Isolierung von freien Nucleotiden aus verschiedenen Geweben

Zusammenfassung Es wurden die freien Nucleotide aus dem säurelöslichen Extrakt des Sarkom 37 (Ascitesform) durch Anionenaustauschchromatographie isoliert. Die Befunde werden mit den Ergebnissen an normalen und anderen Tumorgeweben verglichen.In der Arbeit werden folgende Abkürzungen verwendet Ad Adenosin - A2MP Adenosin-2-monophosphat - A3MP Adenosin-3-monophosphat - AMP Adenosin-5-monophosphat - ADP Adenosin-5-monophosphat - ATP Adenosin-5-triphosphat - G2MP Guasonin-2-monophosphat - G3MP Guanosin-3-monophosphat - GMP Guanosin-5-monophosphat - GDP Guanosin-5-diphosphat - GTP Guanosin-5-triphosphat - C2MP Cytidin-2-monophosphat - C3MP Cytidin-3-monophosphat - CMP Cytidin-5-monophosphat - CDP Cytidin-5-diphosphat - CTP Cytidin-5-triphosphat - U2MP Uridin-2-monophosphat - U3MP Uridin-3-monophosphat - UMP Uridin-5-monophosphat - UDP Uridin-5-diphosphat - UTP Uridin-5-triphosphat - UDPA Uridin-5-diphosphat-N-acetylglucosamin - UDPG Uridin-5-diphosphat-glucose - UDPGa Uridin-5-diphosphatgalaktose - UDPGl Uridin-5-diphosphatglucuronsäure - TPN Triphosphopyridinnucleotid - DPN Diphosphopyridinnucleotid - FAD Flavin-adenin-dinucleotid - HS Harnsäure - RNS Ribonucleinsäure - IMP Inosin-5-monophosphat Mit 6 Textabbildungen.Den soliden Tumor verdanken wir Herrn Dr. Dr.Ch. Hackmann, Farbenfabriken Bayer, Wuppertal-Elberfeld. Dieser Tumor wurde von Herrn Dr.H. Wrba im hiesigen Institut in die Ascitesform übergeführt.     

Activated cyclophosphamide: An enzyme-mechanism-based suicide inactivator of DNA polymerase/3′–5′ exonuclease

Summary DNA polymerase I fromE. coli can toxify activated cyclophosphamide (CP) by means of the 3–5 exonuclease activity associated with the enzyme. Acrolein and an alkylating moiety are released in the process. Preincubation of DNA polymerase I with activated CP for 15–60 min leads to an increasing inhibition of DNA polymerase activity, which can be prevented when preincubation of DNA polymerase I with activated CP is carried out in the presence of 5 AMP, a competitive inhibitor of the 3–5 exonuclease subsite of the enzyme. This demonstrates that toxification of activated CP by the 3–5 exonuclease subsite of DNA polymerase is a prerequisite for the inhibition of DNA polymerase activity. The kinetics and the degree of DNA polymerase inhibition suggest that the alkylating moiety rather than acrolein released from activated CP during toxification is responsible for the inhibition of DNA polymerase. DNA polymerase with associated 3–5 exonuclease activity has also been isolated from eukaryotic cells, and toxification of activated CP by such an enzyme (DNA polymerase from rabbit bone marrow) has been shown previously. Thus we suggest that toxification of activated CP by DNA polymerases/3-5 exonucleases present mainly in proliferating cells might lead to the specific alkylation of macromolecules involved in the cell proliferation process, such as the DNA polymerase subsite of these enzymes and probably also the DNA bound to the enzymes. The relatively high cancerotoxic selectivity and cytotoxic specificity of activated CP could be based on this specific enzyme-mediated alkylation.Supported by the Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg     

Influence of insulin on cyclic 3′,5′-AMP phosphodiesterase activity in liver,skeletal muscle,adipose tissue,and kidney

