The colour pattern of Lineus atrocaeruleus (Nemertia)

The colouration of living specimens of Lineus atrocaeruleus (Lineidae, Nemertea) is bluish or brownish black with many transverse yellow rings. The pigments responsible for the colouration are granular and are elaborated by epidermal and dermal pigment cells, both of which contain uroporphyrins. Granules of melanic nature occur in the dermal pigment cells. The significance of the epidermal pigment cells is discussed.     

Das Verhalten des aminergen Nervensystems während der Regeneration des Vorderdarms von Lineus sanguineus Rathke (Nemertini)

Znsammenfassung An 1–16 Tagen alten Regeneraten wurde mit der OsO₄-KJ- und OsO₄-NaJ-Fixierung sowie einer fluoreszenzmikroskopischen Methode zur Erfassung biogener Amine die Innervation des regenerierenden Vorderdarms histologisch untersucht.Die Vorderdarmanlage entsteht ab dem 4. Regenerationstag. Sie wird von Blastocyten aufgebaut, die dem Mitteldarm und dem Mesenchym entstammen. Im Bereich der Vorderdarmanlage werden Nervenfasern erst ab dem 6. Regenerationstag nachweisbar; ab dem 11. Regenerationstag enthalten sie frühestens ein Catecholamin. Bis zum 16. Regenerationstag entsteht ein aminerger Nervenfaserplexus fast normalen Umfangs. Zu diesem Zeitpunkt können die regenerierten Individuen erstmals Nahrung aufnehmen.Das aminerge Nervensystem beeinflußt das Regenerationsgeschehen nicht. Es ist aber für die Funktionsaufnahme des Vorderdarms sicher von Bedeutung.

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The aminergic nervous system in the regenerating foregut of Lineus sanguineus Rathke (Nemertini)

Summary The aminergic innervation of the regenerating foregut of Lineus sanguineus regenerates, aged 1–16 days, was studied by using OsO₄-KJ and OsO₄-NaJ fixation and fluorescence microscopic methods.The foregut anlage is first observed on the 4th day of regeneration, and consists of blastocytes which originate from the midgut and mesenchyme. Nerve fibers are first detected near the foregut anlage on the 6th day of regeneration. The aminergic nerve plexus are formed from the 11th day onwards and attains a nearly normal size on the 16th day of regeneration; at this time the animals does first able to take in food.The aminergic nervous system did not influence the process of regeneration, but appears to be of importance in the commencement of the foregut function.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Impact of the nemertean Lineus viridis on its polychaete prey on an intertidal sandflat

Interactions of the predator Lineus viridis (O. F. Muller 1774) with infaunal polychaetes were investigated in the German Wadden Sea. L. viridis is the dominant heteronemertean in this intertidal area with a mean abundance of 14 individuals per square meter. Predator enclosure experiments in the field revealed that L. viridis preyed on predatory polychaetes. The preferred prey species was Nereis diversicolor; others were Anaitides maculata. Nephtys hombergii and Harmothoe sarsi. These results were supported by a series of feeding experiments in the laboratory. The rate of consumption was estimated by laboratory and field experiments. The feeding rate of Lineus viridis was estimated by laboratory enclosure experiments to 5.6 g ashfree dry weight (AFDW) per square meter annually.     

Honeycomb structure in the lamina lucida of epidermal basement membrane during metamorphosis of Rana temporaria ornativentris

In this study we examine the structure of the lamina lucida during metamorphosis of Rana temporaria ornativentris. During the metamorphosis of anuran larvae, both the epidermal cells and the dermal connective tissues in the tail regenerate. The basal surface of the epidermis becomes irregular and the epidermal basement membrane detaches from the epidermal cells, showing a widened lamina lucida. In this widened lamina we observed a geometrical honeycomb structure and a ladder structure. Each side of the honeycomb structure was approximately 40 nm and the intervals of the ladder structure were approximately 50 nm. From our observations we believe that the honeycomb and ladder appearances are different aspects of the same structure. At the beginning of metamorphosis anchoring filaments were prominent in the lamina lucida and, when the lamina lucida was tangentially cut, the lamina lucida showed the honeycomb structure. These results suggest that both the honeycomb and the ladder structures observed in the widened lamina lucida originate from constituents of the lamina lucida and become morphologically evident during the epidermal-dermal separation.     

