Brucellae through the food chain: the role of sheep, goats and springbok (Antidorcus marsupialis) as sources of human infections in Namibia.

A confirmed case of human brucellosis motivated an investigation into the potential source of infection in Namibia. Since domestic animals are principal sources of Brucella infection in humans, 1692 serum samples were screened from sheep, goats and cattle from 4 presumably at-risk farms and 900 springbok (Antidorcas marsupialis) serum samples from 29 mixed farming units for Brucella antibodies by the Rose-Bengal test (RBT) and positive cases confirmed by complement fixation test (CFT). To assess the prevalence of human brucellosis, 137 abattoir employees were tested for Brucella antibodies using the standard tube agglutination test (STAT) and by enzyme linked immunosorbent assay (ELISA). Cattle and sheep from all 4 farms were negative by RBT and CFT but 2 of the 4 farms (Ba and C) had 26/42 and 12/285 seropositive goats, respectively. Post mortem examination of seropositive goats revealed no gross pathological lesions typical of brucellosis except enlarged mesenteric and iliac lymph nodes seen in a single buck. Culture for brucellae from organs of seropositive animals was negative. None of the wildlife sera tested positive by either RBT or CFT. Interviews revealed that besides the case that prompted the investigation, a family and another person from other farms with confirmed brucellosis shared a common history of consumption of unpasteurised goat milk, home-made goat cheese and coffee with raw milk and prior contact with goats, suggesting goats as the likely source of infection. All 137 abattoir employees tested negative by STAT, but 3 were positive by ELISA. The 3 abattoir workers were clinically normal and lacked historical connections with clinical cases. Although goats are often associated with B. melitensis, these studies could not explicitly implicate this species owing to cross-reactivity with B. abortus, which can also infect goats. Nevertheless, these data reinforce the need for a better National Control Programme for brucellosis in Namibia.

E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells.

The ability of carcinomas to invade and to metastasize largely depends on the degree of epithelial differentiation within the tumors, i.e., poorly differentiated being more invasive than well-differentiated carcinomas. Here we confirmed this correlation by examining various human cell lines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioid phenotype were noninvasive and expressed the epithelium-specific cell-cell adhesion molecule E-cadherin (also known as Arc-1, uvomorulin, and cell-CAM 120/80), as visualized by immunofluorescence microscopy and by Western and Northern blotting, whereas carcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin mAbs. These findings indicate that the selective loss of E-cadherin expression can generate dedifferentiation and invasiveness of human carcinoma cells, and they suggest further that E-cadherin acts as an invasion suppressor.

Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.

Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation.

A highly sensitive and specific method for the detection of O6-methylguanine (O6-meG), O4-methylthymine (O4-meT), and O4-ethylthymine (O4-etT) has been established by combining prefractionation by high-performance liquid chromatography (HPLC), 32P postlabeling, and immunoprecipitation by monoclonal antibodies (PREPI method). DNA was enzymatically hydrolyzed to 2'-deoxynucleoside-3'-monophosphates (3'-dNps). Each alkyl 3'dNp was separated by reverse-phase HPLC, radiolabeled at the 5' position with [gamma-32P]ATP and polynucleotide kinase. After removing 3'-phosphate for better recognition by the antibodies, the resulting alkyl nucleotides were further fractionated by HPLC and finally precipitated specifically with respective antibodies. The detection limits were 1 fmol for all the alkyl nucleotides analyzed, so that one adduct in 10(8) of its normal counterpart nucleotide can be determined using approximately 100 micrograms (O4-meT and O4-etT) or approximately 150 micrograms (O6-meG) of DNA, i.e., 3-5 x 10(7) cells corresponding to approximately 10 ml of peripheral blood or a few hundred milligrams of tissue. By the use of the PREPI method, three leukocyte and three liver DNA samples from Japanese living in the Tokyo area were analyzed with respect to O-alkyl adduct content. O6-meG was detected in all three of the leukocyte samples (O6-meG:G molar ratio, 1.1 x, 0.8 x, and 1.6 x 10(-8) as molar ratios to guanine). Neither O4-meT nor O4-etT was detected (detection limit, O4-alkylT:thymine molar ratio less than 0.5 x 10(-8)). Among the liver samples analyzed, two cases showed positive O6-meG values (4.2 x and 1.1 x 10(-7) O6-meG:guanine molar ratios). Contrary to the leukocyte DNA, O4-meT (3.9 x, 4.3 x, and 7.5 x 10(-8) as O4-meT:thymine) and O4-etT (1.9 x, 4.9 x, and 8.7 x 10(-8) as O4-etT:thymine) were detected in all the liver samples. These results indicate the validity of the PREPI method for molecular epidemiological studies on DNA alkylation products.

