Fine structure of ureteric duct epithelium in the North Atlantic hagfish (Myxine glutinosa L.)

Summary The epithelial cells lining the ureteric duct in the cyclostome, Myxine glutinosa, have a brush border and show specializations of their apical cytoplasm similar to those observed in absorptive proximal tubule cells in higher vertebrate species. These features and the presence of large and numerous cytosomes, presumed to contain lysosomal enzymes, indicate that the ureteric epithelium has taken over some of the functions of the proximal tubule in the atubular kidney of Myxine. Sparsity of basal cytoplasmic processes and mitochondria in the ureteric duct cells appears to correlate with an inability for active, energy-dependent secretory and ion transport functions.This study has been supported by a grant from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne). The author is indebted to Doctor Bertil Swedmark for permission to use the facilities of Kristineberg Marine Biology Station, Fiskebäckskil, Sweden, where hagfish were caught.     

Ultrastructure of the tubule of the aglomerular teleost Nerophis ophidion

Summary The tubules in the aglomerular kidney of Nerophis ophidion are composed of cells showing different types of specializations of their plasma membrane. All cells possess a luminal brush border composed of microvilli, and show presence of vesicles with 100 Å thick unit membranes — some containing electron dense material —, tubular elements, multivesicular bodies, and plasma membrane invaginations in their apical cytoplasm. These features suggest an absorptive function of the cells. The apical portions of the cells are supplied with typical cilia.Some cells have abundant basilar plasma membrane invaginations usually lacking cytoplasmic organelles. Other cells appear to form interdigitating basilar cytoplasmic processes containing mitochondria; still other cells have smooth basilar cell membranes. These findings are discussed with reference to the known secretory function of the tubules and are compared with tubular fine structure in other species. It was concluded that urine formation by tubular secretion may occur in cells with different types of basilar cell membrane specializations.The occurrence of coated vesicles associated with invaginated basilar plasma membranes indicates transport of proteins (from peritubular blood vessels ?) at these sites.The tubule cells have abundant smooth surfaced endoplasmic reticulum and large and numerous active Golgi zones.Supported by grants from Fonden til Videnskabens Fremme and Therese och Johan Anderssons Minne. Part of this study was done at the Zoologica Stazione, Naples.The assistance of Miss Britt-Marie Petterson and Mr. Magnus Norman is gratefully acknowledged.     

The demonstration of chloride ions in the “chloride cells” of the gills of eels (Anguilla anguilla L.) adapted to sea water

Summary The ultrastructure of chloride cells in the gills of eels kept in artificial sea water and of a control animal kept in fresh water was studied. In addition to glutaraldehydeosmium tetroxide and simple osmium tetroxide fixation, a special method for the demonstration of chloride ions was used (Komnick, 1962, 1963). Based on the principle of silver chloride precipitation in the presence of chloride ions, the procedure showed positive results in the chloride cells of eels adapted to sea water. Smooth-surfaced tubules of the endoplasmic reticulum contained a material of medium to strong electron density, that was often in communication with the plasma membrane. The same material, always of very high density, was present in the intercellular spaces, thus forming conspicuous lines around cells. The silver precipitate was found very often in large quantities in the pits of chloride cells, having thereby the aspect of a secretory product. However, a direct communication between the system of endoplasmic reticulum tubules containing the silver reaction product with the above-mentioned masses of silver chloride was not demonstrated. Schultz (1958) first published an electron micrograph of these cells. Two features, numerous mitochondria and highly developed agranular endoplasmic reticulum, affirmed by all later investigators, have remained the most important criteria for the identification of chloride cells (Kessel and Beams, 1962; Philpott, 1962; Philpott and Copeland, 1963; Rhodin, 1964; Henrikson and Matoltsy, 1968). Kessel and Beams (1962) and Philpott and Copeland (1963) demonstrated pits, or apical cavities (Oberg, 1967) filled with an amorphous granular substance of medium electron density. (Threadgold and Houston, 1964). These cavities are found in animals adapted to sea water only, and correspond to the excretory vesicles described earlier by Copeland (1948). Philpott and Copeland demonstrated numerous vesicles and tubules in the cytoplasm surrounding the apical cavities and opening into them, possibly contributing to their granular material. Several authors attempted to demonstrate a chloride excretory function of chloride cells with the aid of some histochemical reactions. Copeland (1948), Datta Munshi (1964) and Philpott (1966) used silver techniques     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

