Scanning electron microscopy of primary membrane bone

Summary The organization and the development of primary bone were studied in a number of newborn or foetal mammals. Specimens were derived from long bones, calvaria and mandibles. The surface of the mineral component was exposed by treatment with an organic solvent (hot 1,2 ethane diamine or cold sodium hypochlorite solution) and in the case of some internal surfaces (endosteal surfaces of long bone shafts, endocranial aspects of calvaria and the lining of the dental groove or crypts of mandibles) some success was achieved in dissecting the soft tissues from the bone matrix surface. These surface were studied by scanning electron microscopy. Ground sections of similar material were studied by microradiography.Primary membrane bone was found to be most different from adult lamellar bone on rapidly growing (periosteal) surfaces and in the following respects. 1) Mineralization of both the ground substance and collagen occured close to the mineralizing front, so that a higher level was reached rapidly and without a maturation phase. 2) The mineral clusters at the mineralizing front did not reflect the collagen fibre distribution. 3) The collagen fibre bundles at the matrix surface, at least at internal surfaces, were irregular in size and random in orientation.Mineralizing fronts intermediate in character between those of rapidly forming, periosteal primary bone and adult lamellar bone were found on the uncommon internal forming surfaces and in the walls of vascular channels.Most internal surfaces were resorbing. There was a wide range in the size of the resorption lacunae — the many which were only as large as the osteocyte lacunae exposed by the resorption process may have been caused by uninucleated cells.This work has been supported by a grant from the Medical Research Council. The Stereoscan scannning electron microscope was provided by the Science Research Council, and the microradiographic equipment by Mr. R. V. Ely and the Ely-Webster trust. We would like to thank Mr. P. S. Reynolds, Miss A. J. Sutton and Mr. P. F. Farmer for technical and photographic assistance and Mrs. Jeanne Mills for secretarial assistance.     

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.     

On the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastrointestinal tract has been studied, in foetal and adult material, by a technique involving the staining of sections first by an argentaffin method (Gomori-hexamine silver, Schmorl, Diazonium) and subsequently by an argyrophile method (Bodian). A comparison of the cells staining by the two methods shows that all argentaffin cells of the human gastrointestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of HamPerl (1952) regarding the presence of non-argyrophile argentaffin cells.W. H. O. fellow from the Department of Anatomy, Medical College, Rohtak, India. — I am very grateful to Professor J. D. Boyd and to the World Health Organisation for having made it possible for me to carry out this research at the Anatomy School, Cambridge. I am indebted to Dr. G. A. Gresham and his staff for their very willing cooperation in providing material from surgical resections. My thanks are also due to Mr. J. F. Crane for the photographs and to Mr. J. W. Cash and Mr. R. Smith for helpful discussions on staining techniques.     

Scanning electron microscopic studies of cilia

Summary A simple method for the preparation of ciliated epithelia for study with the scanning electron microscope is described. Ciliary groups are well preserved and it is possible to discern individual cilia and work out their numbers and orientation. Following scanning electron microscopical study some of the material was prepared for transmission electron microscopy and the ultrastructure of the tissue was found to be surprisingly well preserved. The tracheal epithelium of the rabbit, the olfactory epithelia of the goldfish and the rabbit, and the sensory epithelia in the statocyst of a cephalopod mollusc were examined with the scanning electron microscope to demonstrate the possibilities of the method. Acknowledgements. We would like to thank Professor J. Z. Young for his continued interest and support. The scanning electron microscope was purchased with a grant provided by the Science Research Council to Dr. Boyde, Mr. R. Willis helped in the initial stages of the study, Mr. G. Savage provided help with the goldfish material, Mr. S. Waterman provided much photographic assistance, and Mrs. N. Finney the secretarial assistance.     

