Electron microscopy and electron microscopical measurements of collagen mineralization in hard tissues

Summary The best focussed members of a through focus series of electron micrographs of ultra-thin sections of freeze-dried forming rat incisor dentine and glutaraldehyde-fixed 10 months old human femoral cortex were selected for measurements. The original micrographs were prepared at magnifications of x20000 or x40000 and were photographically enlarged to a magnification from x200000 to x600000. The number of dot-like calcium phosphate nuclei per hole zone and per overlapping zone of the 64 nm collagen macro-period was determined; we found a range of 6–8 nuclei per hole zone and 3–5 nuclei per overlapping zone. Thus a total of 9–13 nuclei per 64 nm macro-period was found; this is in agreement with the total number of cross bandings per macro-period reported by previous authors. The distances between neighbouring dot-like calcium phosphate nuclei were measured and found to lie in the range of 4.0 to 8.5 nm, which is the range in which most of the distances between the collagen cross-bandings appear. The distances between close, parallel calcium phosphate rows within the collagen fibres were found to lie in the range 4.6–8.0 nm which seems to be a good deal shorter than the distances between the typical microholes of the Hodge-Petruska collagen model. Our results and ideas concerning the active sites of nucleation also are in agreement with previous amino acid sequence analyses in respect of the occurrence of neighbouring polar amino acids which would be free to bind ions.We thank Professor S. Heuck for valuable bone specimens. We thank Frau G. Neinhardt, Frau R. Höhling and Mr. P. S. Reynolds for their valuable technical assistance and Mr. H. Pittorf for building the metal model of Fig. 10. We thank the Deutsche Forschungsgemeinschaft and the Medical Research Council for their financial support.     

Ultrastructural features of adult human tendon

Summary A variety of human tendons have been studied at the electron microscope level. The fibers of these tendons are composed of collagen fibrils that average 1,750 Å and 600 Å in diameter. A third population that measures 100 Å in diameter may represent immature collagen or filaments that are incorporated into tendon elastic fibers. The larger collagen fibrils vary in ratio with respect to one another, and are connected by interfibrillar bridges which in some cases appear to extend through the substance of the fibril. The collagen fibrils of the paratenon are less-well organized than those of the tendon proper and average 600 Å in diameter. Tendons that exhibit the property of lateral stretch (plantaris and palmaris) were compared at the ultrastructural level with tendons that do not have this property. No differences between the two tendon types could be determined in normal or spread preparations, indicating that the differences in physical characteristics are a result of fiber rather than fibril organization.Supported by Edward G. Schlieder Foundation GrantThe authors wish to thank Mrs. Janell Buck and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance, and Mr. Garbis Kerimian for his excellent photographic work     

Transmission microscopy of freeze dried,unstained epiphyseal cartilage of the guinea pig

Summary The question of the initial mineralization in the epiphyseal plate has been investigated to date in specimens prepared by conventional electron microscopical techniques. As conventional techniques can produce artifacts, either a loss of mineral substance or a secondary nucleation, the mineralization process was investigated using freeze dried, vacuum embedded growth cartilage which was neither contrasted nor stained and which had a very short contact with water.The prevailing theory that the first mineralization begins within extracellular matrix vesicles and that the mineralization outside these vesicles is a secondary process was confirmed. Mineralized matrix vesicles were found in the fully mineralized long septa down to the opening zone. In several cases a mineralization could be observed in those transverse septa in which organic substance was present between the cells. The typical radial arrangement of the apatitic needles and platelets in the matrix vesicles could be explained by the formation of a mineralization in an ionotropic gel, the orientation of the matrix macromolecules to be produced by a vectorial influx of calcium ions and phosphate groups coming from different directions. Thin strands of mineral substance with low contrast, which follow the direction of the longitudinal septum, were assumed to be the mineralized collagen fibrils. In several needles dot-like formations were seen and the distance between the middle of neighbouring dots was found to lie mainly in the range 30–56 Å, while the lateral separation distance between the middle of closely packed parallel chains and needles was found to lie mainly in the range 30-42 Å. Parallel periodic structures which could be visualized in apatitic chains and needles 20–40 Å in diameter were assumed to be the 8.2 Å-(100)-lattice planes of apatite, being an indication that these formations already possess criteria of the apatite lattice.We express our thanks to the Deutsche Forschungsgemeinschaft for financial support and to Dr. A. Boyde, London, for valuable discussions.     

