bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological study of the innervation pattern of the rabbit sinoatrial node
Authors: Roberts L. A. , Slocum G. R. , Riley D. A.
Published: February 2005

The pattern of nerves, ganglia, and fine nerve processes in the adult rabbit sinoatrial node, identified by microelectrode recording, was defined by staining histochemically for cholinesterase followed by silver impregnation. A generalized repeatable pattern of innervation was recognized, including (1) a large ganglionic complex inferior to the sinoatrial node; (2) two or three moderately large nerves traversing the sinoatrial node parallel to the crista terminalis; (3) nerves entering the region from the atrial septum, the superior vena cava, and the inferior vena cava; and (4) a fine network of nerve processes, particularly extensive in the morphologically dense small‐cell part of the sinoatrial node. When the site of initial depolarization in the node was located and marked by a broken‐off electrode tip, it was found, after cholinesterase staining, to be characterized by a cluster of cells enclosed in a nest or basket of fine nerves. Similar nested cell clusters were observed elsewhere in the sinoatrial node in this same preparation and in other hearts. A complex interweaving of atrial muscle fibers was observed medial and inferomedial to the sinoatrial node, which may form the anatomical basis for the lack of conduction through this region. The morphological pattern of nerves, ganglia, and myocardial cells described in this study emphasizes the complexity of innervation of the sinoatrial node, including its intrinsic neural elements. Cholinesterase/silver staining can be useful in the definition and comparison of electrophysiologically identified sites within the sinoatrial node.
Electron microscopic study of subcutaneous and intraperitoneal splenules in the mouse
Authors: Johnson Joan , Weiss Leon
Published: February 2005

The development of splenules derived from slices of freshly removed autologous spleen implanted subcutaneously or intraperitoneally was followed by light and electron microscopy from day 2 to day 70. Within 48 hr after transplantation, a rough space filled with blood, unlined by endothelium, formed just under the surface of the splenic fragment. The tissue central to this vascular space was disrupted and necrotic. In the outer portion of the vascular space, fibroblasts appeared and created locules which developed into a highly vascular, hematopoietic red pulp. From the inner portion, blood percolated into the central necrotic tissue.

At 1 week the splenule was divisible into concentric structures. The capsule was outermost. A shell of vascularized, highly hematopoietic red pulp lay within the capsule, having replaced the vascular space. Central to the red pulp lay a band of fibroblasts and macrophages. Next was a layer of fibroblasts in a matrix of degenerating cells, and, at the center, a necrotic core. As fibroblasts and macrophages moved centrad, the red pulp moved with them, expanding and replacing the necrotic tissue.

The splenule differed in character from the original spleen. Splenular red pulp, especially near the surface, was unusually hematopoietic. The circumferential reticulum of white pulp was reduced or absent, and the boundary between red and white pulp was sometimes indistinct. Some white pulp was subcapsular, and the capsule and surrounding connective tissue were infiltrated by lymphocytes. The necrotic core of the splenule was typically surrounded by a zone containing large blood vessels, connective tissue, and adipocytes.
Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer
Authors: Willingham Mark C.
Published: February 2005

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor‐specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron‐microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and reeeptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell‐killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell‐surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.
Immunogold‐surface replica study of ADP‐induced ligand binding and fibrinogen receptor clustering in human platelets
Authors: Isenberg William M. , McEver Rodger P. , Phillips David R. , Shuman Marc A. , Bainton Dorothy F.
Published: February 2005

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma‐membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb‐IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold‐surface replica technique to study the distribution of GPIIb‐IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP‐activated human platelets. We found that the gold probe was monodispersed · over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb‐IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb‐IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding‐competent receptors on ADP‐stimulated platelets were mono‐dispersed. The addition of fibrinogen caused the GPIIb‐IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb‐IIIa binding domains of fibrinogen–namely, the tetrapeptide Arg‐Gly‐Asp‐Ser on the α‐chain or the γ‐chain decapeptide γ402–411. These results show that receptor occupancy causes clustering of GPIIb‐IIIa in activated platelets.
Distribution and movement of membrane‐associated platelet glycoproteins: Use of colloidal gold with correlative video‐enhanced light microscopy, low‐voltage high‐resolution scanning electron microscopy, and high‐voltage transmission electron microscopy
Authors: Albrecht Ralph M. , Goodman Steven L. , Simmons Scott R.
Published: February 2005

