Enzyme immunoassay for pepleomycin, a new bleomycin analog.

An antibody directed toward pepleomycin, a new antitumor antibiotic related structurally to bleomycin, has been produced in rabbits by immunization with a pepleomycin-protein conjugate which was prepared by a novel procedure of coupling pepleomycin to mercaptosuccinylated bovine serum albumin using N-(gamma-maleimidobutyryloxy)succinimide as a coupling agent. The antiserum was monospecific to pepleomycin and showed almost no cross-reactivity with a variety of other bleomycin analogs. An enzyme immunoassay for pepleomycin has been developed utilizing this antiserum and beta-D-galactosidase-labeled pepleomycin. The lower limit of detection by this assay, which involves a double antibody technique for the separation of antibody-bound and free antigen, was 50 pg of pepleomycin per tube. Using this assay, drug levels were easily determined in blood and urine of rabbits following administration of pepleomycin in a single dose of 1.2 mg/kg i.v. This assay is also suitable for measuring pepleomycin in the presence of other drugs since the assay is not significantly affected by any other antineoplastic agents tested. Since pepleomycin is now undergoing clinical trial, the enzyme immunoassay of the drug will be a valuable tool in clinical pharmacological studies.

Enzyme immunoassay for the quantification of mitomycin C using beta-galactosidase as a label.

A mitomycin C (MMC) antibody was produced following immunization of rabbits with a MMC-bovine serum albumin conjugate, which was newly synthesized by coupling MMC to mercaptosuccinylated bovine serum albumin via a cross-linker, N-maleoyl aminobutyric acid. Enzyme labeling of MMC was performed using beta-D-galactosidase (EC 3.2.1.23) via m-maleoyl benzoic acid. An enzyme immunoassay for MMC was developed utilizing these reagents by a double-antibody technique. The standard curve of the assay was linear on a logit-log plot, and the lower limit of detection was 12 nM (0.2 ng/tube) so the enzyme immunoassay was found to be approximately 25 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is practically free from interference by any other anticancer drugs. No significant decrease in MMC immunoreactivity was observed following 24 hr of incubation of the drug in normal human serum or urine at 37 degrees. Using this assay, serum or urine levels of MMC can be determined accurately after administration of the drug to rats at a single dose of 600 micrograms/kg. The sensitivity and specificity of the enzyme immunoassay for MMC should provide a valuable new tool for use in pharmacokinetic and toxicity studies of MMC.

Frequent mutation of beta-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by defective DNA mismatch repair, which results in genetic instability of tumors; however, only a few target genes have been recognized. Our previous study detected a low frequency of APC gene mutation (21%) in colorectal tumors from HNPCC patients, in contrast to a high frequency of APC gene alteration (>70%) in non-HNPCC tumors. Because both beta-catenin and ACP gene mutations have recently been shown to activate the same signaling pathway, we analyzed beta-catenin mutation in HNPCC tumors. A notable frequency of beta-catenin gene mutation (43%, 12 of 28) was found to occur in HNPCC colorectal tumors. Beta-catenin mutations were not detected in tumors with APC mutations. All beta-catenin mutations detected in HNPCC tumors existed within the regulatory domain of beta-catenin. Immunohistochemical staining of tumors with this mutation showed accumulation of beta-catenin protein in nuclei. These and previous data from our laboratory suggest that activation of the beta-catenin-Tcf signaling pathway, through either beta-catenin or APC mutation, contributes to HNPCC colorectal carcinogenesis in approximately 65% of cases.