Summary Influence of insulin on liver glycogen metabolism and on lipolysis appears to be mediated by a decreased intracellular 3,5-AMP concentration. Reduced formation of 3,5-AMP had been shown in adipose tissue incubated with insulin. The influence of insulin on 3,5-AMP degradation has been investigated. — 3,5-AMP phosphodiesterase (PDE) activity was reduced in liver, adipose tissue and, insignificantly, in skeletal muscle of insulin deficient, i.e. alloxan diabetic or starved rats. I.V. injection of a low dose of insulin (0.5 U/kg) or stimulation of endogenous insulin secretion by injection of glucose led to a rapid increase of PDE activity in these tissues. 15 min after insulin injection liver PDE activity was increased. The maximal effect occurred after 30–45 min. Renal PDE activity was not decreased in alloxan diabetes, insulin injection has been found ineffective. —In vitro, there was an activating effect of crystalline insulin on PDE purified from beef heart. Insulin concentration required for duplication of enzyme activity was of the order of 2 · 10^–5 M. Treatment with actinomycin D nearly prevented stimulation of liver PDE by insulin. This may indicate that the action of insulin on PDE activity is essentially based on an increased enzyme synthesis. — Owing to the influence of insulin secretion on liver and adipose tissue 3,5-AMP concentration, glycogen metabolism and lipolysis can be quickly adapted to food intake.

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Der Einfluß von Insulin auf die 3,5-AMP-Phosphodiesterase-Aktivität in Leber, Skeletmuskulatur, Fettgewebe und Niere

Zusammenfassung An der Steigerung der Glykogensynthese der Leber und der Verminderung der Lipolyse durch Insulin ist eine Abnahme der 3,5-AMP-Konzentration wesentlich beteiligt. Die 3,5-AMP-Bildung ist in Fettgewebe, das mit Insulin inkubiert wird, vermindert. Insulin beeinflußt jedoch auch den 3,5-AMP-Abbau. -Die 3,5-AMP-Phosphodiesterase (PDE)-Aktivität des Fettgewebes, der Leber und, in geringerem Grade, der Skeletmuskulatur ist im Insulinmangel vermindert, d.h. bei alloxandiabetischen oder hungernden Ratten. I.v. Injektion von 0,5 E/kg Insulin oder eine erhöhte Abgabe von Insulin aus dem Pankreas nach Glucoseinjektion führen in diesen Geweben zu einem raschen Anstieg der PDE-Aktivität. Dieser ist in der Leber schon 15 min nach Insulingabe nachweisbar und erreicht nach 30–45 min sein Maximum. In der Niere ist kein Einfluß von Insulin auf die PDE-Aktivität nachweisbar. — Aus Rinderherz isolierte PDE wirdin vitro durch Insulin aktiviert, jedoch werden2 · 10^–5 M zur Verdopplung der Aktivität benötigt. Actinomycin D verhindert die Steigerung der Leber-PDE-Aktivität nach Insulininjektion. So kann die Wirkung des Hormons im wesentlichen auf eine gesteigerte PDE-Synthese zurückgeführt werden. — Durch diesen Einfluß der Insulininkretion auf die 3,5-AMP-Konzentration in Leber und Fettgewebe können Glykogenstoffwechsel und Lipolyse rasch an die Nahrungsaufnahme angepaßt werden.

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Influence de l'insuline sur l'activité de la 3,5-AMP-phosphodiestérase dans le foie, le muscle strié, le tissu adipeux et le rein

Résumé L'influence de l'insuline sur le métabolisme du glycogène hépatique et sur la lipolyse semble s'exercer par l'intermédiaire d'une diminution de la concentration de 3,5-AMP intracellulaire. Onamontré une diminution de la formation de 35-AMP dans le tissu adipeux incubé avec de l'insuline. L'influence de l'insuline sur la dégradation du 3,5-AMP est étudiée. — L'activité de la 3,5-AMP-phos-phodiestérase (PDE) est diminuée dans le foie, le tissu adipeux et, de façon non-significative, dans le muscle strié des rats qui manquent d'insuline, c-à-d les rats rendus diabétiques par l'alloxane ou les rats privés de nourriture. L'injection intraveineuse d'une faible dose d'insuline (0.5 U/kg) ou la stimulation de la sécrétion d'insuline endogène par une injection de glucose provoquent une augmentation rapide de l'activité de la phosphodiestérase dans ces tissus. 15 min après l'injection d'insuline, l'activité de la phosphodiesterase du foie est augmentée. L'effet maximum est atteint après 30–45 min. L'activité de la phosphodiestérase rénale n'est pas diminuée dans le diabète alloxanique, l'injection d'insuline s'est avérée inefficace.In vitro, l'insuline cristalline a un effet activant sur la phosphodiestérase purifiée du coeur de boeuf. La concentration d'insuline requise pour doubler l'activité de l'enzyme est de l'ordre de 2 · 10^–5 M. Le traitement avec actinomycin D empêche la stimulation par l'insuline de la PDE dans le foie. Ceci peut indiquer que l'action de l'insuline sur l'activité de la phosphodiestérase est essentiellement basée sur une synthèse accrue de l'enzyme. A cause de l'influence de la sécrétion d'insuline sur la concentration en 3,5-AMP du foie et du tissu adipeux, le métabolisme du glycogène et la lipolyse peuvent s'adapter rapidement à la prise de nourriture.

