bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization
Authors: Iwamoto Masahiro , Yagami Kimitoshi , Shapiro Irving M. , Leboy Phoebe S. , Adams Sherrill L. , Pacifici Maurizio
Published: February 2005

During the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin. A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of ostenection (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17–18 sternum (US cells) and grown in monolayer in standard serum‐containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen‐positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3‐week‐old cultures with RA (10–100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA‐treated cultures. Electron microscopy confirmed the formation of large matrix‐associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells). In these cells, RA failed to induce high levels of APase activity, ON and OP gene expression, and mineralization; instead, it greatly promoted cell proliferation. Thus RA appears to have major, stage‐specific effects on the maturation program of chondrocytes. The retinoid rapidly induces expression of late maturation genes and activates mineralization of the cartilage matrix. © 1994 Wiley‐Liss, Inc.
Adenosine 5′‐triphosphate promotes mineralization in differentiating chick limb‐bud mesenchymal cell cultures
Authors: Boskey Adele L. , Doty Stephen B. , Binderman Itzhak
Published: February 2005

When chick limb‐bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3–4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high‐energy phosphates adenosine 5′‐triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5–5 mM β‐glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells.

In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H‐leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is present. It is suggested that ATP promotes mineral deposition by providing a high‐energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes. © 1994 Wiley‐Liss, Inc.
Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes
Authors: Hunziker Ernst B.
Published: February 2005

Growth plate chondrocytes play a pivotal role in promoting longitudinal bone growth. The current review represents a brief survey of the phenomena involved in this process at the cellular level; it delineates the contributions made by various activities during the course of the chondrocyte life cycle, notably proliferation and hypertrophy, and illustrates how the relative contributions may be modulated according to the particular needs of an organism at critical phases of growth. The cellular mechanisms by which a few well characterized growth‐promoting substances exert their influences are discussed in the light of recent findings pertaining to epiphyseal plate chondrocytes in vivo. © 1994 Wiley‐Liss, Inc.
Improved ultrastructural preservation of rat ciliary body after high pressure freezing and freeze substitution: A perspective view based upon comparison with tissue processed according to a conventional protocol or by osmium tetroxide/microwave fixation
Authors: Eggli Peter S. , Graber Werner
Published: February 2005

Conventional fixation of the delicate, highly folded rat ciliary body and its iridial extension, as well as of vitreal structures, is associated with the induction of a number of artifacts, thus limiting the reliability of morphological interpretations. Improved ultrastructural preservation may be achieved by microwave heating in combination with osmium tetroxide fixation. This protocol, although simple and cheap, yields results, particularly with respect to the extracellular matrix compartment between inner and outer ciliary epithelial cells, which are not greatly inferior to those obtained by implementing the sophisticated high pressure freezing and freeze substitution technique. The latter affords good to very good ultrastructural preservation of epithelium and stromal components, such as blood vessels, neural elements, smooth muscle cells, fibrocytes, and free cells, up to a depth of 50–100 μm from the tissue surface. Its superiority over osmium tetroxide/microwave fixation is revealed in the cytoplasmic, intraorganellar, and vitreal matrix compartments, which incur no obvious losses. © 1994 Wiley‐Liss, Inc.
Fabrication of Au/p‐Si Schottky barrier for EBIC study
Authors: Zhang Xiao , Joy David
Published: February 2005

A simple method for the fabrication of a Au/p‐Si Schottky barrier suitable for electron beam induced current (EBIC) study has been developed. The mechanical and electrical properties of the fabricated Au/p‐Si Schottky barriers have been tested, and EBIC measurements of the dislocation contrast have been conducted using the fabricated Schottky barriers. © 1994 Wiley‐Liss, Inc.
Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy
Authors: Reipert Siegfried , Allen Terence David , Reipert Birgit Maria
Published: February 2005

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent‐free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3–4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in‐lens field emission scanning electron microscope. © 1994 Wiley‐Liss, Inc.
Three‐dimensional organization of biological tissues: A new method for investigation using scanning electron microscopy
Authors: Kolpakov Valeri , Rekhter Mark , Mironov Alexander
Published: February 2005

A new method is described which enables the visualization of hidden cellular surfaces of biological tissues. The method does not depend on the kind of fixative solution, the duration of the fixation, or the quantity of extracellular matrix. The samples are chemically dissociated with potassium ethoxide until the first individual cells appear in the solution. This initial process is followed by phase contrast microscopy, and remaining tissue is investigated with scanning electron microscopy. The method has proved more effective than conventional enzymatic techniques and is a simple, quick, and effective way to investigate the three‐dimensional organization of different normal and pathological tissues. © 1994 Wiley‐Liss, Inc.
Immunohistochemical demonstration of galanin‐, and galanin message‐associated peptide‐like immunoreactivities in sympathetic ganglia and adrenal gland of the guinea pig
Authors: Elfvin L.‐G. , Höukfelt T. , Bartfai T. , Bedecs K.
Published: February 2005

