Heterogeneity of apical glycoconjugates in kidney collecting ducts: Further studies using simultaneous detection of lectin binding sites and immunocytochemical detection of key transport enzymes

Summary In the search for a functional role for the polarized glycoconjugates of rat collecting duct epithelial cells, the relation between binding of various lectins and expression of cellular transport enzyme profile of the cells was studied. For this purpose, principal and intercalated cells of rat kidney collecting duct were identified by morphological criteria and by their immunocytochemically determined content of (Na⁺+K⁺)-ATPase and carbonic anhydrase (CA II), respectively. VariousN-acetylgalactosamine-specific lectins such as those fromHelix pomatia andMaclura pomifera revealed heterogeneity among both principal and intercalated cells, whereas -N-acetylgalactosa nine-specific lectin fromDolichos biflorus andVicia villosa bound preferentially to principal cells. Still another lectin fromArachis hypogaea reacted with most collecting duct cells in the cortex and outer medulla, but only with a subpopulation of cells in the inner medulla. Interestingly, some lectins reacted exclusively with the apical aspect of the collecting duct epithelial cells, whereas others revealed both an apical and basolateral distribution of lectin reactive glycoconjugates. The results thus show subtle differences in the glycocalyx structure of principal and intercalated cells and differences in the intracellular polarization of glycoconjugates of these cells. Thus, lectins may be useful tools in the study of the molecular mechanisms which establish and maintain the polarized functions of principal and intercalated cells.     

Lectin histochemistry of complex carbohydrates in human cervix

Summary Complex carbohydrates in the human cervix were studied histochemically using lectins, conjugated to horseradish peroxidase and correlated procedures. Stratified squamous epithelium of the exocervix and columnar epithelium of the endocervix in some, but not all specimens showed staining for terminal -N-acetyl-d-galactosamine, -d-galactose, -d-galactose and -l-fucose. the staining for -N-acetylgalactosamine and -galactose, the terminal sugars in blood group A and B antigens respectively, corresponded to a large extent with ABO blood type. One exception was the lack of staining for terminal -N-acetylgalactosamine in endocervical secretions in three of nine blood type A patients. A second exception was the staining for terminal -galactose in endocervical secretions in about half of blood type O and A specimens. The type and amount of glycoprotein formed by endocervical columnar cells differed according to location in superficial compared with deep portions of the glands and according to location at the junction with exocervix compared with the more internal regions. Staining of endothelial cells for blood group A and B antigens was confined to subjects of blood type A and B respectively, although three of nine type A specimens showed no lectin reactivity for group, A antigen. Endothelial cells evidenced affinity forUlex europeus I agglutinin demonstrative of fucose in all specimens. Mast cells disclosed lectin affinity consistent with the presence of terminal or internal mannose orN-acetylglucosamine residues. Two blood type O specimens were examined with conjugated lectins at the ultrastructural level. Secretory granules stained for content of terminal -galactose, -galactose and fucose. These results support and concur with biochemical studies of complex carbohydrates in human cervical tissues. They reveal, in addition, the location of the blood group antigens in the human exocervix and endocervix and the marked heterogeneity among endocervical columnar cells in glycoprotein production.     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

Selection and characterization of transferrin receptor mutants using receptor-specific antibodies

Lymphoma cell lines were selected by growth in transferrin receptor-specific antibodies and in transferrin receptor-specific antibody coupled to ricin toxin. Sequential selections were used to isolate lines with multiple mutations affecting the transferrin receptor molecule. Mutant cell lines were characterized by their growth in antibody and their antibody-binding properties. Two basic types of mutations were found. One type resulted in the loss of a binding determinant for the antibody used for selection on one of the two transferrin receptor allelic products. The other type of mutation resulted in the loss of cell-surface expression of the entire gene product of one of the transferrin receptor alleles.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

Relationship between foliar composition of red-pine and jack-pine seedlings and vulnerability to Lophodermium needle-cast disease

Summary The Mn and Al content of needles from two-year-old jack pine (Pinus banksiana) seedlings was found to be more than twice that of red pine (Pinus resinosa). It is speculated that the high Mn and Al content of jack pine seedlings may impart some degree of resistance to needle cast disease caused byLophodermium pinastri. Contribution from the Soils Dept., Univ. of Wis., Madison, in cooperation with and supported in part by the Wisconsin Dept. of Natural Resources. Publication approved by the Director of the Wisconsin Agricultural Experiment Station.     