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Non-Standard Abbreviations G 6 P Glucose-6-phosphate - UDPG UDP-glucose - FFA non-esterifled, free fatty acids - 3,5-AMP cyclic adenosine-3,5-monophosphate - PDE 3,5-AMP phosphodiesterase This study was supported by the Deutsche Forschungsgemeinschaft.Deceased October 31, 1967.     

Changes in the hydroxyproline,hexosamine and sialic acid of the diabetic human and β, β′-iminodipropionitrile-treated rat retinal vascular systems

Summary The retinal vasculature has been isolated from nondiabetic and diabetic post mortem human eyes by controlled trypsin digestion. Chemical analysis demonstrated increases in the hydroxyproline and hexosamine contents in diabetes. There was no general increase in the sialic acid content. These results have been related to histological preparations of sectors of the same retinas. Administration of, -iminodipropionitrile to rats caused a retinopathy characterised by endothelial cell proliferation, increased PAS-positivity and microaneurysms. Chemical analysis of retinal vascular systems from, -iminodipropionitrile-treated rats revealed increases in hydroxyproline, hexosamine and sialic acid contents.

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Änderungen des Gehaltes an Hydroxyprolin, Hexosamin und N- azetyl- Neuraminsäure in den Netzhautgefäen von menschlichen Diabetikern und mit , Iminodiproprionitril behandelten Ratten

Zusammenfassung Die Netzhautgefäße wurden post-mortal aus diabetischen und nichtdiabetischen Augen mit Hilfe einer kontrollierten Trypsin-Behandlung gewonnen. Bei der chemischen Analyse fand sich ein Anstieg des Hydroxyprolin- und Hexosamingehaltes in den diabetischen Augen. Der N-azetyl-Neuraminsäuregehalt war im allgemeinen nicht erhöht. Diese Resultate wurden in Beziehung gebracht zu den histologischen Befunden, die an den einzelnen Sektoren der gleichen Retina erhoben wurden. Verabreichung von, -Iminodiproprionitril an Ratten bewirkte eine Retinopathie unter den Zeichen einer Wucherung der Endothelzellen, verstärkter PAS-Anfärbbarkeit und Mikroaneurysmen. Die chemische Analyse der Netzhautgefäße von mit, -Iminodiproprionitril behandelten Ratten zeigte einen erhöhten Gehalt an Hydroxyprolin, Hexosamin und N-azetyl-Neuraminsäure.

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Variations de l'hydroxyproline, de l'hexosamine et de l'acide sialique dans les systèmes vasculaires rétiniens de l'homme diabétique et du rat traité par le , -iminodipropionitrile

Résumé Le système vasculaire rétinien a été isolé, par digestion trypsique contrôlée, après la mort, à partir d'yeux humains de non-diabétiques et de diabétiques. L'analyse chimique a révélé l'augmentation du contenu en hydroxyproline et en hexosamine, dans le diabète. Il n'y avait pas d'augmentation générale du contenu en acide sialique. Ces résultats ont été rapprochés des préparations histologiques de portions des mêmes rétines. L'administration de, -iminodipropionitrile à des rats provoquait une rétinopathie caractérisée par une prolifération des cellules endothéliales, une PAS-positivité augmentée et des microanévrismes. L'analyse chimique des systèmes vasculaires rétiniens des rats traités par le,-iminodipropionitrile, a révélé l'augmentation du contenu en hydroxyproline, en hexosamine et en acide sialique.

     

Uptake and metabolism of radiolabelled GM1-ganglioside in skin fibroblasts from controls and patients with GM1-gangliosidosis

