Subcellular distribution of epothilones in human tumor cells.

Epothilones are a new class of natural and potent antineoplastic agents that stabilize microtubules. Although 12,13-epoxide derivatives are potent antiproliferative agents, the activities of the corresponding 12,13-olefin analogs are significantly decreased. These data were confirmed for two new analogs, 6-propyl-EpoB (pEB) and 6-propyl-EpoD (pED), in comparison with the natural compounds EpoB/EpoD, by using human A431, MCF7, and MDR1-overexpressing NCI/Adr cells. By using tritiated pEB/pED, compound uptake, release, and nuclear accumulation were investigated in A431 and NCI/Adr cells. In these cells, epothilones can principally be recognized and exported by Verapamil-sensitive efflux pumps, which are not identical to MDR1. The degree of export depends on the structure, olefin vs. epoxide-analog, and also on the intracellular drug concentration. The accumulation of pED used at 3.5 or 70 nM, respectively, was increased in the presence of 10 microM Verapamil in both cell lines 2- to 8-fold. In contrast, the intracellular levels of pEB were affected by Verapamil only at 3.5 nM pEB in NCI/Adr (2-fold) and not in A431 cells. In addition, strong nuclear accumulation was observed for pEB (40-50%) but not paclitaxel or pED (5-15%) in both cell lines. Our study suggests that differences in growth inhibitory efficacy between epoxide and olefin analogs may be based on different mechanisms of drug accumulation and subcellular distribution.

Influence of restricted food intake on estrous cycles and pseudopregnancies in dogs.

OBJECTIVE: To examine effects of restricted food intake on estrous cycle frequency, interestrus interval, and pseudopregnancy prevalence in dogs. ANIMALS: 28 female Labrador Retrievers. PROCEDURE: Dogs were paired by body weight when they were 6 weeks old and fed so that the limit-fed pair-mate received 75% of the amount of food offered to its maintenance-fed counterpart. Estrous cycle, interestrus interval, and pseudopregnancy data were recorded. RESULTS: Mean annual frequency of estrous cycles and duration of interestrus intervals did not differ between feeding groups. Prevalence of clinically evident pseudopregnancy was significantly greater among females that were maintenance fed, although results of endocrinologic testing did not identify a mechanism for this observation. CONCLUSIONS AND CLINICAL RELEVANCE: Pseudopregnancy in dogs can be influenced by physiologic factors related to nutrition. Clinicians should consider a variety of physiologic and environmental factors when evaluating reproductive function in dogs.

The significance of high levels of lipoprotein (a) compared with established risk factors in premature coronary artery disease: differences between men and women.

It was shown in a series of studies that increased lipoprotein (a) concentration is a strong and independent risk factor for coronary artery disease. The goal of this study was to determine the significance of elevated lipoprotein (a) levels for the existence and the early manifestation of coronary artery disease by systematically recording cardiovascular risk factors in diagnostic coronary angiographies in a larger group of patients, whereby particular attention was paid to sex-specific differences. In 1011 consecutive patients who underwent coronary angiography (731 men, 280 women, mean age 59 +/- 10 years), fasting blood samples were taken immediately before the angiographies to determine the levels of cholesterol, low density lipoprotein-, high density lipoprotein-cholesterol, triglycerides and lipoprotein (a). In addition, further risk factors were qualitatively recorded. The data evaluation was carried out using the SPSSx software package univariately and multivariately with stepwise discriminant analysis. In 231 patients (144 men, 87 women) either no or only discrete coronary findings appeared, while in 780 cases (587 men, 193 women) coronary artery disease with stenoses > 50% were found. Women with coronary artery disease were significantly older than men and demonstrated higher lipoprotein levels. Women as well as men with coronary artery disease differed from healthy controls by having higher levels of lipoprotein (a) and other lipoproteins, lipoprotein (a) having the smallest error probability (P < 0.0005). The early manifestation of coronary artery disease (below the 18th age percentile) in men (< 50 years) was connected with significantly higher levels of cholesterol, triglycerides and lipoprotein (a), which emphasized their atherogenic significance in the general view. The most striking finding was that in young women (< 53 years), compared to older women with coronary artery disease--corresponding to the age-determined prevalence--significantly lower concentrations of cholesterol, triglycerides and lipoprotein (a) were found. Possible explanations include later manifestation of coronary artery disease, a steeper increase of the lipids with age, particularly of lipoprotein (a), but also a different valence of the risk factors in women.

