancerous liver and hepatoma is a reflection of the amount of TAT mRNA. Dexamethasone increased the TAT enzyme activity and TAT mRNA concentration in rat livers during chemical carcinogenesis.

The appearance of hepatoma-associated chromosomal non-histone proteins in rat liver after a single dose of 3'-MDAB followed by treatment of phenobarbital.

Previously we have described polyclonal antibodies that recognized a group of nuclear nonhistone proteins whose molecular weights ranged in size from 170 to 220 kDa. These antigenic nonhistone chromosomal proteins are abundant in rat hepatoma chromatin. In this report we discuss the synthesis and cellular localization of these particular proteins during the multistage process of hepatocarcinogenesis. The appearance of these antigenic proteins in rat liver nuclei approximately parallels the appearance of alpha-fetoprotein in the cytosol of hepatocytes. However, the immunoreactivity of antigenic proteins increased steadily even during the prominent dip in the AFP concentration between 50 and 100 days of carcinogenesis. The effect of the tumor promoting agent, phenobarbital, on the synthesis of antigenic nuclear proteins was also studied. The appearance of hepatoma-associated non-histone chromosomal proteins at early stages of tumor promotion during hepatocarcinogenesis was observed. The results of these studies demonstrate that the hepatoma-associated non-histone proteins are expressed not only in hepatoma cells, but also in hepatocyte cells committed to carcinogenesis.