Somatic mutations of LKB1 and beta-catenin genes in gastrointestinal polyps from patients with Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is characterized by multiple gastrointestinal hamartomatous polyps, mucocutaneous melanin deposition, and increased risk of cancer, mainly in the gastrointestinal tract. We examined mutations of the LKB1, beta-catenin, APC, K-ras, and p53 genes in 27 gastrointestinal hamartomatous polyps from 10 patients in nine PJS families. Of these hamartomatous polyps, one intestinal polyp had an adenomatous lesion, and one gastric polyp contained adenomatous and carcinomatous lesions. Germ-line mutations of the LKB1 gene were detected in six PJS families. Somatic mutations of the LKB1 gene were found in 5 polyps, whereas loss of heterozygosity (LOH) at the LKB1 locus at 19p was seen in 14 other polyps. In adenomatous lesions microdissected from hamartomatous polyps, both beta-catenin mutation and 19p LOH were detected. Furthermore, a carcinomatous lesion in a gastric hamartomatous polyp was found to contain a mutation of the p53 gene and LOH at the p53 locus in addition to LOH at the LKB1 locus and a beta-catenin mutation. K-ras mutations were detected in a few polyps, whereas no APC mutation or 5q LOH was detected in hamartomatous polyps. These results suggest that gastrointestinal hamartomatous polyps in PJS patients develop through inactivation of the LKB1 gene by germ-line mutation plus somatic mutation or LOH of the unaffected LKB1 allele, and that additional mutations of the beta-catenin gene and p53 gene convert hamartomatous polyps into adenomatous and carcinomatous lesions.

Alterations of repeated sequences in 5' upstream and coding regions in colorectal tumors from patients with hereditary nonpolyposis colorectal cancer and Turcot syndrome.

One of the characteristics of tumors from patients with germline mutations of DNA mismatch repair genes is instability at microsatellite regions (MSI). We analysed alterations at repeated sequences of coding regions, as well as those of 5' upstream regions, in 29 MSI-High colorectal tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) and Turcot syndrome. We found that repeated sequences in 5' upstream regions were altered in these tumors, at considerable frequencies. The (A)10 repeat in the promoter region (position -178 to approximately -169) of the GAPDH gene was altered in 17% of the tumors. The (A)10(TA)9 in the 5' upstream region (position -318 to approximately -291) of the mitochondrial isoleucyl tRNA synthetase gene (IleRS-A), coded in nuclear DNA, was altered in 59% of the tumors, whereas (A)9 in the 5' upstream region (position -859 to approximately -851) of cytoplasmic isoleucyl tRNA synthetase gene (IleRS-B) was not altered. Alteration at repeated sequences in the coding regions were 72% at TGFbetaRII(A)10, 24% at IGFIIR(G)8, 45% at BAX(G)8, 55% at E2F4(CAG)13, 66% at caspase-5 (A)10, 31% at MBD4(A)10, 55% at hMSH3(A)8 and 34% at hMSH6(C)8. The number of altered genes increased with the advancement of carcinoma according to Dukes categories: mean numbers of altered genes within these 10 genes were 2.6 for Dukes A, 4.7 for Dukes B and 7.8 for Dukes C. The mean number for adenomas was 2.0. These results suggest that the MSI phenotype also causes alteration of 5' upstream regions which may affect apoptosis and some mitochondrial functions in HNPCC and Turcot tumors, and that accumulation of altered genes with repeated sequences is associated with the progression of HNPCC and Turcot colorectal tumors.

Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis.

We have previously detected an increased frequency of loss of heterozygosity (LOH) on chromosome 18q during progression of colorectal carcinomas. To clarify the target of 18qLOH, mutation of Smad4 and Smad2 genes was analysed in 176 colorectal tumors with different stages, including liver metastasis, from 111 sporadic, 52 familial adenomatous polyposis (FAP) and nine hereditary nonpolyposis colorectal cancer (HNPCC) patients. Mutation of other Smad gene families in the TGF-beta signaling pathway was also examined. Twenty-one Smad4 mutations and one Smad2 mutation were detected, whereas mutation of Smad3, 6 and 7 genes was not detected. Smad4 mutations included seven frameshift, one inframe deletion, four nonsense and nine missense mutations, 95% of which resulted in alteration of Smad4 protein regions included in homo-oligomer and hetero-oligomer formation. Frequencies of tumors with Smad4 mutation were 0/40 (0%) in adenoma, 4/39 (10%) in intramucosal carcinoma, 3/44 (7%) in primary invasive carcinoma without distant metastasis, 6/17 (35%) in primary invasive carcinoma with distant metastasis, and 11/36 (31%) in distant metastasis (metastatic/non-metastatic: P=0.006 approximately 0.01). Loss of the other allele was observed in 19 of 20 (95%) invasive and metastasized carcinomas with Smad4 mutations. In four cases both primary and metastasized carcinomas in the same patients showed the same mutations. The present results suggest that Smad4 gene is one of true targets of 18qLOH, and that its inactivation is involved in advanced stages, such as distant metastasis, in human colorectal carcinogenesis.

