The Histochemical Demonstration of Hemoglobin in Blood Cells and Tissue Smears

The demonstration of intracellular hemoglobin in permanent preparations has long been a problem. The affinity of hemoglobin for iron hematoxylin is well known but this stain also colors yolk, chromatin, and other structures and is therefore not a reliable criterion. The presence of hemoglobin has been associated with an acidophil cytoplasm which stains a characteristic color, but a careful inspection of living cells in early hematopoetic or embryological stages demonstrates that hemoglobin is present in the erythrocytes which are quite basophilic. In the course of some research on the blood of embryonic frogs it became desirable to demonstrate the presence of hemoglobin in cells by means of a specific staining reaction.     

Blood Cell Ultrastructure of the Ascidian Botryllus schlosseri L. II. Pigment Cells

Colonies of Botryllus schlosseri L., bred in the laboratory and genetically selected as regards the blue and/or reddish pigments, were used. The following phenotypes were investigated under the electron microscope: (a) blue colonies without reddish pigment; (b) reddish colonies without blue pigment; (c) colonies with both blue and reddish pigments; (d) colonies with neither blue nor reddish pigments. In the pigmented colonies, a specialized blood pigment cell type was recognized that, in giant membrane-limited vacuoles, contained a great number of granules. In general, the granules were similar in size, not individually limited by a membrane and were made up with electrondense material often arranged in concentric rings. Although there could be some variability within the same cell, in each phenotype the granules displayed a characteristic pattern so that the differences in colour of the granules, as seen in vivo, were paralleled by differences in the ultrastructural architecture. In the unpigmented colonies also, granulated vacuolar cells, rare in number but morphologically comparable to the pigment cells, were seen. On the basis of these results, the hypothesis of the existence of a prospective pigment cell and of a common origin for all the pigment cells of B. schlosseri is discussed.     

A Monoclonal Antibody Specific to Embryonic Trunk-Lateral Cells of the Ascidian Halocynthia roretzi Stains Coelomic Cells of Juvenile and Adult Basophilic Blood Cells

In spite of extensive knowledge on the structure and function of ascidian blood cells, little is known about their embryological origin. In the present investigation, the developmental fate of trunk lateral cells (TLCs) was explored using a specific monoclonal antibody. TLCs comprise a group of undefined embryonic cells of the ascidian Halocynthia roretzi , which arise from the A7.6 blastomeres of a 64-cell embryo. The antigenicity first appeared at the middle tailbud stage in a pair of TLC-clusters situated lateral to the brain stem of the bilaterally symmetrical embryo. The position and number of stained cells did not change during later embryogenesis until hatching. After hatching, the stained cells were found in the entire trunk region of the swimming larva. After metamorphosis, cells that expressed the antigen were present within the coelom and within the tunic layer of the juvenile. In addition, the antibody stained adult basophilic blood cells. These observations suggest a relationship of this group of embryonic cells with the prospective blood forming mesenchymal cells.     

Distribution, Histochemical and Enzyme Histochemical Characterization of Mast Cells in Dogs

This study describes the distribution, proteoglycan properties and protease activity of mast cells from 15 different dog organs. In beagles and mixed breed dogs, staining with Alcian Blue-Safranin O revealed mast cells in all the organs examined. However, their numbers varied and they demonstrated unique localization patterns within some of these organs. Berberine sulphate fluorescence-positive mast cells were observed in the submucosa, muscularis and serosa of the intestines, as well as the tongue and liver (within the connective tissue). Mast cells within the intestinal mucosa were negative for, or demonstrated weak, berberine sulphate staining. Heterogeneity of mast cells in terms of the proteoglycans contained within their granules was further confirmed by determination of critical electrolyte concentrations (CECs). The CECs of mast cells within the connective tissue of several organs, including the intestines (submucosal and muscularis-serosal layers) were all greater than 1.0 M. The results from CEC experiments together with berberine staining indicate that heparin was contained within their granules. Relative to the CECs of mast cells in other organs, mast cells in the intestinal mucosa exhibited lower CECs, suggesting that the proteoglycans within their granules were of lower charge density and/or molecular weight. Although mast cells were classified into two groups by proteoglycans within the granules, enzyme histochemical analysis in beagles revealed three subtypes of mast cells: chymase (MC(C)), tryptase (MC(T)) and dual positive (MC(TC)) cells. There was no correlation between the proteoglycan content and enzyme properties of the mast cell granules.     