The normal fine structure and aging changes of the human zonular fibers

Summary The normal zonular fibrils of the human eye do not differ from the fibrils of the zonula Zinnii of the rat. Furthermore, there is no difference between the single zonular fibrils and the fibrils of the vitreous body in rat and man. The average diameter of the human zonular fibrils is 109 Å. They are transversely striated at intervals with a periodicity of 70–150 Å. Over periods of mostly 400–440 å, but also of 500–640 Å were observed. At a few places over periods similar to those of the long spacing-type packing were found. Like in the rat eye, the zonular fibrils of the human eye must be regarded as a special form of collagen.An indirect proof for the collageneous nature of the zonular fibrils is the occurence in advanced age of degenerative alterations which are exclusively observed in connective tissue (hyalinization, elastoid degeneration).Supported by the Deutsche Forschungsgemeinschaft. Acknowledgement: I wish to thank Dr. H. Faßl, Institut für Medizinische Statistik und Dokumentation der Universität Mainz, for the statistical analysis.     

The mechanism of nucleation for in vitro collagen fibril formation.

The formation of collagen fibrils from soluble monomers and aggregates by thermal gelation at neutral pH can be divided into two distinct stages: a nucleation phase and a growth phase. Turbidity studies of the kinetics of the precipitation reaction show that the lag-phase time or nucleation reaction time, t′_l, is markedly temperature dependent while the growth reaction time is temperature independent. The activation energy of the nucleation reaction is essentially constant over the temperature range studied. In monitoring the nucleation-phase reaction by various physicochemical techniques, including viscosity, sedimentation equilibrium, and light scattering, no evidence for the formation of aggregates was observed. Enrichment of the initial collagen solution with aggregates accelerates nucleation, but de novo nuclei formation is still required even in highly aggregated collagen preparations. Removal of pepsin and pronase susceptible peptides lengthens the nucleation reaction time and increases the sensitivity of the rate of nuclei formation to changes in ionic strength. Electron microscope studies show the fibrils formed from the protease-treated collagen to be less well organized. With pepsin-treated collagen, subfibrils and obliquely striated fibrils are seen, showing that while microfibrils are formed interactions between them are modulated by the enzyme susceptible peptides in the same way that these regions modulate nuclei assembly. It appears that pepsin and pronase susceptible peptide regions of collagen play a more prominent role in the in vitro assembly of collagen molecules to form D-stagger nuclei and fibrils than do ionic interactions between helical molecular regions. A mechanism of nucleation of collagen fibrillogenesis is discussed.     

Elektronenmikroskopie und Laserbeugungs-Untersuchungen zur Charakterisierung der organischen Matrix im Speichelstein und Hartgewebe

Zusammenfassung Elektronenmikroskopische Ultradünnschnittbilder vom Speichelstein zeigten — wie solche vom Dentin, Knochen usw. — nadelbzw. kettenförmige Ca-Phosphatbildungen mit globulären Keimen. Wir bestimmten in der Grundsubstanz a) die Länge der Nadeln als Werte für die Länge der kettenartigen Matrix, b) die Abstände zwischen den punktförmigen Keimen innerhalb der Nadeln als Wiedergabe der Abstände zwischen den aktiven Baugruppeneinheiten, die auf der Matrix die Mineralkeimbildung einleiten, und c) die Seitenabstände zwischen dicht zusammenliegenden, parallelen Ketten als Wiedergabe der Seitenabstände zwischen den Hauptsträngen der Matrix (s. Abb. 2). Die Bestimmungen zu b) und c) wurden sowohl über morphologische Vermessungen wie Laserbeugung an den elektronenmikroskopischen Platten durchgeführt. Die unter a) bezeichneten langen Nadeln (damit die Kettenlänge der Matrixleitstruktur) erreichten Werte über 1000 Å. Die unter b) bezeichneten morphologischen Vermessungen zwischen den Keimen ergaben Werte zwischen 40 und 85 Å und die unter c) bezeichneten Seitenabstände Werte zwischen 37 und 75 Å; die Laserbeugung führte zu Werten zwischen 37 und 54 Å, die also bevorzugt im unteren Bereich der morphologischen Vermessungen zu b) und c) lagen. Anhand dieser Meßdaten wurden erste Überlegungen darüber angestellt, ob die Leitstruktur in der organischen Matrix die Eiweiß- oder Polysaccharidkette im Proteinpolysaccharid bzw. Glykoprotein ist. Entsprechende Untersuchungen wurden auch innerhalb der Bakterien und der Bakterienmembran durchgeführt.