The rhabdomeric microvilli of several arthropod compound eyes kept in darkness

Summary Electron microscopical studies were made on the fine structure of the rhabdomeric microvilli of the compound eyes of seven species of arthropods (Procambarus, Neocaridina, Caridina, Potamon, Artemia, Diestrammena, Drosophila) raised in complete darkness for 1–8 months or for successive generations, using various fixation techniques.The rhabdomeric microvilli of the individuals kept in darkness for a long period were regularly arranged as in normal eyes in the material prepared by double fixation with glutaraldehyde and OsO₄, whereas in those fixed solely by OsO₄ various forms of vesiculations were seen. The structural changes of the rhabdomere in darkness, which have been reported by several workers, were conceived to be an artefact caused by OsO₄ fixation.This work is supported by a grant from the U.S. Army Research and Development Group (Far East), Department of the Army (DA-CRD-AG-S92-544-67-G61).The authors wish to express their gratitude to Dr. S. Mori and Mr. T. Kuramoto for their help in supplying some of the materials. We are also indebted to Dr. C. J. C. Rees for correcting English of this paper.     

The fine structure of cephalopod blood vessels

Summary The fine structure of blood vessels of the retina and arms of Octopus and the lip of Sepia is described.There are two main types of vessels. The first type (type 1) has a complete basement membrane, an incomplete lining of endothelial cells formed into finger—like processes, and a complete investment of pericytes surrounding the vessel. These latter cells contain myofilaments. The second type (type 2) is smaller and contains few if any myofilaments, and has a less complexly folded endothelium. This type is subdivided into three forms depending on the number of pericytes and the form of the endothelial lining.Amoebocytes are described and these form a distinct group of cells.The fine structure of hemocyanin is observed in normally fixed material and is correlated with its previously described structure.These observations are related to their possible functional importance.

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Zusammenfassung Die Ultrastruktur von Blutgefäßen der Retina und der Arme von Octopus und der Lippe von Sepia wird beschrieben.Es bestehen vorwiegend zwei Gefäßtypen. Der erste Typ (Typ 1) zeigt eine geschlossene Basalmembran, eine unvollständige Begrenzung durch Endothelzellen, die fingerförmige Fortsätze bilden und eine vollständige Pericytenhülle um das Gefäß. Die letztere enthält Myofilamente. Der zweite Typ (Typ 2) ist kleiner und enthält wenig oder keine Myofilamente. Er besitzt ein weniger komplex gefaltetes Endothel. Dieser Typ wird gemäß der Zahl der Pericyten und der Form der endothelialen Begrenzung in drei Gruppen unterteilt.Deutlich verschiedene Amoebozyten werden beschrieben.Die Ultrastruktur von Haemocyanin ist an normal fixiertem Material zu beobachten. Sie wird zu ihrer in früheren Arbeiten beschriebenen Struktur in Beziehung gebracht.Die Beobachtungen werden in Hinblick auf ihre mögliche funktionelle Bedeutung diskutiert.

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Acknowledgements. We would like to acknowledge the encouragement and advice of Professor J. Z. Young and Dr. E. G. Gray. Mrs. J. I. Astafiev did the drawings and Mr. A. Aldrich and Mr. S. Waterman helped with the photography.     

Development of P-protein in sieve elements ofMimosa pudica

Summary The P-protein in sieve elements of leaves ofMimosa pudica L. is first discernible as fine fibrous material which forms homogeneous aggregates. Ribosomes, rough endoplasmic reticulum, and dictyosomes with associated vesicles occur in the cytoplasm surrounding the aggregates. The plastids and mitochondria are in a parietal position in the parts of the cell where the nascent P-protein accumulates. In a later stage, the fibrillar material is organized into a three-dimensional system of five- and six-sided elongated compartments. The corners of the compartments appear solid at first, then they become electron lucent in the center and assume tubular form. Aggregates of mature P-protein tubules usually occur near the compartmentalized system. Tubules in pentagonal or hexagonal arrangements may be present in the aggregates and may be partly interconnected. The conclusion was drawn that the P-protein tubules are assembled at the corners of compartments within a continuous orderly system. The fully formed tubules occur first in aggregates, the P-protein bodies. Later the aggregates become loose and partly dispersed. Many of the dispersed tubules assume a loose, extended, helical form characteristic of P-protein in older sieve elements.This work was supported in part by National Science Foundation grant GB-5506. I am also grateful to MissHatsume Kosakai and Mr.Robert H.Gill for technical assistance.     

