Immunohistochemical aspects of immunological cross-reaction and masking of epitopes for localization studies on pregnancy-associated plasma protein A

Summary The influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined.Apparently monospecific IgG fraction of the anti-plasma protein applied directly on fixed tissue resulted in staining of connective tissue and a thin apical rim of the syncytiotrophoblast. Further absorption of the antibody with foetal connective tissue abolished this staining reaction. Pre-treatment of the fixed placental tissue with trypsin prior to application of the antibody, which had been absorbed with connective tissue, resulted in staining within the cytoplasm of the syncytiotrophoblast exclusively. Identical staining was seen when this IgG preparation was used directly on frozen placental tissue.The results point to the importance of the specificity of the antibody preparations and of proteolytic unmasking of epitopes when fixed tissues are used for localization studies of pregnancy-associated plasma protein A by immunoperoxidase technique.     

The structure of the basement membrane of human lymph node high endothelial venules: An ultrastructural, histochemical and immunocytochemical study

Summary The structure of the basement membrane of the high endothelium of reactive human lymph nodes was investigated by techniques selective for carbohydrates (periodic acid-Schiff; critical electrolyte concentration staining with Alcian Blue; lectin histochemistry), specific proteins (immunohistochemistry for laminin and fibronectin) and by conventional techniques of light and transmission electron microscopy. Adjacent small lymphocytes were assigned to B and T cell subsets by use of monoclonal antibodies and they were analysed for non-specific esterase,-glucuronidase,-N-acetylglucaminidase and proteolytic activities. The basement membranes were shown to be distinctive and to contain three layers, of differing laminin, glycosaminoglycan and glycoprotein oligosaccharide content. Certain lymphocytes (probably T) contained enzymes potentially able to degrade some components of these basement membranes.     

Arylsulphatase activity associated with phenanthrene induced digestive cell deletion in the marine mussel Mytilus edulis

Summary The acid hydrolase arylsulphatase has been localized at the ultrastructural level in digestive cells of the marine musselMytilus edulis for control and phenanthrene-treated (200µg/l) animals. In untreated mussels the activity was generally restricted to the lysosomal—vacuolar system and the Golgi apparatus. It was associated with all types of vesicle, although not all individual vesicles were reactive. In heterolysosomes which were filled with precipitate the reaction product was most densely associated with the limiting membranes. Lipid inclusions commonly occurred in the digestive cells; these sometimes showed limited reaction for enzyme activity. The striking difference between normal and phenanthrene-treated samples was the presence in all treated animals of reaction product in the inter-cellular spaces and varying degrees of cytoplasmic activity in a number of digestive cells. This is interpreted as a sign of impending cell deletion. Sections for morphological examination showed evidence of increased digestive cell deletion in phenanthrene-treated mussels. The process results in release of membrane-bound bodies into the tubule lumen.     

The use of restriction fragment length polymorphisms in paternity analysis.

This paper examines the utility of restriction fragment length polymorphisms (RFLPs) for paternity analysis. While, on the average, 99% of falsely accused males can be excluded with the standard battery of blood group antigens, red cell enzymes, serum proteins, and HLA antigens, there are still mother-child pairs for whom the exclusion probability is not high. It has been suggested that additional resolution would be available with RFLPs. We have examined the strategic aspects of using RFLPs for paternity analysis, comparing the efficacy and cost of a multimarker haplotypic set with those of a comparable set of unlinked RFLPs, using published frequencies for the beta-globin complex, the serum albumin region, and the growth hormone region. There are four major findings. (1) Greater resolution is obtained with a carefully chosen set of tightly linked RFLPs producing chromosomal haplotypes than with a comparable set (same allele frequencies) of unlinked markers, but only if it is possible to establish linkage phase unambiguously. (2) Assay of linked sets is cheaper than is the assay of unlinked markers, but the cost advantage is optimized with sets of no more than two or three linked markers. (3) Also, with more than two or three tightly linked markers, the haplotypic frequencies are too poorly estimated to provide a reliable measure of the probability of paternity for unexcluded males, given the sample sizes likely to be available in the near future. (4) Optimal resolution, minimal cost, and acceptable accuracy are obtained with several independent sets of no more than two or three tightly linked RFLP markers each. With current technology, RFLP analysis is more expensive for the same level of genetic resolution than is the standard battery, but gradual replacement of the latter can be anticipated as economies of scale reduce the cost of the DNA technology.     

The relationship between maternal age and chromosome size in autosomal trisomy.

