bluuebunny/crossref_metadata_2025 huggingface dataset:
Porcine colonic lymphoglandular complex: Distribution, structure, and epithelium
Authors: Morfitt David C. , Pohlenz Joachim F. L.
Published: February 2005

Lymphoepithelium and cells specialized for uptake and transport of foreign matter are characteristic of antigen sampling organs, Including lymphoglandular complexes (LGCs). Distribution, his‐tologic structure, and epithelial ultrastructure of co‐Ionic lymphoglandular complexes were determined in 5‐ to 13‐week‐old pigs. LGCs averaged 1,231 in number per colon, displayed a characteristic distribution pattern, and were most evenly distributed in colons of older pigs. LGCs consisted of well‐defined submucosal masses composed of lymphatic nodules and internodu‐lar lymphoid tissue penetrated by radially branching extensions of mucosal glands. Epithelial diverticula of each LGC entered the submucosa as a group through a circular collar derived from the muscularis mucosae. LGC epithelium contained goblet cells, cuboidal and columnar enterocytes, enteroendocrine cells, individual and clustered intraepithelial leukocytes, and cells morphologically compatible with follicle‐associated epithelial cells/M cells. We regard the colonic LGC as a distinct mucosal lymphoid organ and suggest a significant role for it in local and systemic immune responses. The porcine colonic LGC may serve as a model for the human LGC.
Formation of secretion granules in the Golgi apparatus of plasma cells in the rat
Authors: Rambourg A. , Clermont Y. , Hermo L. , Chrétien M.
Published: February 2005

The three‐dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanu‐clear Golgi apparatus was formed by a continuous rib‐bonlike structure composed of the following stacked elements. On the cis‐face of the Golgi stack, there was a tubular membranous network referred to as the cis‐element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80‐nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or “peeling off” configuration. These last elements consisted of prosecretory granules attached to flattened, empty‐looking saccules showing buds at their surface; detached, more‐or‐less fenestrated, flattened saccules; and shrivelled residual trans‐tubular networks. In the trans‐region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans‐saccules flows toward the dilated portions which become prosecretory granules. The trans‐most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans‐tubular networks.
Septal collateralization: Demonstration of canine intramyocardial collaterals
Authors: Wilson Jack L. , Scheel Konrad W.
Published: February 2005

The anatomical distribution of intra‐myocardial collateral arteries that develop from the septal to the other major coronary arteries was studied in dogs following gradual Ameroid occlusion of tftt circumflex artery. The septal artery was cannulated and injected with Batson's plastic compound resulting in a cast of the coronary circulation. Collateral vessels radiated from the septal vascular bed to both the circumflex and anterior descending arteries. The collaterals developed from the entire base‐to‐apex extent of the septal artery and were found on both the right and left sides of the septum. Collateral growth appeared to be more concentrated at the apex of the heart. The anatomical details of septal collateral circulation illustrate the importance of intramyocardial collateralization in the dog, which was thought to exhibit primarily epicar‐dial collaterals.
Immunocytochemical localization and biochemical analysis of α and β keratins in the avian lingual epithelium
Authors: Carver Wayne E. , Sawyer Roger H.
Published: February 2005

The α and β keratins are found as 10‐nm and 3‐nm cytoplasmic filaments, respectively. While the a keratins are produced in essentially all vertebrate epithelia (Franke et al.: Exp. Cell Res., 116:429‐445, 1978; Sun et ah: Proc. Natl. Acad. Set. USA, 76:2813–2817, 1979), the β keratins have been demonstrated only in specific epithelial tissues of birds and reptiles (Sawyer et ah: In: Biology of the Integument: Vertebrates. J. Bereiter‐Hahn, A.G. Matoltsy, and K, S. Richards, eds. Springer‐Verlag, Berlin, Vol. 2, pp. 194–238,1986; Land‐mann: In: Biology of the Integument: Vertebrates. J. Bereiter‐Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer‐Verlag, Berlin, Vol. 2, pp. 150–187, 1986). Recently, Homberger and Brush (Zoomorphology, 106:103‐114, 1986) have demonstrated that within the lingual epithelium of parrots, β keratins are expressed exclusively in the anterior ventral region. While it is well established that epidermal‐dermal interactions are important for the regional expression of the β keratin genes in the avian scutate scales and feathers, little is known about the expression of β keratins in other epithelial structures such as the tongue. We have used biochemical and immunocytochemical techniques to analyze the a and β keratins of the lingual epithelium of the chick as an initial step in the characterization of this model system for developmental studies. We have found that α keratins are present throughout the lingual epithelium. The anterior ventral epithelium contains α keratin polypeptides characteristic of skin‐type differentiation, while the epithelium of the dorsal and posterior ventral regions contains α keratin polypeptides characteristic of esophageal‐type differentiation (O'Guin et al.: In: Current Topics in Developmental Biology: The Molecular and Developmental Biology of Keratins. A.A. Moscona and A. Monroy, eds. R.H. Sawyer, vol. ed. Academic Press, New York, Vol. 22, pp. 282–306, 1987). Beta keratins are produced only in the differentiated epithelial cells of the anterior ventral region of the tongue. Immunoelectron microscopy demonstrates that the α and β keratins of the stratum intermedium and corneum of the anterior ventral region are found together in the large filament bundles characteristic of this region. The preexistence of the α keratins in the cells destined to produce β keratins as well as the colocalization of these keratins in the filament bundles of these cells suggests that a functional relationship may exist between the α. and β keratins.
Lectin histochemistry of the embryonic heart: Fucose‐specific lectin binding sites in developing rats and chicks
Authors: Fazel A. R. , Sumida H. , Schulte B. A. , Thompson R. P.
Published: February 2005