Comparative pharmacokinetics of two new steroidal estrogens and ethinylestradiol in postmenopausal women.

It was the aim of the study to compare the pharmacokinetic properties of the two new estrogens, ZK 136295 and ZK 115194, with those of ethinylestradiol (EE2) after single intravenous (60 micrograms) and oral (120 and 240 micrograms) administration in 54 postmenopausal women. In particular, our objective was to examine whether one or both compounds were characterized by an improved oral bioavailability with less inter-subject variability than EE2. Drug serum concentrations were determined using specific radioimmunoassays for EE2 and ZK 136295, and a GC/MS/MS-method for ZK 115194. Following i.v. administration of the new estrogens and of EE2, the drugs were rapidly distributed in the body. The mean terminal half-lives were calculated to be 12.3 +/- 12.4, 28.7 +/- 9.6, and 26.1 +/- 11.1 h for ZK 136295, ZK 115194, and EE2 respectively. After oral administration of 120 micrograms, the absolute bioavailability was calculated to be about 40% for ZK 136295 as well as for EE2 with a high inter-individual variation (variation coefficient: 44 and 67%). By doubling the dose, the systemic availability increased dose-dependently for both drugs to about 70% with the same high inter-individual variation. Following single oral administration of 240 micrograms ZK 115194, the absolute bioavailability amounted to 33 +/- 19%. The present study clearly revealed that although the two new estrogens differed considerably in their pharmacokinetic behavior, they demonstrated a reduced and highly variable systemic availability similar to that of EE2.

PIP: Researchers with the pharmaceutical manufacturer Schering AG in Berlin, Germany, conducted a double-blind clinical trial in 54 healthy, postmenopausal women to compare the pharmokinetic properties of two new estrogens (ZK 136295 and ZK 115194) with those of ethinyl estradiol (EE2). Specifically, they examined whether one or both of the new estrogens improved bioavailability with less inter-subject variability than EE2. The dosage included single intravenous (60 mcg) and oral (120 and 240 mcg) administration. ZK 115195 differs from natural estradiol by 14alpha, 17alpha-bridging, which should prevent early metabolic degradation during absorption and passage through the liver. ZK 136295 is the corresponding derivative of endogenous estriol. All three compounds were rapidly distributed throughout the body. ZK 136295 had the shortest mean terminal half-life; ZK 115194 had the longest (12.3 vs. 28.7 hours); and EE2 had a mean terminal half-life of 26.1 hours. Oral administration of 120 mcg effected an absolute bioavailability of about 40% for ZK 136295 and EE2. The inter-individual variation was high (variation coefficient = 44% and 67%) for both compounds. When the oral dose was 240 mcg, systemic availability increased dose-dependently for ZK 136295 and EE2 to around 70% with the same high inter-individual variation. Oral administration of 240 mcg of ZK 115194 effected an absolute bioavailability of about 33%. These findings show that the 14alpha, 17alpha-bridging of estradiol does not result in a higher bioavailability than that achieved by introduction of a 17alpha-ethinyl group. Yet, increasing the dose of ZK 115194 and of EE2 from 120 to 240 mcg increased their absolute bioavailability two-fold. In conclusion, even though the pharmacokinetic behavior of the two new estrogens differed markedly, the two estrogens exhibited a reduced and highly variable systemic availability similar to that of EE2.

The lack of immunogenicity of iotrolan.

RATIONALE AND OBJECTIVES: To test whether it would be possible to raise antibodies against iotrolan in rabbits and, if possible, to develop a radioimmunoassay for iotrolan. METHODS: A "half-molecular" analogue of iotrolan was synthesized, which contained a carboxylic group. This moiety was coupled via a link to bovine serum albumin. The resultant hapten was suspended in Freund's complete adjuvant and used to immunize rabbits by two subcutaneous and two intramuscular injections followed by monthly booster injections. After bleeding of the animals, the antibodies formed were tested. RESULTS: The rabbits successfully developed antibodies against the hapten. These antibodies were tested for cross-reactivity with iotrolan, the iotrolan half-molecule, and the hapten. Minimal cross-reactivity (below 0.5%) was found for iotrolan and the half-molecule. Only the hapten was found to bind to the antibody. CONCLUSIONS: In the current test setting using a half-molecular analogue, it could be shown that iotrolan is probably not immunogenic. The formation of an antibody against the half-molecule coupled to bovine serum albumin can be explained only by immunogenicity of that part of the molecule, which constitutes the bridge or link to the albumin. This part of the hapten, however, is not representative of iotrolan itself.