Scanning electron microscopy (SEM), especially low‐voltage (1 KeV) high‐resolution SEM, can be used in conjunction with stereo pair high‐voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface‐related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to “prepare” cells for electron microscopy precludes observation in the living state. The use of several types of video‐enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy.

We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold‐labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD‐containing adhesive proteins, with gold‐fibrinogen or gold‐anti‐IIb/IIIa. The initial binding and subsequent movement of gold‐fibrinogen‐IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low‐voltage (1 KeV), high‐resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position‐in‐transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).
Localization of cellular regulatory proteins using postembedding immunogold labeling
Authors: Hand Arthur R. , Jungmann Richard A.
Published: February 2005

Cyclic AMP‐dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP‐binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RI) of differing molecular weight, primary structure, and cyclic AMP‐binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK sub‐units in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP‐binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK sub‐units in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.
Localization of nucleic acid sequences by EM in situ hybridization using colloidal gold labels
Authors: Hamkalo Barbara A. , Narayanswami Sandya , Lundgren Karen
Published: February 2005

It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher‐order chromosome organization and function. A combination of two differentially labelled probes and two different‐sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
Ultrastructural localization of insulin and C‐peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high‐resolution protein A–gold approach
Authors: Bendayan Moïse
Published: February 2005

Insulin and C‐peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light‐microscope level have detected both antigens in the same B cells. With the protein A–gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans‐most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multi‐granular) structures. The fixation procedure used yielded excellent Ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum‐Golgi‐granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans‐cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis‐side, those on the trans‐side, and the trans‐most rigid cisternae. Since insulin and C‐peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C‐peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C‐peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C‐peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.
Localization and quantification of hormones, ligands, and mRNA with affinity‐gold probes
Authors: Childs Gwen V. , Yamauchi Kohen , Unabia Geda
Published: February 2005

The application of immunogold techniques to localize pituitary hormones produces label that can be quantified and correlated with different secretory states. This report focuses on three major applications of the technology. In the first set of studies, immunogold labels for adrenocorticotropin (ACTH) or luteinizing hormone (LHβ) and follicle‐stimulating hormone (FSHβ) were applied to ultrathin sections of pituitaries from adrenalectomized rats or from rats in different stages of the estrous cycle. During the first week after adrenalectomy, ACTH cell area increased. The concentration of immunoperoxidase label (amount of label/area of the cortico‐tropes) decreased. Counts of gold markers showed that there were no changes in the concentration of antigens per granule. Three weeks after adrenalectomy, the amount of immunoperoxidase label increased along with the concentration of that label. The concentration of gold label for ACTH on granules also increased. All changes correlated well with increases in serum ACTH stimulated by adrenalectomy. In the studies of cycling rats, gonadotropes showed increases in the number of gold markers for LHβ or FSHβ per granule area just before an elevation in serum levels. There were also increases in the proportion of granules that contained only LHβ or FSHβ (monohormonal) before the rise in secretion. Thus, nonparallel release of gonadotropins might be attributed to changes in the ratio of gonadotropins packaged per granule. In the second series of studies, avidin‐gold labels were used to identify sites of binding of biotinylated ligands. These studies illustrate and quantify binding by biotinylated gonadotropin‐releasing hormone (GnRH) to ovarian or pituitary target cells. Triple‐labeling protocols (avidin‐peroxidase followed by immunogold) show that the target cells in the pituitary contain gonadotropins. In the third set of studies, avidin gold or avidin peroxidase was used to label sites of hybridization of a biotinylated cRNA probe to gonadotropin β subunit mRNA. The sites of hybridization appear on rough endoplasmic reticulum; however, further work is needed to improve cell ultrastructure and perserve antigens. Triple‐labeling protocols (avidin‐peroxidase followed by immunogold) show the feasibility of the technique as well as the need for further refinement. To summarize, these studies describe multiple applications of gold labels for the localization of antigens, ligands, and mRNA. The labels are sensitive for detection of antigens and ligands and easily quantified. Quantitative analyses show changes in concentration of gold label that correlate well with secretpry states.
Double‐labeling immunogold electron‐microscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries
Authors: Losinski N. E. , Horvath E. , Kovacs K.
Published: February 2005