Glycoconjugates, particularly their sugar side chains, play important roles in embryonic development. Changes in cell‐surface‐associated glyco‐conjugates are known to affect cell differentiation, cellular interactions, and other developmental phenomena during embryogenesis. The embryonic heart goes through a series of complicated morphologic events during development. Of particular interest is morphogenesis of the outflow tract. This region of the embryonic heart originates from more than one cell population and undergoes a complex process of septation during formation of the great vessels. Histochemi‐cal analysis with a series of fucose‐specific lectins conjugated to horseradish peroxidase has revealed the presence of a fucosylated glycoconjugate in the outflow tract of the developing heart. The results reveal further that the expression of the fucosylated glycoconjugate is stage‐dependent and thus probably genetically regulated. The timing and distribution of staining with the lectin OFA suggest that this fucosylated glycocoiyu‐gate may play a role in directing the migration of neural crest cells into the heart and subsequent formation of the conus septum.
Three methods for producing fertile hemopoietic chimeras in mice
Authors: Johnson Sigrid R. , Lala Peeyush K.
Published: February 2005

Three methods for producing semiallogeneic (F1→parental) hemopoietic chimeras with retained or regained fertility are detailed here. Prenatal (PN) chimeras were produced by injecting F1 ([BALB/c ♀ × C3H/HeJ♂] or [CBA/J♂ × C57BL/6♂]) fetal liver (days 13–18) or adult bone marrow cells (106–107 cells/20 μl/embryo) into the yolk‐sac cavities of days 13–17 gestation BALB/c or CBA/J embryos, respectively, and allowing them to be born naturally. Neonatal (NN) chimeras were made by introducing F1 bone marrow cells (1–2 × 107 cells/0.25 ml) into newborn (< 24 hr old) female mice through the anterior facial vein. Female mice were raised to maturity in both cases. Ovary‐transplanted (OT) chimeras were made by first irradiating (9.5 Gy) and repopulating young female adult mice with 107 F1 bone marrow cells, followed by bilateral orthotopic transplantation of syngeneic ovarian tissue six weeks later. Females reconstituted with the above three methods were mated with normal syngeneic males and sacrificed at 11–16 days of pregnancy to evaluate hemopoietic chimerism. This was determined in all cases by a radioautographic evaluation of the extent of donor H‐2 phenotype marker expression on splenic small lymphocytes, after an indirect labelling of single‐cell suspensions with monospecific antibody and [125I]protein‐A. Results indicate that hemopoietic chimerism was best in the PN group (0.3–78.1%, mean = 27.1); intermediate in the OT group (5.8–38.2%, mean = 18.1); and low in the NN group (0–14%, with one exception, which was 83.6%). Observed fertility was best for BALB/c host PN chimeras. Decreased litter size was observed in CBA/J host chimeras of both the PN and NN groups. OT chimeras showed a decreased capacity to become pregnant as well as lowered litter sizes. These data suggest that the prenatal chimera system offers the best method for obtaining both good hemopoietic chimerism and excellent fertility in experimental mice for future studies in reproductive biology and hematology.
Hemopoietic origin of certain decidual cell precursors in murine pregnancy
Authors: Johnson Sigrid R. , Graham Charles H. , Lysiak Jeffrey J. , Lala Peeyush K.
Published: February 2005