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Electron microscopic and laser diffraction measurement and study of the organic matrix of salivary concretions and hard body tissues

Summary Electron micrographs of ultrathin sections of salivary concretions showed needle-like Ca-phosphate formations with globular nuclei. Similar formations were observed in dentine, bone, and other hard tissues. We determined in the ground substance (a) the lengths of the needles as values for the length of the chain-like matrix backbone, (b) the distances between the dotlike nuclei within the needles as distances between the active sites of the chain-like matrix which induce the phosphate nucleation, and (c) the side distances between the close-packed, parallel needles as values for the distances between the parallel matrix backbones. The investigation of (b) and (c) was carried out by morphological measurement as well as by laser diffraction method on electron micrographs. The long needles of (a) (the length of the matrix backbone) reached values of more than 1,000 Å. Measurement of the distances between the small globular nuclei within the needles (b) gave values of 40 to 85 Å, and of those on the side distances of 37–75 Å. The values for (b) and (c) derived by laser diffraction appeared in the range of 37–54 Å, which was the lower range of the morphological values of (b) and (c). The results were analysed to discuss whether the main chain of the matrix is the proteinor the polysaccharide chain in the protein-polysaccharide or the glycoprotein. Corresponding measurements were carried out in areas within the bacteria and in the bacterial membrane.

Herrn Prof. Dr. L. Reimer und Herrn cand. phys. H. G. Heine, Physikalisches Institut der Universität Münster, danken wir für die Durchführung der Laserbeugungs-Untersuchungen und für wertvolle Diskussionen, Fräulein Gisela Rehsöft für sorgfältige Assistenz. Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Electron microscope studies on the structure of collagen fibrils by negative staining

Summary Electron microscope studies on collagen from rat-tail tendon using a negative staining technique have indicated the presence of filaments 15–20 Å in diameter within the fibres. These filaments are thought to correspond to the collagen macromolecule.We would like to thank Prof. R. A. McCance for supplying the specimens of fowl tendon used in this investigation, and Dr. F. H. C. Crick, Dr. S. Fitton-Jackson and Dr. T. Gillman for a number of valuable discussions. One of us (W. J. T.) wishes to thank the Medical Research Council for financial support during this work.     

Electron microscopical and laser diffraction studies of the nucleation and growth of crystals in the organic matrix of dentine

Summary The growing ends of rat incisors were freeze-dried and embedded in methacrylate without contact with any other solution. Dentine from alcohol and formalin fixed human teeth was also embedded in methacrylate. Ultrathin sections were prepared and electron micrographs taken at original magnifications of X20 000 and X40 000. The best focussed pictures from through focus series were selected for photographic enlargement to a total of X200 000. 507 measurements of the distance between dot-like nuclei in the calcium phosphate needles and chains and 536 measurements of the distance between the neighbouring parallel chains and needles were made using a measuring microscope. In addition, the most commonly occurring separation distances between the dot-like nuclei — within individual rows or between neighbouring rows—were measured by laser diffraction of the x20 000 EM negatives.The most commonly occurring range for the distance between the dot-like nuclei and the lateral distance between the rows as determined morphologically was 3.7–6.3 nm. The corresponding value as determined by laser diffraction for the recently formed rat incisor dentine lay in the region 3.0–5.2 nm, whereas the same value reached to 6.5 nm in the case of mature human dentine. The distances between the dot-like nuclei are regarded as representing the distances between active nucleus-inducing centres on a chain-like matrix. From a study of the morphology of the nuclei it is concluded that the plate-like crystallites usually arise through fusion of needle-like rows of dot-like nuclei when these lie close and parallel to one another.We thank Frau G. Neinhardt, Frau R. Höhling and Mr. P. S. Reynolds for their valuable technical assistance. We thank the Deutsche Forschungsgemeinschaft and the Medical Research Council for their financial support.We thank the Landesamt für Forschung des Landes N.R.W. for the laser equipment.     