According to the one cell, one hormone theory, the pituitary gland is composed of 5 cell types which secrete 6 hormones. Recent investigations indicate that this theory must be modified, as there are some bihormonal cells containing 2 hormones, i.e., mammosomatotrophs prolactin‐growth hormone (PRL‐GH). This study was undertaken in order to elucidate whether other adenohypophysial cells are capable of producing 2 hormones and to demonstrate the presence of cells coexpressing PRL‐GH, PRL‐thyrotropin (TSH), or TSH‐GH. Sixteen nontumorous and 16 adenomatous male and female Sprague‐Dawley and Long Evans rat pituitaries were removed immediately after the animals were killed and processed for transmission electron microscopy and the immunogold double‐labeling technique. Coexpression of PRL‐GH, PRL‐TSH, and TSH‐GH was found in both nontumorous and adenomatous pituitaries. Double labeling was present not only in the same cell cytoplasm but also in the same secretory granules. The question of whether these double‐labeled cells represent different cell populations, transitional cell types, or precursor cells requires further investigation.
Use of the protein A–gold immunocytochemical and enzyme‐gold cytochemical techniques in studies of vitellogenesis
Authors: Herbener George H.
Published: February 2005

Vitellogenesis in the frog hepatocyte was investigated by applying the protein A‐gold immunocytochemical and RNase‐gold cytochemical techniques in conjunction with morphometric and biochemical analyses. The morphometric studies demonstrated that the surface density of rough endoplasmic reticulum (RER) and nucleolar size increased more than fourfold and 1.25‐fold, respectively, while the nuclear size and the mitochondrial compartment size remained constant following estrogen treatment. Concurrently, liver RNA concentration increased 2.5‐fold while protein and DNA concentrations did not change. In addition, total plasma protein more than doubled, with vitellogenin accounting for 40% of the final volume. The secretory proteins vitellogenin and protein‐RcX (a nonvitellogenin, estrogen‐induced plasma protein of unknown function, found in the plasma of Rana catesbeiana) were detected immunocytochemically in the RER, Golgi apparatus, and secretory granules in hepatocytes only of estrogen‐treated frogs. Lysosomes also were labeled. These observations established that protein‐RcX was synthesized and secreted by the hepatocyte in parallel with vitellogenin and that both of these export proteins were confined to the secretory pathway and lysosomes. Quantitation of labeling density indicated that the concentration of vitellogenin increased as it progressed along the secretory vector. Albumin was detected immunocytochemically also within these same hepatocyte entities from both untreated and treated animals. In the untreated animals, albumin concentration also increased progressively along the secretory vector. A marked alteration of albumin processing was observed following estrogen treatment While albumin concentration in the RER was unchanged, its concentrations within the Golgi apparatus and secretory granules were lower than those observed in the RER or in counterpart compartments under control conditions. RNase‐gold cytochemistry for total RNA demonstrated a 1.5‐fold increase in labeling density over the nucleolus but no change in RER labeling following estrogen treatment. These labeling data, in combination with the morpho‐metric data, suggest an increase of approximately 80% in the total amount of RNA in the nucleolus and 430% in the RER in response to estrogen. This review thus illustrates the significant contributions which can be made by gold‐probe techniques, alone or in combination with morphometric and biochemical techniques, to investigations of the intracellular processing of secretory proteins.
Cytochemical studies of lipid metabolism: Immunogold probes for lipoprotein lipase and cholesterol
Authors: Blanchette‐Mackie E. Joan , Dwyer Nancy K. , Amende Linn A.
Published: February 2005