The possible hemopoietic origin of certain precursors of uterine decidual cells appearing during normal murine pregnancy was investigated in semiallogeneic hemopoietic chimeras with retained or regained fertility. Chimeras were produced by three different methods in two donor‐host combinations: F1 [BALB/c ♀ × C3H/HeJ ♂] cells introduced into the parental strain BALB/c ♀ hosts or F1[CBA/J ♀ × C57BL/6 ♂] cells introduced into CBA/J ♀ hosts. Prenatal chimeras (PN) were made by reconstituting mouse fetuses (day 13–17) with 106–107 adult bone marrow or fetal liver cells through the yolk sac and they were allowed to be delivered naturally. Neonatal chimeras (NN) were made by injecting 1–2 × 107 adult bone marrow cells into the anterior facial vein of neonatal mice (< 24 hr old). In both cases, experimental animals were raised to maturity. Ovary‐transplanted chimeras (OT) were made by injecting 107 bone marrow cells into lethally irradiated (9.5 Gy) young adult female mice, followed 6 weeks later with bilateral orthotopic transplants of syngeneic ovary grafts to restore fertility. All female chimeras produced by the three different methods were mated with syngeneic male partners to produce normal pregnancy. The extent of chimerism at the cellular level was determined in all cases by a radioautographic identification of the H‐2 phenotype of splenic lymphocytes and decidual cells and macrophages in the collagenase‐dispersed decidua at day 11–16 of normal pregnancy, following a sandwich labelling with monospecific anti‐H‐2 antibodies and 125I– protein A. Morphological discrimination of typical decidual cells from macrophages in the collagenase‐dispersed decidua was carried out on the basis of several distinctive markers: presence of surface Dec‐1 and Thy‐1 and absence of surface F4/80 or latex phagocytosis for decidual cells, in contrast to macrophages which were phagocytic and expressed F4/80 but not Dec‐1 or Thy‐1. While the degree of hemopoietic chimerism (judged by the incidence of donor‐derived lymphocytes in the spleen) varied from animal to animal, in all three groups (PN, NN, and OT) comprising a total of 26 chimeras, the percentage of typical decidual cells expressing donor H‐2 phenotype showed an excellent correlation with that for small lymphocytes in the spleen. These results reveal that at least a sub‐population of typical decidual cells of the pregnant uterus has a hemopoietic genealogy. A possible familial relationship of these cells to granulated metrial gland cells remains unclear.
Contours and distribution of sites that react with antiacetylcholinesterase in chicken intrafusal fibers
Authors: Maier Alfred
Published: February 2005

Serial cross and longitudinal sections of intrafusal fibers from the intracapsular portions of chicken tibialis anterior muscle spindles were incubated with a monoclonal antibody specific for chicken acetylcholinesterase (AchE) and examined by immunofluorescence for the presence of the enzyme on presynaptic and postsynaptic membranes of neuromuscular junctions. The midequatorial sensory region which lacks organized sarcomeres was negative, but immediately distal to it faintly staining regions of AchE localization were observed on intrafusal fibers. In cross sections at the juxtaequator, the outlines of areas that were positive for AchE were either thin and crescentlike or thick and compact. The distribution of both types of localization continued into the polar region. Toward the more distal polar region, the intensity of sites on the postsynaptic membrane that reacted with the anti‐AchE progressively increased. In longitudinal sections, AchE localization was largely limited to two configurations. One was elongate, while the other was more round or oval and often also smaller. Both types might occur on the same, or on different, intrafusal fibers. Examination of silver‐impregnated sections revealed the presence of platelike and of traillike axon terminals. The variety of shapes observed on presynaptic and postsynaptic membranes warrants further study to determine whether chicken muscle spindles are innervated by more than one type of motor neuron.
Mapping gold‐labeled receptors on cell surfaces by backscattered electron imaging and digital image analysis: Studies of the IgE receptor on mast cells
Authors: Stump R. F. , Pfeiffer J. R. , Schneebeck M. C. , Seagrave J. C. , Oliver J. M.
Published: February 2005