Using the indirect immunofluorescence method, the distribution of galanin (GAL)‐ and galanin message‐associated peptide (GMAP)‐like immunoreactivities (LI) were studied in sympathetic ganglia and the adrenal gland of the guinea pig. A rather dense network of GAL‐immunoreactive nerve fibers was found in the inferior mesenteric ganglion (IMG) and in the superior mesenteric pole of the celiac‐superior mesenteric ganglion complex (C‐SMG). The celiac pole of the C‐SMG, the stellate ganglion, and the superior cervical ganglion contained fewer, mostly scattered fibers. SIF‐cells in prevertebral and paravertebral ganglia contained GAL‐LI, as did the adrenal medullary cells. The GAL fibers in the IMG surrounded mainly principal ganglion cells containing somatostatin‐immunoreactivity (SOM‐IR), whereas fewer fibers were seen around neuropeptide Y (NPY) cells and cells in which SOM and NPY coexisted. Application of colchicine or vinblastine onto the IMG did not result in the appearance of GAL‐IR in the principal ganglion cells. In denervation experiments it was revealed that most of the GAL fibers reach the IMG via the lumbar splanchnic nerves.

GAL‐IR appears to be colocalized with substance P (SP) in fibers of the IMG, indicating an origin of the GAL‐containing fibers in dorsal root ganglia (DRG). This conclusion was supported by the finding in lumbar DRGs of GAL‐positive cell bodies that contained SP. The role of GAL in prevertebral ganglia is unclear. It may be suggested that GAL modulates the slow, long‐lasting membrane depolarization of the principal ganglion cells caused by SP in the primary afferents related to the IMG. GMAP‐LI was detected in SIF cells and adrenal medullary cells in which GMAP‐LI parallels the immunoreactivity of GAL. GMAP‐LI was not observed in neuronal cell bodies or nerve fibers of the ganglia. © 1994 Wiley‐Liss, Inc.
Effects of phospholipase A2 inhibitors on the antidepressant‐induced axonal regeneration of noradrenergic locus coeruleus neurons
Authors: Nakamura Shoji
Published: February 2005

In previous experiments, we have shown that antidepressants such as desipramine (DMI) can induce axonal regeneration of noradrenergic locus coeruleus neurons. In this article, we suggest that phospholipase A2 is involved in the molecular mechanism of the antidepressant‐induced regeneration of brain noradrenergic axons. The effects of the PLA2 inhibitors, mepacrine (MEP) or 4‐bromphenacyl bromide (BPB), upon the DMI‐induced regeneration of noradrenergic axons in the rat cerebral cortex were assessed by either histofluorescence or immunohistochemistry using an antibody to dopamine‐beta‐hydroxylase. Symmetrical sites of the frontal cortex in both hemispheres were pretreated with 6‐hydroxydopamine (6‐OHDA). Then, the same cortical site of one hemisphere was infused with DMI by means of osmotic minipumps for more than 2 weeks, while the corresponding cortical site of the other hemisphere was infused with DMI plus MEP or BPB. It was found that the PLA2 inhibitors could attenuate the DMI‐induced regeneration of noradrenergic axons. Thus, if axonal retraction or degeneration of brain noradrenergic neurons is involved in the pathogenesis of clinical depression, elucidating the malfunction of the PLA2 systems may provide substantial evidence to aid in our understanding of the cause of depression at the molecular level. © 1994 Wiley‐Liss, Inc.
Dopaminergic innervation of rat locus coeruleus: A light and electron microscopic immunohistochemical study
Authors: Maeda Toshihiro , Kitahama Kunio , Geffard Michel
Published: February 2005

Dopaminergic innervation of the rat locus coeruleus (LC) was immunohistochemically studied by using monoclonal antibody directed against dopamine (DA) at the light‐ and electron‐microscopic levels. A dense plexus of DA‐immunoreactive (IR) varicose fibers was found not only in the cell body area of the LC but also in the dendritic area. Three hundred and forty DA‐IR terminal boutons were observed. They were distributed in a wide range of diameters of 0.1–1.8 μm but most of them were large (mean value: 0.98 μm). Sixty‐nine percent formed an asymmetric synapse. There were many axo‐spinous connections. Small dendrites less than 0.3 μm in diameter displaying synaptic specialization were mostly dendritic spines (75%). Most of the target dendrites seemed to be noradrenergic in nature. It is suggested that dopaminergic innervation may play a powerful role in control mechanisms of activity of NA‐containing neurons of the LC. © 1994 Wiley‐Liss, Inc.
Three‐dimensional structure of lung elastin demonstrated by scanning electron microscopy/stereo‐pair images
Authors: O'Donnell M. D. , Cottell D. C. , McGeeney K. F.
Published: February 2005

