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PubMed1996

Subcellular localization of angiotensin II immunoreactivity in the rat cerebellar cortex.

We localized angiotensin II (Ang II) immunoreactivity in the rat cerebellar cortex with immunogold staining methods. Perfusion fixation with high amounts of glutaraldehyde and the use of cryoultramicrotomy caused remarkable changes in immunostaining versus formaldehyde/picric acid fixation. With the use of monoclonal and polyclonal anti-Ang II, Ang II immunoreactivity was prominent in cerebellar neurons such as Purkinje, granule, basket, and stellate cells. At the subcellular level, the peptide was clearly localized in nuclei, and in some cell types, such as endothelial and granule cells, it was nearly exclusively present in the transcriptionally active euchromatin. Intracellular Ang II immunoreactivity was also detected in vesicle-like structures in cytoplasm and mitochondria and at cell-cell contacts. Additional experiments with liver and adrenal tissue confirmed the nuclear localization of Ang II immunoreactivity, suggesting a role of Ang II in the regulation of gene transcription.

Adrenal Glands↗
PubMed1991

Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 bp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site.

Base Sequence↗
PubMed2005

Adenocarcinoma of apocrine sweat glands in a mouflon (Ovis musimon).

A free-living mouflon (Ovis musimon) was presented with a mass on the left shoulder. At necropsy, multifocal, slightly protruding whitish spots were noted on the kidneys, and several lymph nodes were abnormal. Histologically, the mass was composed of epithelial cells arranged in tubular and tubulopapillary structures. The cytoplasm of the epithelial cells had numerous periodic acid-Schiff-positive and diastase-resistant granules. Ultrastructurally, the cytoplasm of the neoplastic cells contained numerous pleomorphic secretory granules and microvilli, which partially covered the luminal surface of the tumor cells. Metastatic foci were present in prescapular and mediastinal lymph nodes and kidneys. On the basis of histological and ultrastructural findings, this tumor was diagnosed as a tubulopapillary adenocarcinoma, arising from apocrine sweat glands of the skin.

Adenocarcinoma↗
PubMed2003

Polymorphous low-grade adenocarcinoma of the vulva and vagina: a tumor resembling adenoid cystic carcinoma.

We report the first case of a polymorphous low-grade adenocarcinoma (PLGA) occurring in the vulva and vagina of a 32-year-old woman. This tumor consisted of cellular lobules with distinct cribriform, papillary, and cystic patterns. Owing to its location and its distinct cribriform pattern, this lesion was initially diagnosed as an unusual variant of adenoid cystic carcinoma (ACC). However, this diagnosis was revised to PLGA when it was recognized that the cribriform, papillary and cystic patterns and their concomitant occurrence in the same lesion are characteristic of PLGA. PLGA should be added to the differential diagnosis of vulvar and vaginal neoplasia.

Adenocarcinoma↗
PubMed1985

Involvement of Golgi apparatus and a restructured nuclear envelope during biogenesis and transport of herpes simplex virus glycoproteins.

Following infection of BHK-21 cells with Herpes simplex virus type 1 (HSV-1), progeny nucleocapsids in the nucleus acquire a glycoprotein-rich envelope by budding through host-cell nuclear membranes. To investigate the nature of the glycoprotein products assembled in the virion at the nuclear envelope, infected cells were pulse-labeled with [3H]-mannose, an oligosaccharidal core sugar, or [3H]-fucose, a terminal sugar. After various chase periods, the incorporation of these sugars was monitored by electron microscope radioautography. The results show that HSV glycoproteins accumulate very rapidly in nuclear membranes, where they exist only as core-glycosylated precursors, i.e., containing [3H]-mannose but not [3H]-fucose. [3H]-fucose grains are seen mainly over Golgi membranes and over virions located in the Golgi and in other cytoplasmic vesicular structures. Our data support a model where addition of terminal sugars (e.g., fucose) to HSV-1 glycoprotein precursors can occur at the surface of newly enveloped viral particles as the virions themselves egress from the cell via the Golgi apparatus.

Animals↗
PubMed1988

Glucagon receptors in endothelial and Kupffer cells of mouse liver.

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.

Animals↗
PubMed1988

Intracytoplasmic lumina in meningioma: an ultrastructural and immunohistological study.