Summary The uptake and metabolism of [3-³H-sphingosine]GM1-ganglioside was measured in cultured skin fibroblasts from controls and patients with infantile, juvenile and adult GM1-gangliosidosis. When dissolved in medium with phosphatidylserine, GM1-ganglioside was efficiently taken up by cultured skin fibroblasts and transferred into lysosomes. A linear increase in GM1-ganglioside endocytosis was shown with phosphatidylserine concentrations of up to 40m/ml. A pulse-chase study revealed that [³H]GM1-ganglioside was metabolized to GM2-ganglioside, GM3-ganglioside, ceramide dihexoside, ceramide monohexoside, ceramide and sphingosine. Sphingosine was recycled to sphingomyelin. In a 20-h pulse study, cell lines from patients with GM1-gangliosidosis of infantile, juvenile and adult types hydrolysed 2–5%, 20–44% and 54–58% of the total endocytosed GM1-ganglioside respectively. These values were lower than in control cells (64.17 ± 5.43% (n=10)). The hydrolysis rates of exogenous [³H]GM1-ganglioside in cultured fibroblasts from patients with various types of GM1-gangliosidosis closely reflected the clinical severity.Abbreviations GM1 Gal13GalNAc14(NeuAc23)Gal14Galc-1-ceramide - GM2 GalNAc14(NeuAc23)Gal14Glc-1-ceramide - GM3 NeuAc23Gal14Glc-1-ceramide - CDH Gal14Glc-1-ceramide     

In vivo and in vitro test for growth potential of liver cells from rats during early stage of hepatocarcinogenesis by 3′-methyl-4-dimethylaminoazobenzene

Summary A rapid increase in the fraction of small liver cells was observed in the liver of rats during the early stage of hepatocarcinogenesis by 3-methyl-4-dimethylaminoazobenzene (3-Me-DAB). The change in cell population was represented by the decrease in glucose-6-phosphatase activity and by the increase in number of -glutamyltranspeptidase-positive cells. When DNA synthesis of liver cells from rats fed 3-Me-DAB was measured by autoradiography in primary culture, it began to increase 2 weeks after the start of the carcinogen feeding, reaching a plateau level after 3 weeks. Liver cells from rats fed 3-Me-DAB for 2 weeks or over demonstrated a remarkable resistance to the cytotoxic effect of the carcinogen (0.24 mM) in primary culture. Furthermore, liver cells from rats fed 3-Me-DAB for 3 weeks or over proliferated in the presence of the carcinogen in primary culture. When liver cells from 3-Me-DAB-fed and control rats were transplanted into syngeneic rat spleens, the former cells proliferated more vigorously than did the latter. The growth potential of liver cells from 3-Me-DAB-fed rats tended to be enhanced with time in the carcinogen feeding. Hepatocellular carcinomas developed in the host spleens implanted with liver cells from a rat fed 3-Me-DAB for 8 weeks. As described above, liver cells from rats fed 3-Me-DAB demonstrated much greater proliferative ability than normal control cells in vivo and in vitro.Abbreviations used HCC hepatocellular carcinoma - 3-Me-DAB 3-methyl-4-dimethylaminoazobenzene - GGT -glutamyltranspeptidase     

In vitro and in vivo investigations for the development of cytostatic methylhydrazones

Summary In in vitro short-term (3 h) assays, the -chloroethyl-methyl-hydrazones B 1 and B 2 inhibit the uptake of ³H-thymidine by EAC and L 1210 leukemia cells, B 2 being 5 to 10 times more effective than B 1. The growth inhibitory effect of both compounds was also confirmed in long-term (7 days) clonal assays using agar-containing glass capillaries, B 2 again being more effective than B 1. In contrast to these differences in vitro, in vivo both substances showed remission to the same degree in EAC- and complete resistance in L 1210-bearing mice. The diverging in vitro/in vivo sensitivities were thought to result from differences in the affinity of the methylhydrazones to the tumor cells: using short exposure periods (3 h) B 1 was more inhibitory than B 2 on both EAC and L 1210 colony growth; i.e., the more hydrophilic B 2 could more easily be washed off. To further test the idea of different cell membrane affinities, the methylhydrazones ZB 1 and P 1 with increasing lipophilic properties were synthesized. In vitro, after both pulse and continuous exposure ZB 1 and P 1 showed enforced inhibitory effects on colony growth. In vivo, ZB 1 and P 1 reduced the tumor weight of EAC mice, while only P 1 increased the survival time of L 1210 mice. The results suggest that from the combination of in vitro/in vivo assays mechanistic conclusions can be derived that are valuable for further development of these cystostatics.Abbreviations B 1 N-methyl-N--chloroethyl-benzaldehyde-hydrazone - B 2 N-methyl-N--chloroethyl(p-dimethylamino)benzaldehyde-hydrazone - EAC Ehrlich Ascites Carcinoma - FCS fetal calf serum - ³H-dThd 6-³-H-thymidine - L 1210 Leukemia L 1210 - P 1 N-methyl-N--chloroethyl-pentadiene-benzaldehyde-hydrazone - ZB 1 N-methyl-N--chloroethyl-cinnamoylaldehyde-hydrazone     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Summary Antineoplastic activity of tiazofurin (2--D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC₅₀ of 19.2 M. TAD content in K 562 cells (1.3 nmol/10⁹/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.Abbreviations ALL acute lymphoblastic leukemia - ANLL acute non lymphocytic leukemia - CML chronic myelogenous leukemia - GMP, GDP, GTP guanosine 5-mono-, di-, triphosphate - HPLC high pressure liquid chromatography - IMP inosine 5-monophosphate - IMPD IMP dehydrogenase - NAD nicotinamide-adenine-dinucleotide - NCI National Cancer Institute; - PBS phosphate buffered saline - TAD thiazole-4-carboxamide-adenine dinucleotide - TCA trichloracetic acid - TRMP tiazofurin 5-monophosphate - TRTP tiazofurin 5-triphosphate. The research work outlined in this paper was supported by United States Public Health Service grant R 35 CA 42510 awarded by the National Cancer Institute, Department of Health and Human Services, USA to G. Weber. The results will be presented at the 79th annual meeting of the American Association for Cancer Research in New Orleans, May 25–28, 1988     