The fine structure of the polian vesicles of holothurians

Summary The Polian vesicles are tubules of variable length consisting of four layers: an external peritoneal epithelium, a connective tissue layer, a muscular layer, and an inner epithelium. The two simple epithelia produce a polysaccharide in their Golgi complexes. Their cellular junctions consist of an extensive zonula adhaerens, and an apical septate desmosome. Their surfaces possess microvilli and cilia. A basement membrane is lacking. The muscular layer is composed of paramyosin fibers. The connective tissue layer contains abundant collagen fibers that have a period of 640 Å and are embedded in an acid mucopolysaccharide matrix. Amoebocytes containing waste products and an acid mucopolysaccharide enter the Polian vesicles from the coelom, pass through them, and empty into the lumen along with material secreted by the inner epithelium. While establishing close contact with cells of the muscular layer and inner epithelium the amoebocytes seem to transfer part of their contents to these cells.Polian vesicles appear to be a very primitive excretory organ.Research performed under C.N.R. contract.     

Helical structure in the apical tubules of several absorbing epithelia

Summary Unique and highly ordered structures were discovered in the so-called apical tubules of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes) fixed in situ with a mixture of formaldehyde, glutaraldehyde and osmium tetroxide. The apical tubules were especially numerous in the apical cytoplasm, in addition to the invaginations of the apical plasma membrane, newly formed endocytic vesicles and large endocytic vacuoles. They showed a cylindrical structure (80 nm in diameter) limited by a smooth membrane. Helically wound parallel rows of particles (11 nm in diameter) were found in the apical tubules in close proximity to their limiting membrane. The structure of the helix was determined by following the rows through serial sections and semithin sections, and was found to be a left-handed quadruple helix. These particles surround an electron-lucent cylinder (35 nm in diameter), containing at its center a single row of particles (9 nm in diameter). The apical tubules with the luminal specializations were not seen in continuity with the apical plasma membrane, but were frequently connected with the large endocytic vacuoles, which were present in the deeper levels of the apical cytoplasm. From these observations, it is suggested that the apical tubules are not derivatives of the apical plasma membrane; rather, they represent an intracellular compartment, which is morphologically related to the large endocytic vacuoles.     

On T-tubule openings at the sarcolemma of white fast-twitch muscle fibres in fish and frog

Summary In white muscle fibres of a teleost fish T-tubule openings may occur regularly at all Z-disc levels between adjacent peripheral myofibrils, the T-tubule openings thus occurring at a density of ca. 0.9 m^-2.In frog white fibres, T-tubule openings are infrequently seen in material fixed like the fish material. In material prepared according to the albumin method of Gray (1975, 1976 a, b) which renders the muscle fibres swollen, straight tubules or sometimes chains of vesicles instead are seen opening at the sarcolemmal surface. Such tubules occur at a higher density than expected from experiments with local activation of contraction. Lability and dynamics within the T-system normally and during fixation are discussed.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

On the fine structure of the aglomerular renal tubule in Lophius piscatorius

Summary The fine structure of the secretory tubules in the kidney of the aglomerular goose-fish (Lophius piscatorius) is described. The cells have a pyramidal shape, are joined together by multiple desmosomes, and share as main characteristics: abundant and deep inflections of the basal and lateral cell membranes; coated luminal plasma membranes forming multiple microvilli or a genuine brush border; moderate numbers of comparatively small mitochondria, usually unassociated with the basal and lateral plasma membrane specializations; numerous multivesicular bodies occuring in the apical cytoplasm; abundant large lysosome-like bodies in the intermediate regions of the cytoplasm; and comparatively poor development of endoplasmic reticulum and Golgi apparatus.The observations suggest that the cells perform both absorptive and secretory functions and are metabolically unusually active in autolytic and heterolytic work. Comparisons with other aglomerular species indicate that the ability for active secretory function is not necessarily dependent on a close association between plasma membrane and mitochondria; however, this ability does appear to require a markedly increased basal and/or lateral cell surface created by multiple invaginations of the plasma membrane. The abundance of desmosomes and associated structures appears to represent a unique structural specialization of the goosefish tubule, and indicates that the cells must be firmly anchored to one another to supply a rigid and mechanically continuous lining of the tubule. The multivesicular bodies probably represent endocytic vacuoles which fuse with apical vesicles and invaginate their outer membrane to form the internal vesicles; they appear to transform to ambilysosomes via a function as heterophagosomes and — later — combined hetero- and autophagosomes.Supported by grants from Karolinska Institutet, Fonden til Videnskabens Fremme and Konsul Johannes Fogh-Nielsen og fru Ella Fogh-Nielsens Légat. Part of the study was performed at the Zoological Station at Naples, Italy. The assistance of Mrs. Britt-Marie Karlsson is gratefully acknowledged.     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.     

