HER-2/neu evaluation by immunohistochemistry and fluorescence in situ hybridization in breast cancer: implications for daily laboratory practice.

BACKGROUND: HER-2/neu status was determined by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) methods in more than 300 paraffin-embedded primary breast cancer samples. MATERIALS AND METHODS: HER-2/neu status was determined by FISH using the PathVysion kit (Vysis) and by IHC using either a monoclonal antibody CB11 or a cocktail of antibodies: the monoclonal TAB250 and the polyclonal pAb1. RESULTS: Of the 324 cases evaluable by IHC, 65 out of 318 (20%) and 24 out of 324 (7%) were scored as positive when using the antibody cocktail and the CB11, respectively. HER-2/neu gene amplification occured in 64 out of 324 cases (20%). Concordance of FISH and IHC was found in 285 out of 318 cases (90%) and 278 out of 324 cases (86%) using the cocktail and the CB11, respectively. CONCLUSION: The cost-effectiveness analysis revealed that the use of a sensitive IHC method followed by confirmation of positive results by FISH considerably decreased the FISH costs and may become standard practice for HER-2/neu evaluation.

Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

Chromosomal organization of adrenergic receptor genes.

The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.

Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Human CD19 and CD40L deficiencies impair antibody selection and differentially affect somatic hypermutation.

Individuals with genetic defects in CD40 ligand (CD40L) or B-cell antigen receptor coreceptor molecules CD19 and CD81 suffer from an antibody deficiency. Still, these patients carry low levels of memory B cells and serum antibodies.

Natural hybridization between genera that diverged from each other approximately 60 million years ago.

A fern from the French Pyrenees-×Cystocarpium roskamianum-is a recently formed intergeneric hybrid between parental lineages that diverged from each other approximately 60 million years ago (mya; 95% highest posterior density: 40.2-76.2 mya). This is an extraordinarily deep hybridization event, roughly akin to an elephant hybridizing with a manatee or a human with a lemur. In the context of other reported deep hybrids, this finding suggests that populations of ferns, and other plants with abiotically mediated fertilization, may evolve reproductive incompatibilities more slowly, perhaps because they lack many of the premating isolation mechanisms that characterize most other groups of organisms. This conclusion implies that major features of Earth's biodiversity-such as the relatively small number of species of ferns compared to those of angiosperms-may be, in part, an indirect by-product of this slower "speciation clock" rather than a direct consequence of adaptive innovations by the more diverse lineages.

Understanding Corporate Governance Reform in South Africa: Anglo-American Divergence, the King Reports and Hybridization

This article investigates corporate governance reform in South Africa in the context of the country’s international links with Anglo-American corporate governance and domestic pursuit of socioeconomic development. Two key questions are evaluated. (a) How has divergence within the Anglo-American model influenced corporate governance reform in South Africa? (b) Can South Africa’s historical closeness to the Anglo-American model be combined with increasing attention to stakeholder issues to produce a hybrid “African model” of corporate governance? Evaluating these questions, the following issues are explored in turn: the contrast between shareholder and stakeholder models, divergence between U.S. and U.K. approaches to corporate governance as exemplified by Sarbanes-Oxley, locating a South African approach in context of the Anglo-American model, the King reports and an emerging “African” model of corporate governance, and the role of international and domestic factors in shaping South Africa’s ongoing reform process.

Unidirectional hybridization at a species' range boundary: implications for habitat tracking

Aim Introgressive hybridization between a locally rare species and a more abundant congener can drive population extinction via genetic assimilation, or the replacement of the rare species gene pool with that of the common species. To date, however, few studies have assessed the effects of such processes at the limits of species' distribution ranges. In this study, we have examined the potential for hybridization between range-edge populations of the wintergreen Pyrola minor and sympatric populations of Pyrola grandiflora. Location Qeqertarsuaq, Greenland and Churchill, Manitoba, Canada. Methods Genetic analysis of samples from Greenland and Canada was carried out using a combination of nuclear and chloroplast single nucleotide polymorphisms (SNPs). Results Analysis of nuclear SNPs confirmed hybridization in populations of morphologically intermediate individuals, as well as revealing the existence of cryptic hybrids in ostensibly morphologically pure P. minor populations. Analysis of chloroplast SNPs revealed that this hybridization is unidirectional and suggests that hybrids originate via pollen swamping of P. minor by the more common P. grandiflora. Main conclusions Extensive unidirectional hybridization may lead to the extinction of peripheral populations of P. minor where the two species grow sympatrically. Extinction could occur as a result of genetic assimilation where F1s are fertile, or via the removal of unidirectionally pollinated sterile F1s, or by a combination of these processes. This could compromise the ability of species to respond to climate change via habitat tracking, although the final outcome of these processes may ultimately depend on the rate of global climate change and its effect on the species' distributions. © 2009 Blackwell Publishing Ltd.