Fine Structural Details of Tannin Accumulations in Non-Dividing Cambial Cells

The fine structure of autumn cambial cells of Aesculus hippocastanumand Populus x euramericana reveals the presence of electron-densebodies, other than lipids, in both the cytoplasm and vacuoles.These deposits are identified as tannins following a cytochemicaltest with ferrous sulphate. Small tannin bodies associated withthe strands of ER and inside small vesicles are often evidentin the cytoplasm. The cytoplasmic tannins are deposited intovacuoles by fusion of the tannin-containing vesicles with thetonoplast. The positive reaction of tannin inclusions with periodicacid-thiocarbohydrazide-silver proteinate suggests their polysaccharidicnature. The observations support the role of ER in tannin synthesisand deposition into the central vacuole. Aesculus hippocastanum, Populus x euramericana, cambial cells, tannin, vacuoles     

Fine structure observations on the chromosomes in the spermatids of the Ascidian,Boltenia villosa

Electron micrographs of the relatively young spermatids of the Ascidian, Boltenia villosa, reveal that its chromosomes exist in the form of 120–180 Å fibers which are easily seen to be composed of two subunits of about 50–70 Å each. Fibers of the diameter of such subunits have never been seen as single entities without a mate. Subunits in pairs seen imbedded in areas of fused chromosomes only measure 30–40 Å, probably due to the loss of some protein. It is pointed out that the chromatin goes into the sperm as chromosomes in two-chromatid state, and that each chromatid probably contains only one Watson-Crick double helix.     

Helper Function of Follicle Cells in Sperm-Egg Interactions of the Ascidian, Ciona intestinalis

Studies were made on the involvement in sperm-egg interactions of follicle cells of Ciona intestinalis , which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N-acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA-binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.     

A Quantitative,Comparative Study of Element Variations Found within the Range of Blood Cells from the Tropical Ascidian Phallusia philippinensis,using the Nuclear Microscope

The present study examines the concentrations of vanadium, bromine and sulphur contained within cryofixed/freeze-dried blood cells of the ascidian Phallusia philippinensis. Elemental profiles of seven cell types were obtained using the National University of Singapore nuclear microscope, in order to ascertain the cell types predominantly involved in accumulation. Morula cells were found to contain the following mean values (in ppm dry weight); 7878 vanadium, 34484 bromine and 61078 sulphur. Signet ring cells contained 5191 vanadium, 23945 bromine and 15281 sulphur. Compartment cells had 606 vanadium, 20700 bromine and 24309 sulphur. Other less abundant cell types such as lymphocytes, macrogranular amoebocytes, carotenoid pigment cells and granular amoebocytes were also analysed and found to contain (in ppm) 4384, 6652, 2366 and 10246 vanadium, 19652, 15630, 5964 and 11735 bromine and 13289, 15309, 3106 and 42968 sulphur, respectively. Sulphur occurred in high levels in all cell types, which could indicate its involvement in the vanadium concentration process, while bromine, incorporated into complexes, may be utilised for anti-fouling rather than as a deterrent to predators.     

Fine Structural Changes in Barley Aleurone Cells During Gibberellic Acid-induced Enzyme Secretion

The fine structure of barley aleurone cells was studied in theenzyme secretion phase. An ultrastructural feature of this phaseis the formation of stacked rough endoplasmic reticulum (rER),for such a structure was never found in cells during the enzymesynthesis phase. Other structural features frequently observedin the secretion phase were amoeboid-shaped nuclei, the stackedrER wound round the nucleus and mitochondria, and a stream ofthe stacked rER directed to the plasmamembrane. Hordeum vulgare L, barley, aleurone cells, enzyme secretion, gibberellic acid     

Endocrine Cells in the Oesophagus of the Ascidian Styela clava,a cytochemical and immunofluorescence study