Novel preparation method of immunogen for hydrophobic hapten, enzyme immunoassay for daunomycin and adriamycin.

The present study was undertaken to develop a novel method of preparing a hydrophobic hapten as immunogen. The anticancer drug, daunomycin (DM) was used as prototype and coupled to mercaptosuccinylated bovine serum albumin (MS, BSA) with N-(gamma-maleimidobutyryloxy)succinimide (GMBS). Injection of rabbits with a conjugate (DM-GMBS-MS.BSA) which contained 7.3 DM per BSA molecule produced good levels of anti-DM antibody which was detected by the reaction of diluted antiserum with beta-D-galactosidase-labeled DM. beta-D-galactosidase-labeled DM was used to develop a double antibody enzyme immunoassay for DM and for the DM homologue adriamycin (AM) which reproducibly detected as little as 1 pmole of either drug. A variety of commonly used other anticancer drugs were tested and had little reactivity in this immunoassay. These studies indicate that the anti-DM serum produced is highly specific.

Importance of coronary collaterals for restoration of left ventricular function after intracoronary thrombolysis.

In an evaluation of the role of coronary collaterals in the early period of acute myocardial infarction (AMI), 30 patients with acute total coronary occlusion treated with intracoronary thrombolysis 2 to 8 hours after the onset of symptoms were studied. Only 13 patients with well-developed collaterals in the early period of AMI and successful thrombolysis showed improvement of global and regional ejection fraction (EF) from the acute phase to the chronic phase (global EF from 50% to 71%, p less than 0.001; regional EF from 25.4% to 49.2%, p less than 0.001). In patients with no or less well-developed collaterals and successful thrombolysis, global and regional EF were similar to those in patients in whom thrombolysis was unsuccessful. Among the 19 patients with successful thrombolysis, there was no significant correlation between the duration of ischemia and the improvement of regional EF (r = -0.03, difference not significant). These data suggest that the extent of coronary collateral vessels in the early period of AMI is an important determinant of restoration of left ventricular function after intracoronary thrombolysis.

Effects of percutaneous transluminal coronary angioplasty: intracoronary thrombolysis with urokinase in acute myocardial infarction.

Coronary angiography and percutaneous transluminal coronary angioplasty (PTCA) were performed in 32 patients with evolving acute myocardial infarction. Of the 25 patients with complete occlusion of an infarct-related coronary artery, in 18 (72%) the occluded vessel was successfully opened by an intracoronary infusion of urokinase. With a small dose of urokinase the successful recanalization was achieved in only 25%; with a larger dose it was achieved in 94%. After PTCA, all patients received glucose-insulin-potassium solution for 76 hours. Repeat angiography 42 days later showed a patent coronary artery in 12 (group A) of 18 patients with successful PTCA. In group A, left ventricular ejection fraction increased from 51 +/- 13% to 72 +/- 10% (p less than 0.01) and regional wall shortening from 4.5 +/- 9.5% to 29 +/- 19% (p less than 0.01). In contrast, these variables did not change significantly in patients with unsuccessful PTCA or late reocclusion of an infarct-related vessel (group B). These data suggest that successful PTCA with sustained patency of an infarct-related coronary artery has a beneficial effect on the salvage of the jeopardized myocardium, and glucose-insulin-potassium therapy may enhance the beneficial effect of PTCA.

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for ecotoxicity.

