Showing papers in "Acta Histochemica Et Cytochemica in 1999"

Expression of Thyroid Transcription Factor-1(TTF-1)in Human C Cells and Medullary Thyroid Carcinomas

Abstract: Thyroid transcription factor-1 (TTF-1) has been known to regulate the transcriptional activity of thyroid-specific genes in thyroid follicular cells. We recently identified TTF-1 mRNA expression in rat thyroid C cells. The current study was undertaken to elucidate how TTF-1 is expressed in human C cells and medullary thyroid carcinomas (MTCs), and how this expression influences the functions and clinical behavior of these cells. By immunohistochemistry, the nuclei of normal and hyperplastic C cells distinctively reacted with antibody against TTF-1, whereas the immunostaining intensity in C cells was rather weak and heterogeneous in comparison with that in follicular cells. Identical TTF-1 immunoreactivity was observed in all 15 MTC specimens examined. The reaction intensity did not depend on tumor patterns or cell features. In nonisotopic in situ hybridization, an antisense riboprobe clearly hybridized the cytoplasms of C cells and MTC cells, which concurrently showed immunohistochemical positivity for calcitonin. Northern blot analysis indicated a marked hybridization with TTF-1 mRNA of approximately 2.3 kb in an MTC specimen. Furthermore, the presence of TTF-1 mRNA was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the human MTC cell line, TT. Our results suggest that human thyroid C cells and MTC cells express TTF-1 in connection with their functional ability. Therefore, TTF-1 expression can be a functional marker not only for follicular cells and follicular cell tumors but also for C cells and medullary C cell carcinomas.

Cytochemical study of Prolactin-Releasing Peptide(PrRP)in the rat brain.

Abstract: Strong positive signals for PrRP mRNA and PrRP-like immunoreactivity (PrRP-LI) were detected in the nucleus of the solitary tract and ventral and lateral reticular formation of the caudal medulla oblongata. Weak mRNA signals and immunoreactivity were seen scattered from the hypothalamic dorsomedial nucleus (DMH) to ventromedial nucleus (VMH). Nerve processes and terminals with PrRP-LI were detected from the septal region to the diencephalon. These nerve processes were also clearly visible around capillary walls and in the vicinity of the ependymal cells of the third and lateral ventricles. These observations suggested that PrRP might be secreted into the systemic circulation and cerebrospinal fluid and may play functional roles other than in the release of prolactin from the anterior pituitary.

Thyroglobulin: A Master Regulator of Follicular Function via Transcriptional Suppression of Thyroid Specific Genes

Abstract: Thyroid gland function is thought to be tightly regulated by thyrotropin (TSH). However, the function of each thyroid follicle is heterogeneous despite having the same blood supply of TSH and despite homogeneous thyrocyte expression of the TSH receptor (TSHR). The nature of this heterogeneity is not fully understood. Recent studies showed that thyroglobulin (TG) protein, stored in the follicular lumen, is a potent negative feedback regulator of follicular function. Thus, physiological concentrations of TG significantly suppress thyroidspecific gene expression in cultured thyrocytes and antagonizes the maximal TSH stimulation of thyroid-specific genes: thyroglobulin, thyroid peroxidase, sodium iodide symporter and TSH receptor. In vivo studies are consistent with these results. This regulation is mediated by TG suppression of thyroid-specific transcription factors: thyroid-transcription factors 1 and 2 as well as Pax-8. We propose a model of follicular activity wherein each follicle has its own cycle of thyroid hormone synthesis and secretion and wherein follicular heterogeneity reflects the asynchronous function of individual follicles. We provide evidence that this mechanism may be related to the phenotype of some thyroid diseases.