Summary Immunocytochemical studies have demonstrated the occurrence of an insulin-immunoreactive cell type in the oesophageal epithelium of the Ascidian Styela clava. Staining with aldehyde fuchsin has demonstrated a number of similar small, triangular, cells located on the basement membrane, which may have an endocrine function. Argyrophilic cells have also been found, suggesting the presence of a second endocrine cell type. The absence of argentaffin cells has led us to believe that the cells so far observed do not produce biogenic amines such as 5-HT (5-Hydroxytryptamine). The nature of these cells is discussed with reference to endocrine-like cells found in the digestive tracts of other protochordates.Animals were collected by courtesy of the Admiralty Marine Trials Station, Portsmouth. This research was carried out during the tenure of an S.R.C. grant no. B/RG 82919 to one of us (M.C.T.). —The localisation of polypeptide hormones in the pharynx and gut of protochordates     

唾液葡萄糖与血液葡萄糖的检测与相关性分析

探测糖尿病人的唾液葡萄糖与血液葡萄糖浓度的相关性。采用葡萄糖氧化酶-过氧化物酶法测定唾液中的葡萄糖含量。将唾液和血液葡萄糖的检测结果进行比较,得出相关性为0.764。唾液葡萄糖水平(salivaryglucose level,SGL)和血液葡萄糖水平(blood glucose level,BGL)之间有一个明显的相关,这一关系有望成为对血液葡萄糖水平的一个估计依据。     

Ascidian Larvae: The Role of Test Cells in Preventing Hydrophobicity

Abstract Ascidian test cells co-differentiate on the surface of each ovarian oocyte beneath the vitelline coat. They become vacuolated and later occupy the perivitelline compartment of each egg and embryo. In some species, their vacuoles contain submicroscopic granules or filaments called ‘ornaments’ and acidic glycosaminoglycans. These test cells deposit their products on the surface of the larval tunic in late embryogenesis. In these species, the test cells are lost at hatching. In other species, the test cell vacuoles contain acidic glycosaminoglycans, but no ornaments. In these species, the test cells attach to the larval tunic and probably secrete acidic glycosaminoglycans. We deprived the embryos of seven species of ascidians of their test cells and vitelline coats during midembryogenesis. After completing their development, the larvae of both kinds of species were hydrophobic. They easily become trapped on the surface of sea water in cultures. Normal larvae (controls), bearing test cell secretions, are hydrophilic and never become trapped. We infer that negatively charged secretions of the test cells make normal larvae hydrophilic. Some molgulids with direct development have no test cells, no fins and no swimming larva. We reason that the test cells of these species may have been lost during evolution because they no longer had an important role in preventing hydrophobicity.     

Analysis of Chemotaxis in White Blood Cells

We wish to report the development of an assay system for the study of white blood cells in vitro. With this system we have demonstrated that a yet unidentified substance found in red blood cell membranes and cyclic adenosine monophosphate (cAMP) cause the chemotactic response in white blood cells. We have not yet determined whether the substance released from the membrane is cAMP.     

Structural and Histochemical Changes in Palisade Cells of Prosopis juliflora Seed Coat in Relation to its Water Permeability

Histochemical investigations on the Prosopis juliflora seedcoat indicate the occurrence of a hydrophobic ‘strip’as the primary water barrier. Its position and the structureand histochemistry of the palisade cells of the seed coat differaccording to their location on the seed. These differences maybe responsible for differences in the water permeability ofvarious parts of the seed coat. In particular, parts of theseed coat in which the hydrophobic ‘strip’ is locatedmore superficially tend to be more water impermeable than partslike the chalaza, in which the ‘strip’ is more deeplylocated within the palisade cells. Prosopis juliflora, seed coat impermeability, palisade cells, hydrophobic ‘strip’     

A Fine Structural Analysis of the Statocyst in Turbellaria Acoela

Ferrero, E. (Istituto di Biologia Generale, Universith di Pisa, Pisa, Italy.) A fine structural analysis of the statocyst in Turbellaria Acoela. Zool. Scr. 2 (1): 5–16, 1973.—The fine structure of the statocyst components in the acoelan Convoluta psammophila is described, namely: capsule, parietal cells, lithocyte, and the statolith. The absence of ciliary structures, the highly developed endoplasmic reticulum of the lithocyte and the layered texture of the statolith are remarkable. A functional interpretation of the muscles inserted on the statocyst and of the nerve bundle running nearby is suggested. The morphological similarities and differences between the acoelan statocyst and the statocyst of lower and higher invertebrate phyla are discussed.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.     

Fine structure and differentiation of Ascidian muscle. I. Differentiated caudal musculature of Distaplia occidentalis tadpoles

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.     