The aim of this study was to expose the inflated 3‐D structure of lung elastin. Formic acid digestion followed by freeze‐drying unveiled the lamellar framework. The 3‐D structure of elastin was well preserved within the alveolar septa and ducts, as demonstrated by scanning electron microscopy/stereo‐pair photography. Elastin fibers are seen in the alveolar septa, which are continuous with the lamellae. The removal of collagen fibers and cells by formic acid was visualised as a function of time: The optimum was 48 hours. Transverse sections still retained some collagen fibrils and partially digested cells in addition to elastin as shown by transmission electron microscopy (TEM). Forme acid digestion followed by critical point drying caused damage to the lamellar structures and they appeared to collapse. Sodium hydroxide digestion combined with freeze‐drying did not preserve the 3‐D lamellar structure of elastin, but converted it into flat ribbonlike bands. The main structures remaining following alkali treatment were identified by TEM as collagen fibrils well preserved in their original locations. © 1994 Wiley‐Liss, Inc.
Confocal imaging of dendritic Ca2+ transients in hippocampal brain slices during simultaneous current‐ and voltage‐clamp recording
Authors: Jaffe David B. , Brown Thomas H.
Published: February 2005

Changes in the intracellular Ca2+ concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca2+ ‐sensitive fluorescent indicators. The imaging was performed in thick hippocampal brain slices while simultaneously measuring or controlling electrical activity with sharp microelectrodes or whole‐cell patch‐clamp electrodes. The combination of imaging and electrophysiology was essential for interpreting the changes in [Ca2+]i. We compared the increases in [Ca2+]i produced by either of two methods–direct depolarization of the cell via the somatic electrode or high‐frequency stimulations of synaptic inputs. The increases in [Ca2+]i in the soma and proximal dendrites caused by both methods were of comparable magnitude and they always decayed within seconds in healthy cells. However, the spatial patterns of distal Ca2+ increases were different. Separate sets of synaptic inputs to the same cell resulted in different spatial patterns of [Ca2+]i transients. We isolated and observed what appeared to be a voltage‐independent component of the synaptically mediated [Ca2+]i transients. This work demonstrates that the combination of neurophysiology and simultaneous confocal microscopy is well suited for visualizing and analyzing [Ca2+]i within neurons throughout the CNS and it raises the possibility of routinely relating subcellular [Ca2+]i changes to structural and functional modifications. © 1994 Wiley‐Liss, Inc.
In vivo human corneal confocal microscopy of identical fields of subepithelial nerve plexus, basal epithelial, and wing cells at different times
Authors: Masters Barry R. , Thaer Andreas A.
Published: February 2005

A technique is described to obtain time‐lapse reflected light confocal images of cells in the basal epithelium and adjacent wing cell layer from the in vivo human cornea. The technique is based on the sequential relocation of the uniqúe patterns of the subepithelial nerve plexuses immediately posterior to Bowman's membrane. The patterns of individual subepithelial nerve plexuses, as well as perforation points where nerves traverse Bowman's membrane, serve as fixed landmarks. A real‐time, scanning slit, confocal microscope is used to obtain reflected light images of a subepithelial nerve plexus, and the anterior and adjacent fields of basal and wing cells in the in vivo human cornea. All of the photographs are obtained from single video frames without the necessity of frame averaging or digital image processing. The instrument and relocation technique are prerequisites for applying a time‐lapse observation technique to investigate the dynamics of basal cell proliferation and differentiation in the living eye. © 1994 Wiley‐Liss, Inc.
Understanding the neostriatal microcircuitry: High‐voltage electron microscopy
Authors: Wilson Charles J.
Published: February 2005

High voltage electron microscopy (HVEM) and HVEM tomography of selectively stained cell processes in the neostriatum have offered an alternative to serial thin section reconstruction for accurate 3‐D visualization and measurement of axons, dendrites, and dendritic spines. Tissue preparation is simple and rapid, allowing examination of large numbers of specimens required for quantitation of neuronal morphology. The resolution of the images exceeds that available from any light microscopic technique and is appropriate for measurement of the finest axons and dendritic spine necks. HVEM tomography allows the direct measurement of dendritic surface area, required for computational modeling of synaptic integration. © 1994 Wiley‐Liss, Inc.
Electron microscopic immunolocalization of the 18 and 29 kilodalton secretory proteins in the mouse epididymis: Evidence for differential uptake by clear cells
Authors: Vierula Matti E. , Rankin Tracy L. , Orgebin‐Crist Marie‐Claire
Published: February 2005

