Metabolic activation of pyrrolizidine alkaloids by human, rat and avocado microsomes.

Two pyrrolizidine alkaloids and one pyrrolizidine alkaloid-N-oxide were incubated with microsomal preparations from humans, rat and avocado and the product profiles examined. The alkaloids were converted to dehydroretronecine, the putative toxic metabolite, by both rat and human microsomal preparations. In addition, alkaloid-N-oxides, the major detoxication products from pyrrolizidine alkaloids, were also formed. The pyrrolizidine alkaloid-N-oxide was converted to both dehydroretronecine and the parent alkaloid. This suggests that the toxicity of pyrrolizidine alkaloid-N-oxides could be greater than suggested hitherto as a result of conversion to the toxic metabolite via the parent alkaloid. Quantitative differences in the proportions of products formed by the different microsomal preparations may be of significance in the extrapolation of toxicological data from animal models such as the rat to humans.

Nuclear DNA content of human breast carcinoma: a comparison of results obtained by microspectrophotometry and flow cytometry of paraffin embedded tissue.

This study compares 2 techniques for estimating the nuclear DNA content of tumor cell lines: (i) static cytometry of smears taken from fresh tissue and (ii) flow cytometry of cells extracted from paraffin embedded tissue. Parallel determinations of DNA content, using both techniques, were made on samples of tissue taken from 130 female patients with breast carcinoma. Using a simple classification into diploid and non-diploid groups, the 2 techniques yielded discrepant results in 11% of cases. The most frequent causes of disagreement were (a) the inability of static cytometry to distinguish between a diploid and a near-diploid peak and (b) for flow cytometry, the difficulty of determining whether a minor peak in the tetraploid region represented the G2 peak of a diploid cell line or the G0/G1 peak of a tetraploid cell line. If it is deemed necessary to accurately assess ploidy status, flow cytometry on paraffin embedded tissue, using modern statistical programmes, would seem to be most practical for routine use, but some neoplasms, particularly those with an equivocal ploidy peak in the tetraploid range by this method, will require static cytometry to accurately assess nuclear DNA content. Using this approach, it appears that the disagreement between the 2 techniques would be less than 5%.

Genomic subtractive hybridization to isolate species-specific DNA sequences in insects.

Selective enrichment has been used in a number of instances for the isolation of species-specific sequences in prokaryotes. This paper reports the successful application of the technique to insects. Genomic probes were derived to the target species D. funebris and D. simulans. The method involves the biotinylation of non-target 'driver' DNA prepared from the closely related species D. melanogaster and its hybridization to homologous sequences in the target DNA. Hybrid molecules were removed from the reaction by incubation with streptavidin followed by phenol extraction, leaving a preparation enriched for target fragments. All DNA fragments isolated in the D. funebris experiments proved to be specific to that species. Five out of twenty-four fragments screened in the D. simulans experiments were specific when screened with homologous DNA and genomic DNA from its sibling species, D. melanogaster.

Chloroplast gene expression in lettuce grown under different irradiances.

Chloroplast DNA sequences have been used as hybridisation probes to measure the levels of RNA transcripts present in low- and high-light-grown lettuce (Lactuca sativa L.) plants. The transcript levels for rbc L, psa A, psb A and rDNA are not different between these two light regimes. In contrast, transcript levels for atp BE and pet BD are increased in high-light as a proportion of the chloroplast RNA. Three days after transfer from low-light into high-light, increased transcript levels were found for atp BE, although no change was detected for the psa A or psb A transcripts. In addition, young plants in high-light contain twofold more chloroplast RNA per unit chlorophyll than do low-light plants of equivalent age. Therefore, in these young high-light plants the absolute transcript levels per unit chlorophyll are much greater. With increasing leaf age the RNA per chlorophyll becomes similar for both light conditions. These results are discussed in relation to the photoregulation of chloroplast-encoded gene expression.

A bovine epithelial membrane protein that binds polymeric immunoglobulin and has a structure related to that of bovine secretory component.

A protein of Mr 94000 was isolated from detergent-solubilized bovine intestinal, liver and mammary-gland membranes. It binds immunoglobulin M and also undergoes proteolytic fragmentation in a similar manner to bovine secretory component (Mr 74000). The affinity-purified membrane protein is therefore most likely to be the bovine epithelial receptor for polymeric immunoglobulin. A structural model is proposed.

Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens.

Protein A-colloidal gold immunoelectron microscopy (PAG IEM) has been employed to specifically detect rotavirus and enterovirus antigen in negatively stained fluid specimens. Unlike other IEM methods, PAG IEM can detect not only viral antigen associated with morphologically recognizable particles but also viral antigens of unrecognizable ultrastructure. This rapid and sensitive immunoassay was found to be applicable to virus-infected stool specimens as well as partially purified virus preparations. The sensitivity of viral antigen detection by PAG IEM was 2- to 40-fold greater than direct IEM and 200- to 1,000-fold greater than direct electron microscopy. In addition, PAG IEM appears to offer a more reliable and sensitive alternative to standard IEM for detection and quantitation of viral antibody.

Fragmentation and reduction of bovine secretory component. Preparation of a biologically active fragment and some evidence for a multiple-domain structure.

A tryptic fragment (A) of Mr 25000 was prepared from bovine secretory component. The fragment binds polymeric immunoglobulin, although 9 times less effectively than secretory component on a molar basis. The fragment has four buried half-cystine residues and two exposed half-cystine residues. It gives rise to two fragments of Mr 11000-13000 on prolonged digestion with trypsin, and these do not bind polymeric immunoglobulin. It is proposed that fragment A consists of two immunoglobulin-like domains. Bovine secretory component was found to have 9-11 buried half-cystine residues and four exposed half-cystine residues. Reduction and alkylation of the exposed residues decreases the binding of polymeric immunoglobulin by 3-fold. Initial tryptic cleavage of bovine secretory component gives a fragment (Q) disulphide-bridged to a further fragment (T). Fragment Q is similar in size to a three-domain immunoglobulin fragment, and fragment T is similar in size to a two-domain immunoglobulin fragment. The two-domain fragment A is derived from fragment Q by further tryptic cleavage. The results are compatible with the proposal by Mostov, Friedlander & Blobel [(1984) Nature (London) 308, 37-43] that secretory component consists of multiple immunoglobulin-like domains. The results also indicate that optimal binding of polymeric immunoglobulin involves several domains stabilized by an exposed disulphide bridge.

A comparison of the fragmentation of different species of mammalian immunoglobulin M by trypsin in urea.

Six different species of mammalian IgM that had been preincubated in 4-6 M urea showed marked differences in their fragmentation by trypsin at 25 degrees C. These differences are most probably due to different degrees of conformational change in urea particularly as regards the C mu 2 domains. The fragments obtained by digestion with trypsin in urea were often different to those obtained by digestion in aqueous buffer at 37 or 55 degrees C. In particular the production of (Fc*)5 fragment containing C mu 2 domains was favoured. In the case of human and mouse IgM the fragmentation could be controlled sufficiently to produce series of molecules which differed in their numbers of Fab arms and/or C mu 2 domains.