The response of cells to signalling molecules such as hormones, growth factors, and immune mediators that bind to cell‐surface receptors depends in part on the density and distribution of the relevant receptors. We have developed methods to map the distribution of IgE receptors on RBL‐2H3 mast cells at high resolution in the scanning electron microscope (SEM). The key elements of our procedure are a new fixative that preserves receptor binding activity; a family of colloidal gold‐conjugated probes that bind directly or indirectly to the IgE‐receptor complex; an SEM with detectors for both secondary and backscattered electrons (to observe surface topography and gold particles, respectively); and an image processor that can average, digitize, and store these images. Topographical maps are generated by processing and superimposing the digitized images. The methods we describe can be applied to study the density and distribution of any membrane receptor that can be labeled with colloidal gold particles.
Topographical and planar distribution of Helix pomatia lectin‐binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: Demonstration of membrane heterogeneity
Authors: Kan Frederick W. K. , Bendayan Moïse
Published: February 2005

The lectin‐gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze‐fracture preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen‐granule content, labeling of the acinar lumen was intense. Fracture‐label preparations revealed preferential partition of HPL‐binding sites to the exoplasmic half of the zymogen‐granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture‐face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin‐section fracture‐label approach. In addition, favorable profiles of thin sections of freeze‐fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture‐face. We conclude that (1) zymogen granules contain little HPL‐binding glycoconjugates, (2) HPL‐binding sites are preferentially associated with the exoplasmic half of the zymogen‐granule and plasma membranes, and (3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL‐binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory‐granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.
Immunogold detection of glycoprotein antigens in sea urchin embryos
Authors: Benson Nancy C. , Benson Stephen C. , Wilt Fred
Published: February 2005

Four developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.
Detection of gold probes with video‐enhanced contrast microscopy: Nanovid microscopy
Authors: De Brabander M. , Geerts H. , Nuydens R. , Nuyens R.
Published: February 2005

This contribution describes a new microscopic technique aimed at visualizing colloidal gold particles of 20‐40 nm diameter as dynamic markers at the light microscopic level. The technique, called nanovid microscopy, is based on the use of contrast enhancement by video techniques and digital image processing. Two applications on living cells are discussed. The first is the receptor‐mediated endocytosis of the transferrin receptor, which for the first time can be followed in real time. A second application documents the motion of cell‐surface glycoproteins on PTK2‐cells.

In addition, nanovid microscopy allows the number of gold particles in immunogold staining to be quantified easily. With this tool, the translocation of specific molecules in living cells becomes one of the most enjoyable activities to observe.
Gold labeling of cell monolayers grown on dextran beads
Authors: Hogan M. E. , Hassouna H. I. , Klomparens K. L.
Published: February 2005

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these mono‐layers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM.

The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell‐attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 μm, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal.

With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer.

The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.
Use of colloidal gold in diagnostic surgical pathology
Authors: Warhol Michael J.
Published: February 2005

Colloidal gold immuno‐electron microscopy is a powerful tool for defining antigenicity at the subcellular level. Such studies permit correlation with cell fractionation studies. They also allow one to assess the specificity of a particular antibody.

The most useful reagent for immuno‐electron microscopy is colloidal gold stabilized by a binding protein, either staphyloccocal protein A or immunoglobulin. This method permits highly discrete labeling, and the system is useful for most antibodies used in diagnostic pathology.
Applications of immunogold‐silver enhancement: Testing of monoclonal antibodies and detection of human cytomegalovirus in histologic specimens
Authors: Dankner Wayne M. , Spector Stephen A.
Published: February 2005

Human cytomegalovirus (HCM‐V) is an important pathogen in neonates, transplant patients, and individuals with acquired immunodeficiency syndrome (AIDS). Reliable techniques for the detection of this virus in clinical specimens would aid in improving methods for diagnosis and increasing our understanding of viral pathogenesis. We evaluated the utility of immunogold‐silver enhancement to determine the specificity of murine monoclonal antibodies directed against HCMV proteins and applied these antibodies to the detection of HCMV in histologic material. Nine antibodies were tested in a tissue‐culture system to determine the location of staining. All were found to be active in frozen tissue, but only two of these antibodies were reactive in formalin‐fixed tissue. We also evaluated a novel tissue fixative technique (AMeX; Fixation and dehydration with acetone) that has been used to maintain antigenicity of T‐lymphocyte cell markers. All antibodies remained reactive against their respective HCMV proteins in tissue fixed by this technique. However, dehydration of tissue may limit the usefulness of AMeX fixation. Immunogold‐silver enhancement is a useful and reliable histochemical technique for detection of HCMV antigens in pathologic specimens.
Use of immunogold preembedding technique to detect hepatitis A viral antigen in infected cells
Authors: Cromeans Theresa , Humphrey Charles , Sobsey Mark , Fields Howard
Published: February 2005