Simple repetitive DNA sequences are transcribed in early embryonal stages of Xenopus laevis.

We have isolated and sequenced three cDNA clones from early embryonal stages of Xenopus laevis containing highly repetitive sequences. By means of poly A+ RNA hybridization we could show that simple repeats of the type AC/TG are ubiquitously transcribed in the ovary and in early embryonal stages of Xenopus laevis.

Effects of fenofibrate on angiographically examined coronary atherosclerosis and left ventricular function in hypercholesterolemic patients.

Within the framework of a prospective lipid-lowering intervention study 44 patients were treated over a period of 3 years with a lipid-lowering diet and 200-400 mg fenofibrate daily. The intervention led to statistically significant decreases in total cholesterol (Chol), low density lipoprotein cholesterol (LDL-Chol) and triglycerides levels, and to a significant increase in high density lipoprotein cholesterol (HDL-Chol) levels. Despite intervention, in 8 patients the HDL-Chol levels decreased by up to 20 mg/dl, where these were mainly patients with high initial values. Likewise, the triglycerides of 4 patients whose initial levels were relatively low increased (up to 49 mg/dl) and the LDL-Chol levels of 8 patients whose initial levels were also low increased (up to 49 mg/dl). Only minor success was achieved through the 6-week diet, but this was still slightly significant for Chol and LDL-Chol levels. A total of 21 patients underwent repeat angiography within 3 years for clinical reasons. For the evaluation of the angiographic progress a total of 98 minor and moderate stenoses was measured using digital image processing and automatic contour finding. The change in the angiographic parameters 'percent diameter reduction' (%DR) and 'percent plaque area' (%PA) correlated with on-treatment LDL-Chol levels (%DR change with LDL-Chol: r = 0.67, P = 0.0005; %DR change with Chol: r = 0.61, P = 0.002; %PA change with LDL-Chol: r = 0.40, P = 0.037; %PA change with Chol: r = 0.38, P = 0.044), while for HDL-Chol and triglycerides no influence on the angiographic progress could be demonstrated. On the basis of the reproducibility of the measuring methods the patients were classified in the categories 'regression', 'unchanged' and 'progression'. The patients classified as 'regression' (parameter: %DR change) showed an LDL-Chol mean value of 162 +/- 9 mg/dl, whereas those classified as 'unchanged' or 'progression' showed values of 189 +/- 25 mg/dl and 199 +/- 21 mg/dl, respectively (P = 0.014). A negative correlation appeared between the angiographic progress parameters and the initial degree of stenosis. The left ventricular ejection fraction in the second angiography showed relationships to lipoprotein levels and angiographic progress parameters.

[Quantitative coronary angiography: progression and regression of coronary stenoses--an intervention study with fenofibrate]. / Quantitative Koronarangiographie: Progression und Regression von Koronarstenosen--Eine Interventionsstudie mit Fenofibrat.

In order to examine the effect of fenofibrate on coronary narrowings, within the framework of a prospective intervention study, we treated a total of 44 hypercholesterolemic patients (who were in our clinic to undergo PTCA) with diet and fenofibrate (200-400 mg/day) over a period of 3 years. After a mean interval of 21 months, control angiographies were performed in nearly identical projections for 21 patients on clinical grounds. The minor and medium-grade narrowings of the reangiographed patients at the beginning and at the end of the intervention interval were measured by means of digital image processing and automatic contour detection. The measuring parameters were percent diameter reduction (% DR) and percent plaque area (%PA). With regard to their angiographic progression, the 21 reangiographed patients of the intervention group were compared to a comparison group consisting likewise of 21 patients of similar age and sex distribution and persistently high lipid and lipoprotein levels. During the intervention period, the reangiographed patients of the intervention group showed the following changes of the lipid and lipoprotein levels in contrast to the outset values: cholesterol -19 +/- 8%, LDL -20 +/- 14%, HDL +19 +/- 44%, triglycerides -30 +/- 31%.(ABSTRACT TRUNCATED AT 250 WORDS)

Progression and regression of minor coronary arterial narrowings by quantitative angiography after fenofibrate therapy.