Tapping-mode atomic force microscopy in fluid of hydrated extracellular matrix

Fragments of native, hydrated rat tail tendon were imaged by tapping-mode atomic force microscopy while immersed in fluid. The specimens were soft and sensitive to the operating parameters, and with minimal imaging pressure the collagen fibrils appeared covered by irregular blobs or by filamentous material. A slight increase in pressure caused the underlying fibril surface to appear, with an evident D-period, gap- and overlap-zones and three intraperiod ridges. Fibrils often ran parallel and in phase, implying some coupling mechanism. Longitudinal subfibrils, 8-9 nm thick, occasionally appeared. The simultaneous acquisition of the "tapping amplitude" along with the usual "height" channel clearly confirmed the presence of longitudinal subfibrils, indicative of the inner architecture of the fibril.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

The perinuclear microtubule system in the green algaAcetabularia: anchor or motility device?

Summary Migrating secondary nuclei inAcetabularia are tightly associated with actin bundles and possess a comet-like tail composed of microtubules. When secondary nuclei begin to settle in preparation for cyst morphogenesis, the tails expand into radially symmetrical arrays of microtubules. Concomitantly, nuclei become gradually dissociated from the actin bundles and eventually stop moving, even though the actin bundles remain intact and persist through this stage. If, however, the radial perinuclear microtubule arrays are destroyed by inhibitors, the nuclei reassociate with the actin bundles and regain their motile activity. Because this movement is sensitive to Cytochalasin D, we propose that actin is required for nuclear movements, whereas microtubules most likely function as a trailing anchor that begins to act as a braking device above a certain threshold in the number and length of perinuclear microtubules.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Ultrastructural immunocytochemical localization of luteinizing hormone-releasing hormone (LH-RH) in the median eminence of the guinea pig

Summary LH-RH was localized at the ultrastructural level in axons and nerve terminals of the median eminence of the male guinea pig. LH-RH positive neuronal profiles were most concentrated in the medial-dorsal aspect of the infundibular stalk and in the post-infundibular median eminence at the level immediately following separation of the stalk from the base of the brain. LH-RH containing axon profiles were most abundant in the palisade zone; nerve terminals in contact with the hypophysial portal vasculature were relatively rare. The hormone was present within granules that measured 900–1,200 Å in axons of the palisade zone and 400–800 Å in nerve terminals abutting on the portal plexus. The differently sized granules represent heterogeneous populations.Supported in part by U.S. Public Health Service grant HD-09636 from the National Institutes of Health and RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center Publication No. 15-031The authors wish to thank Dr. Sandy Sorrentino, Jr. for the gift of antiserum to LH-RH and Dr. Ludwig Sternberger for the peroxidase.antiperoxidase complex     

Analysis of the crystal arrangement in collagen fibrils of mineralizing turkey tibia tendon