Localization of virus and viral antigen in cell cultures infected with a rapidly replicating isolate of strain HM‐175 of hepatitis A virus (HAV; pHM‐175) was accomplished by using immunogold probes. Cells infected under one‐step growth curve conditions were prefixed with 2% paraformaldehyde and 0.1–0.001% saponin at appropriate times postinfection for detection of maximum virus and viral antigen. An indirect labeling technique was employed using monoclonal antibody to HAV followed by 5 nm gold‐antimouse IgG conjugate. Cells were then fixed by standard electron microscopy techniques and thin sectioned. This prefixation technique allowed penetration of the immunogold probes and moderate preservation of ultrastructure. Within infected cell cytoplasm, numerous antigenic sites were labeled with six to 200 gold particles. Two types of cells were infected with HAV and somewhat different results were obtained with the two cell types. In BS‐C‐1 cells, where a cytopathic effect (CPE) was not observed, myelin figures were immunogold labeled or frequently were located near immunogold‐labeled sites. Vesicles containing viruslike particles (14–22 nm) were also observed. A significant observation in infected FRhK‐4 cells was the presence of multivesicular bodies labeled with immunogold. Microfilaments were commonly seen near the multivesicular bodies. Our results demonstrate that the choice of prefixation method for immunogold labeling should be empirically determined for the cell type and condition.
Immunogold‐silver labeling in histology
Authors: In't Veld Peter A.
Published: February 2005

Immunogold‐silver staining is a relatively new technique in light‐microscopic immunocytochemistry. It is based on silver enhancement of 1 to 10 nm gold probes immunologically bound to antigenic determinants in tissue sections. The detection efficiency of this third‐generation detection technique is compared to that of the peroxidase‐based ABC technique in routinely processed, paraffin‐embedded, human biopsy material.
Immunogold‐labeled microtubule crossbridges in platinum‐carbon replicas of the marginal band of erythrocyte cytoskeletons
Authors: Centonze Victoria E. , Ruben George C. , Sloboda Roger D.
Published: February 2005

Cytoskeletons of erythrocytes from the toad Bufo marinus are composed of a surface‐associated cytoskeleton that encapsulates the annular bundle of microtubules known as the marginal band (MB) and the centrally located nucleus. As seen by phase‐contrast microscopy, the microtubules (MTs) of the MB remain tightly bun‐led after cell lysis without the need for added stabilizing factors. The integrity of this structure suggested that in addition to MTs other components were present in the MB and were responsible for its stability. Thin (< 18 nm) platinum‐carbon (Pt‐C) replicas of freeze‐dried cytoskeletons prepared by using a modified Balzers 300 system provided a novel method of sample reparation for a high‐resolution study of the ultrastructure of the MB. Electron micrographs of replicas revealed that, the MTs of the MB displayed numerous filamentous projections which, hen viewed in stereo, appear as side‐arm connections between adjacent MTs. Immunofluorescence data show that monospecific antibodies to tubulin and to MT‐associated protein 2 (MAP2) from brain each detect cross‐reactive material in the MB. The combination of immunogold cytochemistry with Pt‐C replication provided the increased resolution required to identify the individual structures recognized by antibodies to tubulin and MAP2. As expected, antitubulin labeled the MTs of the MB. However, anti‐MAP2 antibodies were localized specifically to the cross‐ridging filaments between adjacent MTs. Thus, a IAP2‐like protein was identified in situ as the crossbridging filament that bundles MTs to form a table MB.
On‐grid immunogold labeling of glial intermediate filaments in epoxy‐embedded tissue
Authors: Johnson Anne B. , Bettica Annemarie
Published: February 2005

On‐grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold‐labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy‐embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde‐fixed, epoxy‐embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on‐grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween‐20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy‐embedded material to immunolabel thin structures like intermediate filaments.
Light and electron microscopic observations on the cervical epithelium of the rabbit: II
Authors: Odor D. Louise , Horacek Mark J. , Blandau Richard J.
Published: February 2005

The endocervical epithelium of long‐term ovariectomized rabbits treated for 1–10 days with 5 μg of estradiol benzoate every 12 hr has been studied by light and electron microcopy. In addition, morphometric data on ciliated and nonciliated cells of rabbits treated for 2, 6, and 10 days are compared to those on untreated ovariectomized, estrous, and ovulatory rabbits.