Ectoenzymes on the surface of cells from human lymphoblastoid lines: 5′-Nucleotidase and phosphatase

Summary Activities of the ecto-enzymes 5-nucleotidase (5-N) and phosphatase were determined on the surface of intact cells from 15 different established lines of human B- and T-lymphoblasts. Whereas all the lines express phosphatase, 10 of the lines were negative for 5-N. 5-N-negative cell lines are found among B as well as T cells, and they do not carry cryptic enzyme activity. In a 5-N-positive line activity of this enzyme is correlated with growth showing a peak during the logarithmic phase. On the other hand, inhibition of 5-N does not change the growth curve of this line. Neuraminidase treatment of the cell surface brings about an increase in phosphatase but not in 5-N activity. 5-N of two B-cell lines and of human peripheral blood lymphocytes shows complete crossreactivity with an antiserum obtained against human placental 5-N. However, the enzyme of one lymphoma line with B-cell properties (EHR-A-Ramos) does not cross-react with this serum. The results are discussed with respect to suitability of these lymphoblast lines as model systems for the study of immunodeficiencies.With a financial support of the Deutsche Forschungsgemeinschaft (grants SFB 37, Gu 123 and Gu 153)     

Demonstration of a relatively hepatoselective effect of covalent insulin dimers on glucose metabolism in dogs

Summary Insulin analogues with relatively greater effect on hepatic glucose production than peripheral glucose disposal could offer a more physiological approach to the treatment of diabetes mellitus. The fact that proinsulin exhibits this property to a minor degree may suggest that analogues with increased molecular size may be less able than insulin to obtain access to peripheral receptor sites. Covalent insulin dimers have previously been shown to possess lower hypoglycaemic potencies than predicted by their in vivo receptor binding affinities. Reduced rates of diffusion to peripheral target tissues-might be an explanation for the lower in vivo potency compared to insulin. To test the relative hepatic and peripheral effects of covalent insulin dimers, glucose clamp procedures with D-[3-³H] glucose tracer infusions were used in anaesthetised greyhounds to establish dose-response curves for rates of hepatic glucose production and glucose disposal with insulin, N^B1, N^{B 1},-suberoyl-insulin dimer, and N^B29, N^{B 29},-suberoyl-insulin dimer. With N^B1, N^{B 1},-suberoyl-insulin dimer molar potencies relative to insulin were 68%, (34–133) (mean and 95% fiducial limits), for inhibition of hepatic glucose production and 14.7%, (10.3–20.9) for glucose disposal. With N^B29,N^{B 29},-suberoyl-insulin dimer potencies were 75%, (31–184) and 2.5%, (1.5–4.3), for inhibition of hepatic glucose production and for glucose disposal, respectively. The demonstration that both dimers exhibit a significantly greater effect on glucose production than on glucose disposal supports the suggestion that analogues with increased molecular size may exhibit reduced ability to gain access to peripheral target cells.Abbreviations B1-B 1D N^B1,N^{B 1},-suberoyl-insulin dimer - B29-B 29D N^B29,N^{B 29},-suberoyl-insulin dimer - Ra hepatic glucose production rate - Rd peripheral glucose disposal rate - M_r relative molecular weight - MCR metabolic clearance rate - ANOVA analysis of variance     