In previous studies we reported the synthesis, secretion, and immunolocalization at the light microscopic level of two mouse epididymal proteins, MEP 7 and MEP 10 [Rankin et al. (1992b), Biol. Reprod., 46:747–766]. MEP 7 is the mouse homologue of the rat metalloproteins, AEG/D and E, and MEP 10 is the mouse homologue of the rat retinoic acid binding proteins, B and C. We now describe the immunolocalization of MEP 7 and MEP 10 in the mouse epididymis at the electron microscopic level. MEP 7 was localized in the Golgi apparatus, in small electron‐lucent secretory vesicles, and on microvilli of the principal cells from the distal caput epididymidis to the cauda. The luminal contents were also immunoreactive in these regions of the epididymis. Although some gold particles were associated with the sperm surface, there was no selective concentration of these particles. In addition, MEP 7 was localized in large (600 nm) supranuclear endocytic vesicles and in infranuclear lysosomes. MEP 10 immunoreactivity was also seen on the microvilli of the principal cells of the distal caput and corpus and the luminal contents from the distal caput to the cauda epididymidis. There was no association of gold particles with the sperm surface. In contrast to MEP 7, there was no detectable MEP 10 immunoreactivity on the organelles of the principal cells involved in protein secretion or endocytosis.

Clear cells also demonstrated immunoreactivity to MEP 7 and MEP 10. However, the intensity of immunolabeling, and the number of clear cells labeled, was greater with MEP 10 than MEP 7. In the case of MEP 7, the gold particles were located on the large supranuclear endocytic vesicles and on some infranuclear lysosomes, from the proximal corpus to the middle cauda, while in the case of MEP 10, gold particles were predominantly present in infranuclear lysosomes from the distal caput to the middle cauda.

These results suggest that the principal cells are involved in both the secretion and endocytosis of MEP 7. The MEP 10 and MEP 7 proteins present in the lumen of the mouse epididymis are endocytosed from the lumen and degraded in the clear cells. However, the process of endocytosis by the clear cells of these two proteins appears to be different. © 1995 Wiley‐Liss, Inc.
Direct observations of arrangements of carbonate groups in oxycarbonate superconductors by high‐resolution electron microscopy
Authors: Matsui Yoshio , Akimitsu Jun
Published: February 2005

Arrangements of CO3 groups in various types of oxycarbonate superconductors are examined by high‐resolution transmission electron microscopy (HRTEM). Every other B‐site of basic perovskite structure is replaced with CO3 groups in the first carbonate superconductor, (Ba0.56Sr0.44)2Cu1.1(CO3)0.9Oy. The 123‐related oxycarbonate superconductor obtained in a Y‐Ca‐Sr‐Cu‐O system, (Y0.475Ca0.475Sr0.05)Sr2Cu2.4(CO3)0.6Oy, has a superstructure with 2α periodicity due to ordered replacements of Cu‐site with CO3 groups. The non‐superconducting counterpart with 123‐related structure, (Y0.84Sr0.16)2Sr2Cu2.6(CO3)0.4Oy, on the other hand, shows more disordered arrangements of CO3 groups with nearly 3α periodicity. Similar superstructures, due to ordered replacements of Cu sites with CO3 groups, are also observed in the 223‐related oxycarbonate superconductors, (Ln,Ce)2Sr2Cu2.5(CO3)0.5Oy (Ln = Ho, Dy). Homologous series of compounds, (CaSr)n+1Cun(CO3)Oy (n = 1–5), consist of alternate stacking of Sr2Cu(CO3)Oy and SrCuO2 (infinite‐layer) types of blocks. They become superconductive by additionally doping the BO3 group. Another homologous series of Bi‐based oxycarbonate superconductors, (Bi,Pb)2Sr2n+2Cun+1 (CO3)nOy (n = 1–3), contain alternate CuO2 and CO3 layers in between the two (BiO)2 layers. Both mercury (Hg)‐and thallium (TI)‐based oxycarbonate superconductors, MBa2Sr2Cu2(CO3)Oy (M = Hg or TI) show quite unique modulation structures, where both HgO (or TlO) and CO3 layers repeat in the same plane, along [110] in the Hg compound and [100] in the Tl compound, to form longperiod superstructures with wavy distortion of atom planes. © 1995 Wiley‐Liss, Inc.
Preparation of oxide superconductor specimens for TEM examination
Authors: Fendorf Mark , Powers Michael , Gronsky Ronald
Published: February 2005