Summary Mineralized pieces of tendons from the tibio-tarsus of turkeys were (i) shock-frozen, freeze-dried, embedded and cut without staining, or (ii) fixed, embedded and stained after sectioning. Micrographs were taken with an electron microscope on longitudinally cut sections. The center-to-center distances of neighboring apatitic needles within collagen fibrils were measured. For shock-frozen and freeze-dried specimens, the average of these distances is 4.7 nm and the most frequent value 4.2 nm; for fixed and stained specimens, 3.8 nm and 3.6 nm, respectively. Laser diffraction of the electron micrographs showed a dumbbell-like intensity pattern (two diffuse maxima of intensity on the equator, one on each side of the central spot), giving an average distance of about 6 nm. This value represents the upper range of the direct measurements. The measurements demonstrate that the arrangement of the collagen microfibrils is mainly preserved during mineralization. However, using laser diffraction, distances of 9–11 nm were also observed. Such large distances can also be demonstrated by X-ray diffraction on collagen fibrils stained under special conditions. This may indicate that special conditions of apatitic mineralization or staining may alter the arrangement of the microfibrils.The authors thank the Deutsche Forschungsgemeinschaft for financial support     

Model membranes: Spherical shells bounded by one bimolecular layer of phospholipids

Summary The conditions are described which lead to the formation of small spherical vesicles bounded by singlelipid membranes. These membranes appear as triple-layered structures in electron micrographs and thus resemble theunit membrane configuration of cellular boundaries. The theory of light scattering by spherical shells is found applicable to the vesicles described here; size determinations by electron microscopy (r=195 Å) and by light scattering (r=225 Å) give reasonably close values. The refractive index of the membranes as determined by differential refractometry is found to be 1.46 for green incident light of wavelength 5461 Å.The results of this work were presented in part at the Symposium on Biophysical Aspects of Permeability, Jerusalem, Israel, July 2–9, 1968.I gratefully acknowledge the help provided me during this work by Dr Walter Stoeckenius, and I also thank Dr Dominic Dziewiatkowski for the permission to use his light scattering photometer.This work was supported by grant No. GB-4871 from the National Science Foundation.     

Ciliated neurons and different types of synapses in anterior hypothalamic nuclei of reptiles

Summary The magnocellular paraventricular and supraoptic nuclei and the parvocellular preoptic and periventricular nuclei have been studied by light and electron microscopy in Emys orbicularis, Lacerta agilis and Elaphe longissima. The ultrastructure of cerebrospinal fluid (CSF)-contacting neurons was described in the preoptic and periventricular nuclei of Emys and Lacerta species. Single 9×2+0 cilia similar to those of the CSF-contacting dendritic terminals were found on perikarya of non CSF-contacting nerve cells, in all four investigated nuclei. The cilia project from funnel-like invaginations of the perikarya into the intercellular space. In the neurons of the nuclei studied, granular vesicles were found, their size being mainly 1,600 Å in the paraventricular nucleus, about 1,800 Å in the supraoptic nucleus, 1,100 Å in the periventricular nucleus and 800 Å, or up to 1,250 Å in the preoptic nucleus. In general, the neurons possess synapses of the axo-somatic, axo-somatic spine, axo-dendritic and axo-dendritic spine types. In the supraoptic nucleus, multiple interdigitated synapses were observed. Presynaptically, either synaptic vesicles only, or synaptic vesicles and dense core vesicles of different sizes (600 to 800 Å, about 1,100 Å, 1250 Å, and up to 2,000 Å) were found. It is discussed whether the above described 9×2+0 cilia may represent some kind of hypothalamic sensory structure that earlier physiological studies postulated to exist. The ciliated hypothalamic perikarya are considered by the authors to be a more differentiated form of the CSF-contacting neurons. The different types of synapses indicate multilateral connections of the nerve cells of the nuclei studied.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70th birthday     

Cytokinesis and cytochalasin-induced furrow regression in the first-cleavage zygote of Xenopus laevis

