Occluding junctions in the epithelia of the gut-associated lymphoid tissue (GALT) of the rabbit ileum and caecum

Summary The zonulae occludentes of the dome epithelia and adjacent non-dome epithelia in four locations of the gut-associated lymphoid tissue (GALT) in the rabbit ileum and caecum (Peyer's patches, sacculus rotundus, caecal lymphoid patches, appendix) were studied in freeze-fracture replicas. In all locations the zonulae occludentes of the dome epithelium are composed of more junctional strands than in the corresponding non-dome epithelium. In the dome epithelia of Peyer's and caecal lymphoid patches the zonulae occludentes show considerable structural variation; the number of superimposed strands is 10 (range 5–18). In the dome epithelia of sacculus rotundus and appendix, in addition to zonulae occludentes, extended networks of junctional strands (fasciae occludentes) are present particularly between M-cells and enterocytes. The zonulae occludentes consist of 8 to 9 (range 5–15) superimposed strands; the fasciae occludentes extend up to a depth of 20m on the lateral membranes. The presence of the fasciae occludentes correlates with the appearance of regularly shaped clusters of lymphocytes, which are most developed in the dome epithelia of sacculus rotundus and appendix. These results suggest (1) that in contrast to the dome epithelia of Peyer's and caecal lymphoid patches those of sacculus rotundus and appendix are compartmentalized, and (2) that the mobility of lymphocytes and diffusion of antigens in the dome epithelia of sacculus rotundus and appendix is restricted.     

Ultrastructure of the nongranulated cells and morphology of the extracellular spaces in the pars distalis of adult and pouch-young tammar wallabies (Macropus eugenii)

Summary Differentiated nongranulated (folliculo-stellate) cells were observed in the centre and periphery of the cords of cells in the pars distalis of the adult tammar wallaby (Macropus eugenii). The nongranulated cells formed follicles containing a small lumen; the cell apices were joined by junctional complexes including zonulae adhaerentes and maculae adhaerentes (desmosomes). Follicles of granulated cells were also occasionally found. Follicles were rarely observed in the adult, but were numerous and generally larger in pouch-young wallabies. Moreover, whereas the involvement of granulated cells in the follicles of the adult was infrequent, they were common components of follicular structures in the pouch-young. The marginal cells at the periphery of the pars distalis in contact with the hypophysial cleft had many of the cytological characteristics of the nongranulated cells in the central pars distalis. In both adult and pouch-young wallabies, nongranulated cells at the periphery of the cell cords were juxtaposed such that they formed sinusoid-like spaces or lacunae, presumably as part of the extravascular channel system. The observations are discussed in light of the proposed phagocytotic, metabolite and/or hormone transport and stem cell roles attributed to these cells.This work was supported by a travel grant and grant-in-aid of research from the N.S.E.R.C. (J.F.L.) and from the A.R.G.C. and the N.I.H. (M.B.R.)     

Freeze-fracture analysis of junctional complexes in the nephron of the garter snake,Thamnophis sirtalis

Summary Zonulae occludentes are shown by freeze-fracture to be pleomorphic along the garter snake nephron. In the neck and proximal segments the occluding junctions are moderately complex with frequent discontinuities in their junctional fibrils. Junctional depth and complexity are maximal in the distal and collecting segments and discontinuities in fibrils are absent. Comparison of these results with similar observations on other tissues indicates that the zonulae occludentes in the neck and proximal segments are intermediate to leaky and that they may be very tight in the distal and collecting segments. These findings suggest that in the garter snake nephron transepithelial flow of fluid may occur primarily by passive diffusion through the zonulae occludentes in the neck and proximal segments and by cell-mediated osmotic flow in the distal and collecting segments. Gap junctions occur only in the proximal tubule and are probably involved in low resistance, intercellular movement of ions.Supported by the National Research Council of Canada. The authors wish to acknowledge the generous provision of freeze-fracture facilities by Dr. M.W. Brightman, NINDS, National Institutes of Health, Bethesda, Maryland, U.S.A.     

