Freeze-fracture replication of organized tissue without cryoprotection.
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Fresh pieces of rat liver and pancreas were rapidly frozen without prior chemical fixation or cryoprotection, and replicated folloing freeze-fracture. Replicas revealed small peripheral areas free of ice crystals or damage and, within such areas, general ultrastructural morphology was essentially similar to that seen in conventionally processed material. On fracture faces of plasma and nuclear membranes a population of less prominent particles in addition to conventional membrane-associated particles was seen, and smooth areas devoid of particles of any type were seen on some nuclear membranes. These smooth areas did not appear to be similar to smooth areas allegedly arising as artifacts of conventional processing. Tight junctions and gap junctions appeared as they do in cryoprotected specimens. The results provide a base-line for assessing the possible effects of processing steps or agents on the ultrastructure of organized tissues as revealed in freeze-fracture replicas. (+info)
The development of M cells in Peyer's patches is restricted to specialized dome-associated crypts.
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It is controversial whether the membranous (M) cells of the Peyer's patches represent a separate cell line or develop from enterocytes under the influence of lymphocytes on the domes. To answer this question, the crypts that produce the dome epithelial cells were studied and the distribution of M cells over the domes was determined in mice. The Ulex europaeus agglutinin was used to detect M cells in mouse Peyer's patches. Confocal microscopy with lectin-gold labeling on ultrathin sections, scanning electron microscopy, and laminin immuno-histochemistry were combined to characterize the cellular composition and the structure of the dome-associated crypts and the dome epithelium. In addition, the sites of lymphocyte invasion into the dome epithelium were studied after removal of the epithelium using scanning electron microscopy. The domes of Peyer's patches were supplied with epithelial cells that derived from two types of crypt: specialized dome-associated crypts and ordinary crypts differing not only in shape, size, and cellular composition but also in the presence of M cell precursors. When epithelial cells derived from ordinary crypts entered the domes, they formed converging radial strips devoid of M cells. In contrast to the M cells, the sites where lymphocytes invaded the dome epithelium were not arranged in radial strips, but randomly distributed over the domes. M cell development is restricted to specialized dome-associated crypts. Only dome epithelial cells that derive from these specialized crypts differentiate into M cells. It is concluded that M cells represent a separate cell line that is induced in the dome-associated crypts by still unknown, probably diffusible lymphoid factors. (+info)
Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast.
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The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis. (+info)
The human papillomavirus type 11 upstream regulatory region triggers hair-follicle-specific gene expression in transgenic mice.
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We have generated transgenic mice carrying the URR of the human papillomavirus type 11 ligated in front of the Escherichia coli beta-galactosidase coding region sequence. Using X-Gal staining to demonstrate beta-galactosidase production, we observed a hair-specific transcription of the reporter gene. This transcription was limited to the epithelial cells of the hair bulge region. The transgene was developmentally regulated, as no LacZ staining was demonstrated during embryogenesis and specific staining was first observed after birth. Surprisingly, dexamethasone and ultraviolet B, but not phorbol myristate acetate or progesterone treatment of the animals resulted in an increase in number and intensity of hair follicles expressing the reporter gene. (+info)
Infrared spectra of basal cell carcinomas are distinct from non-tumor-bearing skin components.
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Infrared spectroscopy, by probing the molecular vibration of chemical bonds, directly indicates tissue biochemistry. An expanding body of literature suggests that infrared spectra distinguish diseased from normal tissue. The authors used infrared spectroscopy to examine basal cell carcinoma to explore distinctive characteristics of basal cell carcinoma versus normal skin samples and other skin neoplasms. Spectra of epidermis, tumor, follicle sheath, and dermis were acquired from unstained frozen sections, and analyzed qualitatively, by t-tests and by linear discriminant analyses. Dermal spectra were significantly different from the other skin components mainly due to absorptions from collagen in dermis. Spectra of normal epidermis and basal cell carcinoma were significantly different by virtue of subtle differences in protein structure and nucleic acid content. Linear discriminant analysis characterized spectra as arising from basal cell carcinoma, epidermis, or follicle sheath with 98.7% accuracy. Use of linear discriminant analysis accurately classified spectra as arising from epidermis overlying basal cell carcinoma versus epidermis overlying nontumor-bearing skin in 98.0% of cases. Spectra of basal cell carcinoma, squamous cell carcinoma, nevi, and malignant melanoma were qualitatively similar. Distinction of basal cell carcinoma, squamous cell carcinoma, and melanocytic lesions by linear discriminant analyses, however, was 93.5% accurate. Therefore, spectral separation of abnormal versus normal tissue was achieved with high sensitivity and specificity, which points to infrared spectroscopy as a potentially useful screening tool for cutaneous neoplasia. (+info)
Expression and localization of Rab3D in rat parotid gland.
