George N. Thomopoulos

Age-related thickening of basement membrane in stria vascularis capillaries

Ultrastructural examination was undertaken to investigate the pathogenesis of age-related atrophy of the stria vascularis (StV). Basement membrane (BM) thickness was increased in 65-85% of strial capillaries in gerbils aged 33 months or older and often exceeded by several-fold that observed in young controls. In an early stage of thickening the BM expanded slightly around the full capillary profile, after which nodular expansions of BM encircling slender cell processes were often observed at or near one or both poles of the elliptical vessel profile. As widening progressed, the BM consisted of 2-3 layers separated by cell processes in the nodules but fewer strata elsewhere. Association of slender processes of both endothelial cells and pericytes with focal thickening outside the process suggested their participation in genesis of the capillary lesion. In later stages of atrophy, pericytes degenerated and disappeared, while endothelial cells remained intact. Eventually, thick multilayered BM devoid of endothelial cells surrounded a narrow lumen occluded by debris. The age-related change in BM in the inner ear was confined to StV capillaries. Degenerative changes in StV epithelial cells occurred apparently as a secondary consequence of the capillary lesion. The pathologic alterations in marginal cells included extrusion of blebs from the luminal surface, separation and loss of basolateral interfoldings, alteration and depletion of mitochondria and nuclear pyknosis. At the end-stage of degeneration, the StV consisted of a simple or multiple layer of squamous cells lining the scala media.

Structural and Functional Impairment of Mitochondria in Adriamycin-Induced Cardiomyopathy in Mice: Suppression of Cytochrome c Oxidase II Gene Expression

The use of adriamycin (ADR) in cancer chemotherapy has been limited due to its cumulative cardiovascular toxicity. Earlier observations that ADR interacts with mitochondrial cytochrome c oxidase (COX) and suppresses its enzyme activity led us to investigate ADRs action on the cardiovascular functions and heart mitochondrial morphology in Balb-c mice i.p. treated with ADR for several weeks. At various times during treatment, the animals were assessed for cardiovascular functions by electrocardiography and for heart tissue damage by electron microscopy. In parallel, total RNA was extracted from samples of dissected heart and analyzed by Northern blot hybridization to determine the steady-state level of three RNA transcripts encoded by the COXII, COXIII, and COXIV genes. Similarly, samples obtained from the liver of the same animals were analyzed for comparative studies. Our results indicated that 1) treatment of mice with ADR caused cardiovascular arrhythmias characterized by bradycardia, extension of ventricular depolarization time (tQRS), and failure of QRS at high concentrations (10-14 mg/kg body weight cumulative dose); 2) the heart mitochondria underwent swelling, fusion, dissolution, and/or disruption of mitochondrial cristae after several weeks of treatment. Such abnormalities were not observed in the mitochondria of liver tissue; and 3) among the three genes of COX enzyme examined, only COXII gene expression was suppressed by ADR treatment, mainly after 8 weeks in both heart and liver. Knowing that heart mitochondria represent almost 40% of heart muscle by weight, we conclude that the deteriorating effects of ADR on cardiovascular function involve mitochondrial structural and functional impairment.

Novel membranous structures in apical and basal compartments of inner hair cells

Postfixation with a ferrocyanide‐osmium tetroxide solution preserved a dense network of canaliculi extending from the apical to the upper lateral plasma membrane in cochlear inner hair cells (IHCs). Numerous Golgi bodies intermingled with this apical canalicular reticulum (CR). Osmium‐ferrocyanide treatment also disclosed several previously unreported structures below the IHC nucleus. The first consisted of stacks of six or eight and sets of three parallel cisternae of rough endoplasmic reticulum spanning between clustered mitochondria. Some parallel cisternae ended with segmentation where they contacted mitochondria, and others terminated by transforming into blebs or continuing into canaliculi. A second feature was comprised of a complex of segmented cisternae and branching canaliculi with clustered mitochondria. Branching minicanaliculi with associated vesicles neighbored the complexes. A fourth entity consisted of synaptic‐like vesicles that largely filled the subnuclear cytosol and congregated at synapses. An additional infranuclear structure was composed of slender canaliculi that collected near or streamed to plasmalemma, often next to a synapse. A paradoxical absence of rough endoplasmic reticulum above and Golgi zones below the nucleus provided evidence of atypical mechanisms for generating the membrane in CR and forming synaptic vesicles. The observations offer the view that IHCs are compartmentalized into an apical mechanoreceptor half and a basal half that affects neurotransmission. The apical CR provided a possible structural basis for sequestering the K+ known to influx apically and for directing its diffusion to the site of known efflux across the lateral plasmalemma. The codistribution of parallel cisternae, canalicular‐mitochondrial complexes, and synaptic‐like vesicles, all of which are unique to IHCs, implicated the cisternae and complexes in the genesis of the vesicles. J. Comp. Neurol. 409:424–437, 1999.