Stepwise engineering of a <Emphasis Type="Italic">Pichia pastoris</Emphasis> D-amino acid oxidase whole cell catalyst

Background  

Trigonopsis variabilis D -amino acid oxidase (Tv DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of Tv DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris Tv DAO whole-cell biocatalyst.     

Changes in the fine structure of the human testicular interstitial cells after treatment with human gonadotrophins

Summary Study of the fine structure of the human interstitial cells after prolonged stimulation with human gonadotrophin reveals a striking increase in the quantity of the agranular endoplasmic reticulum. This is accompanied by an increase in the number of mitochondria which exhibit more extensive cristae, collections of intramitochondrial lipid and aggregations of electron-dense granular deposits. A rise is also evident in the number of lipofuscin pigment deposits and granular membrane-bounded bodies, both of which exhibit acid phosphatase activity. These changes after gonadotrophic stimulation are discussed in relation to steroid biosynthesis.In the pretreatment biopsies of these patients aged between 25–35 years, some interstitial cells contain intranuclear crystals which exhibit a hexagonal structure. The relationship of these intranuclear crystals to the cytoplasmic crystals of Reinke is discussed.The author is indebted to Dr. J. W. Johnstone and Dr. A. Long for the human material used in this study. Thanks are also due to Dr. H. P. Taft for helpful suggestions in the management of these patients, to Professor B. Hudson for the estimations of plasma testosterone and to Dr. J. B. Brown for the supply of human pituitary gonadotrophin and the estimations of urinary oestrogens. The technical help of Mr. T. Mezciems and the photographic assistance of Mr. J. S. Simmons F. R. P. S. and Miss S. Flett is gratefully acknowledged.     

Fabrication of collagen hybridized elastic PLCL for tissue engineering

Biodegradable elastic poly(l-lactide-co-ε-caprolactone) (PLCL) (50:50) copolymer was blended with collagen (0.05, 0.1 and 0.2% w/w) in an acidic dioxane solution to form a collagen/PLCL hybrid material suitable for tissue engineering applications. Stability and dispersivity of collagen on collagen/PLCL hybrid films and collagen coated PLCL films under mechanical stress were determined by a collagen release test and water contact angle measurement. Hybrid films had a higher stability than collagen-coated PLCL films. Elastic recovery as well as high interconnectivity and uniform pore morphology of the hybrid scaffolds were not affected by the collagen concentration. Fibroblasts (NIH-3T3) cell culture test was performed for cell growth and viability evaluation. Collagen concentration had little affect on the initial cell adhesion after 4 h cell culture; but after 48 h cell culture, increased cell proliferation on the hybrid films was observed. The hybrid material can be applied as a scaffold for vessel and cartilage regeneration for mechano-active tissue engineering.     

Development of the acrosomic system of the spermatozoon in the Norwegian lemming (Lemmus lemmus)

Summary An electronmicroscopic study on the development of the acrosome system of the spermatozoon in the Norwegian lemming (Lemmus lemmus) has been performed. The proacrosomal granules were found to be few and to consist of a dense center and a less dense periphery, and the acrosomal vesicle to contain stainable material of the same structure but of lower density than that of the acrosomal granule. Like in the guinea pig, two zones of different density exist throughout acrosome development and are visible also in mature spermatozoa. A large osmiophilic formation consisting of saccular, tubular, and lamellar structures, was found between the apex of the condensed nucleus and the acrosome and was identified as perforatorium.We are greatly thankful to Dr. Antti Telkkä, and Mr. Mauri Nyholm, M. Sc. for expert advise in electronmicroscopy and Mr. P. Lehtimäki for photographic help. For the supply of the lemmings we are indebted to Prof. K. Lagerspetz, and Mr. O. Hissa, M. Sc. This work is related to a series of studies on the biology of the Norwegian lemming (for previous works see Asp et al.).     