We have investigated a wide variety of oxide superconductors and report here on a number of techniques that can be effectively used to prepare transmission electron microscopy (TEM) specimens from these materials. Crushing, cleaving, ion milling, ultramicrotomy, and jet polishing all were successfully utilized, and details of each technique, as well as equipment used, are described. Selection among these methods depends both on the starting form of the material and the information required. Ion milling and crushing generally give the best results and have the widest applicability in our particular work, while crushing and cleaving involve the least equipment cost. In some cases, particularly with ion milling and jet polishing, small variations in the details of preparation have a dramatic effect on the success rate. We have found it to be a great advantage that the same techniques can be applied in a similar manner to a whole range of oxide materials, even (with some refinements and special precautions) to those that are extremely oxygen or moisture sensitive. © 1995 Wiley‐Liss, Inc.
Interaction of colloidal gold‐labelled glucosylated albumin with endothelial cell monolayers: Comparison between cryofixation and glutaraldehyde fixation
Authors: Hoying James B. , Chen Shih‐Chieh , Williams Stuart K.
Published: February 2005

Bovine aortic endothelial cells (BAEC) were exposed to glucosylated albumin‐gold complexes (GgA), and the distribution of the tracers was compared after cryofixation and after glutaraldehyde fixation. Morphometric analysis revealed differences in the GgA distribution depending upon the method of fixation used. In BAEC monolayers cryofixed after 3 min of incubation with GgA, tracer was observed in predominately apically located vesicular elements. After 16 min of incubation, all vesicular elements were labelled, and multivesicular bodies were the prominent labelled structure. In contrast, chemically fixed monolayers exhibited a heterogeneous distribution of GgA within vesicular profiles after 3 min and 16 min of GgA incubation. The differences in tracer distribution depending upon the fixation method must be resolved before the mechanism of vesiclemediated endothelial cell transport function is defined and universally accepted. © 1995 Wiley‐Liss, Inc.
Microstructures in high‐Tc Bi(Pb)‐family 2212 superconductors as revealed by scanning and transmission electron microscopy
Authors: Horiuchi Shigeo , Hirasaka Masao , Tsutsumi Masayuki , Kosuda Kosuke , Szerer M. Y. , Ben‐Dor Lina
Published: February 2005

Microstructures of Bi(Pb)‐family 2212 superconductors, which were prepared by a sol‐gel method with three different compositions, were examined mainly by scanning and transmission electron microscopy. The magnetization of the specimens strongly depends on the ratio between Bi and Pb content, while Tc is almost constant. In specimen 1, prepared with the nominal composition of Bi/Pb = 9/1, small grains of 2212 phase are formed with a minor fraction of some impurity phases. In specimen 2, with Bi/Pb = 17/3, which is optimum from the viewpoint of magnetization, large grains of the 2212 phase are formed during heating at 800°C, also with the impurity phases. In specimen 3, with Bi/Pb = 8/2, the 2212 grains are divided by layers of (Bi0.86, Pb0.14) (Ca0.7, Sr0.3)Ox. Moreover, plate‐like 2212 crystals are severely bent so that small cracks appear often with an inclusion of amorphous substance being rich in Ca and Pb. These layers and cracks must degrade the magnetization. A modulated structure of Bi‐type is formed in the 2212 grains of specimens 1 and 2, while not only Bi‐type but also Pb‐type are formed in specimen 3. The wavelength of Bi‐type is different for each specimen. © 1995 Wiley‐Liss, Inc.
Organ culture of benign, aging, and hyperplastic human prostate
Authors: Pretlow Thomas G. , Yang Bin , Pretlow Theresa P.
Published: February 2005

Organ culture of the human prostate began in the 1970s and was modeled after the work of Lasnitzki and her collaborators in the mouse two decades earlier. In organ culture of human prostates, one sees a rapid increase in epithelial cells and decrease in stromal cells during the first 3–5 days of culture. While modulation of many phenotypic properties occurs, these cultures provide a simple and rapid way to achieve large numbers of human prostatic epithelial cells in cultured tissues that are markedly depleted of stromal cells. There is some evidence that organ cultures are maintained in slightly better functional states in the presence of androgens; however, most of this evidence is less than quantitative. Most organ culture of prostates has been accomplished with tissues from unspecified locations within the prostate; interpretation of cultures carried out in this fashion has been less complete than would have been possible if they had been carried out from specific anatomic locations within the prostate. Careful pathological characterization of locations contiguous to the cultured tissue is mandatory if cultures are to be interpreted meaningfully. © 1995 Wiley‐Liss, Inc.
Instructive induction of prostate growth and differentiation by a defined urogenital sinus mesenchyme
Authors: Timms Barry G. , Lee Curtis W. , Aumüller Gerhard , Seitz Jurgen
Published: February 2005

Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe‐specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe‐specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal‐epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium. © 1995 Wiley‐Liss, Inc.
Analysis of cell death and cell proliferation in embryonic stages, normal adult, and aging prostates in human and animals
Authors: Sensibar Julia A.
Published: February 2005