There is growing public pressure to minimize the use of vertebrates in ecotoxicity testing; therefore, effective alternatives to toxicity tests causing suffering are being sought. This report discusses alternatives and differs in some respects from the reports of the other three groups because the primary concern is with harmful effects of chemicals at the level of population and above rather than with harmful effects upon individuals. It is concluded that progress toward the objective of minimizing testing that causes suffering would be served by the following initiatives--a clearer definition of goals and strategies when undertaking testing procedures; development of alternative assays, including in vitro test systems, that are based on new technology; development of nondestructive assays for vertebrates (e.g., biomarkers) that do not cause suffering; selection of most appropriate species, strains, and developmental stages for testing procedures (but no additional species for basic testing); better integrated and more flexible testing procedures incorporating biomarker responses, ecophysiological concepts, and ecological end points (progress in this direction depends upon expert judgment). In general, testing procedures could be made more realistic, taking into account problems with mixtures, and with volatile or insoluble chemicals.

Influence of persistence period of an insecticide on recovery patterns of a zooplankton community in experimental ponds.

An insecticide, carbaryl, was applied singly or repeatedly to experimental ponds in order to control the residue of the chemical, and the subsequent changes in the zooplankton community were investigated. In ponds where a single application of carbaryl, which degraded rapidly in the water, was made, cladocerans were reduced, but recovered soon and suppressed rotifers through competition. On the other hand, in ponds receiving repeated chemical applications, the treatment suppressed cladocerans for longer and induced the occurrence of abundant rotifers. The rotifer abundance after the treatment seemed to depend on the persistence period of the chemical. From these findings it can be hypothesized that applications of chemicals which have different degradation rates induce different zooplankton community structures.

Effects of a carbamate insecticide, carbaryl, on the summer phyto- and zooplankton communities in ponds.

A carbamate insecticide, carbaryl (1-naphthyl-N-methylcarbamate), was applied in concrete ponds and the effects on plankton communities were studied. In a control pond, Cladocera declined following the increase in the density of inedible algae after a cladoceran peak. Once the density of Cladocera became low, Chaoborus larvae suppressed the increase of Cladocera and consequently supported the rotifer dominance in the zooplankton community by their selective predation on cladocerans. In a treated pond, the plankton community and its succession were similar to those in the control pond until the chemical application. 1 ppm of carbaryl killed all zooplankton and Chaoborus larvae. Cladocera reappeared soon and increased rapidly due to the absence of Chaoborus larvae. Consequently, rotifer populations were suppressed. Thus, the chemical application altered the dominance of rotifers to that of cladocerans. The same phenomenon was observed again after the second chemical application 12 days after. Although apparent direct effects of the chemical application on phytoplankton were not found, the phytoplankton community structure changed following the changes in the zooplankton density.

Effects of carbaryl on the spring zooplankton communities in ponds.

A carbamate insecticide, carbaryl, was applied in spring to concrete ponds to study its effects on zooplankton communities. The population density of Cladocera (Daphnia spp.) was nearly constant before application of the chemical. Carbaryl at 1 ppm killed all zooplankton species, including Chaoborus larvae. After treatment, cladocerans never recovered to their previous level. The relatively rapid recovery of a predator, Chaoborus, seemed to interfere with recovery of the cladoceran populations. The lower water temperature occurring in spring was thought to favour the former because of its influence on growth rates.

Effects of temephos on zooplankton communities in enclosures in a shallow eutrophic lake.

An organophosphorus insecticide, temephos, was applied to large-volume (105 m3) enclosures set up in a shallow eutrophic lake. Application of the chemical at a target concentration of 500 microg litre(-1) eliminated almost all zooplankters. No recovery of cladocerans was evident at the termination of the experiment (47th day after the treatment). Copepods showed a slight recovery after having been absent for 26 days in one enclosure and 40 days in another. The residual chemical remaining in the water until the final day may have suppressed the recovery of the crustacean zooplankters. The rotifer community was reconstructed 16-20 days after the treatment. However, the species composition of this community differed from that of the rotifer community in the control enclosures. Rotifer species might therefore show differences in susceptibility to temephos.