Macrophage scavenger receptors : structure, function and tissue distribution

Abstract: Macrophage scavenger receptor (MSR) is one of the major receptors of macrophages. It plays an important role in the pathological deposition of cholesterol in macrophage-derived foam cells during atherogenesis through receptor-mediated uptake of modified low density lipoproteins. MSR is also important for macrophages to recognize and eliminate pathogenic microorganisms. Targeted disruption of the MSR gene resulted in a reduction in the size of atherosclerotic lesions in atherosclerosis model mice. MSR-knockout mice were more susceptible to infection. Hepatic granuloma formation induced by a single intravenous injection of heat-killed Corynebacterium parvum was significantly delayed in MSR-knockout mice. Using MSR-knockout mice as immunization animals, five monoclonal antibodies against human MSR were successfully produced. Immunohistochemistry using these antibodies revealed a restricted distribution of MSR protein on tissue macrophages in various tissues and organs. These antibodies will provide a new approach to study the role of MSR in normal and various pathological conditions in humans.

Proteoglycans in perineuronal nets

Abstract: Proteoglycans are one of the major constituents of the extracellular matrix and cell membranes. In the brain, there are two major proteoglycans, chondroitin sulfate proteoglycans and heparan sulfate proteoglycans. In the adult mammalian central nervous system, some species of chondroitin sulfate proteoglycan are localized in the ‘perineuronal net’ around a restricted number of neurons. The ‘perineuronal net’ is a reticular structure covering the cell bodies and proximal dendrites of certain neurons. Various glycoproteins, proteoglycans and hyaluronic acid are constituents of the perineuronal nets. Recently, an N-terminal proteolytic fragment of neurocan named neurocan-130 was found to be a member of the perineuronal net-constituting molecules. Neurocan is a nervous tissue-unique chondroitin sulfate proteoglycan whose expression and proteolytic cleavage are developmentally regulated. Most of the perineuronal netconstituting molecules are considered to extracellularly exist and some are considered to be localized at the surface of glial cell processes enwrapping the neural cell body. However, neurocan-130 was detected in the cytoplasm of the glial cell processes. Perineuronal nets could be involved in synapse stabilization or neuronal maturation because they appear in the vicinity of the synapses relatively late in neuronal development. The presence of perineuronal nets around a limited number of cells may reflect some functional heterogeneity of the neuron and/or glia.

PCR In Situ Amplification and Catalyzed Signal Amplification: Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization

Abstract: The method of in situ hybridization (ISH) using non-radioactive detection systems has become a very important molecular tool in research and diagnosis. However, nonradioactive ISH techniques generally detect only relatively abundant DNA or RNA molecules. The methods of target amplification, which multiply target molecules, or the methods of signal amplification, which increase the sensitivity of visualization, have been introduced for the detection of rare targets. Polymerase chain reaction (PCR) has been employed in most approaches to target amplification. Although the general principle of PCR in situ amplification is simple, its practical methods have revealed poor amplification efficiency and reproducibility as well as target localization. We have evaluated PCR in situ amplification methods to detect single copies of viral DNA with an experimental model of cervical carcinoma cell lines, and compared them with catalyzed signal amplification methods that we have developed using hapten-conjugated tyramide. Both amplification methods demonstrated the capability of detecting a single copy of DNA. In this overview, the applications and limitations of both target and signal amplification are summarized.

Extinction of Organelles in Differentiating Epidermis

Abstract: In the dorsal skin of fetal rats, the epidermis on day 16 of gestation consisted of a single or a few layers of undifferentiated cells, and it developed to display the full strata, i e the stratum (s) corneum (horny layer), s granulosum (granular layer), s spinosum (spinous layer), and s germinativum (basal layer) by the day of birth Perinuclear electron lucent special areas, which were also detectable by conventional staining under light microscope, appeared in the cells of the s spinosum or the s granulosum on the day of birth We call such an area the “peri-nuclear compartment (PNC) ” The PNC was not partitioned by membranous structures either Typical PNCs became recognizable on the day of birth The cell nuclei seemed to be digested inside of the PNC of the cells located in the outermost area of the s granulosum or sometimes in the boundary between the s granulosum and the s corneum In the initial phase of nuclear extinction, morphology of the nucleus undergoing degeneration resembles that of apoptotic cells In comparison to the morphological degeneration of the nucleus, DNA fragmentation was suggested to occur earlier Localization of cathepsin B and cathepsin D in the s spinosum and s granulosum as revealed by immunohistochemistry, together with an electron microscopic study on the appearance of autophagosomes in those areas, suggested a contribution of autophagy to the digestion of organelles including mitochondria and endoplasmic reticula in the terminal differ-entiation of epidermal cells