Homeostasis in the prostate is recognized to be maintained by a complex interplay between the opposing actions of cell proliferation and Cell death. Growth regulatory factors that promote or inhibit cell proliferation and promote cellular death have been identified in the prostate. The integration of these forces involves cellular cooperation between the prostatic stroma and epithelium. Hormone‐regulated production of growth regulatory factors by one cell type may determine growth stimulation, inhibition, or cell death in a reciprocal cell partner. Imbalance between net cell proliferation and net cell death rates may result in abnormal growth leading to BPH. Additional study of the growth regulatory factors associated with distal vs. proximal epithelial cells and stroma and comparison of growth factor expression by the neonatal, postnatal growing, adult quiescent, and aging prostates will likely provide further insight into the regulation of prostate cell division and death. © 1995 Wiley‐Liss, Inc.
Muscle‐specific gene expression during myogensis in the mouse
Authors: Ontell Marcia , Ontell Martin P. , Buckingham Margaret
Published: February 2005

Over the past decade, significant advances in molecular biological techniques have substantially increased our understanding of in vivo myogenesis, supplementing the information that previously had been obtained from classical embryological and morphological studies of muscle development. In this review, we have attempted to correlate morphogenetic events in developing murine muscle with the expression of genes encoding the MyoD family of myogenic regulatory factors and the contractile proteins. Differences in the pattern of expression of these genes in murine myotomal and limb muscle are discussed in the context of muscle cell lineage and environmetal factors. The differences in gene expression in these two types of muscle suggest that no single coordinated pattern of gene activation is required during the initial formation of the muscles of the mouse. © 1995 Wiley‐Liss, Inc.
Expression of myosin heavy chain isoforms and myogenesis of intrafusal fibres in rat muscle spindles
Authors: Soukup Tomáš , Pedrosa‐Domellöf Fatima , Thornell Lars‐Eric
Published: February 2005

This review concerns the pattern of expression and regulation of myosin heavy chain (MHC) isoforms in intrafusal fibres of rat muscle spindles detected by immunocytochemistry. The three types of intrafusal fibres—nuclear bag1, nuclear bag2, and nuclear chain fibres—are unique in co‐expressing several MHCs including special isoforms such as slow tonic and α cardiac‐like MHC and isoforms typical of muscle development, such as embryonic and neonatal MHC. The distinct intrafusal fibre types appear sequentially during rat hind limb development, the nuclear bag2 precursors being first identifiable at 17–18 days in utero as the only primary myotubes expressing slow tonic MHC. Sensory innervation is required for the expression of “spindle‐specific” MHC isoforms. Motor innervation contributes to the diversity in distribution of the different MHCs along the length of the nuclear bag fibres. It is suggested that unique populations of myoblasts are destined to become intrafusal fibres during development in the rat hind limb muscles and that the regional heterogeneity in MHC expression is related both to sensory and motor innervation and to the properties of the myoblast lineages. These distinct features make intrafusal fibres an attractive in situ model for investigating myogenesis, myofibrillogenesis, and the mechanisms regulating MHC expression. © 1995 Wiley‐Liss, Inc.
Postembedding colloidal‐gold immunocytochemistry of noncollagenous extracellular matrix proteins in mineralized tissues
Authors: McKee M. D. , Nanci A.
Published: February 2005

Immunocytochemistry is a powerful tool for investigating protein secretion, extracellular matrix assembly, and cell‐matrix and matrix‐matrix/mineral relationships. When applied to the tissues of bones (bone and calcified cartilage) and teeth (dentin, cementum, and enamel), where calcium phosphate–containing extracellular matrices are the predominant structural component related to their weight‐bearing and masticatory roles, respectively, data from immunocytochemical studies have been prominent in advancing our understanding of mineralized tissue modeling and remodeling. The present review on the application of postembedding, colloidal‐gold immunocytochemistry to mineralized tissues focuses on the advantages of this approach and relates them to conceptual, theoretical, and experimental data currently available discussing matrix‐mineral interactions and extracellular matrix formation and turnover in these tissues. More specifically, data are summarized regarding the distribution and role of noncollagenous proteins in different mineralized tissues, particularly in the context of how they interface with mineral, and how this relationship might be affected by the various tissue‐processing steps and immunocytochemical strategies commonly implemented to examine the distribution and function of tissue proteins. Furthermore, a technical discussion is presented that outlines several different possibilities for epitope exposure in mineralized tissues during preparation of thin sections for transmission electron microscopy. Cell biological concepts of protein secretion by cells of the mineralized tissues, and subsequent extracellular matrix assembly and organization, are illustrated by examples of high‐resolution, colloidal‐gold immunolabeling for osteopontin, bone sialoprotein, and osteocalcin in the collagen‐based mineralized tissues and for enamel protein (amelogenin) in enamel. © 1995 Wiley‐Liss, Inc.
Immunocytochemistry of the muscle cell cytoskeleton
Authors: Stromer Marvin H.
Published: February 2005

