Alveolar epithelial surface area-volume relationship in isolated rat lungs.
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In vitro studies of the alveolar epithelial response to deformation require knowledge of the in situ mechanical environment of these cells. Because of the presence of tissue folding and crumpling, previous measurements of the alveolar surface area available for gas exchange are not equivalent to the epithelial surface area. To identify epithelial deformations in uniformly inflated lungs representative of the in vivo condition, we studied isolated Sprague-Dawley rat lungs (n = 31) fixed by perfusion with glutaraldehyde on deflation after cycling three times at high lung volume (10-25 cmH2O). The epithelial basement membrane in 45 electron micrographs (x12,000)/rat was traced, digitally scanned, and analyzed. Epithelial basement membrane surface area (EBMSA) was computed from a morphometric relationship. EBMSA was found to increase 5, 16, 12, and 40% relative to EBMSA at 24% total lung capacity at lung volumes of 42, 60, 82, and 100% total lung capacity, respectively. The increases in EBMSA suggest that epithelial cells undergo significant deformations with large inflations and that alveolar basement membrane deformation may contribute to lung recoil at high lung pressures. (+info)
Analytical detection and quantitation of strychnine in chemically fixed organ tissues.
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This study reports the results of the detection and quantitation of strychnine in formalin-fixed tissues and in the formalin solutions in which the tissues were fixed. The toxicological analyses were performed on formalin-fixed liver and kidney samples and formalin solutions (10% buffered pH 7) in which the same samples from a case of acute strychnine poisoning were preserved. The analyses carried out at the time of autopsy on body fluid and tissues (bile, 2.40 mg/L; stomach contents, 14.2 mg; liver, 6.68 mg/kg; kidney, 2.68 mg/kg) allowed the identification of this substance as cause of death. The tissue samples were preserved in formalin solutions for 8 weeks. The analyses performed on formalin-fixed tissues (liver and kidney) and on formalin solutions, in which the same tissues were preserved, permitted the detection and quantitation of strychnine (liver, 1.59 mg/kg; formalin from the liver, 1.80 mg/L; kidney, 0.98 mg/kg; formalin from the kidney, 1.11 mg/L). The results indicate that this particular toxic substance also shows good stability in biological specimens subjected to chemical fixation. (+info)
Reduction of BRCA1 protein expression in Japanese sporadic breast carcinomas and its frequent loss in BRCA1-associated cases.
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BRCA1 is a tumor suppressor gene that is responsible for hereditary breast and ovarian cancer syndrome. To clarify the possible involvement of the BRCA1 protein in mammary carcinogenesis in sporadic and hereditary forms, we have analyzed the BRCA1 protein expression pattern in five breast epithelial cell lines, including a BRCA1-deficient cell line, and 162 breast cancer tissue samples [including 108 sporadic, 35 hereditary (BRCA1 status unknown), and 19 BRCA1-associated cases] from Japanese women. Twelve anti-BRCA1 antibodies were tested by fixation conditions, in which nuclear localization of BRCA1 protein was preserved, and by specificity of the antibodies, which was evaluated in BRCA1-deficient cancer cells. Using monoclonal antibodies applicable to immunohistochemical analysis of paraffin-embedded tissue sections, we found high-level expression of BRCA1 protein in normal mammary epithelium and various degrees of reduced expression in breast cancer cells. Of the 19 BRCA1-associated breast cancer tissues, 15 (79%) showed reduction (8 cases) or complete loss (7 cases) of nuclear expression. Thirty (28%) of 108 sporadic and 6 (17%) of 35 hereditary carcinomas showed reduced BRCA1 protein expression. Reduction of BRCA1 protein expression in sporadic carcinomas was associated with solid-tubular phenotype, with poor tubular differentiation, and with an overexpression of c-erbB-2 protein, which is one of the prognostic factors in breast cancer. Our data suggest that reduced expression of BRCA1 protein may play an important role in mammary carcinogenesis, not only in BRCA1-associated breast carcinomas, but also in sporadic carcinomas, and also suggest that mechanisms other than mutation may be involved in its reduced expression. (+info)
A comparison of three methods of orienting cervical punch biopsies.