Summary The egg cleavage and the cytochalasin effect has been investigated in the first-cleavage zygotes of Xenopus laevis.—Furrow formation results from the joint action of surface constriction, junction formation, and ingrowth of new membrane. During the constriction phase lanthanum-binding exudate is deposited in the furrow gap. This material is distributed in dispersed patches (Ø 200 Å) giving rise to a bur surface which coats interdigitating cell protrusions. At places where protrusions meet they form 160 Å wide adherent junctions which provisionally fix the contracted furrow. At the end of the constriction phase (which ultimately accounts for 15 per cent of the reduction in egg diameter in the plane of cleavage) the layer of 100 Å filaments beneath the furrow bottom is split by local ingrowth of new membrane, and the filaments take up lateral positions. Furrow ingrowth proceeds by bilateral insertion of new membrane.The application of 7.5 g/ml cytochalasin B (CCB) leads to furrow regression without blocking contractility. CCB primarily affects the cell surface, and only indirectly affects the microfilament system. It interferes with cell junction formation and deranges furrow ingrowth. In the absence of stable 160 Å wide, adherent junctions the new membrane grows outwards instead of inwards. The results are discussed with reference to furrow regression induced by other membrane-destabilizing agents such as phospholipase C. Comparison reveals that CCB in addition facilitates the insertion of new cell membrane—. To interpret the biological effects of cytochalasin an alternative working hypothesis is presented, which meets the objections that can be raised against the concept that cytochalasin B specifically interferes with thin microfilaments.Dedicated with deep respect to Prof. Dr. Chr. P. Raven at the occasion of his 65th birthday.I thank Dr. S. B. Carter for a supply of cytochalasin B. I am grateful to my fellow-staff members of the Hubrecht Laboratory, and in particular to Prof. P. D. Nieuwkoop, for constructive criticism and valuable suggestions. I should like to thank Mr. E. van Voorst for his technical assistance, and Miss Eva Bartová, Mr. L. Boom and Mr. R. Tokaya for preparing the prints and the drawing. I am also indebted to Dr. J. Faber for editorial assistance, and to Drs. P. H. Ververgaert for his help in carrying out the densitometric measurements.     

The fine structure of the hypothalamic secretory neurons of the White-crowned Sparrow,Zonotrichia leucophrys gambelii (Passeriformes: Fringillidae)

Summary The fine structures of the neurons and neuropils of the magnocellular supraoptic nucleus and the parvocellular nuclei of the rostral hypothalamus, including the suprachiasmatic and medial, lateral and periventricular preoptic nuclei, and the neuronal apparatus of the organum vasculosum laminae terminalis, have been examined in the male White-crowned Sparrow, Zonotrichia leucophrys gambelii, by correlated light and electron microscopy.The magnocellular supraoptic nucleus is characterized by large neurosecretory perikarya which contain a well developed Golgi complex and densecored granules 1,500–2,200 Å in diameter. The neuropil displays axons, dendrites and glial fibers. Some axonal profiles contain dense-cored vesicles 800–1,000 Å in diameter and clear vesicles 500 Å in diameter. Axo-somatic and axo-dendritic synapses are conspicuous in this nuclear region.The suprachiasmatic nucleus is characterized by an accumulation of small neurons with moderately developed cellular organelles and some dense-cored granules, approximately 1,000 Å in diameter. The profiles of axons within the neuropil contain dense-cored granules 800–1,000 Å in diameter and clear vesicles 500 Å in diameter.The neurons of the medial preoptic nucleus are relatively large and exhibit well developed cellular organelles and dense-cored granules 1,300 to 1,500 Å in diameter. Granular materials are formed within the Golgi complex. The medial preoptic nucleus is rich in secretory perikarya.Occasionally, neurons with granules 1,500–2,200 Å in diameter are encountered in the lateral preoptic and periventricular preoptic nuclei. They may be considered as scattered elements of the magnocellular (supraoptic and paraventricular) system.The organum vasculosum laminae terminalis consists of three layers, i.e., ependymal, internal and external zones, and exhibits a vascular arrangement similar to that of the median eminence. The perikarya of the parvocellular neurons and their axons in the internal zone contain numerous secretory granules ranging from 1,300 to 1,500 Å in diameter.This investigation was supported by Grant No. 5R040 Japan-U.S. Cooperative Science Program of the Japan Society for the Promotion of Science to Professor H. Kobayashi and Professor S.-I. Mikami, by a Scientific Research Grant No. 56019 from the Ministry of Education of Japan to S.-I. Mikami, by support from the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Biologie der Zeitmessung) to Prof. A. Oksche and by Grant No. GF 33334, U.S.-Japan Cooperative Science Program of the National Science Foundation to Prof. D.S. Farner.Herrn Professor Dr. Dres h.c. Wolfgang Bargmann zu seinem 70. Geburtstag am 27. Januar 1976 gewidmet.     