In this article, cytochemical methods are presented for the study of lipid metabolism both in normal cells and in mutant cells with genetic disorders characterized by abnormal lipid metabolism. The benefit of using an immunocytochemical approach to the study of lipase in tissues is discussed, and a review is presented of the results on immunolocalization of lipoprotein lipase in cardiac tissue of normal mice. Immunocytochemical techniques are applied to the study of lysosomal proliferation in hepatocytes from liver of mutant mice with a genetic defect responsible for the lack of hepatic lipase and lipoprotein lipase activity in these animals. Localization of lipids in tissues with structural techniques has been an area of great interest to our laboratory for many years. Attention is called to the development of a technique for the visualization of fatty acids as a function of their ionization state and the production of fatty‐acid myelin figures in membranes. Results on the use of filipin to detect unesterified cholesterol in membranes are reviewed. Filipin produces fluorescent filipin‐cholesterol complexes but also perturbs cell membranes. Application of this cytochemical probe, in combination with immunocytochemistry of lysosomes, produced useful information on defects in low‐density lipoprotein‐derived cholesterol translocation in mutant human fibroblasts. Initial results on the application of immunological techniques to the study of cholesterol in lipid model systems indicate a novel approach, which may be applicable to specialized cell systems. Recent advances in cryoultramicrotomy and development of immunoprobes present valuable opportunities for the structural assessment of lipids and lipases in cell organelles and cell membranes.
Comparison of detecting sensitivities of different sizes of gold particles with electron‐microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model
Authors: Gu Jiang , D'Andrea Michael
Published: February 2005

The detecting sensitivities of different‐sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha‐ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 μm2 nongranule area from that in 1 μm2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha‐ and beta‐ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha‐ and the beta‐ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.
Quantitative aspects of immunogold labeling in embedded and in nonembedded sections
Authors: Slot Jan W. , Posthuma George , Chang Lin‐Yi , Crapo James D. , Geuze Hans J.
Published: February 2005

We tried to control immunolabeling conditions so that information about antigen concentration could be achieved by quantifying labeling patterns. Working with immunogold labeling procedures in ultrathin cryosections, we observed that differential penetration of immunoreagents causes considerable differences in labeling efficiency between various cell structures. Therefore, in these nonembedded sections, labeling densities can only be used to measure variations in antigen concentration within one cell structure. After embedding the tissue in 30% polyacrylamide (PAA), differences in penetration were negated. The equalizing effect of PAA on the labeling efficiency enabled us to design a simple immunocytochemical method by which concentrations of a protein can be measured in situ at subcellular levels, provided that no variations in he protein's structural conformation occur that would affect its immunoreactivity.

In spite of a higher sensitivity observed for Ig‐gold, we preferred to use protein A–gold in our system because of the low nonspecific labeling and the more precise antigen detection by the later immunomarker.
Structure, composition, and assembly of basement membrane
Authors: Leblond C. P. , Inoue S.
Published: February 2005

Basement membranes are thin layers of matrix separating parenchymal cells from connective tissue. Their ultrastructure consists of a three‐dimensional network of irregular, fuzzy strands referred to as “cords”; the cord thickness averages 3–4 nm.

Immunostaining reveals that the cords are composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin. Collagen IV has been identified as a filament of variable thickness persisting after the other components have been removed by plas‐min digestion or salt extraction. Heparan sulfate proteoglycan appears as sets of two parallel lines, referred to as “double tracks,” which run at the surface of the cords. Laminin is detected in the cords as diffuse material within which thin wavy lines may be distinguished. The entactin and fibronectin present within the cords have not been identified as visible structures.