Application of Real-Time Confocal Microscopy to Observations of ATP-Induced Ca2+-Oscillatory Fluctuations in Intact Corneal Epithelial Cells

Abstract: Digital imaging of intracellular calcium ion concentration ([Ca2+]i) dynamics has been a fundamental technique in cell biology. The present study examines the possibility of using the confocal microscope for [Ca2+]i imaging of living tissue. We analyzed the ATP-induced [Ca2+]i dynamics of rabbit corneal epithelium as an experimental model. After loading the cells with Indo-1, a real-time confocal microscope (Nikon RCM/Ab) revealed individual images. ATP (100μM) in the perfusate elicited an increase in [Ca2+]i. Superficial cells showed a biphasic change in [Ca2+]i composed of an initial spike phase followed by a persistent plateau phase. On the other hand, oscillatory fluctuations were evident in the mid-wing cell layer, and they were often synchronized, indicating intercellular communication. The spikes in the wing cells did not affect the [Ca2+]i dynamics of the superficial cells. In conclusion, the optical slicing effect of confocal microscopy is suitable for observing thick specimens, such as living tissues composed of various cell types. This is the first report to describe [Ca2+]i dynamics in intact epithelial tissue specimens, and to reveal heterogeneous responses of different cell layers.

W4-3 Dimerization and Nuclear Entry of mPER Proteins in Mammalian Cells

Abstract: Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner.

Three Dimensional Protein Localization Using High Voltage Electron Microscopy

Abstract: Three-dimensional (3D) protein localization at the electron microscopic level is of great benefit when determining the precise distribution of a given protein relative to complex subcellular structures. We have developed several 3D ultrastructural labeling approaches for use with higher voltage electron microscopy. High voltage electron microscopy allows for the use of much thicker sections than conventional transmission electron microscopy. Many of our staining protocols are based on the photooxidation of diaminobenzidine (DAB) by fluorescent markers. As a fluorescent dye is excited in the presence of DAB, the reactive oxygen generated gradually oxidizes DAB into an insoluble polymer which can be rendered electron dense. We previously demonstrated that photooxidation by the fluorophore eosin can be used for high resolution immunolocalization. Because of its small size, eosin-conjugated reagents penetrate well into tissue slices, providing labeling through a reasonable depth of tissue. When combined with high voltage electron microscopy using thick sections, the 3D distribution of a variety of proteins can be visualized. Analysis and interpretation of these 3D labeling patterns is further facilitated by the use of electron tomography, which allows for the derivation of 3D structure from a series of images taken at different orientations. In this review, several examples of 3D protein localization using these technologies are presented.

Growth hormone-releasing hormone(GHRH)expression in pituitary somatotroph adenomas studied by immunohistochemistry and in situ hybridization using catalyzed signal amplification system

Abstract: Growth hormone-releasing hormone (GHRH) is a well-known hypothalamic hormone that stimulates the synthesis and release of growth hormone (GH) as well as the proliferation of GH-producing cells in the anterior pituitary gland. Recent reports have shown GHRH synthesis in pituitary somatotroph adenomas, but GHRH immunoreactivity has not been shown in previous studies. To confirm the role of locally generated GHRH for the progression of somatotroph adenomas, we investigated the expression of GHRH in 25 pituitary somatotroph adenomas immunohistochemically, through the use of both conventional avidin-biotin-complex (ABC) method and novel catalyzed signal amplified (CSA) system. In addition, we investigated the expression of GHRH mRNA and GHRH receptor mRNA with in situ hybridization (ISH) using the CSA system. The weak immunopositivity of GHRH was observed in only 2 adenomas (8.0%) of 25 somatotroph adenomas using the ABC method. In contrast, 15 adenomas (60.0%) of 25 somatotroph adenomas were immunopositive for GHRH, as shown by CSA system. Very few of nonsomatotroph adenomas were immunopositive for GHRH using the CSA system. The expression of GHRH mRNA was confirmed, using the CSA-ISH system in 13 adenomas (72.2%) of 18 somatotroph adenomas. In 11 adenomas (61.1%) of 18 somatotrophic adenomas, the expression of GHRH receptor mRNA was demonstrated using the CSA-ISH system. This is a first report that clarified histopathologically GHRH production in pituitary somatotrophic adenomas. The demonstration of GHRH and its receptor expression is meaningful in clarifying the autocrine or paracrine regulation of GHRH in GH production and progression of pituitary somatotroph adenomas.