Three surgically removed meningotheliomatous meningiomas with hyaline inclusions or pseudopsammoma bodies were studied. Ultrastructurally, the lumina seemed to be predominantly intracytoplasmic, defined by a systemic unit membrane; they displayed abundant microvilli and contained granular or filamentous material, vacuoles, vesicular bodies, and lamellar structure. The cytoplasm surrounding the intracytoplasmic lumina was electron-dense and contained tonofilaments and desmosomal junctions. All three meningiomas showed expression of carcinoembryonic antigen, cytokeratin, and epithelial membrane antigen in the cells surrounding the hyaline bodies. Keratin, alpha-1-antitrypsin, and immunoglobulin M showed weak positive staining. There was widespread vimentin except in the cells with hyaline inclusions. Glial fibrillary acidic protein and S100 were negative. These results provide additional confirmation of immunohistochemical data that can serve as evidence for epithelial and secretory differentiation in meningiomas.

Aged↗
PubMed1998

Coexpression of both types of desmosomal cadherin and plakoglobin confers strong intercellular adhesion.

Desmosomes are unique intercellular junctions in that they invariably contain two types of transmembrane cadherin molecule, desmocollins and desmogleins. In addition they possess a distinct cytoplasmic plaque structure containing a few major proteins including desmoplakins and the armadillo family member plakoglobin. Desmosomal cadherins are putative cell-cell adhesion molecules and we have tested their adhesive capacity using a transfection approach in mouse L cells. We find that L cells expressing either one or both of the desmosomal cadherins desmocollin 2a or desmoglein 1 display weak cell-cell adhesion activity that is Ca2+-dependent. Both homophilic and heterophilic adhesion could be detected. However, co-expression of plakoglobin with both desmosomal cadherins, but not with desmoglein 1 alone, resulted in a dramatic potentiation of cell-cell aggregation and the accumulation of detergent-insoluble desmosomal proteins at points of cell-cell contact. The effect of plakoglobin seems to be due directly to its interaction with the desmosomal cadherins rather than to its signalling function. The data suggest that the desmosome may obligatorily contain two cadherins and is consistent with a model in which desmocollins and desmogleins may form side by side heterodimers in contrast to the classical cadherins that are homodimeric. Plakoglobin may function by potentiating dimer formation, accretion of dimers to cell-cell contact sites or desmosomal cadherin stability.

Animals↗
PubMed2004

Expression and visualization of a human interferon-beta-enhanced green fluorescent protein chimeric molecule in cultured cells.

We have constructed a recombinant cDNA encoding the chimeric protein between human IFN-beta (HuIFN-beta) and enhanced green fluorescent protein (EGFP) to elucidate the intracellular localization of IFN-beta. Transient expression of the chimeric molecule, HuIFN-beta-EGFP, in L cells demonstrated that the chimeric molecule secreted from the cells had an intact biological activity as far as antiviral effect was concerned. Immunostaining of the transfected cells using anti-HuIFN-beta antibody demonstrated that green-fluorescence was co-localized with the IFN signal and its profile was similar to IFN signals in the cells transfected with HuIFN-beta expressing plasmid DNA. These results indicate that the HuIFN-beta-EGFP chimeric gene was expressed as a chimeric protein and the chimera was transported via the regular secretory pathway in the cells. In other cell types, the fluorescence derived from the chimeric protein was also seen on cytoplasmic vesicular structures. These results suggest that HuIFN-beta-EGFP will be a useful tool to investigate the intracellular trafficking processes of HuIFN-beta in a variety of cell types.

Animals↗
PubMed2005

MicroRNA function: multiple mechanisms for a tiny RNA?

MicroRNAs are sequence-specific regulators of post-transcriptional gene expression in many eukaryotes. They are believed to control the expression of thousands of target mRNAs, with each mRNA believed to be targeted by multiple microRNAs. Recent studies have uncovered various mechanisms by which microRNAs down-regulate their target mRNAs and have linked a well-known subcellular structure, the cytoplasmic processing bodies (PBs) to the microRNA pathway. The finding that microRNAs are misexpressed in cancers has reinforced the idea that their regulatory roles are very important.

Animals↗
PubMed2004

Morphologic characteristics of pulmonary macrophages in cetaceans: particular reference to pulmonary intravascular macrophages as a newly identified type.

We examined the morphologic characteristics of pulmonary macrophages in 42 specimens of Odontoceti (Globicephala macrorhynchus, Grampus griseus, Tursiops truncatus, Stenella attenuata, Stenella coeruleoalba, Berardius bairdii), using light and electron microscopes as well as immunohistochemistry with SRA-E5. SRA-E5-positive alveolar macrophages and pulmonary interstitial macrophages contained graphitic soots, indicating the clearance of airborne, aspirated foreign bodies. Pulmonary intravascular macrophages (PIMs), positive with SRA-E5, were present within pulmonary capillaries, attaching to applied endothelial cells by cell junctions. They showed cytoplasmic tubular structures of micropinocytosis vermiformis and erythrophagocytosis, indicating their contributory role in the clearance of blood-borne particles. The uptake of pathogens by PIMs may be associated with the inducement of acute lung injury, especially bacterial infectious pneumonia. This study revealed for the first time the presence of PIMs in cetaceans.