Investigations on the relationship between DNA ethenobase adduct levels in several organs of vinyl chloride-exposed rats and cancer susceptibility.

The levels of 1,N6-ethenodeoxyadenosine (epsilon dAdo) and 3,N4-ethenodeoxycytidine (epsilon dCyd) were measured in DNA of several target organs of vinyl chloride (VC)-exposed rats. Seven-day-old (group I) and 13-week-old (group II) BD VI rats were exposed during 2 weeks to 500 ppm VC in air (7 hr per day and 7 days per week). epsilon dAdo and epsilon dCyd were measured by a combination of prepurification of DNA hydrolysates by HPLC and competitive radioimmunoassay using specific murine monoclonal antibodies. Both ethenodeoxynucleosides were detected in liver, lungs and brain (levels ranging from 0.6 x 10(-7) to 1.3 x 10(-7) for epsilon dAdo/2'-deoxyadenosine and from 1.95 x 10(-7) to 4.92 x 10(-7) for epsilon dCyd/2'-deoxycytidine) but not in kidneys of group I rats. In group II rats, only liver DNA was analysed and the levels of each adduct were six times lower than in young (group II) rats. These findings are in good agreement with the organotropism and the age-related sensitivity of VC-induced carcinogenesis in rodents.

Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies.

Conventional radiochromatographic procedures for the quantitation of carcinogen/mutagen-induced structural DNA modifications have a number of limitations. Thus, these techniques for the most part require application of radioactively labeled carcinogens and the use of relatively large amounts of DNA for analysis at low levels of DNA modification. Radiochromatographic methods also preclude analyses at the level of single cells and DNA molecules. Recently developed immunoanalytical methods have improved this situation considerably. Monoclonal antibodies (Mab) characterized by a high substrate specificity and affinity, in combination with radio- and enzyme-immunoassays, or with "immuno-slot-blot" techniques, now permit the detection of femtomole to subfemtomole amounts of, e.g., alkyldeoxynucleosides in small samples of DNA isolated from tissues or cultured cells previously exposed to nonradioactive N-nitroso compounds. Furthermore, selected Mab can be used to quantitate by direct immunofluorescence (with the aid of computer-based image analysis of electronically intensified fluorescence signals), specific alkyldeoxynucleosides in the nuclear DNA of single cells. With this method, the detection limit for the alkylation product O6-ethyldeoxyguanosine (O6-EtdGuo) is presently of the order of 10(2) -10(3) O6-EtdGuo residues per diploid mammalian genome. Individual cells can thus be monitored for the presence of specific carcinogen-DNA adducts, and with respect to their capacity for enzymatic removal of such modified structures from DNA (as exemplified here by the kinetics of the enzymatic elimination of O6-EtdGuo from the DNA of malignant neurogenic rat cell lines). In combination with transmission electron microscopy, Mab also permit direct visualization (via Mab binding sites) of specific carcinogen-modified structures in individual DNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prophylaxis of dental caries using sugar substitutes]. / Zur Prophylaxe der Zahnkaries durch Zuckersubstitute.

Among the three measures, which are capable of producing a preventive effect against caries only when applied combined, i.e. adequate fluoride supply, proper mouth hygiene and healthy nutrition, the latter is dealt with in greater detail. The use of sugar substitutes is discussed under the aspects of caries prevention, substitute composition and production technology as well as from a medical point of view. Among the presently available sugar substitutes with nutritive value are mentioned Xylite, Lycasine, Mannite, Sorbite, Palatinite, the non-calorific substitutes such as the natural Aspartame as well as the synthetic sweetening agents Saccharine and Cyclamate. The possibilities and limitations of using these sugar substitutes in the prevention of caries in adults and children are presented.

[Experimental infection of mice, gerbils, and rats with mycoplasms from canine pericardium and cardial value (author's transl)]. / Experimentelle Infektion von Mäusen, Gerbils und Ratten mit Mykoplasmen aus Herzbeutel und Herzklappe von Hunden.