Cytologic evidence for mechanisms of K+ transport and genesis of Hensen bodies and subsurface cisternae in outer hair cells

A system commonly termed the tubulocisternal endoplasmic reticulum (TCER), but designated here the canalicular reticulum (CR), occurs selectively in ion‐transporting epithelia, in which it is interpreted as facilitating the transcellular diffusion of ions. Mechanoelectrical transduction in the cochlear outer hair cells (OHCs) depends on the apical influx and the subsequent basolateral efflux of K+. Cytologic structures that possibly mediate K+ transport in gerbil OHCs were investigated here.

Increased laminin deposition in capillaries of the stria vascularis of quiet-aged gerbils.

The distribution of laminin (LA) and type IV collagen (IV-C) in the gerbil inner ear was investigated by light and electron microscopic immunohistochemistry. Changes in protein expression were assessed from birth to old age to determine the relation of these constituents to maturation of the cochlea and development of presbyacusis. The distribution of LA paralleled that of IV-C during postnatal development, and both were visualized in the basement membrane (BM) of endothelial, epithelial and spiral ganglion cells in neonatal and young adult gerbils. Immunopositive BM underlying the stria vascularis disappeared at 8-12 days after birth coincident with the development and maturation of the strial capillaries. Immunoreactivity for LA afforded an index to the thickness of the BM and was found to increase with age only in the BM of strial capillaries. At 6 months of age, occasional strial capillaries in the apex of the cochlea showed thickening of the LA-positive BM. Abnormal deposition of LA in strial capillary BM spread to lower turns and increased in prevalence with advancing age, affecting apical and basal more than middle cochlear turns. Thickening of the capillary BM appeared to precede capillary obstruction which eventuated in complete strial atrophy. Staining for IV-C in the walls of the strial capillaries did not increase with age. The data show that LA and IV-C play important roles in postnatal development of the cochlea and that LA deposition increases with age only in the BM of strial capillaries.

Structural evidence for ion transport and tectorial membrane maintenance in the gerbil limbus

Cells medial to the tunnel of Corti were examined to assess fine structural features relevant to their proposed role in cochlear K(+) homeostasis. A dense network of canaliculi referred to as canalicular reticulum (CR) resided in the foot body of inner pillar cells, where it bordered and could resorb ions released from inner radial and spiral nerves. Lateral interdental cells (IDCs) formed columns which connected the inner sulcus epithelium with the base of the tectorial membranes (TM) middle zone. A spout-like neck in cells at the top of lateral IDC columns housed a dense concentration of CR which resembled that characteristic of ion transporting epithelia and appeared to be located here for transporting ions and fluid toward the TM. Clustered IDCs in the center of the limbus connected underlying limbal stroma with the TMs limbal zone and appeared capable of transporting ions from stroma to TM. Abundant CR in limbal stellate fibrocytes evidenced their capacity to transport ions and fluid, presumably from inner sulcus epithelium toward central IDCs. The most medial IDCs possibly function as the terminus of an ion cycling path from scala vestibuli to endolymph. Light fibrocytes situated between supralimbal fibrocytes and medial IDCs appeared to serve as a link in this pathway. The limbal zone of the TM overlying central IDCs consisted of three distinct regions which offered a structural basis for transformation of an amorphous matrix supplied by central IDCs into the protofibrils of the membranes middle zone.

The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. I. Morphology and periodic acid-thiocarbohydrazide-silver proteinate staining of vicinal diols.

SummaryThe influence of fixation and embedding medium on the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining reactivity in the mouse intestine was studied. It was found that the combination of osmium tetroxide and epoxy resins was the least sensitive for the demonstration of complex carbohydrate with the PA-TCH-SP method. Post-osmication reduced, but did not abolish, PA-TCH-SP reactivity (except for the Golgi complex) when non-epoxy resins were used. The staining pattern of a particular organelle differed depending on the embedding medium used. Golgi cisternae exhibited the most intense PA-TCH-SP reactivity in non-osmicated tissues embedded in non-epoxy resins. Post-osmication of tissues was required to reveal the fine structure of the glycocalyx as well as to preserve the fine structure of tissues embedded in styrene-methacrylate and styrene-Rigolac 2004. The choice of fixation procedures and embedding media in a given study should be governed primarily by the sites of interest.