Critical Electrolyte Concentration of Chicken Erythrocyte Chromatin

Abstract: This study examined whether chicken erythrocyte chromatin, whose great physicochemical stability is promoted by histone H5, has a critical electrolyte concentration (CEC) value comparable to that of other chromatin types with special condensed packing states but devoid of H5. The affinity for toluidine blue molecules under Mg2+ competitive binding action was thus investigated. The CEC value was found to be 0.12M, which is close to that of heterochromatin from mouse liver cells and to sperm chromatin with histone H1 variants. Acetic ethanol fixative was found to affect the CEC, possibly by influencing the chromatin packing state and DNA phosphate availability through dissociation of proteins from the DNA. The phenomenon of programmed cell death was considered to contribute to high CEC values by inducing special states of chromatin condensation.

Sugar Transporters in Polarized Epithelial Cells

Abstract: Sugar transporters are integral membrane proteins responsible for the transport of sugars across the cellular membranes. They play important roles in the transfer of sugars across different compartments separated by epithelia in the body. Preferential localization of them in specific plasma membrane domains of epithelial cells is crucial in the vectorial transfer of sugars. Modification of the transporter genes with the molecular biology techniques and visualization of the expressed gene products by immunohistochemical methods have shed light on the molecular mechanism underlying the specific localization of the transporter molecules.

The automatic thin sectioning and staining system for light microscopy : the tests by two trial products

Abstract: The preparation of tissue sections for light microscopy is absolutely necessary in pathology. However, this process is still mainly performed manually. The preparation of many samples from a large number of specimens requires meticulous skills. In addition, it is very difficult to maintain a constant accuracy of the producedure.To solve these problems we propose two new test machineries for automatic highspeed sample preparations. The first apparatus is conceptually based on attaching pressure-sensitive adhesive tape on the surface of a paraffin block prior to sectioning. This whole process could be repeated automatically. The sectioning is performed by a newly developed high-precision automatic microtome. Using the proposed machine, cut sections could be continuously mounted on the tape and stained automatically.We also developed the second machine to mount sections on the object glass automatically by using static electricity. The principle of this method is based on electrostatically charging the surface of a paraffin block by which insulation tape was attracted and attached to the surface of the block. After sectioning the block, the section remains adhered on the insulation tape by electrostatic forces. Subsequently, the section is transferred to the object glass by using static electricity as well. In the use of such methods, tissue section on conventional object glass could be made fully automatic.

In Situ Localization of Gene-Specific Transcription Regulatory Factors by Southwestern Histochemistry

Abstract: To understand better the regulation of gene expression, an analysis of the expression of trans-acting transcription regulatory factors in individual cells is essential. Although cellular analysis of specific mRNA expression is successfully performed by in situ hybridization, the results are not enough to tell whether an elevated level of certain mRNA is due to an increased rate of transcription of the gene or due to a decreased rate of degradation of the mRNA. Recently, we have developed a new method, Southwestern histochemistry, which allows us to localize transcription regulatory factors in situ. In principle, double-stranded (ds) DNA harboring specific response element base sequences is synthesized and labeled with a hapten such as thymine-thymine (T-T) dimer and digoxigenin (Dig). After the reaction of frozen tissue sections with the probe ds DNA, the signal is detected enzyme-immunohistochemically by horseradish peroxidase (HRP)-labeled anti-T-T dimer or anti-Dig, respectively. As a model system, we localized estrogen receptor (ER) in mouse uterine tissue sections by immunohistochemistry with 1D5 monoclonal antibody and Southwestern histochemistry using T-T dimerized estrogen responsive element (ERE) ds DNA of vitellogenin. Consequently, very similar localization of ER was obtained by both methods. This result, together with other examples, further indicates the usefulness of Southwestern histochemistry as a method to localize specific transcription regulatory factors in situ.