Ultrastructural investigation on the cell membranes of the vomeronasal organ in the rat: A freeze-etching study

Summary The free surfaces and cell contacts in the epithelia of the vomeronasal organ of the rat were investigated by freeze-etching. The microvilli of receptor cells show a lower density of intramembranous particles (IMP) than the microvilli in the receptor-free epithelium. The ratio between the IMP on P and E-face is approximately 111 in the receptor terminals, and 3.51 in the cilia and microvilli of the receptor-free epithelium. Although atypical in length and only poorly equipped with rootlet fibers, the cilia of the receptor-free epithelium are furnished with typical ciliary necklace structures of up to 10 rows of membrane particles. Differences in the density of IMP on the P-faces of different cilia are probably due to continual ciliogenesis and also due to the different age of cilia in the receptor-free epithelium. Zonulae occludentes show different configurations in the neuroepithelium and in the receptor-free epithelium. In the former, they show a tendency to cross-link and form facet-like patterns, reflecting a constant morphology and relative stability for this apical region. In the receptor-free epithelium the junctional rows of zonulae occludentes display only loosely interconnected networks and a tendency to orient parallel to each other and to the free surface. In addition to zonulae occludentes, typical square aggregations of IMP are observed in the receptor-free epithelium. They are not exclusively restricted to the zone of intensive cell contacts by means of fine interdigitating cell processes, and their function has yet to be identified experimentally.This paper is dedicated to Dr. David G. MoultonPortions of this work are from a thesis in preparation by F.M. Supported by the Deutsche Forschungsgemeinschaft (SFB 114)     

Intercellular junctions in the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela)

Summary Intercellular junctions in the uterine epithelium of the ovoviviparous urodele Salamandra salamandra were studied in pregnant and non-pregnant females by freeze-fracture technique. Junctional complexes consist of zonulae occludentes (tight junctions) and numerous maculae adhaerentes (desmosomes); z. adhaerentes and nexuses (gap junctions) could not be identified. Tight junctions are of the flexible type exhibiting loosely interconnected fibrils. The fibrillary network appears stretched more often in pregnant females possibly due to the mechanical stress of pregnancy. The structure and occurrence of the junctions identified, especially that of the tight junctions, is discussed with regard to the functions of the uterus during pregnancy.Zusammenfassung Mit Hilfe der Gefrierbruchtechnik wurden im Uterus-epithel trächtiger und nichtträchtiger Feuersalamanderweibchen (Salamandra salamandra) Zonulae occludentes und Maculae adhaerentes, jedoch keine Z. adhaerentes sowie Nexus identifiziert. Die Z. occludentes sind flexibel. Ihr fibrilläres Netzwerk ist bei trächtigen Weibchen öfter gestreckt; das ist möglicherweise auf die stärkere Ausdehnung des Uterusgewebes während der Trächtigkeit zurückzuführen. Das Vorkommen der verschiedenen Kontakt-strukturen, namentlich das der Z. occludentes, wird im Hinblick auf die Funktionen des Uterus während der Trächtigkeit diskutiert.We are indebted to Mrs. K. Ott for excellent technical assistance and to Miss Dr. U. Beigel for linguistic help     

Gap junctions between pinealocytes

Summary The intercellular junctions between the pinealocytes of male rats were investigated by freeze-fracture and conventional electron microscopy.Our findings reveal that the intercellular contacts between pineal cells, formerly described as zonulae adhaerentes or zonulae occludentes, are in fact gap junctions which are difficult to characterize in thin sections due to their peculiar geometrical arrangement, which is in the form of fenestrated communicating zonules.The arrangement of these communicating zonules around rudimentary lumina of pineal clusters and rare transitions between tight and gap junctions may point to phylogenetic transformations of occluding into communicating zonules, corresponding with the change of the pineal gland from a sensory to a secretory organ. Alternatively, these tight-to-gap junctional transitions may reflect the periodic (circadian or seasonal) activity of the pineal gland.These Studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

Fine structural study of gas secretion in the physoclistous swim bladder of Fundulus heteroclitus and Gadus callarias and in the euphysoclistous swim bladder of Opsanus tau