Gene functional annotation by statistical analysis of biomedical articles

BACKGROUND Functional annotation of genes is an important task in biology since it facilitates the characterization of genes relationships and the understanding of biochemical pathways. The various gene functions can be described by standardized and structured vocabularies, called bio-ontologies. The assignment of bio-ontology terms to genes is carried out by means of applying certain methods to datasets extracted from biomedical articles. These methods originate from data mining and machine learning and include maximum entropy or support vector machines (SVM). PURPOSE The aim of this paper is to propose an alternative to the existing methods for functionally annotating genes. The methodology involves building of classification models, validation and graphical representations of the results and reduction of the dimensions of the dataset. METHODS Classification models are constructed by Linear discriminant analysis (LDA). The validation of the models is based on statistical analysis and interpretation of the results involving techniques like hold-out samples, test datasets and metrics like confusion matrix, accuracy, recall, precision and F-measure. Graphical representations, such as boxplots, Andrews curves and scatterplots of the variables resulting from the classification models are also used for validating and interpreting the results. RESULTS The proposed methodology was applied to a dataset extracted from biomedical articles for 12 Gene Ontology terms. The validation of the LDA models and the comparison with the SVM show that LDA (mean F-measure 75.4%) outperforms the SVM (mean F-measure 68.7%) for the specific data. CONCLUSION The application of certain statistical methods can be beneficial for functional gene annotation from biomedical articles. Apart from the good performance the results can be interpreted and give insight of the bio-text data structure.

Immunoglobulin deposition in thickened basement membranes of aging strial capillaries

The presence of immunoglobulins in the thickened basement membrane (BM) of aging strial capillaries was investigated as a possible indicator of autoimmunity in the genesis of atypical BM. Cochleas from young and old Mongolian gerbils raised in quiet were examined by immunostaining at the light microscopic level for IgG and IgM and for the BM components laminin (La) and type IV collagen (IV-C). Another age-graded series of cochleas was stained for IgG at the ultrastructural level. No immunoreactive IgG was detected in specimens from animals less than 6 months old. In contrast, 2 of 12 cochleas from 20- to 28-month-old gerbils and 11 of 20 cochleas from gerbils 30 months or older showed positive staining for IgG in strial capillary BM. IgM was not detected at any age. At the electron microscope level, no immunoreactive IgG was detected in the stria of cochleas younger than 30 months. However, labeling demonstrative of IgG was observed in the thickened BM of some strial capillaries in all six cochleas from gerbils older than 33 months. Lysosome-like granules in endothelial cells and the superiormost marginal cells also stained for content of IgG as did fibrillar material in edematous regions in the intrastrial space. In addition to showing accumulation of IgG, the findings confirm our prior demonstration of increased La deposition in the thickened strial capillary BM of all cochleas from old gerbils. The BM alterations appear confined to strial capillaries in old gerbils, since morphological observations and immunostaining for La and IgG failed to detect changes in BMs at any other site in a wide survey of aged gerbil organs including vessels in other regions of the affected cochleas. The results point more towards the development of an age-dependent permeability to IgG selectively in strial capillaries than to autoimmunity as an explanation of the IgG in BM.

Localization of organ of Corti protein II in the adult and developing gerbil cochlea

The distribution of organ of Corti protein II (OCP-II) was assessed in the developing and mature gerbil cochlea by light and electron microscopic immunohistochemistry. In the adult cochlea, OCP-II was expressed only in certain epithelial cells which included all supporting cells of the organ of Corti, inner and outer sulcus cells and interdental cells. Inner and outer hair cells lacked immunoreactivity. The highest gold particle labeling density was seen overlying intracellular regions devoid of organelles. In the developing inner ear, OCP-II was first detected at 2 days after birth (DAB) with the strongest staining in immature Deiters, inner phalangeal and pillar cells. Immunostaining intensity increased gradually in cells lying laterally and medially to the more centrally located supporting cells and reached adult levels in all reactive cell types around 18 DAB. The results demonstrated conclusively that OCP-II is a cytosolic protein and fail to support its role as a transcription factor postulated on the basis of its homology with p15 or a role in the control of the cycle as suggested by its near-identity with p19Skp1, a cyclin A/CDK2-associated protein. The continued high level of expression in the mature cochlea argues against OCP-IIs involvement in regulating the development and differentiation of epithelial cells. The proteins unique distribution and its gradual increase in expression prior to and during the onset and maturation of hearing, however, support its potential function in the recycling of K+ effluxed from hair cells and neurons back to endolymph.