Changes in Uterine Microvessels as a Possible Pathogenic Factor in the Development of Adenomyosis Induced by Pituitary Grafting in Mice

Abstract: Changes in uterine blood vessels were evaluated quantitatively by an immunohistochemical study using an antibody to von Willebrand factor (vWF) during the development of experimentally induced adenomyosis in mice. In all mice treated with pituitary grafting, which is a useful method of inducing adenomyosis, slight adenomyosis was found near the mesometrium 4 weeks after the operation. In the uteri with adenomyosis, the mean surface area and minor axis of blood vessels increased significantly in the endometrium and showed a tendency to increase in the myometrium, while the mean number of blood vessels in both endometrium and myometrium decreased significantly compared with those in the normal uteri. The mean percentage of total surface area of blood vessels to mean total tissue area of either endometrium or myometrium was not different in the two groups. Remarkably dilated venules in the endometrium were frequently found in pituitary-grafted mice. In the myometrium, the mean percentage of surface area and mean number of blood vessels were greater in the mesometrial side than in the antimesometrial side in both the control and pituitary-grafted mice. Thus, pituitary grafting significantly increased the number of dilated venules and decreased the number of microvessels in the uterus, suggesting that vascular changes may be one of the important etiological factors for the development of adenomyosis.

Morphological Aspects of Superoxide Dismutase-1 Mutation in Amyotrophic Lateral Sclerosis and its Transgenic Mouse Model

Abstract: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Recent investigations have obtained evidence that mutations in the gene for superoxide dismutase-1 (SOD1) are detected in a subset of familial ALS patients, and that mutant SOD1-expressing transgenic mice develop ALS-like clinicopathological features. Several in vitro studies suggest that SOD1-mutated ALS is caused by a newly acquired neurotoxicity of mutant SOD1, but not by reduced enzyme activity. From the viewpoint of morphology, we analyzed the immunolocalization of SOD1 and some other substances in spinal cords from familial ALS patients with SOD1 Ala4→Val mutation. The spinal cords of the ALS patients demonstrated the characteristic neuronal hyaline inclusions (NHIs) immunoreactive with antibodies to ubiquitin and phosphorylated neurofilament protein (NFP) in the lower motor neurons and cordlike swollen axons. The NHIs contained the epitopes of SOD1 and Ne-carboxymethyllysine (CML), one of the major advanced glycation endproducts (AGE), whereas there was no focal accumulation of SOD1 and CML in control individuals. Immunoelectron microscopy depicted the SOD1 and CML determinants on the granule-associated thick linear structures that mainly compose the NHIs. We also performed a similar study on mice carrying a transgene for Gly93→Ala mutant SOD1. The spinal cords of the transgenic mice were characterized by the appearance of NHIs resembling those of familial ALS and by vacuolar degeneration. The mouse NHIs were immunoreactive for ubiquitin, phophorylated NFP, SOD1 and CML. Our findings of the coexistence of SOD1 and AGE in both the human and mouse NHIs indicate that certain substances are implicated in glycoxidation in the presence of oxidative stress originating from mutant SOD1 and finally deposited in the NHIs, suggesting a pathogenic role of the oxidative processes in motor neuron degeneration in vivo.