Summary The Haldane-Koch-Scholander-Kuhn-Steen theory of salting out countercurrent multiplication effect of the rete mirabile now accounts for release of most gases in the fish swim bladder. Evidence presented here indicates that final release is by microbubbles from the secretory epithelium. There is only one specific cell type in a highly vascularized epithelium. It is characterized by complex folds in the paravascular zone and by gas forming bodies which seem to form from plentiful Golgi material. The bodies are formed with dark amorphous matrix that becomes patterned (tubular or lamellar), finally froths and then is released to the gas surface. Residual material may form myelin-like layers on the lumenal surface. Active cells are also characterized by surface villi and subsurface, parallel cisternal spaces. Gas may be formed by cells not touching the gas surface and released through intercellular spaces. There are discontinuous desmosomes (maculae adhaerentes) near the gas surface and there are no tight junctions (zonulae occludentes). Gas release as bubbles would explain Wittenbergs observations that the gases found in swim bladders have ratios more closely related to their solubility coefficients in water than to ambient partial pressures. A surfactant may be present to lower the surface tension of the microbubbles. The carrier in the cytoplasm would have to be an iron-protein (or perhaps peroxidase) compound capable of binding molecular oxygen.Supported by grant-in-aid from the national Science Foundation (GB-676) and from the USPHS (General Medical Sciences Institute, GM-06836).     

Structural changes in the zonulae occludentes of the chloride cells of young adult lampreys following acclimation to seawater

Summary Thin sections and freeze-fracture replicas have been used to study the structure of the zonulae occludentes of the branchial chloride cells in young adults of the anadromous lamprey Geotria australis, caught during their downstream migration to the sea and after acclimation to full-strength seawater (35). The chloride cells in the epithelium of the gill filaments of both freshwater- and seawater-acclimated animals form extensive multicellular complexes. In freshwater animals, the majority of chloride cells (64%) are covered by pavement cells and are thus not exposed to the external environment. Most of the other chloride cells are separated from each other by pavement cells or their processes. The zonulae occludentes between chloride cells and pavement cells and between adjacent chloride cells are extensive and characterised by a network of 4 (range 3–7) superimposed strands. In seawater-acclimated animals, the pavement cells cover only 30% of the chloride cells and their processes no longer occur between chloride cells. Whereas the zonulae occludentes between chloride cells and pavement cells are still extensive, those between chloride cells are shallow and comprise only a single strand or two parallel strands. The zonulae occludentes between the chloride cells of lampreys acclimated to seawater are similar to those in the gills of teleosts in seawater, and are thus considered to be leaky and to provide a low-resistance paracellular pathway for the passive transepithelial movement of Na⁺.     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

Cell junctions with funnels in the olfactory mucosa of frogs (Rana temporaria L.)

Summary A characteristic structure in the apical junctional belt of the olfactory epithelium in Rana temporaria is visible in freeze-fracture preparations. This structure is described as a funnel with channel across the junctional belt. It is supposed to represent a possible way for discarding used molecules after stimulation, and to allow the stimulation of free nerve endings in the depth of olfactory epithelia.I wish to thank Prof. C.F. Bardele and Mr. H. Schoppmann for their kind support and technical help     

Synthesis of deoxygalactose-containing sialyl LeX ganglioside analogues to elucidate the structure necessary for selectin recognition

Sialyl Lewis X ganglioside analogues containing 4-deoxy-, 6-deoxy-, and 4,6-dideoxy-d-galactopyranose in place ofd-galactopyranose have been synthesized. Glycosylations of 2-(trimethylsilyl)ethyl 2,6-di-O-benzyl--d-galactopyranoside and 2-(trimethylsilyl)ethyl -d-fucopyranoside with the phenyl 2-thioglycoside derivative of sialic acid, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid (TfOH) as the promoter in acetonitrile, gave the desired 2-(trimethylsilyl)ethyl sialyl--(23)--d-galactopyranoside and--d-fucopyranoside, respectively. The sialylgalactose derivative obtained was then modified to 4-deoxy and 4,6-dideoxy derivatives. These were converted, byO-benzoylation, transformation of the 2-(trimethylsilyl)ethyl group to trichloroacetimidates, and introduction of the methylthio group with methylthiomethysilane, into the corresponding glycosyl donors, which were then coupled with 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(13)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(13)-2,4,6- tri-O-benzyl--d-galactopyranoside in the presence of dimethyl(methylthio)sulfonium triflate (DMTST). The resulting pentasaccharides were each converted to the corresponding -trichloroacetimidates, which, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the desired sphingosine derivatives. Selective reduction of the azide group,N-acylation with octadecanoic acid,O-deacylation, and saponification of the methyl ester afforded the target compounds.Synthetic Studies on Sialoglycoconjugates, Part 79.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Histochemistry of some acid hydrolases in striated muscles of the rat