Funktionelle Morphologie des Geruchsorgans und Histologie der Kopfanhänge der NasenmuräneRhinomuraena ambonensis (Teleostei,Anguilliformes)

Zusammenfassung 1. An drei, wahrscheinlich adulten Exemplaren der Nasenmuräne,Rhinomuraena ambonensis Barbour, wurden die funktionelle Morphologie des Geruchsorgans sowie dessen Histologie und die der bartelartigen Kopfanhänge untersucht.2. Die Ränder der vorderen Riechöffnungen sind zu trichterförmigen Hautlappen ausgewachsen. Die in ihrer Ausrichtung und Form durch Körperbewegungen kaum beeinflußbaren Trichter leiten auch im Ruhezustand des Tieres Wasser in die Riechhöhle, da ihre weite Öffnung im Strömungsbereich des Atemwassers liegt. Auf diese Weise erfolgt eine effektive Ventilation der Riechhöhle.3. Im mehrschichtigen Epithel der Trichter liegen Sekretzellen und vereinzelt Sinnesknospen; unmittelbar über den Epithelbildungszellen verlaufen Nervenfasern. Im Bindegewebe, das beidseitig von Epithel bedeckt ist, liegen Gefäße, Nervenbündel (Nervus trigeminus) und Pigmentzellen. Muskelgewebe fehlt. Neben dem Bindegewebe gibt es kein anderes Stützgewebe.4. Die beiden Riechhöhlen sind von extremer Ausdehnung. Die Zahl der lamellenartigen Riechfalten, die in zwei Doppelreihen nach dem 90°-Typ liegen, beträgt ca. 200. Zwischen den Innenrändern der Falten einer Doppelreihe, dorsal von der Mittelraphe, erstreckt sich eine zylindrische Rinne, die der Duftwasserpassage dient.5. Die Riechfalten sind bis auf die lateralen, dorsalen Teile und Teile der ventralen Faltenansätze kontinuierlich mit Riechepithel bedeckt. Der histologische Aufbau des Riechepithels entspricht dem des Aals. Die Rezeptorendichte schwankt zwischen 2–3×10⁴ pro mm². Im Verhältnis zur Riechhöhlenausdehnung ist die Riechfeldgröße erheblich.6. Tuscheexperimente zeigten, daß — ausgelöst durch die in die Längsrinne der Riechhöhle ragenden fingerförmigen Innenränder der Riechfalten — Mikroturbulenzen entstehen, die neben der Flimmerbewegung durch Kinocilien für die Mikroventilation des Faltensystems sorgen.7. Das auf der Symphyse des Oberkiefers zwischen den Trichterbasen sitzende Rostrum ist eine Hautprojektion, deren bindegewebiger Zentralteil ein mehrschichtiges Epithel trägt. Im Bindegewebe sind Gefäße, feine Nerven und Pigmentzellen eingebettet. Im Epithel finden sich geschmacksknospenähnliche Sinnesorgane. Prinzipiell haben die drei Mandibularbarteln den gleichen histologischen Aufbau wie das Rostrum. Auch ihr Epithel weist Sinnesknospen auf, die wie jene des Rostrums und der Trichter vielleicht als chemische Sinnesorgane beim Nahrungstest fungieren.8. Nach dem Ergebnis vorliegender Untersuchungen wirdRhinomuraena ambonensis in die Gruppe der makrosmatischen Knochenfische eingereiht.

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Functional morphology of the olfactory organ and histology of the head appendages of the moray eelRhinomuraena ambonensis (Teleostei, Anguilliformes)

In three individuals of the moray eelRhinomuraena ambonensis (Barbour), the functional morphology and histology of the olfactory organ as well as the histology of the different head appendages were investigated. The edges of the anterior nares are extremely extended, forming a funnel-like wide opening through which the odour water passes. Even when the fish does not move, an effective ventilation of the olfactory chambers is maintained by a permanent water current towards the funnel opening induced by the peristaltic movements of the musculature of the gill chamber (principle ofBernoulli). The histological structure of the head appendages (funnels, rostrum and mandibulary barbels, which are all skin projections) is described. The epithelia of all these appendages bear sensory organs which are similar to taste buds. The two olfactory organs are of extremely large size. Each chamber contains about 100 olfactory lamellae which are arranged in two lines (90°-type;Holl 1965). A cylindrical cavity extends between the inner edges of the olfactory lamellae through which the water runs to the posterior nare. Interlamellar microventilation is produced by numerous turbulences which are caused by the edges of the olfactory lamellae and by the kinocilia of the olfactory epithelium. The histological structure of the olfactory epithelium is similar to that ofAnguilla anguilla. The different results demonstrate thatRhinomuraena ambonensis is probably a macrosmatic fish.

     

Standardization of the Feulgen-Schiff technique

Summary Four fuchsin analogues (Pararosaniline, Rosaniline, Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches.Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 m.Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Immunohistochemical localization of vimentin in the gerbil inner ear

Cells containing immunoreactive vimentin-type intermediate filaments (IF) were identified in paraffin sections and whole-mount preparations of the gerbil inner ear. Most connective tissue cells showed positive immunostaining, although one unusual class of stromal cell lacked vimentin. Several different types of epithelial cells contained high levels of vimentin. In the cochlea, Deiters' cells, inner phalangeal cells, Boettcher's cells, some outer sulcus cells, and the intermediate cells of the stria vascularis showed strong immunoreactivity. Strial basal cells exhibited weaker and less consistent staining. Neither inner nor outer hair cells were stained. In the vestibular system, hair cells with a morphology and location more characteristic of type I than of type II cells showed strong immunostaining for vimentin. Supporting cells in vestibular neurosensory epithelium stained with less intensity. These results were surprising because epithelial cells in vivo only rarely express vimentin-type IF. Although the functional significance of vimentin remains to be established, its presence in some but not other highly specialized cell types provides an excellent marker for investigating the lineage and morphogenesis of the complex inner ear tissues.