In Finland insulin gene region encoded susceptibility to IDDM exerts maximum effect when there is low HLA-DR associated risk

Summary An association between insulin-dependent diabetes mellitus (IDDM) and polymorphisms of the insulin gene on chromosome 11p15 (INS) is a consistent finding in Europid populations. While one study suggested that the INS association is restricted to HLA-DR4-positive individuals, studies in other Europid populations have shown the disease-associated INS genotype to confer susceptibility independently of HLA-DR. We have investigated the role of INS in susceptibility to IDDM in Finland, which has the highest incidence of diabetes mellitus in the world, at two polymorphic restriction sites, 5 and 3 to the insulin gene. From the DiMe (Childhood Diabetes in Finland) Study we studied 154 diabetic children without regard to HLA-DR type; 108 DR4 positive/non-DR3 diabetic children; 39 DR3 positive/non-DR4 diabetic children; 30 DR4/DR3 positive diabetic children; 31 non-DR4/non-DR3 diabetic children; 96 matched DiMe control subjects and 86 other healthy, non-diabetic Finnish control subjects. We found an overall association between IDDM and INS in the high-risk Finnish population only with the 5 polymorphism and identified an INS haplotype negatively associated with IDDM in Finland. However, among diabetic subjects with a reduced HLA-associated susceptibility (non-DR4/non-DR3) both 3 and 5 INS loci showed an association with IDDM (p values 0.02 and 0.0002, respectively). Thus, in the Finnish population insulin gene-encoded susceptibility to IDDM exerts a maximum effect in those with reduced HLA-associated risk.Abbreviations INS Insulin gene region - VNTR variable number tandem repeats - PCR polymerase chain reaction - RR relative risk - CI confidence intervals - IDDM insulin-dependent diabetes mellitus     

Characterization of secretin release in secretin cell-enriched preparation isolated from canine duodenal mucosa

The release of secretin was studied in secretin cell-enriched preparations isolated from canine duodenal mucosa. The crude enterocytes were isolated by treating the duodenal mucosa sequentially with collagenase and ethylenediaminetetraacetic acid. Secretin cell-enriched fraction was prepared by centrifugation of the crude enterocytes in a counterflow elutriation rotor to obtain a final preparation containing 3.2±0.3 pmol/10⁶ cell of immunoreactive secretin, which was 13-fold greater than the crude cell preparation (N=5). The cells were incubated in Hanks' balanced salt solution for 20 min at 37°C under 95% O₂/5% CO₂ before adding various agents and further incubated for various periods of time. The amounts of secretin released into the medium and retained by the cells were then determined by a specific radioimmunoassay. The release of immunoreactive secretin was increased dose-dependently over the control by dibutyryl cyclic-3, 5-adenosine monophosphate, forskolin, 4-12-O-tetradecanoylphorbol-13-acetate, the synthetic serine protease inhibitor, camostat, and the calcium ionophore, A23187. The effects of forskolin, the phorbol ester, and A23187 were time-dependent and not observed at 4°C. The release of immunoreactive secretin was also stimulated by KCl in high concentration and by sodium oleate. The effect of A23187 was abolished in a Ca²⁺-free medium, while those of dibutyryl cyclic-3, 5-adenosine monophosphate and forskolin were potentiated by 3-isobutyl-l-methylxanthine, which did not have a significant effect when added alone. These results indicate that the release of secretin is regulated by both Ca²⁺- and cyclic-3, 5-adenosine monophosphate-dependent mechanisms. In addition, cellular protein kinase C activity may play an important role in regulation of secretion release.     

Investigations on metabolism,genotoxic effects and carcinogenicity of 2,2′-dichlorodiethylether