Summary The distribution of acid phosphatase, -n-acetylglucosaminidase, -glucuronidase, and acid -galactosidase was studied in mm. extensor digitorum longus, soleus, and diaphragm of rats. Using the technic of semipermeable membranes activities of these enzymes were demonstrated beside cells of the interstitial tissue in muscle fibers themselves as well. Acid phosphatase displayed the highest activity which appeared in many small dots dispersed in the fiber. The activity of acid phosphatase was about 1.2 x higher in the m. soleus than in the m. extensor digitorum longus. In the latter muscle a somewhat higher activity was often found in muscle fibers displaying a higher staining for NADH tetrazolium reductase. The activity of -n-acetylglucosaminidase was slightly lower, that of -glucuronidase very weak but still discernible. The activity of acid -galactosidase was not ascertained in the majority of fibers. The ratio of activities measured in an area of the same size in cells of the interstitial tissue and in muscle fibers amounted in average to 2.6: 1 in the case of acid phosphatase, 2.5:1 in the case of -n-acetylglucosaminidase, 5.7: 1 in the case of -glucuronidase, and 44.3:1 in the case of acid -galactosidase. The importance of the histochemical technic in studies concerned with acid hydrolases in striated muscle fibers in normal and pathological conditions is pointed out.     

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.     

The NGF complex from the African ratMastomys natalensis

The 7S NGF complex from the male mouse submaxillary gland consists of the , and subunits in the ratio ₂₂. The (NGF) subunit contains all the known biolocial activity of 7S NGF. The and subunits are both members of glandular kallikrein gene family, yet only subunit has protease activity. The subunit plays a role in the processing of preproNGF to its mature form, while the role of the subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the or subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the ratMastomys natalensis in order to more fully understand the roles of the and subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio ₂. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.Special issue dedicated to Dr. Lawrence Austin     

Kinetic study on the β-glucosidase-catalysed reaction of Trichoderma viride cellulase

A kinetic study of the -glucosidase-catalysed reaction of a commercial cellulase preparation from Trichoderma viride is described. The K _m and V _max values of the -glucosidase system were: (a) 0.5 mm and 6.6 mol/min, respectively, using p-nitrophenyl -d-glucopyranoside (pNPG) as substrate; and (b) 2.5 mm and 8.1 mol/min, respectively, using cellobiose as subtrate. The glucose effect on initial reaction velocity agrees with a mixed-inhibition pattern. The inhibition constant (K _i) values were, 0.53 and 0.39 mm with nNPG and cellobiose as substrates, respectively. The temperature and pH optima were determined. Correspondence to: A. Romeu     

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     

Intercellular junctions in the water-blood barrier of the gill lamella in the adult lamprey (Geotria australis,Lampetra fluviatilis)

Thin sections and freeze-fracture replicas of the water-blood barrier in the gill lamellae of adult lampreys (Geotria australis, Lampetra fluviatilis) demonstrate that the occluding junctions between epithelial pavement cells differ markedly from those between endothelial pillar cells in the structure and arrangement of their strands. The zonulae occludentes between pavement cells typically consist of complex networks of 4–6 strands, the mean number of which undergoes a small but significant decline when the animal is acclimated to seawater. In comparison, the occluding junctions between pillar cells are less elaborate and may represent maculae or fasciae, rather than zonulae occludentes. They do not apparently undergo a change when the animal enters saltwater. The results indicate that the main part of the paracellular diffusion barrier to proteins and ions is located in the epithelium rather than the endothelium. Communicating (gap) junctions are present between adjacent pavement cells, between pavement and basal cells and between pillar cells. These findings suggest that the epithelial cells and the pillar cells in the water-blood barrier of lampreys both form functional syncytia. The results are discussed in the context of ion-transporting epithelia in other aquatic vertebrates.This paper is dedicated to Professor H. Leonhardt on the occasion of his 75^th birthday     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.