Animals↗
PubMed2006

Analysis of ribosomal protein gene structures: implications for intron evolution.

Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.

Amino Acid Sequence↗
PubMed1975

[Genetic relationship of two mutant genes which producing three different syndromes in the mouse (author's transl)].

We determined that leaner gene (la) is located in the linkage group XVIII and closely linked to Es-1, which is known to be located closely to tottering gene (tg). Double heterozygote (la/tg) produced by mating between la heterozygote and tg heterozygote showed an intermediate syndrome between those seen in tottering (tg/tg) and leaner (la/la) mice. Both leaner and tottering mice showed neuromuscular disorders, but their clinical and pathological characteristics were different. Leaner mice were found to represent a so-called cerebellar mutant having the reduced size of cerebellum and severe cytoarchitectonic abnormalities with focal losses of Purkinje and granular layer cells. Tottering was, however, another mutation having epileptiform seizures, and it was characterized pathologically by cellular losses and shrinkage as well as vesiculations of cytoplasmic membranous structures in the cerebellum. The double heterozygote was shown to have both pathologic characteristics seen in each homozygote, and also showed shrinkage of Purkinje cells and vesiculation of the endoplasmic reticulum and Golgi apparatus. These clinical and pathological findings supported the genetic data suggesting that la and tg constitute an allele.

Alleles↗
PubMed2003

Mouthrinses with alcohol: cytotoxic effects on human gingival fibroblasts in vitro.

BACKGROUND: Mouthrinses are widely utilized in daily oral and dental hygiene to control plaque. However, most commercially available mouthrinses contain alcohol as an excipient. Most studies have focused on the clinical side effects related to the alcoholic fraction of mouthrinses, overlooking alcohol metabolism in the mouth. Due to this oral enzymatic process, the well-recognized toxic compound acetaldehyde is emitted in the mouth. Since gingival fibroblasts play a key role in oral connective tissue health maintenance, we investigated the effects of different doses of acetaldehyde on human gingival fibroblasts (HGFs) in order to better define the effects of alcohol-containing mouthrinses on oral tissue. METHODS: Cultured HGFs were exposed to different concentrations of acetaldehyde (10(-4) M to 10(-2) M). The cell adhesion rate was measured after a 3-hour incubation period, and cell viability over a 5-day period. In order to assess the reversibility of the damage produced by acetaldehyde, treatment was interrupted at critical doses (10(-3) M and 3 x 10(-3) M), and cell viability was evaluated on the third and fifth day of incubation. The HGF cytoskeleton was studied by immunocytochemical technique, and internal cell structures were observed with transmission electron microscopy to evaluate the morphological changes due to acetaldehyde. RESULTS: The results showed that acetaldehyde produced a dose- and time-dependent inhibition on cell adhesion and viability, together with disruption of cytoskeletal structures and cytoplasmic organelles. Nevertheless, these quantitative and qualitative damages were reversible when the treatment was interrupted. CONCLUSIONS: Although more knowledge is necessary, our results suggest that these deleterious effects may also occur in vivo. Pending further investigations, clinicians should be alerted to the potentially adverse effect of alcohol-containing mouthrinses and, to promote oral health, patients should be warned about improper use of these products.

Acetaldehyde↗
PubMed2005

Analysis of eosinophilic round bodies formed after injection of enamel matrix derivative into the backs of rats.

BACKGROUND: Enamel matrix derivative (EMD) is used in dental clinics for the regeneration of alveolar bone. Its effects have not yet been clarified, although it induces eosinophilic round bodies (ERBs) and cartilage formation at the injection site. The objective of this experiment was to examine the histopathologic and biochemical properties of ERBs formed after EMD injection. METHODS: The backs of Sprague-Dawley rats injected with various concentrations of EMD were examined histopathologically. For biochemical examinations, ERBs were microdissected out from the sections. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), and database analysis of ERBs were carried out. RESULTS: The histopathological findings were consistent with a foreign body reaction. Numerous ERBs were observed 7 days after injection of 30.0 mg/ml EMD. Histopathologically, ERBs did not contain polysaccharide, amyloid, or hemosiderin. The cells surrounding ERBs were not macrophages or vascular endothelial cells. SDS-PAGE of the microdissected ERBs revealed an intense band at around the 40-kDa region. MALDI-TOF MS showed that the spectrum for ERBs has only a single strong ion intensity. Analysis of the amino acid sequence revealed that the ERBs were composed of various molecular fragments, which all contained an identical seven amino acid sequence. In addition, these peptides are a component of amelogenin. CONCLUSIONS: A high concentration of EMD induces ERBs that consist of a 40-kDa protein which includes a constituent part of amelogenin. The ERBs (or remaining EMD) might promote mesenchymal cell differentiation into hard tissue-forming cells around the EMD injection site.

