Subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 assessed by immunohistochemistry.

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.     

Distribution of mRNAs Encoding the Peroxisome Proliferator-Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System

Abstract: We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.     

Refined mapping of the gene for thiamine-responsive megaloblastic anemia syndrome and evidence for genetic homogeneity

Thiamine-responsive megaloblastic anemia (TRMA, also known as Rogers syndrome, OMIM 249270) is a rare autosomal recessive disorder characterized by a triad of megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Patients respond, to varying degrees, to treatment with megadoses of thiamine. We have recently shown genetic linkage of the TRMA gene to a 16-centimorgan (cM) region on 1q23.2–1q23.3 based on the analysis of four large, inbred families of Alaskan, Italian, and Israeli-Arab origin. Here we narrow the TRMA interval down to 4 cM based on genetic recombination, homozygosity mapping, and linkage disequilibrium (highest LOD score of 12.5 at D1S2799, at a recombination fraction of 0). We provide further evidence that the TRMA gene is located in this region and confirm the homogeneity of the disease. In this analysis, we genotyped seven additional families of diverse ethnic origin (Pakistani, Indian, Italian, Brazilian, and Japanese), and analyzed additional markers in two previously reported families showing evidence of linkage disequilibrium in a large area of their haplotypes. The multi-system manifestations of TRMA suggest that thiamine has a pivotal role in a multiplicity of physiological processes. Mapping the TRMA gene and understanding the molecular basis of the disease might, thus, shed light on the role of thiamine in common disorders such as deafness, anemia, and diabetes. Received: 16 April 1998 / Accepted: 6 July 1998     

Response of protozoans to detergent-enzymes

Summary These studies were divided into two parts: (1) time until death curves in which Paramecium caudatum Ehrenberg was exposed to selected concentrations of the detergent-enzyme Axion, and (2) exposure of fresh-water protozoan communities to selected concentrations of Axion. The former were carried out in test tubes, the latter in plastic troughs with a constantly flowing fluid. Both types of bioassays were carried out with filtered water from Douglas Lake, Michigan, U.S.A. at a temperature of about 25° C. One hundred percent of an exposed population of Paramecium caudatum was killed in 20 to 30 minutes at a concentration of 100 ppm of Axion. No appreciable change in the controls was noted during these tests. The bioassays with fresh-water protozoan communities were carried out in plastic troughs with a constant flow of Douglas Lake water. During the exposure period the lake water flow was stopped and a flow of Axion solution was substituted for approximately three hours. After this exposure period lake water flow were restored. The number of species was determined before and at intervals following exposure. Appropriate controls were maintained. A three hour exposure to 56 ppm (introduced concentration) of Axion caused a 35 percent reduction in the number of species in less than five hours with recovery to the original number in approximately 145 hours; a three hour exposure to 75 ppm caused a 54% reduction in number of species in less than five hours with recovery to the original number in approximately 240 hours; 100 ppm produced a 55 percent reduction with recovery in approximately 200 hours; 125 ppm produced a 48% reduction with recovery in approximately 148 hours; 200 ppm caused a 78% reduction with recovery in about 140 hours. Response patterns vary as do those of bioassays with single species. However, it is important to note that each test community had different types of species present, and it is quite likely that some communities had a higher percentage of sensitive species than others. Since this variation also occurs in nature the tests are probably representative of the differences that exist when natural communities are exposed to wastes. Variation in control troughs was less than ± 10 percent in the number of species.