Fine structure of cilia in rat cerebral cortex

Summary Cilia have been demonstrated on granular neurons and astroglial cells in the fascia dentata, a part of the hippocampal region, in the rat. Every granular cell seems to possess one cilium, which shows an 8+1 pattern in the greater part of its length. This 8+1 pattern is shown to result from the displacement of one peripheral doublet of a 9+0 cilium into the middle of the cilium. The neuronal cilia have a two-centriole basal organization, and fine rootlets radiate from the basal body proper into the cytoplasm. The possible function and significance of these cilia are discussed on the basis of earlier literature.This study was supported in part by Grant NB 02215 of The National Institute of Neurological Diseases and Blindness, U.S. Public Health Service, in part by The Norwegian Research Council for Science and the Humanities. This aid is gratefully acknowledged. I am greatly indebted to Dr. Th. Blackstad for encouragement and advice during this study, to Mrs. J. L. Vaaland, Mr. B. V. Johansen and Mr. E. Risnes for technical assistance, and to Dr. B. Afzelius for valuable discussions.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Cytologic and metabolic parameters of pineal inhibition by continuous light in the rat (Rattus norvegicus)

Summary Adult rats (Rattus norvegicus) were subjected to continuous light or control conditions (14 hours light/day) for six weeks or longer, and quantitative cytological and metabolic studies were made of the pineal organs. After 11 weeks of continuous light, the pineal parenchymal cell's largest nucleolar eosinophilic mass is significantly reduced in diameter, especially in the medulla of the organ. Evidence of metabolic inhibition includes reduction of pineal glycogen content, succinic dehydrogenase activity, and respiration in the absence of exogenous substrates. Pineal ATP content, P³²-phosphate uptake and 5-hydroxy indole acetic acid content did not appear to be affected. Pineal serotonin content and melatonin-forming activity in the continuously lighted animals were measured but could not be interpreted metabolically, due to the diurnal fluctuations of these in control animals. Results provided here and elsewhere suggest that pineal inhibition by continuous light involves primarily the citric acid cycle, the accumulation of metabolites and lipid, and the synthesis of protein.This investigation was supported by grant GM-05219 from the National Institutes of Health, U.S. Public Health Service.I am grateful to Mrs. Virginia Green Bowers, Mrs. Ann Richards, Mr. Peter Charles Baker and Mr. Jorge Antonio Alvarado for laboratory assistance, and to Dr. Richard Strohman and Mr. David Epel, for advice on the determination of ATP.     

Hypothalamic neurosecretion and photoinduced testicular growth in the tree sparrow,Spizella arborea

Summary To assess the role of the hypothalamic neurosecretory system in the mechanism of photoperiodic control of testicular growth in Spizella arborea, we have examined the effect of (1) photostimulation on the density of paraldehyde-fuchsin-stainable (PAF+) material in the neurovascular zone of the median eminence, and (2) interruption of the hypothalamo-hypophysial (neurosecretory) tract on the photoperiodic testicular response. Birds were captured during midwinter and retained on 8-hr daily photoperiods several months prior to the experiment. Interruption of the neurosecretory tract was effected by a small knife fashioned from stainless steel wire; radiography was employed to facilitate stereotaxic placement of the knife assembly.Extending the daily photoperiod from 8 to 20 hrs induced rapid testicular growth in photosensitive Spizella arborea, but had no consistent effect on the density of PAF+ material in the palisade layer of the anterior median eminence. Whereas some terminal controls showed moderate depletion after 21 days' exposure to 20-hr daily photoperiods, others retained as much or more stainable material than initial controls in which testicular growth was not induced. Moreover, testicular growth was induced at essentially identical rates whether the PAF+ innervation of the anterior median eminence was intact or eliminated to varying degrees. The finding that the rate of photoperiodically induced testicular growth is independent of the density or pattern of accumulation of PAF+material in the neurovascular zone of the median eminence, coupled with the inconstant effect of photostimulation on the density of stainable material therein, strongly suggests that the eminential component of the hypothalamic neurosecretory system is not a part of the mechanism that controls photoperiodic testicular growth in S. arborea.Contribution No. 980, Division of Biology, Kansas Agricultural Experiment Station, Manhattan 66502. This investigation was supported in part by the Undergraduate Research Participation Program (GY-771) of the National Science Foundation. We are grateful to French's Pet Bird Laboratory, Rochester, N.Y., for the parakeet foods, to Richard S. Donham, Michael D. Ruff, Thomas G. Shane, and Philip G. Watt for assisting with field operations, to Mr. Gary R. Dierking for preparing the drawing, and to Mr. Don Pihlaja for assisting with the photomicrography.     