The percentage of ciliated cells increases after ovariectomy to 76.3% and that of secretory cells decreases to 23.7% as compared to estrous controls. Treatment of ovariectomized rabbits with estradiol results in a gradual increase in ciliated and secretory cell area, height, and nuclear area. After 10 days of treatment, cell areas are significantly larger than those in the ovulatory or estrous controls; cell height and nuclear areas have returned to preovariectomized levels; and the percentages of ciliated and secretory cells have reached those of estrous levels. Estradiol stimulates mitotic division of secretory cells but affects ciliogenesis minimally.

In ciliated cells, estradiol treatment results in a modest increase in polysomes and granular endo‐plasmic reticulum and in striking increases in the size of the Golgi complex and in the number of lipofuscin bodies as compared to those in the ovariectomized controls.

In secretory cells, estradiol treatment brings about an increase in the numbers of polysomes, Golgi complexes, and cisternae of the granular en‐doplasmic reticulum, in the sizes of the nucleoli, and in the amount of euchromatin. Secretory granules appear in some cells after 2 days of estradiol stimulation and increase in number through 10 days of treatment. Perinuclear granules are more pleomorphic and heterogeneous in structure and more numerous in the 6‐ to 10‐day‐estradiol‐treated than in ovulatory animals, and they may function as lysosomes degrading excess secretory product. Deep apical concavities of the secretory cells occur most often after 2 and 6 days of treatment.
Mediobasal prosencephalic defects, including holoprosencephaly and cyclopia, in relation to the development of the human forebrain
Authors: Müller Fabiola , O'Rahilly Ronan
Published: February 2005

Four very early synophthalmic embryos were studied in serial sections and reconstructed graphically by the point‐plotting method. Three belonged to stage 16 (5 weeks) and one to stages 19/20 (7 weeks). Recently completed accounts and reconstructions of the normal brains of staged human embryos served as controls for comparison with the abnormal examples. The embryos shared in common: holoprosenceph‐aly, arhinencephaly sensu stricto (absence of olfactory nerve fibers, bulbs, and tracts), presence of a proboscis, synophthalmia with two lens vesicles, a retarded telencephalic wall, absence of the mediobasal part of the telencephalon (the future septal area and the commissural plate: future anterior commissure and corpus callosum), irregularity of the diencephalon, mensural changes in the brain, absence of the rostral part of the noto‐chord and consequent cranial defects, and small ganglia of the cranial nerves. Where it could be determined (at least in the three less advanced specimens), the adenohypophysial primordium was either small and isolated or was absent; a ten‐torial condensation appeared to be missing; and disturbances of the primordia of the orbital muscles and their innervation were noted. The corpus striatum is single and corresponds to only the di‐encephalic part (medial eminence) of normal embryos. Interference with induction by the pre‐chordal plate at or before stage 8 (18 days) would be expected to affect the future mediobasal part of the neural plate (median prosencephalic dysgen‐esis) and the future optic primordium (cyclopia sensu stricto). Insufficient formation of material from the prechordal plate would account for disorders of the orbital musculature and, possibly, for inadequacy of the tentorium cerebelli. Disturbance a couple of days later (stage 9) would result in synophthalmia. Cyclopia and synophthalmia entail arhinencephaly and holoprosencephaly, both of which may arise independently. Defective distribution of the cephalic mesenchyme points to a derangement of the mesencephalic neural crest (stages 10 and 11), causing such features as an incomplete chondrocranium and reduction in size of the ganglia of the cranial nerves. Failure of bilateral division of the telencephalon would occur at or before 4 weeks (stages 13 and 14). It is concluded that all the above conditions arise during the first 4 postovulatory weeks.
Individual rabbit cardiac myocytes have different thresholds for alpha myosin heavy chain regulation by thyroid hormone
Authors: Lin Zhaoying W. , Wenderoth Mary P. , Eisenberg Brenda R.
Published: February 2005

Myocytes in adult rabbit ventricle express an α and a β form of myosin heavy chain (MHC). The α‐MHC distribution detected with indirect immunofluorescence has been found in different proportions in adjacent myocytes producing a mosaic staining pattern. The basis for cell‐specific expression of the α‐MHC isoform is not known. Since thyroid hormone is a major regulator of myosin gene expression, we varied the plasma thyroid level and followed the α‐MHC content within a population of myocytes. Ventricular myocytes were induced to become 100% β‐MHC by placing the rabbits on a 0.15% propylthiouracil diet for 70 days. L‐triiodothyronine (LT3) over a dose range of 1 to 10 μg/kg/day was delivered by an osmotic minipump for 5 days, with actual serum levels confirmed by LT3 radioimmunoassay to be in the range of from 115 to 1,230 ng/dl. The amount of α‐MHC that returned was estimated in randomly selected cells by measuring the relative intensity of the fluorescence‐tagged secondary antibody. The normal mosaic pattern of α‐MHC expression in the left ventricle returned with an LT3 dose of 2–5 μg/kg/day. The first myocytes to express α‐MHC were in the subepicardium and did so at a LT3 serum level of 115 of ng/dl. All myocytes of the ventricular wall expressed α‐MHC at serum levels above 1,230 ng/dl. These data are interpreted to show that the variation of myosin isoform content seen in the adult heart is indicative of heterogeneity of thyroid sensitivity, with the threshold for serum LT3 being between 115 and 370 ng/dl.
Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: I. Normal development
Authors: Brody Joel R. , Cunha Gerald R.
Published: February 2005