Clinical experience with a new rate-responsive TX pacemaker.

In this report, we describe our initial experience with a new rate-responsive TX pacemaker in 10 patients. This pacemaker system uses a conventional transvenous ventricular electrode, which senses the evoked QT interval after a ventricular paced beat, as an indicator of physiological metabolic demand. Implantation of the TX pacemaker does not differ from that of conventional VVI units, and two special modes are available after implantation: TX and VVI mode. Bicycle ergometer tests showed that significantly higher rate response and exercise ability were achieved during the TX mode compared with the VVI mode (mean maximal heart rate: 115 +/- 13 bpm vs 96 +/- 21 bpm, p less than 0.01, and mean maximal exercise time; 10.7 +/- 3.9 min vs 8.0 +/- 3.8 min, p less than 0.01, respectively). In one patient, cardiac index was measured by a thermodilution method during the bicycle ergometer test, which confirmed that an adequate increase in cardiac index (rest: 3.38 liters/min/m2, vs exercise; 7.34 liters/min/m2) was obtained by an increase of the pacing rate (rest: 72 bpm vs exercise: 128 bpm). Our results show that physiological rate-responsive pacing using the QT interval provides a simple means of increasing the heart rate and cardiac index.

Myocardial infarction after acute carbon monoxide poisoning: case report.

A 28-year-old man with acute myocardial infarction after carbon monoxide poisoning is reported. He had chest pain after the exposure to carbon monoxide. The electrocardiogram, serum enzymes, and technetium-99m pyrophosphate scintigrams showed anterior myocardial infarction. The coronary angiogram, which was performed one month after the onset, showed no visible atherosclerotic lesion. As to the cause of myocardial infarction, it is assumed that carbon monoxide reduced the oxygen supply to the myocardium and might induce coronary artery spasm with or without accompanying coronary thrombosis.

Takayasu's arteritis with collateral circulation from the right coronary artery to intracranial vessels--a case report.

A forty-four-year-old woman with Takayasu's arteritis and involvement of the aortic arch and its main branches complained of precordial pain on effort. Exercise electrocardiograms revealed significant ST segment depression in leads II, III, aVF, and V. Coronary arteriograms demonstrated no stenosis. However, the right coronary arteriogram revealed collateral circulation arising from the sinus node artery to the bilateral vertebral arteries and the left internal carotid artery. The collateral circulation was considered to be an important route of blood flow supply to the brain and, at the same time, a cause of coronary steal syndrome and, consequently, of angina pectoris.

A case of acute myocardial infarction. Intracoronary thrombosis in two major coronary arteries due to hormone therapy.

A fifty-four-year-old woman was admitted to the hospital for a sensation of tightness in the chest of one hour's duration. She had undergone surgery for breast cancer two years previously and had been taking 30 mg of tamoxifen and 1200 mg of medroxyprogesterone daily after surgery. Emergency coronary angiography on admission revealed thrombi in both the right coronary artery and the left anterior descending coronary artery. Tissue-type plasminogen activator was injected into both coronary arteries, resulting in diminution of thrombus size. Repeat coronary angiography on the next day disclosed no thrombus in either artery and no significant stenosis. Electrocardiographic and laboratory data indicated myocardial infarction. These findings strongly suggest that the combination hormone therapy altered the patient's blood coagulability and played an important role in the formation of the intracoronary thrombi and subsequent acute myocardial infarction.

Angiographic and pathologic evidence of hemorrhage into the myocardium after coronary reperfusion.

Severe myocardial hemorrhage can occur as a potential adverse effect of reperfusion therapy in evolving myocardial infarction. This report describes a 83-year-old man, who showed angiographic evidence of extravasation of contrast medium from the reperfused right coronary artery into the inferoposterior left ventricular wall. At autopsy, severe hemorrhage was transmurally observed in the inferoposterior wall of the left ventricle. The finding of extravasation is a useful angiographic sign of the production of hemorrhage during coronary reperfusion therapy, and great attention should be focused to the existence of this sign to prevent further hemorrhage.