The pattern of maternal age-specific incidence of autosomal trisomy in spontaneous abortions was examined for each chromosome for which a sufficient number of trisomies was observed. This included chromosomes 2, 4, 7-10, 13-16, 18, and 20-22. The rate of increase after age 30 for each of the small chromosomes (groups D-G) was similar, with the exception of chromosome 16, which showed a significantly shallower rate. The C group chromosomes tended to have an intermediate rate of increase after age 30, with the exception of chromosome 7, which had a pattern similar to the smaller chromosomes. The larger chromosomes (2 and 4) had the smallest rate of increase. There was a significant relationship between chromosome size and rate of increase after age 30 (after excluding chromosome 16), but not with rate of increase before age 30. The results suggest that autosomal trisomies may be of heterogeneous origin, with a maternal age-related factor associated with chromosome size and other sources unrelated to chromosome size. Additional evidence for and against this hypothesis is discussed.     

Immunochemical studies on lactate dehydrogenase A subunit deficiencies.

In lactate dehydrogenase (LDH) A subunit deficiency, is there not only a lack of activity but also a lack of subunit production? We demonstrated three remarkable points to answer this question: There are no proteins that immunologically react with anti-A subunits. There are no heterotetramers that react with anti-B subunits. B subunits seem to be genetically produced at normal level, and all of them form only one isoenzyme, LDH-B4. From these points, we concluded that there is a complete lack of A subunit production or production of incomplete A subunits that can neither react with anti-A subunits nor form heterotetramers.     

An autosomal dominant form of adolescent multinodular goiter.

Eighteen members of an extended pedigree have been found to have a form of euthyroid adolescent multinodular goiter. Histological examination showed multiple adenomata with areas of epithelial hyperplasia, hemorrhage, and calcification. In two subjects there were focal areas of epithelial hyperplasia reminiscent of low-grade papillary carcinoma, but capsular and vascular invasion was not found. The pattern of inheritance appeared to be autosomal dominant, with diminished penetrance in males. Although the patients were euthyroid, the likely basis for this disorder is an abnormality in thyroglobulin structure and function.     

Polymorphisms of mitochondrially encoded proteins.

Polymorphisms of mitochondrially encoded proteins can be detected in human lymphocytes by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Using an SDS-polyacrylamide 8 M urea system, 17 mitochondrially encoded proteins are distinguishable. Three of these (ME-6, ME-8, and ME-17) were polymorphic among 92 individuals screened, and these polymorphisms are reported here for the first time. With SDS-polyacrylamide electrophoresis without urea, 18 mitochondrial proteins are detectable. One of these (MV-1) varied in two of 31 individuals tested. This polymorphism has been identified previously in HeLa cells. Maternal inheritance of the ME-8 polymorphism was demonstrated by three informative families.     

Evaluation of Diverse Antisera, Conjugates, and Support Media for Detecting Bradyrhizobium japonicum by Indirect Enzyme-Linked Immunosorbent Assay

We evaluated three antisera and four enzyme conjugates for the detection of Bradyrhizobium japonicum by an indirect enzyme-linked immunosorbent assay in microtiter plates. Nitrocellulose membrane sheets were then evaluated as an alternative support medium by using some combinations. Partially purified immunoglobulin G (IgG) or unpurified antisera to strain USDA 110 raised in rabbits, goats, or sheep was reacted in microtiter plates with alkaline phosphatase conjugated to protein A, goat anti-rabbit (GAR), sheep anti-rabbit (SAR), or rabbit anti-goat (RAG) IgG. Cultures or nodules containing homologous rhizobia were detected with equal sensitivity when protein A, GAR, or SAR was reacted with 5 μg of protein IgG per ml or a 1:800 titer of antisera from rabbits, but not goats or sheep. RAG reacted with IgG or antisera from goats or sheep. The detection limit was 2 × 10⁵ rhizobia per well. Rhizobia were spotted on nitrocellulose sheets as an alternative support medium, followed by soaking in 5 μg of protein per ml as IgG and 1:4,000 dilutions of protein A or GAR conjugate. Rhizobia in serogroup 110 were detected with the dye combination Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), and rhizobia in serogroup 122 were detected with fast red-naphthol phosphate (FR-NP). At the conclusion of the 5-h assay, purple (NBT-BCIP) or red (FR-NP) spots were visible in positive reactions. The sensitivity of detection was about 1,000 rhizobial cells or 3 μg of nodules tissue.