Analysis of apoptotic cells in surgical pathology materials

Abstract: Analysis of tissue turnover, the balance between cell proliferation and cell death, can provide important information about our understanding of the biological characteristics of various tissues and their functions. Especially it is very important to study apoptosis, one of the modes of cell death, in surgical pathology specimens. It is very important to note that the whole concept of cell death including apoptosis and oncosis is based on the morphological findings. Therefore, an analysis of morphological features of the cells undergoing the processes of various modes of cell death is very important. DNA fragmentation can be detected in situ by labeling 3′-OH ends with biotinylated deoxyuridine triphosphate (dUTP) through the action of terminal deoxynucleotidyl transferase (TdT).Since then, this method, subsequently termed 3′-OH nick end labeling, or TdT-mediated dUTP-biotin nick end labeling (TUNEL), has been widely used to detect cells with DNA fragmentation, especially in surgical pathology materials of human disorders. This TUNEL method is now being incorporated into many pathology laboratories throughout the world. However, increasing evidence suggest that TUNEL method is by no means specific for the detection of apoptosis and this method may also detect cells which are committed to, but not yet in the process of apoptosis. Therefore, it is very important to correlate the findings of TUNEL with morphological features of surgical pathology specimens. In addition, it is also important to note that over-fixation is associated with false negative results and insufficient fixation is likely to be associated with false positive results. Therefore, the processing of the specimens including fixation and others is critical in performing TUNEL and its interpretation.

The use of alkaline EDTA solution improves heat-induced epitope retrieval for immunohistochemical localization of MIB-1 antigen

Abstract: This study was carried out to show whether the removal of tissue-bound calcium ions during a heat-induced epitope retrieval (HIER) procedure improved immunohistochemical staining of the tissues. In addition, the proteolytic susceptibilities of the fixed tissues before and after various epitope retrieval procedures were also evaluated. Immuno-histochemical staining for MIB-1 antigen from paraffin-embedded cases of tonsillitis, gastric adenocarcinomas, and breast carcinomas was performed following heat-treatments for 1, 2, 3, and 5 min in the presence of various buffers such as 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCI buffer (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0), each of which has different calcium chelating capabilities. In addition, the immuno-histochemical staining was performed following 0.05% and 0.1% trypsinizations of the tissues before and after the epitope retrieval. The trypsin-treated tissues were also analyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The immunohistochemical staining of MIB-1 was most prominent when 1 mM EDTA solution (pH 8.0) was employed during HIER. Although trypsin digestion of the tissues before and after HIER generally increased the staining intensity, the cellular morphology of the background was poorly preserved. In addition, no significant differences were observed with gel electrophoresis of the trypsin digested tissues prepared in the various epitope retrieval solutions. Taken together, it can be suggested that the EDTA solution is more effective than other buffers in exposing epitopes presumably by removing tissue-bound calcium ions. Also, an additional trypsin treatment is not necessary even before and after the HIER as long as the EDTA solution is used.

Comparative Expression of E-cadherin, α and β Catenin in Salivary Gland Tumors

Abstract: To study the expression and role of adhesion molecules in normal salivary glands and their tumors, we have studied simultaneously the expression of E-cadherin, and associated cytoplasmic proteins, α-catenin and β-catenin. Paraffin sections were evaluated using the streptavidin biotin peroxidase complex (SBPC) method for E-cadherin, and the SBPC method with tyramide signal amplification (TCA) for α- and β-catenin.Normal submandibular (n=10), parotid (n=4) and oral minor glands (n=4) showed cell membrane staining for E-cadherin and a positive reaction in intercalated and striated ducts for α - and β -catenin. Pleomorphic adenoma (n = 17) showed cell membrane staining for E-cadherin and α-catenin in luminal and non-luminal cells, but were negative for β-catenin. E-cadherin was also focally or irregularly positive in modified myoepithelial cells. Warthin's tumors (n = 4) expressed Ecadherin only and were negative for a- and β-catenins. Papillary adenocarcinoma (n = 4) showed predominant staining for E-cadherin, with no expression in the other tumor cells. Adenoid cystic carcinoma (n = 4) showed only cell membrane positivity for E-cadherin, with an absence of α- and β-catenins in the luminal cells. Non-luminal tumor cells of adenoid cystic carcinoma were negative for E-cadherin and positive for α- and β-catenins. The results were concluded as follows, 1) there was uniform cell membrane staining for E-cadherin in normal and benign tumor epithelium, but α- and β-catenins showed different expression and heterogeneous localization, 2) loss of E-cadherin reactivity was seen in malignant lesions, 3) there was no correlation between the distribution of Ecadherin, α-catenin and β-catenin in benign or malignant tumors of salivary gland origins. The results show variability and heterogeneous expression for E-cadherin, α- and β-catenins in salivary gland tumors.