Summary Either 40 mole or 160 mole 2,2-DDE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in the 24-h urine specimens. Comparative investigations were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. In a further step, inhalation experiments were performed to determine urinary excretion of the two metabolites after an 8-h exposure of male Wistar rats to 10, 50, 100, and 500 ppm 2,2-DDE and to 50, 200, und 1000 ppm vinyl chloride.A long-term study was conducted to investigate the possible carcinogenicity of 2,2-DDE in male and female Sprague-Dawley rats following s.c. injections of 4.36 mole and 13.1 mole 2,2-DDE in DMSO per week. The evaluation of tumor development in treated groups and controls were based on macroscopic inspection and histological examinations of the suspect organs and tissues.Analysis of the metabolites showed that HEMA excretion was much lower than the excretion of TdGA following the uptake of 2,2-DDE, 2-chloroethanol and 2-chloroacetaldehyde diethyl acetal. Contrary to these, vinyl chloride uptake resulted in a higher urinary excretion of HEMA than TdGA.There was no appreciable increase in the number of tumors detected in 2,2-DDE-treated animals when compared with untreated or DMSO-treated groups.Since irradiation of 2,2-DDE with UV did not elevate mutagenic activity of the compound against Salmonella typhimurium TA100, the high mutagenicity of the compound found in a desiccator cannot be due to the liberation of mutagenic compounds produced under the influence of UV light.Abbreviations 2,2-DDE 2,2-dichlorodiethylether - DMSO dimethylsulfoxide - HEMA hydroxyethyl mercapturic acid - TdGA thiodiglycolic acid     

Allele-specific amplification of genomic DNA for detection of deletion mutations: Identification of a French-Canadian tay-sachs mutation

Summary A rapid and efficient method for the detection of a 7.6-kb deletion in the-hexosaminidase A-subunit gene, a mutant allele causing Tay-Sachs disease in French Canadians, is described. The protocol involves PCR (polymerase chain reaction) amplification of target sequences on normal and mutant chromosomes. Three amplification primers, a single 5 primer complementary to normal and mutant DNA templates and two 3 primers specific for normal and mutant DNA templates are required. The primers direct amplification of two unique fragments (normal and mutant) that are easily separated by gel electrophoresis. Allele-specific oligonucleotide hybridization using normal and mutant probes to genomic DNA samples from normal, heterozygous and homozygous individuals confirms these results and is consistent with results of genotypic classification of individuals using Southern analysis. The method is applicable to detection of deletion mutations in cases where some deletion-flanking sequence is known.     

Time Intervals (3′ or 5′) Between Dose Steps Can Influence Methacholine Challenge Test

Bronchial hyperresponsiveness (BHR) is a common feature in the majority of asthmatic subjects and methacholine is the most frequently used agent for the test. The influence of 3 or 5 min time intervals between doses steps in a double methacholine challenge test (MCH-3 or MCH-5) was investigated. Using the MCH-3 challenge, 52 intermittent asthmatics were classified as having moderate (BHR-M; 18 subjects), mild (BHR-m; 19 subjects), or bordeline (BHR-B; 15 subjects) BHR. The cumulative dose and the PD20FEV₁ were higher for MCH-5 compared with MCH-3 in BHR-m (p < 0.05) and BHR-B (p < 0.05) but not in the BHR-M group. Also the dose response slopes, FEV₁% decline/cumulative methacholine dose, calculated for the two challenge tests were statistically different only in BHR-m (p < 0.05) and BHR-B (p < 0.01). At MCH-5, there were 16 subjects with BHR-M, 18 with BHR-m, 12 with BHR-B and 6 subjects with normal reactivity. Results may suggest that in the group of BHR-m and BHR-B subjects, at MCH-5 compared with MCH-3, the cumulative effect of the administered drug, quickly metabolized by cholinesterase, is not complete, thus leading to an incorrect estimation of bronchial hyperresponsiveness degree. It is hoped that time interval between doses be standardized to ensure maximum comparability within and between subjects in challenge tests.     

Differences in the regional distribution and response to ischaemia of adenosine-regulating enzymes in the heart

Summary The activities of the adenosine-generating enzyme 5-nucleotidase and the adenosine-degrading enzyme adenosine deaminase were determined for four regions of rat hearts prior to and following 10–60 min of ischaemia. Whereas adenosine deaminase was uniformly active throughout the heart, 5-nucleotidase was twice as active in atrial than in ventricular myocardium, and more active in the right than in the left ventricles in normoxic tissues. In isolated heart preparations normoxic perfusion decreased adenosine deaminase and increased 5-nucleotidase activity compared to levelsin vivo. Global ischaemia for 10 min elevated adenosine deaminase activity but had no effect on 5-nucleotidase activity. However, 30 min of ischaemia decreased 5-nucleotidase activity by 50% in all regions of the heart. These changed levels were not altered by 10 min of reperfusion. The fall in 5-nucleotidase activity with ischaemia occurred only in the 90% of this enzyme which is membrane-bound. The reasons for the marked differences in distribution and responses to ischaemia of these two enzymes have yet to be elucidated but metabolic inhibitors seem unlikely to be involved.     