The muscle cell cytoskeleton is defined for this review as any structure or protein primarily involved in linking or connecting protein filaments to each other or to anchoring sites. In striated muscle, the M line connects thick filaments at their centers to adjacent thick filaments. Titin forms elastic filaments that extend from the M line to the Z line and may contribute to the resting tension properties of striated muscle. Nebulin forms inextensible filaments in skeletal muscle that are closely associated with thin filaments and that may provide a length template for thin filaments. Z lines anchor thin filaments from adjacent sarcomeres via the actin‐binding function of α‐actinin. Other proteins located at the Z line include Cap Z, Z‐nin, Z protein, and zeugmatin. Intermediate filaments connect myofibrils to each other at the level of the Z line and to the sarcolemma at the Z‐ and possibly the M‐line levels. Immunolocalization has identified the adhesion plaque proteins spectrin, vinculin, dystrophin, ankyrin, and talin at subsarcolemmal sites where they may be involved with filament attachment. Smooth muscle cell cytoskeletons are believed to include membrane associated dense bodies (MADBs), intermediate filaments, cytoplasmic dense bodies (CDBs), and perhaps a subset of actin filaments. MADBs contain a menu of attachment plaque proteins and anchor both thin filaments and intermediate filaments to the sarcolemma. CDBs are intracellular analogs of striated muscle Z lines and anchor thin filaments and intermediate filaments. © 1995 Wiley‐Liss, Inc.
Uranium‐contaminated soils: Ultramicrotomy and electron beam analysis
Authors: Buck Edgar C. , Dietz Nancy L. , Bates John K.
Published: February 2005

Uranium‐contaminated soils from the U.S. Department of Energy (DOE) Fernald Site, Ohio, have been examined by a combination of backscattered electron imaging (BSE) and analytical electron microscopy with electron diffraction (AEM). The inhomogeneous distribution of particulate uranium phases in the soil required the development of a method for using ultramicrotomy to prepare transmission electron microscopy (TEM) thin sections from the SEM mounts. A water‐miscible resin was selected that allowed comparison between SEM and TEM images, permitting representative sampling of the soil. Uranium was found in iron oxides, silicates (soddyite), phosphates (autunites), and uraninite (UO2+x). No uranium was detected in association with phyllosilicates in the soil. © 1995 Wiley‐Liss, Inc.
Em‐tomography of section collapse, a non‐linear phenomenon
Authors: van Marle J. , Dietrich A. , Jonges K. , Jonges R. , de Moor E. , Vink A. , Boon P. , van Veen H.
Published: February 2005

Using back projection for reconstruction and tilt series of Epon or Lowicryl embedded and sectioned material, we demonstrated: (1) a reduction in thickness of 50% for Epon and 80% for Lowicryl sections, and (2) a non‐uniform density distribution along the electron‐optical axis in sections. The highest density was found at the vacuum exposed side of the section. The formvar side of the section showed a similar increase in density, but not to the same extent. Minimalization of electron exposure, even without pre‐exposure, did not affect the reconstructed thickness, nor did it affect the non‐uniform density distribution. However, parallax measurements showed that at 150K, collapse of Epon sections does not take place. For EM‐tomography of plastic embedded material our findings imply that at the top and bottom portion of the sections the dimensions of the reconstructed structures are distorted, but that in the middle portion the dimensions are reliably retained. © 1995 Wiley‐Liss, Inc.
Comparative methodological investigations on the cytochemical localization of calcium in brain and inner ear of cichlid fish
Authors: Vöhringer Petra , Nindl Gabi , Aich Brigitta , Körtje Karl‐Heinz , Rahmann Hinrich
Published: February 2005

Four different methods for calcium precipitation are compared in the optic tectum and the inner ear of the cichlid fish, Oreochromis mossambicus. Several parameters are investigated concerning their influences on the reaction product. Three procedures (bichromate, fluoride, and oxalate‐pyroantimonate) produce fine‐grained deposits, often flocculent in the latter method. The fourth method (potassium‐pyroantimonate) generates predominantly coarse‐grained reaction product. The calcium content of the deposits is always proven with energy‐filtering transmission electron microscopy (EFTEM). In both tissues fine‐grained reaction product is found in endoplasmic reticulum and synaptic vesicles, and in addition in some mitochondria and at the cytoskeleton. The coarse‐grained deposits of the potassium‐pyroantimonate method have a more unspecific distribution. This is the only method which produces extracellular deposits in the inner ear, whereas in the optic tectum extracellular precipitates are always present except with the oxalate‐pyroantimonate procedure. Two factors have an influence on the reaction product: the duration of fixation and the type of resin. The prolongation of the fixation time up to 24 hours leads to an increase of the reaction product, which also becomes coarse‐grained. These observations are corroborated by quantification with image analysis. Furthermore the use of an epoxy resin compared to acrylic resins decreases the amount of reaction product produced. We show that the application of several methods is meaningful in order to understand the calcium properties of the investigated tissue, but it is necessary to optimize a certain method for a given tissue. © 1995 Wiley‐Liss, Inc.
Ocular lens gap junctions: Protein expression, assembly, and structure‐function analysis
Authors: Kistler Joerg , Evans Clive , Donaldson Paul , Bullivant Stanley , Bond Jacqui , Eastwood Sharon , Roos Marc , Dong Yimin , Gruijters Terry , Engel Andreas
Published: February 2005