Confocal Light Microscopy of Brain Cells and Tissue: Image Analysis & Quantitation

Abstract: The brain is an inherently three-dimensional (3-D) structure with tissue regions that occupy large complex volumes and cells that have complicated morphologies, e. g. branching fields of dendrites and axons extending in all three dimensions. The linear dimensions range from micrometers to tens of millimeters. The Confocal light microscope can image relatively large volumes of tissue, and is therefore an ideal imaging tool for studying the brain and its cells. We have used histochemical and immunohistochemical labels to delineate structure at the tissue, cellular, and subcellular levels. The 3-D images are analyzed by custom software that automatically segments and counts nuclei over large tissue regions, automatically montages any number of 3-D images, and traces branching structures such as neuronal processes or blood vessels. Examples of nuclear detection in the rat hippocampus, where the cell density is high and nuclear images are close together, and of tracings of the dendritic field of a dye-filled neuron from the visual cortex of the kitten are shown. Complete penetration of thick tissue sections by large immunolabeling molecular complexes is demonstrated in tissue double-labeled for glial fibrillary protein and laminin. The ability to correlate multiple labels in 3-D space is another strong point for Confocal light microscopy. The influence of deconvolution on the quality of 3-D images is also demonstrated.

Immunohistochemical Studies on Paraquat-Induced Damage to Neuronal and Glial Cells in Rat Hippocampus

Abstract: Paraquat has previously been shown to reduce the viability of rat C6 glioma cells, suggesting that this drug may be toxic to normal glial cells. However, there is no direct evidence for the cytotoxic effect of paraquat on glial cell in vivo. To investigate the toxic effect of paraquat, the damage to hippocampal neurons and astrocytes in paraquat-poisoned rat brain was immunohistochemically examined using microtubule associate protein-2 (MAP-2) and glial fibrillary acidic protein (GFAP) as a histological marker. Paraquat poisoning reduced the number of astrocytes in rat hippocampus in a concentration-dependent manner. In addition, paraquat caused the damage to hippocampal neurons at relatively lower doses (25 and 50mg/kg), and this neuronal damage was unexpectedly less pronounced at a higher dose (100mg/kg). These findings indicate that paraquat is toxic to astrocytes in vivo, and furthermore suggests that the toxic effect of paraquat on glial cells may be different from that on neuronal cells in the rat brain.

Mitochondrial NADH-quinone Oxidoreductase (NQOm) Produces Reactive Oxygen Species in the Chemical Injury of Rat Liver Mitochondria

Abstract: The generation of reactive oxygen species in the mitochondria was investigated using electron microscopic cytochemistry and energy-filtering transmission electron microscopy in reference to the toxic mediator of paraquat poisoning. When isolated intact mitochondria from rat livers were reacted with paraquat in the presence of NADH, mitochondrial NADH-quinone oxidoreductase generated superoxide anions to cause the destruction of mitochondria which resulted in cell death. Cerium chloride captured hydrogen peroxide and formed cerium perhydroxide precipitates on the outer surface of the mitochondrial outer membrane. This localization was also clearly identified with electron spectroscopic imaging based on electron energy loss spectral analysis.

Amyloid β Peptides and Presenilins in the Pathogenesis of Alzheimer's Disease

Abstract: Amyloid β peptides, especially those ending at the 42nd residue (A β 42), is the initially and predominantly deposited A β species in the brains of patients with Alzheimer's disease and Down's syndrome. The pathogenetic significance of overproduction and/or deposition of A β 42 is highlighted because mutations in multiple different genes responsible for early-onset familial Alzheimer's disease, i.e. those in βAPP, presenilin 1 and 2 genes, led to an increase in the secretion and deposition of Aβ42.