Histochemical and Morphological Characteristics of Canine Cardiac Mast Cells

Cardiac mast cells have been recently isolated and characterized in humans, however canine cardiac mast cells have not been investigated. The objective of this study is to describe the histological and morphological characteristics of canine cardiac mast cells and examine the potential usefulness of canine models in investigating the role of mast cells in cardiovascular pathology. Canine cardiac mast cells could be easily identified by staining with Toluidine Blue or FITC-avidin. Using Toluidine Blue staining, we demonstrated fewer mast cells in formalin-fixed samples than in specimens fixed in Carnoy's, thus identifying a formalin-sensitive mast cell population in the canine heart. Mast cells were equally distributed in atria and ventricles with approximately 50% showing a perivascular location. Using enzyme-histochemical techniques, we detected tryptase and chymase activity in canine cardiac mast cells. Ultrastructural studies identified mast cells as granular cells with an eccentric non-segmented nucleus. Immunohistochemistry with the macrophage specific antibody AM-3K demonstrated that resident cardiac macrophages were 1.9 times more numerous than mast cells, also showing a predominantly perivascular (60%) location. Perivascular macrophages were more often periarteriolar, whereas perivascular mast cells were more often located along small veins and capillaries. Due to their ability to release cytokines and growth factors and their strategic perivascular location, resident cardiac inflammatory cells, such as mast cells and macrophages, may be important in pathological processes causing myocardial inflammation and fibrosis. Furthermore, mast cell-derived chymase, an important angiotensin II-forming enzyme may have a significant role in regulating the cardiac renin-angiotensin system.     

Monoclonal Antibodies against Differentiating Mesenchyme Cells in Larvae of the Ascidian Halocynthia roretzi

Mechanisms of cell specification of mesenchyme during ascidian embryogenesis are poorly understood. This is because no good molecular markers have been available to evaluate differentiation of the mesenchyme cells. To obtain molecular markers of mesenchyme differentiation, we established monoclonal antibodies, Mch-1 and Mch-3, that recognize antigens present in the mesenchyme cells of the larva of Halocynthia roretzi. The antigens recognized by both antibodies start to be detectable in the mesenchyme cells at the late tailbud stage. The Mch-3 antibody specifically recognized all mesenchyme cells of the larva, whereas the Mch-1 antibody stained the cells only in the anterior portions of mesenchyme clusters in the trunk region of the larva. The Mch-1 antibody also stained trunk lateral cells. In addition, both antibodies recognized the mesenchyme cells in the ventro-lateral boundary between endoderm and epidermis that are migrating to the anterior head region of the larva. The partial embryos that originated from the mesenchymelineage cells at the 8-cell stage expressed the Mch-1 and Mch-3 antigens. The Mch-1 and Mch-3 antibodies will be useful as immunological probes for studying the specification mechanisms of mesenchyme cells.     

Fine Structural Analysis of Animal Cell Surfaces: Membranes and Cell Surface Topography

Current research on the mechanism of transmembrane regulationof topographic modulation at the cell surface is described forthe sea urchin egg and Sarcoma 180 ascites tumor cells of SwissWebster white mice. The transmembrane system is characterizedin terms of three components: glycocalyx, membrane, and cytofibrillarstructures. The importance of membrane molecular architectureper se relative to the other surface components is assessedin terms of freeze fracture analysis of both cell types as wellas concanavalin A (ConA)-mediated long term agglutination, cytochalasinB effects, and other drug-induced changes at Sarcoma 180 cellsurfaces. A quantitative and qualitative assessment of intramembranousparticle (IMP) sizes and density distributions reveals intrinsicstructural changes of the fusing membranes at cortical reactionduring sea urchin egg fertilization and also with the post-fertilizedaccumulation of surface microvilli. Comparable changes in IMPare noted for microvillus retraction and membrane smoothingin Sarcoma 180 cells under a variety of experimental conditions.On the other hand, chemical perturbation of S-180 cell surfacesreveals a rather non-ubiquitous, though identifiable, involvementof microfilaments and no microtubule involvement in these topographicchanges. These observations suggest that the plasma membraneis a dynamic structure poised between "restrictive" and "lessrestrictive" states of fluidity or deformability and, hence,is a determinant component in topographic change.