Mycoplasms isolated from heart valves (A. laidlawii (A 42 Hzbtl)) and pericardium (M. canis (A 56 Hzkl)) of dogs were investigated for infectivity of rats, mice and gerbils (Meriones unguiculatus). In mice the species M. spumans (PG 13), M. maculosum (PG 15), M. edwardii (PG 24) and M. molare (H 542) were investigated too. The mycoplasmas were inoculated by oral, subcutaneous, intravenous and intraperitoneal route. Intraperitoneally the mycoplasmas were given without and with complete and incomplete Freund adjuvants. The injected Mycoplasmas did not produce any disease and could not be reisolated from rats and gerbils. From mice A. laidlawii (A 42 Hzbtl), M. canis (A 56 Hzkl) and M. edwardii (PG 24) could be reisolated after intraperitoneal administration in emulsion with complete and incomplete Freund adjuvants. Whereas M. canis (A 56 Hzkl) and M. edwardii (PG 24) could be reisolated from lung, liver and spleen only, A. laidlawii (A 42 Hzbtl) was present in heart muscle too. In histological investigations the mice which got A. laidlawii (A 42 Hzbtl) intraperitoneally with complete Freund adjuvants showed focal lymphocytic infiltrations in the myocardium.

Cocultivation studies with cells of patients bearing fragile X chromosomes.

Fourteen cocultivation studies were carried out with cells of our patients with fragile X, one obligate and two possible female heterozygotes, two female controls, and a rabbit. In all cocultivations the number of fragile X chromosomes was sharply reduced in the patient cells. The strongest effect was causes by the animal cells. A distinct difference between the two controls in the reducing ability was observed. No such difference was found between the obligate and possible heterozygotes on the one hand and the controls on the other. To test the influence of the residual serum in the mixed blood cultures, the serum of a patient's blood sample was replaced by the serum of a control. The frequency of fragile X chromosomes was not decreased by this procedure. Therefore a soluble factor is supposed to exist which is produced by normal or heterozygote cells in culture and which reduces the expression of fragile sites in patient cells.

The expression of fragile X chromosomes in members of the same family at different times of examination.

Three brothers with fragile X chromosomes were repeatedly examined using the same culture and preparation techniques. It was observed that a given individual showed a very constant frequency of fragile sites at his X chromosomes, whereas large differences in the fragile X counts occurred between the three brothers.

Second-harmonic generation with partially poled polymers.

The poling of polymers leads in general to inhomogeneous polarization distributions within the polymer film. These polarization distributions affect any nonlinear-optic experiment based on second-order nonlinearities. Second-harmonic generation measurements are presented for a partially poled ferroelectric polyvinylidene fluoride film. By partial poling, we mean that the film is polarized only within a part of the film thickness. The measured angle-dependent second-harmonic signal is explained only if the polarization distribution as measured by the piezoelectric pressure pulse technique is taken into account.

Quantification of specific DNA O-alkylation products in individual cells by monoclonal antibodies and digital imaging of intensified nuclear fluorescence.

We report the establishment of a standardized, monoclonal antibody (Mab)-based immunocytological assay (quantitative ICA) for the visualization and quantification of low levels of specific DNA O-alkylation products in individual cells by electronically intensified, indirect or direct immunofluorescence. In terms of specific binding to alkali-denatured nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-BudGuo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(6)-3.0 x 10(10) l/mol, were found to be best suited for ICA. At present, > or = 200 O6-EtdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of > or = 8.4 x 10(-8)), > or = 400 O6-BudGuo residues (O6-BudGuo/dGuo, > or = 1.7 x 10(-7)), > or = 1800 O4-EtdThd residues (O4-EtdThd/dThd, > or = 7.5 x 10(-7)) and > or = 4800 O6-MedGuo residues (O6-MedGuo/dGuo, > or = 2.0 x 10(-6)), can be quantified per diploid genome. Using a SIT video camera in combination with multiparameter image digital analysis, DNA adduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimmunoassay (RIA) for hydrolysates of DNA isolated from the respective cells exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were superimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alkylating carcinogens or chemotherapeutic agents and for DNA repair measurements in individual (e.g. human) cells. Work is currently underway to extend the spectrum of carcinogen--DNA adduct-specific Mabs suited for quantitative ICA.