The fine structure of cephalopod blood vessels III. Vessel innervation

Summary The cephalic aorta of Octopus vulgaris has a fairly complete endothelium lining the lumen, a thick complete basement membrane, a layer of circularly orientated and a layer of longitudinally orientated muscle fibres. Presumptive synaptic endings are of two types. In the circular muscle, axons containing vesicles, contact club-shaped projections of the muscle. The gap between the pre- and postsynaptic membranes is less than 100 Å and in some places apparently forms a tight junction. The second type of ending has been found in the longitudinal muscle; here axons full of vesicles end on the muscle. The ending is enclosed by a mesaxon of muscle and the synaptic gap is approximately 100 Å. In the smaller blood vessels, axons end on myofilament-containing pericytes of blood vessels (equivalent to small arterioles). The endings contain vesicles and have a synaptic gap of 100 Å. Only some of the pericytes seem to be innervated and transmission between one pericyte to another may be mediated by specialized junctions between the cells. The smaller non-myofilament containing vessels (equivalent to capillaries) are not thought to be innervated.We would like to thank Professor J. Z. Young and Dr. E. G. Gray for advice and encouragement, Mrs. Jane Astafiev for drawing Figs. 1 and 12, Mr. S. Waterman for photography, and Miss Cheryl Martin for secretarial assistance.     

The ultrastructure of the male and female prostate of Praomys (Mastomys) natalensis

Summary Male ventral and female prostates of Praomys (Mastomys) natalensis were examined with the electron microscope. The findings support and add to information obtained with the light microscope on tissues from normal, castrated and ovariectomised animals.Our results indicate that although the female prostate may be considered a homologue of the male ventral prostate anatomically and histologically, there are differences in sub-cellular morphology and hormone dependence.Cells of the intact ventral prostate of the male are characterised by prominent dilated Golgi vesicles and electron-dense mature secretory granules seen in the apical region of the cell. In the cells of the female prostate these features are absent. These morphological differences reflect the influence of hormones upon the cells, as shown by the reduction of the dilated Golgi vesicles in the castrated male and conversely, their occasional presence in the cells of the oestrous female.Comparison of castrated and ovariectomised animals shows that the male ventral prostate is much more dependent on androgens than the female is on ovarian hormones.There are several modes of secretion in the male ventral and the female prostate. These are by acellular and cellular blebbing, by a variety of secretory vesicles into the acinar lumina, and by a system of double walled vesicles not previously described.We are grateful to Dr. R.C.B. Pugh of the Department of Pathology, St. Peter's Hospitals for helpful discussion, to Mr. P. Chaloner and Miss P. Gunter for technical assistance and also the Department of Medical Art of the Institute of Urology     

The fine structure of the olfactory tract in the teleost Carassius carassius L.

Summary Electronmicrographic montages of the olfactory tract at two levels in each of two fish (Carassius carassius L.) were constructed and fibre diameters measured using a Zeiss TGZ 3 particle size analyzer. Medial and lateral tract divisions, rhinocele and dorsal tela were identified. Ciliated ependymal cells line the rhinocele. Meninges form the outer covering of both tract divisions and the tela roofing the central canal.The lateral tract consists of 10–14 fasciculi in which myelinated nerve fibres are prominent. These fibres range in diameter between 0.2 and 1.8 (mean 0.7 ) consistent with conduction velocities averaging 0.6 m/sec recorded in the carp lateral olfactory tract.The medial division of the olfactory tract contains two larger fasciculi within which are numerous fine unmyelinated nerve fibres (mean diameter 0.17 ) arranged in bundles partly enveloped by glial cell processes. Myelinated nerve fibres are unevenly distributed within both fasciculi and have mean diameters of 0.6 .An interesting observation is the consistent presence of synapses within the largest bundle of the medial tract at all levels.Supported by Grant 5 Ro5 TW00154-03 from the National Institutes of Health, United States Public Health Service.The authors are indebted to the Fisheries and Wildlife Department who generously provided the fish from Snob's Creek Fish Hatchery, and gratefully acknowledge the technical assistance of Mr. T. Armitage, Mr. J. Simmons and Miss D. Harrison.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