Recent advances in understanding lens fiber gap junction formation are reviewed. These include studies of junctional protein expression in the embryonic lens, and of age related changes affecting gap junction structure and composition in the adult lens. An in vitro assembly system based on detergent solubilized pore complexes and endogenous lipids has been developed to provide information on the molecular interactions involved in gap junction formation and to provide material for structure analysis. Important information on the electrical properties of lens gap junction channels is obtained using electrophysiological techniques including planar lipid bilayer analysis and patch clamping. © 1995 Wiley‐Liss, Inc.
Structural and molecular determinants of intercellular coupling in cardiac myocytes
Authors: Lee Kanter H. , Beyer Eric C. , Saffitz Jeffrey E.
Published: February 2005

Electrical activation of the heart requires intercellular transfer of current through gap junctions connecting individual cardiac myocytes. Using a combination of light and electron microscopic techniques and molecular approaches, we have characterized the number, size, and spatial distribution of intercellular connections at gap junctions in cardiac myocytes and have also cloned, sequenced, and elucidated the subcellular distribution of three physiologically distinct gap junction channel proteins. In this review, we present evidence to suggest that the spatial distribution of myocyte interconnections and the molecular composition of gap junction channels may confer distinct conduction properties on specific tissues of the mammalian heart such as atrial and ventricular myocardium, and the nodes and bundles of the cardiac conduction system. © 1995 Wiley‐Liss, Inc.
Role of gap junctions in the development of the preimplantation mouse embryo
Authors: Becker David L. , Davies Catherine S.
Published: February 2005

We have taken several approaches to study the role of gap junctional communication during preimplantation mouse development. Firstly, the normal expression pattern of gap junctions has been characterized using immunostaining in conjunction with laser scanning confocal microscopy. Changes in junctional distribution have been correlated with developmental events. We have gone on to study development and junctional organization in mice which naturally exhibit reduced cell to cell communication (DDK syndrome), and in normal mice in which gap junction permeability has been artificially manipulated. Furthermore, anti‐peptide antibodies hae been tested for their ability to block gap junction communication and for the effects of such a block on subsequent development. Collectively, the results demonstrate that gap junctional communication plays an important role in the maintenance of compaction and the differentiation of an organized epithelium within an embryo, features which are vital for preimplantation development to progress successfully. © 1995 Wiley‐Liss, Inc.
Quantitative junctional permeability measurements using the confocal microscope
Authors: Miller Lan
Published: February 2005

This paper describes the use of a photobleach method and confocal microscopy to compare the cell‐to‐cell transfer rate of 5,6 carboxyfluorescein in dissociated embryonic chick lens cells with those in the anterior epithelium of the whole embryonic chick lens. The average cell‐to‐cell transfer rates obtained were 7.9 × 10−3 sec−1 in the dissociated cells and 2.6 × 10−3 sec−1 in the anterior epithelium in an intact lens. © 1995 Wiley‐Liss, Inc.
Lectin binding and identification of sialic acid acceptor sugars in rabbit oviduct under hormone administration
Authors: Menghi Giovanna , Scocco Paola , Materazzi Giovanni
Published: February 2005

Localization of individual glycosidic residues and sialic acid acceptor sugars was investigated by conjugated lectins in the rabbit oviduct under physiological hormonal conditions and human chorionic gonadotropin (HCG) administration. Ampulla and isthmus were found to exhibit lectin binding profiles typical of each hormonal stage. Two different sialylated glycomolecules were identified within the epithelial lining; in particular, sialoglycoconjugates characterized by the terminal sequence sialic acid‐galactose were visualized in the secretory cells and the sialic acid‐N‐acetylgalactosamine terminal disaccharides were localized on both ciliated and secretory cells of the entire oviduct. Surface and cytoplasmic sialoglycoconjugates were also found to exhibit a differential behaviour inside the two oviduct tracts examined. Present findings further supported the idea that in ampulla and isthmus, the greatest modifications consequent to hormone treatment take place at different times. © 1995 Wiley‐Liss, Inc.