Some structural features of an insoluble α-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular α-D-glucans from sucrose: alternan and dextran. An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan. Methylation analysis shows it contains linear (1→3) and (1→6) linkages as well as (1→2) and (1→3) branch linkages. The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue. Treatment of the soluble dextranase-limit fraction with an α(1→2) debranching enzyme led to further dextranase susceptibility. Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L. mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans. Received 05 November 1998/ Accepted in revised form 31 March 1999     

Accumulation of organelles at the ends of interrupted axons

Summary Proximal and distal stumps of the sciatic nerve of rats were examined with the light and electron microscope in the course of 48 hours following nerve crush. On both sides of the lesion organelles accumulate in axons beyond regions disorganized by injury. A stretch of clear axoplasm filled with fine granules usually separates the cone of accumulating particles from the damaged part of the fibre. From two hours onwards closely packed vesicles, tubules, mitochondria and other organelles form dense pellets which fill up the whole lumen of the fibre. Further away from the fibre tip organelles are stranded at the circumference only, whereas the central core is occupied by neurofilaments. In a number of fibres no pellets are observed and only a moderately increased network of axoplasmic reticulum is seen at the fibre ends.Measurements on isolated fibres have shown that the length of the pellet increases with time on both sides of the lesion up to 18 hours after crush; thereafter the elongation is arrested in the distal stump, while in the proximal stump it continues further at a slower rate.The authors wish to acknowledge gratefully the technical assistance of Mrs. M. Sobotková, Mr. M. Doubek, Mrs. J. Waryszewska and Mrs. B. Lwowska.     

Preparation and Some Properties of Type I Collagen from Fish Scales

Soluble collagen from fish (sardine) scales was yielded at about 5% with 0.5 m acetic acid after demineralization with EDTA, while a great portion of the collagen remained insoluble. The solubility of this insoluble collagen was about 20% at 45°C (denaturation temperature of soluble collagen) for 24 h. The remaining 80% of the insoluble collagen was denatured in the form of insoluble gelatin, and that may be an interesting food material.     

A possible correlation between ultrastructure and function in the thin descending and ascending limbs of the loop of Henle of rabbit kidney

Summary An ultrastructural study of the thin loops of Henle has been made in the renal papilla of the rabbit. Animals in different states of water balance were used but no morphological difference was observed in the loops obtained from animals in different experimental groupings. The cytoplasm of the squamous cells lining the limbs was characterised by a paucity of organelles. Descending and ascending limbs were distinguishable. A distinct morphological difference was seen in the junctional regions of cell processes of the descending and ascending thin limbs of the loop. The ascending limb processes were joined by continuous tight junctions whereas the descending limb junctional regions invariably showed a space of at least 70 Å between adjacent processes. It is suggested that there may be a correlation between the structure of these junctional regions and the different permeability characteristics of the two limbs. The thin ascending limb must, on physiological evidence, be relatively impermeable with reference to the thin descending limb.The author wishes to thank Professor F. R. Johnson for his advice and assistance, and Mr. R. F. Birchenough, Mr. P. L. Hyam and Mr. J. Manston for valuable technical assistance.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.