To study the effects of fenofibrate, a lipid-lowering medication, on patients with coronary artery disease, 191 minor coronary narrowings in 42 patients with coronary artery disease were analyzed by quantitative coronary angiography using computer-assisted contour detection. Computed parameters were percent diameter reduction and percent plaque area. A prospectively formed intervention group of 21 patients treated with special diet and fenofibrate (200 to 400 mg/day) was checked every 6 weeks with regard to risk factors. After a mean interval of 21 months, coronary angiography was repeated, using the same x-ray system and nearly identical projections. The intervention group was angiographically compared at follow-up with an untreated comparison group, also comprising 21 patients. Both groups had high initial serum cholesterol (mean 311 mg/dl) and low-density lipoprotein (LDL) cholesterol levels (mean 235 mg/dl). Only among the treated patients did lipid levels change significantly: cholesterol, -19%; LDL cholesterol, -20%; high-density lipoprotein cholesterol, +19%; and triglycerides, -30%. At angiographic follow-up, the changes in percent diameter reduction and percent plaque area correlated positively with the mean serum and LDL cholesterol levels of the intervention group. Significant differences were found in the change in percent plaque area between both groups. The intervention subgroup with angiographic regressions (11 patients) had significantly lower serum and LDL cholesterol levels than the intervention subgroup with angiographic progressions (10 patients). These results indicate the beneficial effect of fenofibrate on minor coronary narrowings. Because of its high reproducibility in measuring minor narrowings, quantitative coronary angiography proved to be a suitable method for angiographic follow-up.

Undulin, an extracellular matrix glycoprotein associated with collagen fibrils.

Undulin, a novel noncollagenous extracellular matrix protein, was isolated from skin and placenta. In polyacrylamide gels most of the unreduced protein migrates with Mr above 1,000,000 yielding bands A (Mr 270,000), B1 (Mr 190,000), and B2 (Mr 180,000) after reduction. Undulin is biochemically and immunochemically distinct from other previously characterized large matrix glycoproteins. Immunoblotting using monoclonal antibodies suggests that bands A and B are closely related. Electron microscopy reveals undulin as structures consisting of an approximately 80-nm-long-tail with a nodule on one end and with one or two shorter arms on the other. Ultrastructurally immunolabeled undulin is found mainly between densely packed mature collagen fibrils. Indirect immunofluorescence shows bundles of uniform wavy fibers in dense connective tissues superimposable on a subpopulation of type I collagen structures. This suggests that undulin serves a specific yet unknown function in the supramolecular organization of collagen fibrils in soft tissues.

[Studies of the in vitro toxicity of D-penicillamine in the Chinese hamster ovary system]. / In-vitro-Toxizitätsuntersuchungen von D-Penicillamin am CHO-System.

The amino-thiol-containing compound D-penicillamine is used as a disease-modifying drug in the treatment of rheumatoid arthritis. In the present study the effects of D-penicillamine on Chinese hamster ovary cells (CHO) were evaluated. It could be shown that only high doses, i.e. doses exceeding the therapeutically occurring plasma concentration many times, were toxic to the cells, leading to an early death of the cells. High doses (1,500 micrograms/ml) of D-penicillamine caused an inhibition of the endogenous pyrimidine synthesis of the CHO and lead to a drastic reduction of the cellular protein biosynthesis. The observations made are discussed with respect to formerly published data about mutagenicity and carcinogenicity of D-penicillamine.

DNA-binding activity is associated with purified myb proteins from AMV and E26 viruses and is temperature-sensitive for E26 ts mutants.

Oncogene protein products from avian myeloblastosis virus, p48v-myb, and from avian leukemia virus E26, p135gag-myb-ets, are located predominantly in the nucleus of nonproducer bone marrow cell clones, as revealed by indirect immunofluorescence. Both oncogene proteins were purified by immunoaffinity chromatography using monoclonal antibodies against p19 and immunoglobulins specific for myb, which was expressed in bacteria for antibody production. The purified proteins bind to DNA in vitro. In contrast, purified p135gag-myb-ets proteins from several mutants of E26 virus, temperature-sensitive for myeloblast transformation, either lost their abilities to bind to DNA or exhibited highly thermolabile DNA-protein interactions in vitro. DNA binding of AMV and E26 oncogene proteins is inhibited by myb-specific immunoglobulins. Our results suggest that lesions in the myb oncogene affect transformation as well as DNA binding of myb proteins in vitro.