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AIMS: To compare methods of orienting cervical punch biopsies, since well oriented biopsies are needed to optimise the diagnosis and grading of cervical intraepithelial neoplasia. METHODS: The orientation, the presence and preservation of the squamocolumnar junction, and the presence or absence of the surface layers of the squamous epithelium were compared in 345 cervical biopsies that had either been attached to paper (n = 112), floated directly into 10% formalin (n = 107), or floated into a solution of 10% formalin including 0.05% eosin (n = 126) in the colposcopy clinic. RESULTS: When the specimens were mounted on filter paper before fixation, they were more likely to be optimally oriented, to have a preserved squamocolumnar junction, and to have intact surface epithelium than specimens that were handled using the other methods, even when the specimens floated off the paper in transit. CONCLUSIONS: Cervical biopsy specimens should be mounted on paper before fixation and submission to the laboratory. (+info)
Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.
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Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies but the extraction of high quality nucleic acid may be problematic. We have optimised DNA extraction by comparing 10 protocols, including a commercially available kit and a novel method that utilises a thermal cycler. The thermal cycler and Chelex-100 extraction method yielded DNA capable of amplification by PCR from every block and 61% of sections versus 54% using microwave and Chelex-100, 15% with classical xylene-based extraction and 60% of sections using the kit. Successful RNA extraction was observed, by beta-actin amplification, in 83.7% sections for samples treated by the thermal cycler and Chelex-100 method. Thermal cycler and Chelex-100 extraction of nucleic acid is reliable, quick and inexpensive. (+info)
Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.
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The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function. (+info)
An anatomic atlas of the medulla oblongata of the adult goat.
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An anatomic atlas of the goat brain stem was developed for use in studies that analyze medullary neuronal groups, and factors that influence variability in the location of neuronal groups were determined. The medullas of 31 adult goats (weight, 17-88 kg) were fixed, harvested, frozen, serially sectioned, stained with 0.5% neutral red, and examined with a light microscope. Obex, the point at which the central canal opens into the fourth ventricle, was taken as the zero reference point from which the rostrocaudal and mediolateral coordinates of medullary neuronal groups were determined, whereas dorsoventral coordinates were calculated from the medullary surface. Histological variations with goat body weight were quantified, and linear regression analysis provided adjustment factors for weight in all three dimensions. Similar analysis of percentage of shrinkage on fixation and processing provided adjustment factors for precise coordinates of medullary neuronal groups. For accurate location of neuronal groups, body weight and histological procedure should be taken into account. The present study provided adjustment factors for body weight and standard histological processing to locate most major medullary neuronal groups in the adult goat. (+info)
A key function of non-planar membranes and their associated microtubular ribbons in contractile vacuole membrane dynamics is revealed by electrophysiologically controlled fixation of Paramecium.
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The contractile vacuole complex of the fresh water protozoan Paramecium multimicronucleatum exhibits periodic exocytotic activity. This keeps cytosolic osmolarity at a constant value. The contractile vacuole, the central exocytotic vesicle of the complex, becomes disconnected from its surrounding radial arms and rounds before its fluid content is expelled. We previously proposed a hypothesis that the rounding of the contractile vacuole corresponds to an increase in its membrane tension and that a periodic increase in membrane tension governs the exocytotic cycle. We also proposed a hypothesis that transformation of excess planar membrane of the contractile vacuole into 40 nm diameter tubules, that remain continuous with the contractile vacuole membrane, is a primary cause for the tension development in the planar membrane. In order to investigate tension development further, we have examined electron microscopically the contractile vacuole membrane at the rounding phase. To do this, we developed a computer-aided system to fix the cell precisely at the time that the contractile vacuole exhibited rounding. In this system a decrease in the electrical potential across the contractile vacuole membrane that accompanied the vacuole's rounding was monitored through a fine-tipped microelectrode inserted directly into the in vivo contractile vacuole. A decrease in membrane potential was used to generate an electric signal that activated an injector for injecting a fixative through a microcapillary against the cell at the precise time of rounding. Subsequent electron micrographs of the contractile vacuole membrane clearly demonstrated that numerous approximately 40 nm membrane-bound tubules formed in the vicinity of the vacuole's microtubule ribbons when the vacuole showed rounding. This finding suggested that membrane tubulation was the cause for topographical isolation of excess membrane from the planar membrane during the periodic rounding of the contractile vacuole. This together with stereo-pair images of the contractile vacuole complex membranes suggested that the microtubule ribbons were intimately involved in enhancing this membrane tubulation activity. Electron micrographs of the contractile vacuole complexes also showed that decorated tubules came to lie abnormally close to the contractile vacuole in these impaled cells. This suggested that the contractile vacuole was capable of utilizing the smooth spongiome membrane that lies around the ampullae and the collecting canals to increase its size. (+info)