Amelogenin↗
PubMed2004

Subcellular distribution of di-(2-ethylhexyl)phthalate in rat testis.

Subcellular distribution of di-(2-ethylhexyl)phthalate (DEHP) in the testis was studied by single oral administration of [3,4,5,6-(3)H]-phthalic acid di-(2-ethylhexyl) ester (DEHP-3H) or phthalic acid di-(2-ethyl[1-(3)H]hexyl) ester (3H-DEHP) to 8-week-old male rats. Autoradiographs and electron microscopic autoradiographs were prepared from the testis, liver and kidney at 6 and 24 hr after administration and distribution of radioactive materials in the tissues were observed. In the autoradiographic specimen at 6 hr after administration of DEHP 3H-labeled at phthalic acid moiety (DEHP-3H), many grains were observed in the testis, mainly at the basal area of seminiferous tubules at the stages IX to I of the spermatogenic cycle. Electron microscopic autoradiographs taken at the same time revealed that localization of grains were in the smooth-surfaced endoplasmic reticulum and mitochondria of Sertoli cells. A few grains were also present at the Golgi apparatus and lysosome of Sertoli cells, and at the interfaces between the Sertoli cells or between Sertoli cells and spermatocytes, and in the cytoplasm of spermatocytes. Autoradiographs of the liver revealed grains in the centrilobular hepatocytes, localized at mitochondria, rough-surfaced endoplasmic reticulum and peroxisomes. In the kidney, the radioactivity was localized at the brush border of the tubular cells in the pars recta of proximal tubules. In the 24-hr specimen, the grain density in the seminiferous tubules obviously decreased. On the other hand, by autoradiography with DEHP 3H-labeled at the alcohol (3H-DEHP), only a few grains were observed in autoradiographs of the testes at 6 hr after administration. No grains were noted in autoradiographs of the liver and kidney with 3H-DEHP. The results showed that the phthalic acid ester was splitted rapidly in the body and only the phthalic acid moiety distributed into the cells.

Administration, Oral↗
PubMed1981

Leukoplakia-like lesions developing in patients with oral discoid lupus erythematosus.

In a long-term follow-up of 52 patients with oral lesions of discoid lupus erythematosus (DLE) 8 patients showed a gradual transition of the typical DLE lesions to lesions which closely resemble leukoplakia. Histopathologically and immunopathologically the leukoplakia-like lesions generally showed features similar to those in leukoplakias not preceded by DLE. Ultrastructurally, the leukoplakia-like lesions showed the presence of cytoplasmic tubular structures in vascular endothelium in two out of three patients examined. The leukoplakia-like stage is considered to be an inactive or scar-like stage, which may be the mucous membrane counterpart of the atrophic scars following DLE lesions of the skin. A primary examination of a DLE patient presenting oral lesions at the leukoplakia-like stage may result in an incorrect diagnosis. Therefore, when investigating patients with leukoplakias, they should be questioned on previous or present skin lesions as well as symptoms of systemic lupus erythematosus.

Adult↗
PubMedDate not supplied

Ultrastructural and morphometric investigation of human brain capillaries in normal and peritumoral tissues.

Capillaries of peritumoral and normal brain tissues were ultrastructurally and morphometrically investigated to evaluate the changes in peritumoral capillaries connected with the tumor-associated vasogenic edema. The endothelial cells of peritumoral capillaries showed varying thickness, electron-lucent cytoplasm, and structurally normal tight junctions. The basal lamina was thickened, rarefied, and vacuolated. The pericytes were provided with pinocytotic vesicles and phagocytic bodies. The astrocytic glia appeared empty or swollen, with few glycogen granules and a disarranged cytoskeleton; well-preserved glia was occasionally observed. The brain tissue was slightly edematous. No statistically significant differences were observed between normal and peritumoral capillaries as regards diameter, wall thickness, endothelial thickness, and endothelial vesicle density. Instead, the peritumoral capillaries displayed three times as many endothelial surface-connected vesicles, a markedly thicker basal lamina, and significantly reduced extension of pericytic and glial investments. The kind and severity of the vascular modifications, compared with the slight edematous appearance of the nervous tissue, strengthen the hypothesis that peritumoral capillaries could be involved in the edema resolution process.

Astrocytoma↗