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Biomedical subjects

J Lüthy

Publications and source records attributed to J Lüthy.

At least 19 recordsLinked to original sources
PubMed1992

Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.

Animals↗
PubMed1992

Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles.

The use of additional primers in the standard two primer polymerase chain reaction (PCR) is described. This modification allows detection of a target gene in a single reaction, and identification of the amplification product obtained or recognition of a specific allele. The oligonucleotides used are internal to the original amplification primers and amplification-compatible with one of the original primers. Annealing of an additional primer to the target gene as well as to the primary amplification product will lead to the appearance of an additional smaller amplification fragment upon agarose gel electrophoresis of PCR products. Use of one or more allele-specific oligonucleotides as additional primers, in addition to two gene-specific primers, will allow recognition of different alleles of the target gene in a single PCR without further analysis except gel electrophoresis. The general applicability of the method was determined with several PCR assays for the detection of pathogenic bacteria.

Alleles↗
PubMed1992

Use of polymerase chain reaction for detection of Listeria monocytogenes in food.

A previously described polymerase chain reaction (PCR) assay (B. Furrer, U. Candrian, C. Höfelein, and J. Lüthy, J. Appl. Bacteriol. 70:372-379, 1991) was used to analyze food for the presence of Listeria monocytogenes. Food samples were artificially contaminated to develop two procedures to detect the organism following enrichment steps. Procedure A was based on dilution of the enrichment broth followed by lysis of the bacteria and direct analysis of the lysate with PCR. With procedure A and artificially contaminated food samples, it was possible to detect fewer than 10 bacteria per 10 g of food. In procedure B, centrifugation was used to concentrate bacteria before lysis and PCR. With procedure A, 330 naturally contaminated food samples of several types were analyzed. Twenty samples were found to be positive for L. monocytogenes, which was in agreement with the classical culture technique. By using procedure B on a subset of 100 food samples, 14 were found to be positive by PCR whereas the classical culture method detected only 13. Analysis times, including enrichment steps, were 56 and 32 h with procedures A and B, respectively.

Animals↗
PubMed1991

Detection of Escherichia coli and identification of enterotoxigenic strains by primer-directed enzymatic amplification of specific DNA sequences.

The polymerase chain reaction (PCR) was used to amplify DNA sequences from the malB operon of Escherichia coli. All E. coli strains tested yielded the specific DNA fragment. No amplification products were obtained with other Enterobacteriaceae. E. coli strains which produce enterotoxins were identified with additional primer pairs specific for the genes coding for the heat-labile toxin type I (LTI) and the heat-stable toxin type I (STI). Amplification products were identified by DNA-DNA hybridization. Alternatively, restriction endonuclease analysis was used for identification and to distinguish between different alleles of the enterotoxin genes. The detection limit was 10 bacteria. The PCR systems were validated by testing 27 E. coli of known enterotoxigenic properties. The PCR results were consistent with factual toxin production as determined by immunoassays. In addition, 58 E. coli strains isolated from soft cheese and mayonnaise were analyzed by PCR. One strain from a cheese sample was found to have the genetic information for STI production. This strain produced STI as determined by enzyme-linked immunosorbent assay.

Animals↗
PubMed1991

Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli.

A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.

Alleles↗
PubMed1990

Detection and identification of E. coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment.

The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI). Amplification primers were designed to detect E. coli strains of human as well as porcine origin. This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI). In addition, three clinical E. coli isolates of human and one of porcine origin were tested. All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method. All strains yielded the expected 275 bp DNA fragment after enzymatic amplification. This fragment was further identified by allele specific oligonucleotide hybridization. Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E. coli isolated from humans and pigs. The detection limit determined in water with the ATCC 37218 strain was 20 bacteria. The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI. All of the strains tested showed this polymorphism as expected. Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively. This method may be adapted to environmental and food samples.

Animals↗
PubMed1989

ELISA with enzyme amplification for sensitive detection of staphylococcal enterotoxins in food.

A highly sensitive amplified ELISA in microtitre plates for the detection of staphylococcal enterotoxins compared favourably with a commercially available kit. The amplified ELISA demonstrated a reduced effect of the food sample matrix and an ease of handling. Using the amplified ELISA, the influence of different parameters (pH, time, temperature, re-extraction, concentration procedure) on the recovery efficiency and assay reliability was tested on foods implicated in food poisoning outbreaks as well as on spiked foods containing low levels of enterotoxins.

Enterotoxins↗
PubMed1986

The mutagenic activity of agaritine--a constituent of the cultivated mushroom Agaricus bisporus--and its derivatives detected with the Salmonella/mammalian microsome assay (Ames Test).