Myometrial development from the prenatal to adult period was examined in rats and mice (1) by histologic and immunocytochemical methods with anti‐actin, ‐vimentin, and ‐laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells; and (2) by morphometric procedures to assess quantitatively the expression of cellular orientation in the emerging inner circular myometrial layer. Uterine mesenchymal cells initially were uniformly vimentin‐positive, undifferentiated, and randomly oriented during the late fetal period. By the early neonatal period, three mesenchymal layers became recognizable histologically, the middle one of which (prospective circular myometrium) developed distinct circular orientation and differentiated into a layer composed of actin‐positive smooth muscle cells. The cells of the inner mesenchymal layer initially exhibited radial orientation. By 10 days postpartum, the outer longitudinal mesenchymal layer differentiated into bundles of smooth muscle cells representing the longitudinal myometrium. The inner mesenchymal layer remained vimentin‐positive and differentiated into the randomly ordered endometrial stroma. The cells of the middle and outer mesenchymal layers that were destined to form myometrium initially expressed vimentin throughout and then coexpressed vimentin and actin, but with time vimentin staining disappeared in the maturing smooth muscle cells as they expressed actin.
Trophoblast differentiation during the transition from trophoblastic plate to lacunar stage of implantation in the rhesus monkey and human
Authors: Enders Allen C.
Published: February 2005

The transition from the tropho‐blastic plate stage to the early lacunar stage was examined in a series of implantation sites from the rhesus monkey, timed on the basis of the preovulatory estrogen peak, and prepared for transmission electron microscopy. This transition was compared with specimens from stage 5a, b, and c in the Carnegie collection of human embryos. The transition was marked by the differentiation of a new type of syncytial trophoblast—namely, a unilaminar microvillous polarized syncytium, which developed throughout the trophoblastic plate, forming characteristic intrasyncytial clefts. The rapid development of this type of syncytium created a nonclotting chamber for maternal blood wherever trophoblast intrusion into maternal vessels created confluence. Although the nature of the material in the Carnegie series precluded cytological characterization of the trophoblast, there is evidence that a similar transition occurs in human trophoblast and that in the human also the appearance of lacunae marks a change from an early invasive trophoblast to a situation in which growth is more significant.
Reconstitution of endoplasmic reticulum in rapidly dividing cells of early Xenopus embryos
Authors: Dominguez J. Manuel , Paiement Jacques
Published: February 2005

The cytology of early blastomeres of Xenopus laevis embryos was examined. Particular attention was given to the organization of the nuclear envelope of karyomeres (chromosome vesicles) and the endoplasmic reticulum (ER) at different stages in early cleavage cycles of frog development. Nuclear envelope formation was observed to occur rapidly around individual chromosomes during early anaphase, and karyomeres fused subsequently to yield the final nucleus during telophase. Endoplasmic reticulum in the perinuclear cytoplasm was observed to be vesicular during metaphase and cisternal in form during telophase. Following microinjection of rat liver rough microsomes into early blastomeres, heterologous ER components were identified by electron microscope immunocyto‐chemistry. The foreign ER was observed as large, reconstituted cisternae at stages in the cell cycle when the nuclear envelope was intact. Therefore, transplanted ER maintained the capacity to reconstitute in the cytoplasm of a rapidly dividing cell. In an attempt to better assess ER structure at the metaphase stage of the cell cycle, we next slowed down the division process by treating Xenopus embryos with anti‐microtubule agents. Treatment with critical concentrations of colchicine, nocodazole, or vinblastine led to cleavage arrest but not to inhibition of the nuclear cycle. Following such treatment, homologous ER was observed in a vesicular form at all stages of the nuclear cycle. Heterologous ER, however, identified by immunocyto‐chemistry in microinjected cells treated with nocodazole, displayed both vesicular and cisternal forms. We conclude that microinjected ER membranes exhibit cell‐cycle‐specific behavior, which is different from that of the host cell ER.
Endothelial attachment and plasmalemmal apposition in the transcellular movement of intravascular leukemic cells entering the myeloid parenchyma
Authors: De Bruyn Peter P. H. , Cho Yongock , Michelson Sonia
Published: February 2005