A comparison and evaluation of some phytosociological techniques

Summary In three areas of vegetation (dune, mountain heath and salt marsh) the following phytosociological techniques have been tested and compared, using the same data: the Braun-Blanquet method; association and inverse analysis ofWilliams &Lambert; cluster analysis (agglomerative classification) based on different coefficients of similarity; and ordination (principal components analysis performed on matrices of different coefficients).The Braun-Blanquet method is considered to combine several advantages of the other methods and to be most economical in terms of efficiency (ratio of time input to information emerging).

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Zusammenfassung Auf drei verschiedenen Vegetationsflächen (Bergheide, Küstendünen und Salzwiesen) sind die folgenden pflanzensoziologischen Methoden geprüft und verglichen worden: die Methode von Braun-Blanquet; association analysis vonWilliams &Lambert (1959, 1961); Ordination (principal components analysis); und cluster analysis (Sokal &Sneath, 1963). Die letzteren beiden wurden mit verschiedenen Ähnlichkeitskoeffizienten geprüft.Auf Grund solchen Erfahrungen, zeigte sich die Braun-Blanquetische Methode leistungsfähiger als die anderen Methoden (d.h. optimale Einsicht in der Vegetation pro Arbeitsstunde). Sie vereinigte viele Vorteile der anderen Methoden.

     

Mesothelial intercellular junctions and pathways

Summary Freeze-etch preparations of mesothelial cells taken from the peritoneum of mouse reveal the presence of vesicles invaginating the apical and the basal cell surfaces. These vesicles are scarcely seen within the cytoplasm. Long tortuous tubular profiles extend for considerable distance within the cytoplasm and are frequently associated with the vesicles. The possible nature and role of the vesicles and the tubules in transport phenomena across the mesothelial barrier, are discussed in relation to the pore theory advanced by physiologists and the stomata concept observed by early German and contemporary anatomists. Occludens junctions of the leaky type are seen though their macular or zonular nature is yet to be established.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Purification and characterization of the apical plasma membrane of the rat pancreatic acinar cell

Summary A method is described for the rapid purification of the apical plasma membrane from the rat pancreatic acinar cell. It makes use of wheat germ agglutinin affinity chromatography to selectively bind vesicles with N-acetyl glucosamine present at their surface. Particular conditions (150 mm NaCl) had then to be used to keep membrane vesicles in the coveted orientation, i.e. as right-side-out vesicles. Due to its specific apical location in many epithelial cells, -glutamyltranspeptidase was chosen to monitor the purification procedure. The final fraction was enriched in -glutamyltranspeptidase by a factor of 75 relative to the homogenate. Na,K-ATPase, a strict basolateral membrane marker, was not detectable in the fraction. No membranes originating from other compartments, more particularly expected from zymogen granules, or from other cell types, did contaminate the preparation. As expected for an epithelial cell apical plasmalemma, lipid composition showed a very high ratio of glycolipids (37.5%). The absence of membrane-bound GP-2, and the exceptionally high specific activity of -glutamyltranspeptidase suggest that the apical membrane would not be made up by the exocytosis of secretory granule, but instead by the fusion of specialized secretory vesicles very likely originating from the constitutive secretory pathway. In conclusion, this report describes a method of obtaining a fraction highly enriched in the secretory apex of the pancreatic exocrine cell that would be directly involved in exocytosis with zymogen granules and also in local anion transport.The authors would like to thank Dr. Andrew W. Shyjan (Yale University, New Haven, CT) for his kind gift of anti-Na,K-ATPase 1 subunit, Dr. Yannick Laperche (INSERM, Hôpital Mondor, Paris) for his gift of anti--GT, and finally Mr. Gilles P. Grondin for the production of antibodies against amylase. D.L. is supported by NSERC of Canada, FCAR of Québec, and the Canadian Cystic Fibrosis Foundation.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.