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Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An approximately 200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells. (+info)
Active gelatinase B is identified by histozymography in the cartilage resorption sites of developing long bones.
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In order to determine which proteinases mediate the resorption of endochondral cartilage in the course of long bone development, a novel assay called "histozymography" has been developed. In this assay, frozen sections of tibial head from 21-day-old rats are placed for 4 hr at room temperature on light-exposed photographic emulsion (composed of silver grains embedded in gelatin). We report a localized but complete digestion of emulsion gelatin facing two tissue sites which are, therefore, presumed to contain an active proteinase. One of the sites is localized at the growth plate surface forming the epiphysis/metaphysis interface. The other consists of small patches located within the epiphysis at the edge of the marrow space. Both sites are engaged in the resorption of endochondral cartilage. In both sites, inhibitor tests have established that the involved proteinase is a gelatinase. Furthermore, the use of neutralizing antibodies against gelatinase A or B have demonstrated that only those that are specific for the latter block the reaction. That gelatinase B is present in the two sites has been confirmed by light microscopic immunohistochemistry. Finally, when immunoelectron microscopy is used for fine localization of the cartilage structures that form the epiphysis/metaphysis interface, the enzyme is detected within the 0.5-microm thick edge of the cartilage, and outside the cartilage, it is present in debris composed of type II collagen-rich fibrils in various states of digestion. It is concluded that gelatinase B attacks the edge of an endochondral cartilage and helps to solubilize the type II-collagen-rich fibrillar framework, which is then released as debris for further digestion. This final step opens the way to invasion by capillaries, thereby making possible the replacement of cartilage by bone. Dev Dyn 1999;215:190-205. (+info)
Apoptotic chondrocyte death in rheumatoid arthritis.
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OBJECTIVE: Recently, chondrocytes were shown to undergo apoptosis by the addition of nitric oxide and by coupling of Fas/Fas ligand in vitro, suggesting the possibility that chondrocytes have an inherent programmed cell death pathway that operates in adult cartilage. Chondrocyte apoptosis was verified in situ in articular cartilage samples from humans with osteoarthritis (OA) and from an animal model of OA. The present study investigates apoptotic chondrocyte death and the expression of Bcl-2 and Fas in rheumatoid arthritis (RA) cartilage. METHODS: Cartilage samples were obtained from 13 RA patients at the time of joint replacement surgery and from 8 normal subjects at autopsy. Apoptotic chondrocytes were observed and counted in hematoxylin and eosin-stained cartilage specimens. Apoptosis was verified by TUNEL, electron microscopy, and DNA ladder assay. Bcl-2 and Fas expression were evaluated by immunohistochemistry. RESULTS: Apoptotic cells were frequently observed in RA cartilage, whereas normal cartilage rarely showed apoptotic cells (3.01% versus 0.15%, respectively), a finding that was further confirmed by TUNEL staining. On electron microscopy, numerous apoptotic cells with typical chromatin condensation were observed in RA cartilage. DNA from RA cartilage also revealed 180-basepair nucleosome ladders on electrophoresis. Bcl-2 expression was significantly lower in RA cartilage than in normal cartilage (23.3% versus 43.1%, respectively), whereas Fas expression was not statistically different. CONCLUSION: Apoptotic chondrocyte death and decreased Bcl-2 expression were verified in RA cartilage. They might provide a novel model system for the research of cartilage breakdown and joint destruction in RA. (+info)