The plasmalemmal relationship between metastases‐forming leukemia cells and myeloid sinus endothelium during the transmural passage of the leukemia cells has been studied in rat bone marrow. After the myeloid vascular system was freed from normal circulating blood cells, the bone marrow was perfused with a suspension of leukemia cells derived from an ascites tumor. The bone marrow was then fixed by perfusion with double aldehyde with and without the addition of tannic acid.

Leukemia cells were seen adhering to the adluminal aspect of the sinus endothelium and in all stages of endothelial penetration. The penetration of the sinus wall was independent of endothelial junctions; i.e., the transmural passage into the myeloid parenchyma was transcellular. At these sites, there were restricted areas of close plasmalemmal appositions of the two cell types where the intraplasmalemmal space was reduced to 2.3 nm. This space was interrupted by electron densities of 5 nm diameter and spaced 9 nm center to center. These close plasmalemmal appositions extended over distances ranging from 150 nm to 200 nm. It is suggested on the basis of the structural similarity that these heptalaminar complexes of close plasmalemmal apposition represent the structural equivalent of gap junctions and may be sites of intercellular communication requisite for transmural passage.

When tannic acid was added to the fixative, there were extended areas of apparent fusion of the plasmalemmas of the two cell types, at the sites both of adhesion and of endothelial penetration. This fusion was limited to the outer leaflets of the two plasmalemmas, resulting in a single pentalaminar complex. These pentalaminar complexes extended over decidedly longer distances than the presumed gap junctions seen in the non‐tannic‐acid‐fixed material. The tannic acid material did not show the heptalaminar gap junction type of plasmalemmal apposition. It is believed likely that the tannic‐acid‐induced pentalaminar complexes may incorporate the smaller heptalaminar ones. The factors underlying the plasmalemmal configurational differences between the tannic acid and non‐tannic‐acid material remain undetermined.
Role of nerve and muscle factors in the development of rat muscle spindles
Authors: Kucera Jan , Walro Jon M. , Reichler Judith
Published: February 2005

The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent‐muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (α) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (γ) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when γ axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not γ motor innervation. However, transient innervation of future bag2 fibers by α axons suggests that both sensory and α motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.
Ultrastructural architecture of pulmonary small‐granule cell clusters in adult syrian golden hamster
Authors: Pearsall Allan Dorland
Published: February 2005

Throughout the epithelial lining of the respiratory system is a class of cells with characteristics similar to Amine Precursor Uptake and Decarboxylation (APUD) polypeptide hormone‐producing cells. In the intrapulmonary airways, these small‐granule cells (SGCs) occur either singly or in organized clusters. Although no specific peptide has yet been identified, subclasses have been postulated based on granule geometry or light microscopic staining. The present study characterizes the architectonic and cellular organization of clustered SGCs in the adult Syrian golden hamster. Two morphologically distinct cells can be defined in such clusters, “light” and “dark.” This distinction was based primarily on differences in the electron density of the cytoplasmic matrix rather than on the remarkable variations in cellular organelles or dense‐core secretory vesicles. Both cell types were normal as judged by uniform spherical nuclei, chromatin organization, and distribution of cellular organelles. The “dark” cells, however, presented the profile of a cell actively involved in synthesis with a markedly dilated perinuclear cisterna and endoplasmic reticulum. Additionally, the “dark” cells contained membrane‐delimited structures containing concentric membranous whorls, clear vacuoles, and lipofuscin granules. Occasionally, cells were observed to contain features of both cell types, suggesting that they may represent a continuum of common cell lineage. Accordingly, in the absence of additional morphologic or biochemical data, the “light” and “dark” cells most probably correspond to different stages of functional activity or age‐related changes of a single type of cell. Unmyelinated nerve endings were occasionally interposed between cells, but synaptic specializations were not observed. Beneath the clusters, nerve fibers were also present, but they were never observed to penetrate the basal lamina or contact any of the SGCs. Of equal occurrence were elements of the vascular system and smooth muscle, suggesting that some SGCs in the adult hamster may function in a paracrine or endocrine manner. Such knowledge is essential to any study attempting to delineate the functional role or roles of these enigmatic organoids.