Inhibition of DNA binding of purified p55v-myc in vitro by antibodies against bacterially expressed myc protein and a synthetic peptide.

To identify viral myc proteins, we have prepared myc-specific antibodies: (i) against a synthetic peptide corresponding to the nine carboxy-terminal amino acids of the viral myc (C9); (ii) against a bacterially expressed viral myc protein obtained by inserting the SalI-BamHI fragment of the viral MC29 DNA clone in the expression vector pPLc24. Both antisera recognize a protein of 55 000 mol. wt., p55v-myc, in MH2- and OK10-transformed fibroblasts. The protein is located in the nucleus, as shown by indirect immunofluorescence and cell fractionation. Antibodies against the C9 peptide were used to purify the p55v-myc by immunoaffinity column purification (3000-fold) from OK10- and MH2-transformed fibroblasts. p55v-myc binds to double-stranded DNA in vitro as does p110gag-myc. DNA binding in vitro is inhibited by the immunoglobulin fraction of antibodies against the bacterially expressed myc protein. Furthermore, a synthetic peptide consisting of 16 amino acids (C16) was used to isolate specific immunoglobulins which also inhibit DNA binding in vitro. OK10 codes, in addition to p55v-myc, for a p200gag-pol-myc polyprotein. The majority of this protein is located in the cytoplasm (79%). The purified protein binds to single-stranded RNA in vitro, unlike other gag-myc or myc proteins.

Decreased DNA-binding ability of purified transformation-specific proteins from deletion mutants of the acute avian leukemia virus MC29.

Avian myelocytomatosis virus MC29 is a highly oncogenic replication-defective retrovirus that predominantly affects hematopoietic cells and causes acute leukemia in vivo and that transforms hematopoietic cells as well as fibroblasts in vitro. The transformation-specific sequence, v-myc, is expressed as part of a fusion protein that contains the viral structural protein p19. By use of monoclonal antibodies against p19 we showed that the v-myc-encoded protein is located in the nucleus of MC29-transformed fibroblasts and that after purification over an immunoaffinity column the protein binds to double-stranded DNA. In this report we describe the analysis of the v-myc gene product from MC29-transformed bone marrow cells. The immunoaffinity column-purified protein from these cells also bound to DNA and was indistinguishable from the purified protein from MC29-transformed fibroblasts. In addition, the v-myc gene products from fibroblasts transformed by three nonconditional mutants of MC29--which transform hematopoietic cells with a markedly decreased efficiency in vivo and in vitro but still transform fibroblasts in vitro, expressing deleted v-myc proteins--were analyzed. In contrast to the wild-type protein, the purified mutant proteins had decreased DNA-binding abilities. Furthermore, a preferential binding of the wild-type protein to poly(dG) . poly(dC) duplexes was observed. Such a specificity was lost with a mutant protein. These results provide evidence that the interaction of the v-myc protein with DNA may be directly involved in transformation of the hematopoietic target cells. Further, the transformation-specific fusion proteins purified from cells transformed by avian erythroblastosis virus, which belongs to a different class of acute leukemia viruses, and by Fujinami sarcoma virus were found not to be DNA-binding proteins, suggesting the existence of different transformation mechanisms.

Analysis of two phosphoproteins related to pp60src from Schmidt-Ruppin D virus particles.

During endogenous phosphorylation of partially purified pp60src from virus particles, besides pp60src two additional phosphoproteins, 45K and 42K, were found. These proteins copurify with pp60src. They were shown to be proteolytic degradation products due to the action of the virus-associated protease p15. All three phosphoproteins were present in particles of two different sarcoma virus strains, Schmidt-Ruppin D and Prague C, indicating that this phenomenon is general rather than strain-specific. The degradation rate of pp60src was reduced by the presence of 3 mM-ZnCl2, which acts as a protease inhibitor.