Ultrastructure of the neurosecretory system of the Colorado potato beetle,Leptinotarsa decemlineata (Say)

Summary The ultrastructure of seven types of neurosecretory cells (NSC) in the medial and lateral groups of the protocerebrum is described. The differences among cell types established earlier by light microscopy parallel differences in size and appearance of the neurosecretory particles observed in electron micrographs. No relationship was found between the affinity for Gomori's paraldehyde fuchsin stain and the nature of the particles.The secretions of the A-, A₁-, and C-types of NSC of the medial group are characterized by electron-dense neurosecretory granules of 1250 Å dia., medium-dense granules of 2100 Å, and electron-lucent vesicles of 1700 Å, respectively. The L-type NSCof the lateral group contain smaller (1300 Å) or larger (1700 Å) neurosecretory granules. The medial B- and E-types of NSC and the lateral L_B-type contain granulated vesicles (1200 Å) of the same appearance. These cell types differ in other respects and most likely have separate functions.The author wishes to thank the Laboratory of Virology of the Agricultural University for the use of the electron microscope, Mr. J. Groenewegen and Miss J. van Rinsum for technical assistance, and Professor J. Lattin for correcting the English text. Part of the work has been done while the author was in the service of the Netherlands Organization for the Advancement of Pure Research (ZWO, grant 942-48), and the National Council for Agricultural Research (TNO).     

Three-dimensional organization of the collagen fibrils in the rat sciatic nerve as revealed by transmission- and scanning electron microscopy

Summary The organization of collagen fibrils in the rat sciatic nerve was studied by scanning electron microscopy after digestion of cellular elements by sodium hydroxide treatment, and by conventional transmission electron microscopy. The epineurium consisted mainly of thick bundles of collagen fibrils measuring about 10–20 m in width; they were wavy and ran slightly obliquely to the nerve axis. Between these collagen bundles, a very coarse meshwork of randomly oriented collagen fibrils was present. In the perineurium, collagen fibrils occupied the interspaces between the concentrically arranged perineurial cells; in each interspace, they formed a sheet of characteristic lacework elaborately interwoven by thin (about 3 m or less in width) bundles of collagen fibrils. In the subperineurial region, there was a distinct sheet of densely woven collagen fibrils between the perineurium and underlying endoneurial fibroblasts. In the endoneurium, collagen fibrils surrounded individual nerve fibers in two layers as scaffolds: the inner layer was made up of a delicate meshwork of very fine collagen fibrils, and the outer one consisted of longitudinally oriented bundles of about 1–3 m in width. The collagen fibril arrangement described above may protect the nerve fibers against external forces.     

Supramolecular recognition: association of palladium molecular clefts with planar platinum complexes

The crystal structures of two new molecular recognition adducts formed between a dicationic, di-terpyridyl-Pd-Cl molecular cleft and two square planar platinum complexes are reported. In both structures, the planar platinum-containing guests are located within the molecular cleft formed by the two parallel disposed terpyridyl-Pd-Cl⁺ units of the receptor. The crystal structure of the adduct formed between the molecular cleft and a neutral platinum complex has interplanar distances between the host and guest of 3.24 Å, a distance shorter than that usually ascribed to π-stacking interaction (∼3.45 Å). The short distance is likely the result of metal-metal interaction between the host and guest. The second adduct, that between the dicationic molecular receptor and an anionic platinum complex, also bears the guest within the molecular cleft. The interplanar distances between the cationic terpyridyl-Pd-Cl units of the host and the anionic guest (3.21 and 3.29 Å) are also shorter than typical π-stacking distances but no metal-metal interaction is present. Coulombic attraction between the host and guest is believed to be responsible for the short interplanar separation. These data are discussed in relation to analogous systems that associate through π-π and metal-metal interaction.