The ability of laminin, heparan sulfate proteoglycan, fibronectin, and entactin to bind to collagen IV has been demonstrated by visualization with rotary shadowing and/or biochemical studies. Incubation of three of these substances–collagen IV, laminin (with small entactin contamination), and proteoglycan–at 35°C for 1 hr resulted in a precipitate that was sectioned for electron microscopic examination and processed for gold im‐munolabeling for each of the three incubated substances. Three structures are present in the precipitate: (1) a lacework, exclusively composed of heparan sulfate proteoglycan in the form of two parallel lines, similar to double tracks; (2) semi‐solid, irregular accumulations, composed of the three initial substances distributed on a cord network; and (3) convoluted sheets, which are also composed of the three initial substances distributed on a cord network but which, in addition, have the uniform appearance and thickness of the lamina densa of basement membrane. Hence these sheets are closely similar to the main component of authentic basement membranes.
In vivo ciliogenesis in human fetal tracheal epithelium
Authors: Gaillard Dominique A. , Lallement Aude V. , Petit Annie F. , Puchelle Edith S.
Published: February 2005

Development of ciliated (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18–19 weeks (stage II), centriolo‐genesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22–23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P < .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.
Ultrastructure of the pronephric kidney in upstream migrant sea lamprey, Petromyzon marinus L
Authors: Ellis L. C. , Youson J. H.
Published: February 2005

The pronephric kidneys were examined in upstream migrant sea lampreys,Petromyzon marinus L., by transmission and scanning electron microscopy. Each prolcphios consists of an enlarged renal corpuscle (glomus) and ciliated nephrostomes, but there are no renal tubules. The renal corpuscle contains an extensive mesangium, which consists of a highly fibrous extracellular matrix, numerous mesangial cells, granulocytes, and macrophages. The extracellular matrix contains microfibrils with a morphology similar to amyloid P microfibrils, fibrils with a periodicity similar to fibrin, and abundant collagen. Often these fibrillar components are aggregated in the region of the basement membrane, giving it a thickened appearance. Some podocytes of the visceral epithelium appear swollen, and their cytoplasm contains numerous vacuolar inclusions, and many have only primary major processes with only a few or no foot processes. The morphological features of the pronephric kidney of the lamprey at this time in the life cycle reflect the regression of this organ, but some features also resemble those seen in renal pathologies of higher vertebrates.
Characterization by morphometric model of liver regeneration in the rat
Authors: Vizzotto Laura , Romani Federico , Ferrario Virgilio F. , Degna Carlo Tommasini , Aseni Paolo
Published: February 2005

A new stereologlcal approach to the study of liver regeneration in the rat is described. The method employs a morphometric model consisting of number and size, as well as surface and volume fractions of hepatocytes, their nuclei, cytoplasm, and their relation to the sinusoidal bed. With this technique, it was found that 8 hr after partial hepatectomy, the number of nuclei increased as did the nucleus/cytoplasm ratio, suggesting an early hyperplastic phase. These data, which have not been reported in other studies, underline the importance of evaluating the complex phenomenon of liver regeneration using a stereological approach, which is more representative of all morphometric changes occuring inside and outside the hepatocyte.
Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: II. Effects of DES on development
Authors: Brody Joel R. , Cunha Gerald R.
Published: February 2005

The effects of diethylstilbestrol (DES) treatment on myometrial development from the prenatal to adult period were examined in rats and mice by histologic and immunocytochemical methods using anti‐actin, ‐vimentin, and ‐laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells, and by morphometric procedures to assess quantitatively the effect of DES on the expression of cellular orientation in the emerging inner circular myometrial layer. Neonatal rats and mice were treated with DES from day 0 (day of birth) to day 2 with dosages known to perturb myometrial development. Neonatal treatment with DES increased the degree of circular orientation within the uterine mesenchyme, an effect detectable following as little as 24 hr of DES treatment. This effect on spatial organization of the mesenchyme was followed by an increase in the thickness of the actin‐positive middle layer (prospective circular myometrium) of uterine mesenchyme during days 3–15; from day 15 onward, however, the circular myometrial layer began to fragment into irregular bundles of smooth muscle, and the longitudinal myometrial layer became thinner and more irregularly organized than controls. Vimentin localization in rats treated with DES neonatally was more intense than in controls within the circularly orientated uterine mesenchyme at 5 days. By 60 days the circular and longitudinal myometrial layers of DES‐treated animals showed strands and bundles of vimentin‐positive cells, which were not present in controls. Both rats and mice show comparable effects of DES treatment.
Association of fibronectin with the microfibrils of connective tissue
Authors: Inoue S. , Leblond C. P. , Rico P. , Grant D.
Published: February 2005

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase–antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion.

Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves.

Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective‐tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.
Postnatal growth of pulmonary acini and alveoli in normal and oxygen‐exposed rats studied by serial section reconstructions
Authors: Randell S. H. , Mercer R. R. , Young S. L.
Published: February 2005

Three‐dimensional reconstructions from serial sections were used to examine postnatal lung development of rats reared in air (control) or oxygen. From birth to age 21 days, control lung volume increased ninefold, and the average volume of each ventilatory unit (all airspaces distal to a single respiratory bronchiole) increased seven times. There were approximately 5,000 ventilatory units at birth and on day 21, indicating that the lung grew by enlargement and subdivision of ventilatory units and not by their multiplication. Growth in hyperoxia (>97%) for 7 days had no effect on the number of ventilatory units but, compared to controls, total lung volume and ventilatory unit volume were reduced 32% and 16%, respectively. At birth there were 0.6 × 106 alveoli, and at age 7 days in controls alveolar number increased 16‐fold while the average volume of a single alveolus fell to one‐sixth that at birth. Exposure to hyperoxia for 7 days stopped alveolarization; the surface area to volume ratio (Sa/V) of the ventilatory unit was lower, alveolar number was the same as at birth, and the alveoli present were large. At age 21 days, after 14 days of recovery in air, lung volume and ventilatory unit volume were greater than in controls but the Sa/V of the ventilatory unit was still depressed 20%. Alveoli from oxygen‐exposed lungs were larger than in controls, and a greater size distribution coefficient showed them to be more variable. A shape coefficient for alveoli did not change as a function of the animal's age or oxygen treatment; it demonstrated proportional growth of alveolar height and diameter.
An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA‐synthesizing (S) stage of the cycle
Authors: El‐Alfy Mohamed , Leblond Charles Philippe
Published: February 2005

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H‐thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde‐formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography.

Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: (1) numerous chromatin “specks” ranging in size from about 5 to 70 nm and averaging 47 nm; (2) a few roughly circular or elongated chromatin “packets” measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20–30 or more per nuclear profile; their average diameter is 128 nm. During mid‐prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid‐ and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleo‐plasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps.

Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro‐, meta‐, ana‐, and telophase.

Finally, radioautography 20 min after 3H‐thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid‐prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid‐prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid‐prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: (1) a slow, moderate condensation of the chromatin packets during early and mid‐prophase and (2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.
Effects of catecholamines on fetal rat cardiocytes in vitro
Authors: Marino Thomas A. , Walter Ruth Ann , D'Ambra Kimberly , Mercer W. Edward
Published: February 2005

Norepinephrine stimulates the growth in size of non‐dividing, neonatal cardiac muscle cells, and it can stimulate the growth in numbers of dividing hepatocytes and endothelial cells in culture. The objective of this study was to test the hypothesis that in dividing fetal cardiocytes, norepinephrine would stimulate growth in cell number rather than in cell size. Fourteen‐day fetal heart cells were placed in serum‐free or serum‐supplemented cultures in the presence or absence of norepinephrine (NE), NE plus propranolol, or isoproterenol for 4 days. Almost 90% of the cardiocytes in serum‐supplemented medium were in the cell cycle as determined by proliferating cell nuclear antigen (PCNA) antibody staining during this period. In addition, between days 2 and 4 of culture, 35% and 40% of these cardiocytes were labeled with 3H‐thymidine. After 4 days the cardiocytes increased in cell number in the serum‐supplemented NE cultures as compared to serum‐free cultures. In contrast, there was no significant change in cardiocyte volume between any of the groups examined. It was concluded that in dividing muscle cell populations the effect of norepinephrine was to enhance cell proliferation rather than to stimulate cell growth in size.
Morphology of the prairie dog gallbladder: Normal characteristics and changes during early lithogenesis
Authors: Haley‐Russell Diane , Husband Kathryn J. , Moody Frank G.
Published: February 2005