The transforming protein of the MC29-related virus CMII is a nuclear DNA-binding protein whereas MH2 codes for a cytoplasmic RNA-DNA binding polyprotein.

The acute avian leukemia viruses MH2 and CMII belong to the group of avian myelocytomatosis viruses, the prototype virus of which is MC29. This group of viruses is characterized by myc-specific oncogenes which are presumably expressed as gag-myc polyproteins. These polyproteins are synthesized in non-producer cells transformed by MH2 and CMII and have mol. wts. of 100 000 (p100) and 90 000 (p90), respectively. Monoclonal antibodies against the N terminus of gag, p19, were used to localize the protein in MH2- and CMII-transformed non-producer fibroblasts. Immunofluorescence and cell fractionation indicated that greater than 90% of p100 from MH2 was located in the cytoplasm, whereas greater than 70% of p90 from CMII resided in the nucleus. Isolation of p100 and p90 by immunoaffinity chromatography resulted in an approximately 2000-fold purification of the two polyproteins. Both of them, as well as p110 of MC29, bound to double-stranded DNA of chick fibroblasts in vitro. However, only the MH2-specific polyprotein p100 bound to RNA in vitro. Such a binding was not observed for p90 or p110, or for the purified gag precursor Pr76. Another polyprotein, gag-erbA, from avian erythroblastosis virus, which is also located in the cytoplasm, did not bind to RNA. Our results indicate that the CMII-specific polyprotein p90 behaved indistinguishably from the p110 of MC29. However, the MH2-specific polyprotein p100 exhibited unique and novel properties which were distinct from a gag-myc-type protein.

Association of gag-myc proteins from avian myelocytomatosis virus wild-type and mutants with chromatin.

The localization of the transformation-specific proteins was analyzed in quail embryo fibroblast cell lines transformed by wild-type avian myelocytomatosis virus MC29 and by three of its deletion mutants, Q10A , Q10C , and Q10H , with altered transforming capacities, and in a chicken fibroblast cell line transformed by the avian erythroblastosis virus (AEV). These viruses code for polyproteins consisting of part of the gag gene and of a transformation-specific region, myc for MC29 and erb A for AEV. Analysis by indirect immunofluorescence using monoclonal antibodies against p19, the N-terminal region of the polyprotein, showed that the gag-myc proteins in cells transformed by the wild-type MC29 as well as by the three deletion mutants are located in the nucleus. In contrast, cells transformed by AEV, which express the gag-erb A protein, give rise to cytoplasmic fluorescence. Fractionation of cells into nuclear and cytoplasmic fractions and analysis by immunoprecipitation and gel electrophoresis confirmed these results. About 60% of the gag-myc proteins of wild-type as well as of mutant origin were found in the nucleus, while 90% of the gag-erb A protein was present in the cytoplasm. Also, pulse-chase analysis indicated that the gag-myc protein rapidly accumulates in the nucleus in just 30 min. Further, it was shown that the wild-type and also mutant gag-myc proteins are associated with isolated chromatin. Association to chromatin was also observed for the gag-myc protein from MC29-transformed bone marrow cells, which are believed to be the target cells for MC29 virus in vivo.

Biochemical characterization of transformation-specific proteins of acute avian leukemia and sarcoma viruses.

The biological and biochemical properties of the transformation-specific proteins of three avian oncornaviruses with different oncogenic potentials were compared, namely the gag-myc protein of the avian myelocytomatosis virus MC29, the gag-erb A protein of the avian erythroblastosis virus AEV, and the gag-fps protein of Fujinami sarcoma virus FSV. These oncogenes were analyzed in transformed fibroblasts that expressed only the transforming proteins but showed no virus replication. Monoclonal antibodies against the viral structural protein p19, which is the N-terminus of the proteins, were used for indirect immunofluorescence, for immunoprecipitation of the proteins from subcellular fractions, and for immunoaffinity column chromatography. With this last method a 3000-fold purification of the proteins was obtained. By indirect immunofluorescence it was shown that the gag-myc protein was located in the nucleus, and bound to DNA after purification. The gag-erb A protein was not nuclear but probably located in the cytoplasm and did not bind to DNA after purification. Neither of the two proteins exhibited protein kinase activity. In contrast, the gag-fps protein did not bind to DNA but showed protein kinase activity after purification. It was not located in the nucleus either.