Monoclonal antibody-based immunoanalytical methods for detection of carcinogen-modified DNA components.

Hybridoma cell lines secreting monoclonal antibodies (Mab) directed against the products formed by reaction of alkylating N-nitroso carcinogens with DNA have been established by fusion of rat or mouse myeloma cells, respectively, with spleen cells of rats or mice immunized either with conjugates of various alkyl-ribonucleosides with suitable carrier proteins, or with alkylated DNA electrostatically complexed to carrier proteins. Due to their high affinity and specificity, some of these Mab detect very low amounts of the respective alkyl-deoxynucleosides (e.g., O6-methyl-2'-deoxyguanosine, O6-ethyl-2'-deoxyguanosine, O6-n-butyl-2'-deoxyguanosine, O6-isopropyl-2'-deoxyguanosine, O4-methyl-2'deoxythymidine, O4-ethyl-2'-deoxythymidine) and can be used in various types of immunoassays. With a competitive radioimmunoassay (RIA), specific DNA alkylation products can be quantitated in hydrolysates of cellular DNA, in body fluids, or in urine. The RIA is routinely applicable, reproducible, and sufficiently sensitive to permit the quantitation of femtomole amounts of modified nucleosides in small samples of DNA. When the alkyl-deoxynucleosides in question are separated from bulk DNA by high-performance liquid chromatography prior to analysis by RIA, very low levels of modification in DNA can be detected. The immuno-slot-blot (ISB), a noncompetitive solid-phase immunoassay, is more sensitive than the RIA. For analysis by ISB, alkylated DNA is heat-denatured and immobilized on nitrocellulose filters prior to exposure to the respective Mab and subsequent binding of a second (125I-labelled or biotinylated) antibody. In immunocytological analysis (ICA), the binding of Mab to alkyl-deoxynucleosides is visualized in individual cells by immunostaining of denatured nuclear DNA in situ (direct immunofluorescence; peroxidase-staining).(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies for the specific detection of 3-alkyladenines in nucleic acids and body fluids.

We describe an immunoanalytical procedure for the detection and quantitation of 3-alkyladenines in biological samples with the use of anti-(3-alkyladenine) monoclonal antibodies (Mab). A new hapten-protein conjugate, 3-ethyl-8-(3-carboxypropyl)-adenine, was used for immunization of BALB/c mice after conjugation to carrier proteins via the carboxyl group. Eighty-nine hybridomas were established which secrete anti-(3-alkyladenine) Mab with antibody affinity constants ranging from 1 x 10(7) to 5 x 10(9) l/mol for 3-ethyladenine (3-EtAde). One of these Mab (EM-6-47) had detection limits of 30 fmol for 3-EtAde, 17 fmol for 3-n-butyladenine (3-BuAde) and 475 fmol for 3-methyladenine (3-MeAde) respectively, at 25% inhibition of tracer-antibody binding. The binding pattern of Mab EM-6-47 revealed high specificity for adenine substituted at N-3 with different alkyl residues and no, or very low, cross-reactivity with other alkylated or unmodified nucleic acid components or structurally related compounds. 3-MeAde and 3-EtAde can be well separated from nucleic acids, and from rat and human urine samples, using HPLC with two successive stationary phases. Using Mab EM-6-47 in conjunction with a competitive RIA, both 3-MeAde and 3-EtAde were detected in the range of 100-300 ng (3-MeAde) and 2-10 ng (3-EtAde) in urine samples (10 +/- 2 ml) of untreated rats collected over a 24 h period. Only 3-MeAde (range 1.3-24.20 micrograms) was found in human urine samples. The concentration of 3-EtAde in rat urine increased significantly during the 24 h following a single i.v. application of N-ethyl-N-nitrosourea. After i.p. application of known amounts of 3-MeAde and 3-EtAde, greater than 90% of 3-MeAde and greater than 70% of 3-EtAde were excreted in rat urine within the subsequent 24 h. The concentration of 3-alkyladenines in body fluids (urine) may thus provide a useful indicator of environmental exposure to nucleic acid-reactive agents, and the immunoanalytical procedure described here permits the sensitive determination of adenines carrying different substituents at N-3.

Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.