Purified agaritine (N'-(gamma-L(+)-glutamyl)-p-hydroxymethylphenylhydrazine) isolated from Agaricus bisporus, p-hydrazinobenzoic acid (its presumptive precursor) and some agaritine-degradation products were tested for mutagenic activity with the Salmonella/mammalian microsome assay (Ames test). Consistent with the literature, agaritine showed a distinct direct-acting mutagenicity with the strain TA1537 (30 revertants/mumol) and with TA97. Incubation of agaritine at alkaline pH increased the mutagenic effect. Pre-incubation of agaritine with gamma-glutamyl transferase (GT) during 10 h at room temperature (pH 8.2) even enhanced the mutagenicity by a factor of 8 to 16 depending on the strain. In accordance with this finding, synthetic p-hydroxymethylphenylhydrazine (the presumptive product of the GT catalyzed degradation) showed also a distinct direct-acting mutagenicity, but the increase was only about 3- to 6- times compared with agaritine. The hypothetical ultimate mutagenic metabolite of agaritine, the p-hydroxymethylbenzenediazonium ion, a compound occurring naturally in A. bisporus, showed the highest mutagenic activity (with TA1537 approximately 300 to 1,000 revertants/mumol).

Agaricales↗
PubMed1985

[The occurrence of goitrogenic substances in milk. 1. Release of goitrin in the milk of cows fed on rapeseed extract cakes].

A total of six cows, divided into 3 groups, were fed various amounts of rape cake containing 6 g of goitrin/kg over a period of 7 days. The cows were milked twice a day and the goitrin content of the heated milk samples were determined by a HPLC-method within 2 h. When rape cake was fed at 0.39, 1.9 and 3.9% resp. of the total feed this resulted in medium goitrin values of 37, 163 and 707 micrograms/l milk. These values correspond to a transfer of about 0.1% of the original progoitrin content in the feed. 12 h after the last rape feeding the amount of goitrin in the milk was below the detection limit of 7 ppb. The toxicological significance of these findings are dicussed.

Animal Feed↗
PubMed1985

In vivo covalent binding of retronecine-labelled [3H]seneciphylline and [3H]senecionine to DNA of rat liver, lung and kidney.

Retronecine-labelled [3H]seneciphylline ([3H]SPH) and [3H]senecionine ([3H]SON) of high specific radioactivity (22 and 49 mCi/mmol, respectively) were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine as precursor. [2,3-3H]Putrescine was synthesized by Gabriel synthesis of 1,4-diamino-2-butene from 1,4-dibromo-2-butene and catalytic hydrogenation of the product with tritium gas. Rats of both sexes were treated with the labelled pyrrolizidine alkaloids (PAs) (75-215 microCi SPH or 40-485 microCi SON/kg body wt.) and killed after 6 h or 4-5 days. SON-treated females excreted 83.4 +/- 0.2% of applied radioactivity in faeces and urine within 4 days whereas equally treated males excreted 90.9 +/- 3.2% in the same time. Excretion of 3H-activity from SPH-treated females was completed within 5 days (104.7 +/- 6.4%). Corresponding with these results, tissue levels were highest in SON-treated females. DNA and proteins were isolated from liver, lungs and kidneys and covalent binding of the alkaloids to DNA was determined. A Covalent Binding Index (CBI, mumol alkaloid bound per mol nucleotides/mmol alkaloid administered per kg body wt.) of 210 +/- 12 was found for the liver from SON-treated females whereas binding to liver DNA of males was lower by a factor of 4. The DNA damage determined six hours after treatment persisted during the following 4 days. Administration of [3H]SPH to female and male rats resulted in a CBI of 69 +/- 7 and 73/92, respectively, for the liver DNA. Furthermore we found binding of both alkaloids to DNA of lungs and kidneys in male and female rats. The in vivo formation of [3H]SON derived DNA adducts could be proved by HPLC analysis of hydrolyzed DNA.

Animals↗
PubMed1976

Stereology of liver biopsies from healthy volunteers.

The stereological model and the base-line data of normal human liver needle biopsy-specimens are presented. Four reference systems were introduced: 1 cm3 of liver tissue, 1 cm3 of hepatocyte, 1 cm3 of hepatocytic cytoplasm and the volume of an average "mononuclear" hepatocyte. The sampling was done at three levels of magnification (1,000 X, 5,000 X and 10,000 X). A lobular differentiation was not considered. The baseline data show strikingly small variations (s.e. less than 10%) within the individual biopsy specimen and within the group of four biopsies. There is no principal difference between human beings presented here, rats, mice and dogs. Only the mean individual volume of human hepatocytes is clearly larger than in rodents. The problems and limitations of stereological work on liver biopsy specimens are discussed.

Animals↗