Direct visualization and cellular localization of D1 and D2 dopamine receptors in rat forebrain by use of fluorescent ligands.

The regional and cellular localization of the two subtypes of dopamine receptors, D1 and D2, have been ascertained in rat forebrain by use of fluorescent dopaminergic antagonist ligands. (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3- methyl-[1H]-3-benzazepin-7-ol, the 4'-amino derivative of the high-affinity D1-specific antagonist SCH 23390, and the D2 selective antagonist N-(p-aminophenethyl)spiperone were chemically derivatized using the fluorescent compound tetramethylrhodamine. The modification of these antagonist ligands has allowed the specific, cellular resolution of the D1 and D2 receptor binding sites in intact, highly organized regions of forebrain slices in a very rapid experimental time frame. The regional localization of receptors labeled by the fluorescent probes is in agreement with previous receptor autoradiography studies. Moreover, the specific cellular binding patterns for both receptors can now be compared and contrasted to one another in the same tissue by using these fluorescent ligands. D1 receptor sites are most evident within the striatum and exhibit regions of intense "patch" fluorescence corresponding to receptor reactivity in cells and their processes. The distribution of D1 receptor binding is highly analogous to the pattern of dopamine terminal histofluorescence in the caudate nucleus. D2 receptor sites are less prevalent overall and may be localized to a subpopulation of the D1 fluorescent neurons in the caudate nucleus and nucleus accumbens regions.     

Improving the management of bladder cancer with fluorescence cystoscopy.

Endoscopic visualization (cystoscopy) and transurethral resection are effective, well-tolerated diagnostic and treatment techniques for bladder cancer. However, it is widely recognized that cystoscopy can miss biologically important lesions, such as carcinoma in situ. Attempts to improve the effectiveness of cystoscopy are not new, but initial methods were impractical and had limited efficacy. Fluorescence cystoscopy became feasible with the discovery that intravesical administration of aminolevulinic acid (ALA) made bladder cancers fluoresce when exposed to blue light. More recently, the creation of a hexyl ester of ALA (HAL) made this technique practical, because HAL significantly shortens the amount of time needed for drug exposure prior to cystoscopy. Not surprisingly, studies have shown that fluorescence cystoscopy can reveal carcinoma in situ that is visually occult under conventional (white-light) cystoscopy. An unexpected finding was that fluorescence cystoscopy also enhanced the detection of papillary tumors. Studies with ALA have shown that resection of bladder cancer with fluorescence results in improved disease-free survival compared to conventional resection under white light. This report summarizes some of the recent studies of fluorescence cystoscopy in bladder cancer.     

Mice lacking the transcription factor RelB develop T cell-dependent skin lesions similar to human atopic dermatitis

Mice with a targeted disruption of the Rel / NF-kappaB family member RelB develop a complex inflammatory phenotype and hematopoietic abnormalities. RelB-deficient (relB(- / -)) mice were clinically normal until 4 - 10 weeks after birth when thickening of the skin and hair loss developed. Histological and immunohistochemical evaluation of relB(- / -) skin lesions revealed hyperkeratosis and marked epidermal hyperplasia. Many CD4(+) T cells and eosinophils mixed with lesser numbers of CD8(+) T cells and neutrophils were present in the dermis. There was a moderate increase of MHC class II-positive dermal dendritic cells and dermal mast cells. Increased expression of Th2 cytokines correlated with increased mRNA levels of eotaxin and CCR3 in relB(- / -) skin. The dermatitis did not develop in the offspring of relB(- / -) mice crossed with transgenic mice that lack peripheral T cells, demonstrating that the skin lesions were T cell dependent. The dermatitis observed in RelB-deficient mice had many similarities with atopic dermatitis in human patients including infiltrating CD4(+) T cells and eosinophils in the skin, increased number of eosinophils in the blood and increased serum IgE. Thus, the relB(- / -) mouse should be a useful model to study the pathogenesis of this common allergic human disease.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

The integrity of sperm DNA is crucial for the maintenance of genetic health. A major source of damage is reactive oxygen species (ROS) generation; therefore, antioxidants may afford protection to sperm DNA. The objectives of the study were, first, to measure the effects of antioxidant supplementation in vitro on endogenous DNA damage in spermatozoa using the single cell gel electrophoresis (comet) assay and, second, to assess the effect of antioxidant supplementation given prior to X-ray irradiation on induced DNA damage. Spermatozoa from 150 patients were prepared by Percoll centrifugation in the presence of ascorbic acid (300, 600 microM), alpha tocopherol (30, 60 microM), urate (200, 400 microM), or acetyl cysteine (5, 10 microM). DNA damage was induced by 30 Gy X-irradiation. DNA strand breakage was measured using the comet assay. Sperm DNA was protected from DNA damage by ascorbic acid (600 microM), alpha tocopherol (30 and 60 microM) and urate (400 microM). These antioxidants provided protection from subsequent DNA damage by X-ray irradiation. In contrast, acetyl cysteine or ascorbate and alpha tocopherol together induced further DNA damage. Supplementation in vitro with the antioxidants ascorbate, urate and alpha tocopherol separately has beneficial effects for sperm DNA integrity.      

Effects of CD8 depletion on retinal soluble antigen induced experimental autoimmune uveoretinitis.

During the later stages of soluble-antigen (sAg)-induced experimental autoimmune uveoretinitis (EAU), an increase in the relative number of CD8+ lymphocytes has been observed at the site of inflammation in the retina. It has been suggested that these late-appearing CD8+ cells might down-regulate this acute disease process. To determine the role of the CD8+ cells in EAU, Lewis rats were depleted of CD8+ cells prior to and during disease and the enucleated eyes examined histologically. The spleen cells from CD8-depleted rats were also examined for their ability to respond to concanavalin A (Con A) and to allogeneic targets as determined by mixed lymphocyte reaction (MLR) and cytotoxicity assays. The results suggest that depleting CD8+ cells had no effect on the course of disease and that CD8+ cells do not play a crucial role in the immunoregulation of EAU.     

Instability of normal (CTG)n alleles in the DM kinase gene.

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Stainable hepatic iron in 341 African American adults at coroner/medical examiner autopsy

Background  

Results of previous autopsy studies indicate that increased hepatic iron stores or hepatic iron overload is common in African Americans dying in hospitals, but there are no reports of hepatic iron content in other cohorts of African Americans.