Synthesis and evaluation of catechol analogs of diethylstilbestrol on a hormone-dependent human mammary carcinoma implanted in nude mice

Summary 3,4-Bis(3,4-diacetoxyphenyl)hex-3-ene (5a), 4(4-acetoxyphenyl)-3(3,4-diacetoxyphenyl) hex-3-ene (5b), and 4(3-acetoxyphenyl)-3(3,4-diacetoxyphenyl)hex-3-ene (5c) were synthesized according to the method of Dodds et al. (1939). All compounds inhibited the interaction of ³H-estradiol with its receptor. The relative binding affinity (RBA) values increased in the order: 5a (1.0)<5c (7.6)<5b (21.7). In the immature mouse uterine weight bioassay, the uterotrophic activity of 5a-c was only weak. 5a and 5c, but not 5b, exhibited significant antiuterotrophic properties. All compounds significantly inhibited the growth of a postmenopausal hormone-dependent human mammary carcinoma serially implanted in nude mice.Abbreviations COMT catechol-O-methyl transferase - ETOH ethanol - E2 estradiol - RBA relative binding affinity (E2=100) - LH luteinizing hormone - DMBA 9,10-dimethyl-1,2-benzanthracene Supported by grants from the Deutsche Forschungsgemeinschaft und Verband der Chemischen Industrie-Fonds der Chemischen Industrie     

A polymorphism of the NFKBIA gene is associated with Crohn''s disease patients lacking a predisposing allele of the CARD15 gene

Background and aims Nuclear factor kappa-B (NFB) plays a crucial role in diseases associated with dysregulated immune response. NFB inhibitor downregulates the activity of NFB.Patients and methods To evaluate the contribution of the NFB inhibitor gene in Crohn's disease single nucleotide polymorphisms in the 3-UTR and at position –420 in the promoter were studied in 259 patients with Crohn's disease genotyped for the variations of the CARD15 gene in comparison to 441 healthy controls. Additionally we screened the coding region of the NFB inhibitor gene for polymorphisms by SSCP analysis.Results In comparison to controls the A allele and the AA genotype frequencies of the single nucleotide polymorphisms in the 3-UTR were significantly increased only in Crohn's disease patients without a variation in the CARD15 gene. Similarly, the difference between patients harboring no predisposing CARD15 alleles and patients harboring such a variation was significant.Conclusion The findings indicate that the phenotype Crohn's disease is to be substructured with respect to genetic susceptibility.     

Androgen-linked alkylating agents: biological activity in methylnitrosourea-induced rat mammary carcinoma

Summary This article gives a comprehensive survey on the anticancer activity of nitrosoureas linked to steroidal androgens in methylnitrosourea (MMU)-induced rat mammary carcinoma. cis-Androsterone, testosterone, 19-nortestosterone and 5--dihydrotestosterone were used as carrier hormones and were linked to various cytotoxic N-[N-(2-chloroethyl)-N-nitrosocarbamoyl] (CNC)-aminoacids and to N-(2-hydroxyethyl)-N-(2-chloroethyl)-N-nitrosourea hemisuccinate (HECNU-hemisuccinate). In the MNU-model used esters of dihydrotestosterone (DHT) invariably were more active and less toxic than those of testosterone, nortestosterone and cis-androsterone. Within the DHT esters of CNC-aminoacids those of CNC-glycine, CNC-methionine and CNC-alanine showed the highest antineoplastic activities and superiority compared with equimolar dosages of their unlinked mixtures. Additionally, CNC-alanine-DHT ester had the highest therapeutic ratio of all agents investigated. HECNU-hemisuccinate-DHT ester, on the other hand, achieved even higher antitumor activity at the optimal dose but had a narrower therapeutic ratio.No obvious correlation between antineoplastic efficacy and receptor binding affinity could be demonstrated, but, to be active, a conjugate apparently had to have some receptor binding affinity for both androgen and progesterone receptors. The results obtained indicate that linking antineoplastic agents to transport molecules with affinity to steroid receptors is a highly promising approach to obtain drugs with specific activity in steroid receptor containing tumors.Abbreviations CNC N-[N-(2-chloroethyl)-N-nitrosocarbamoyl] - DHT dihydrotestosterone - HECNU N-(2-hydroxyethyl)-N-(2-chloroethyl)-N-nitrosourea - MNU methylnitrosourea Dedicated to Professor Dr. D. Schmähl on the occasion of his 65th birthday