Studies were undertaken to describe the normal structure of the prairie dog gallbladder and adjacent cystic duct, and then to determine sequential changes that occurred as abnormalities in bile composition developed during high cholesterol feeding. Control animals were fed a diet with trace cholesterol, while experimental animals were fed a diet enriched with 1.2% cholesterol for 1, 2, 3, or 4 weeks. Light microscopy and scanning and transmission electron microscopy were used to characterize morphologic changes at each time interval. Biliary lipid composition was altered in all experimental groups, evidenced by significant decreases in bile‐acid‐to‐cholesterol ratios. Cholesterol crystals appeared in experimental bile at 1 and 2 weeks, while stones formed at 3 and 4 weeks. The cystic duct and neck of the gallbladder occasionally displayed goblet cells. Little mucus was demonstrable in principal cells of the gallbladder, but much more in those lining the cystic duct. After 2 weeks of lithogenic diet, there was an increase in mucus content and secretion from all areas, as well as an influx of polymorphonuclear and mononuclear leukocytes. Accumulation of plasma cells in the lamina propria was an especially prominent feature of experimental tissues. These results suggest that (1) there is regional heterogeneity in the mucus content of the gallbladder and cystic duct of the prairie dog, and (2) both regions respond to lithogenesis with mucus hypersecretion and acute and chronic inflammatory changes prior to the appearance of cholesterol gallstones.
Endocardial surface and atrial morphological changes during development and aging
Authors: Gilloteaux Jacques , Linz David
Published: February 2005

Light, scanning, and transmission electron microscopic observations related to morphological changes of the right atrium as well as the atrial endocardium during development (15th embryonic day and 1 day old) and aging (560 days old) in the Syrian hamster were described and correlated. From the fetus to the adult, the atrial endocardium differentiates in parallel with, or in response to, the subjacent proliferating myocytes in the atrial wall and the trabeculae. Simultaneously, the atrium compartmentalizes grossly into a main chamber and an appendicular region. There is a progressive differentiation from a rudimentary, open chamber with primitive mural ridges in the fetal atria to a distinct, separate, atrial main chamber and appendage with a dense network of trabeculae in the adult. The fetal and neonatal endocardial, endothelial cells are convex with a central nuclear bulging and attenuated cytoplasmic extensions; the adult endocardium shows a squamous endothelium. Two cell surface specializations were observed in all age groups: microvilli and blebs or cytoplasmic protrusions. The general atrial morphology and surface endocardial changes were correlated with growth and the role of the endocardial endothelium as a barrier which controls metabolic exchanges, including the transport of atrial natriuretic factor, between the myocytes and the blood. This endothelial function appears to be essential in the fetal and neonatal age groups since no blood vessels are detected in these groups.
Errors in bone remodeling: Toward a unified theory of metabolic bone disease
Authors: Burr David B. , Martin R. Bruce
Published: February 2005

Recent evidence suggests that skeletal adaptation is organized by a functional unit that includes cells of diverse origin working in coordination. Genetic and metabolic factors control and regulate the processes of modeling and remodeling, only rarely acting on the isolated individual functions of specific cell lines. Errors in the genetic or metabolic regulation of the functional unit affect the entire process of skeletal adaptation rather than specific elements of it. Viewed in this way, some metabolic bone diseases can be understood as relatively simple errors in factors that control the coordinated activities of the entire functional unit. This paper reviews the modeling and remodeling processes and demonstrates how abnormal morphological characteristics of bone tissue can be viewed as products of specific errors in the adaptive process.