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20 days on 3 μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β₂-integrins, but not β1- or β₃-integrins. Migration from the subendothelial compartment was supported by β1- and β₂-integrins for all cultures, but blockade of β₃-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β₃-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine ?-NH₂ groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Human glomerular basement membrane. Heterogeneity of antigenic determinants

Human glomerular basement membrane was solubilized by digestion with proteolytic enzymes and immunoreactive components were quantitated and characterized by using rabbit antibodies raised against the particulate membrane. A number of antigens were demonstrated but they did not separate on gel filtration. However, two antigenic components in a collagenase digest of the membrane could be separated and isolated by Sepharose 6B chromatography. Chemical characterization suggests that both fragments are noncollagenous glycopeptides (molecular weights approx. 1 000 000 and 60 000–200 000, respectively).     

Invadopodia and basement membrane invasion in vivo

Over 20 years ago, protrusive, F-actin-based membrane structures, termed invadopodia, were identified in highly metastatic cancer cell lines. Invadopodia penetrate artificial or explanted extracellular matrices in 2D culture conditions and have been hypothesized to facilitate the migration of cancer cells through basement membrane, a thin, dense, barrier-like matrix surrounding most tissues. Despite intensive study, the identification of invadopodia in vivo has remained elusive and until now their possible roles during invasion or even existence have remained unclear. Studies in remarkably different cellular contexts—mouse tumor models, zebrafish intestinal epithelia, and C. elegans organogenesis—have recently identified invadopodia structures associated with basement membrane invasion. These studies are providing the first in vivo insight into the regulation, function, and role of these fascinating subcellular devices with critical importance to both development and human disease.     

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8–12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [³⁵S]sulfate into basement membrane from which this non-collagenous ³⁵S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (M_r greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [³⁵S]-sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.     

Die Erregungsübertragung bei Lineus sanguineus Rathke (Nemertini)

Zusammenfassung Mit histochemischer Methodik läßt sich in den Geweben von Lineus sanguineus (Nemertini) Acetylcholinesterase (ACHE; Spezifitätskontrolle: Eserin- und iso-OMPA-Test) nachweisen. Das Enzym kommt im zentralen und peripheren Nervensystem (Ganglienzellen, Neuropilem, Nervenfasern) vor. Daneben sind die Epithelien der Frontalorgane und Kopfspalten, die Cerebralorgane, die Muskulatur und die Blutzellen ACHE-reaktiv. Die Enzymverteilung läßt auf einen cholinergen Transmittermechanismus schließen.Die Befunde werden in Zusammenhang mit Befunden am aminergen Nervensystem von Lineus (Reutter, 1969) besprochen. Möglicherweise werden doppelt innervierte Sinnesorgane (z.B. Frontalorgane) cholinerg erregt und aminerg kontrolliert, analog zum Parasympathicus-Sympathicus-System der Wirbeltiere. Die ACHE-Aktivität im Bereich der Muskulatur, die frei von aminergen Fasern ist, läßt darauf schließen, daß sie cholinerg erregt wird.

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Impulse transmission in Lineus sanguineus Rathke (Nemertini)II. The cholinergic nervous system

Summary With histochemical methods acetylcholinesterase (ACHE; the specifity of which was tested with eserine and iso-OMPA) was demonstrated within several tissues of Lineus sanguineus (Nemertini). The enzyme is mainly located in the central and in the peripheral nervous system (neurons, neuropileme, and nerve fibers). The epithelia of the frontal organs and of the lateral head folds, the cerebral organs, the musculature and the blood cells are ACHE-reactive, too. The distribution of the enzyme may indicate cholinergic neurotransmission.The results are discussed in view to the adrenergic nervous system of Lineus (Reutter, 1969). Possibly double innervated organs (i.e. the frontal organs) are stimulated cholinergically and are controlled aminergically. This will be in analogy to the parasympathetic-sympathetic system of vertebrates. The activity of ACHE within the musculature, where no aminergic nerve fibers are found, indicates its cholinergic stimulation.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Extracellular silk fibres in Stannophyllum (Rhizopodea: Protozoa)

Summary The agglutinated shell or capsule of Stannophyllum zonarium, a deep sea rhizopod, contains a mass of extracellular proteinaceous fibres called linellae. They are 2–3 m diameter, weakly positively birefringent and on examination by X-ray diffraction give an amorphous type of diagram. An amino acid analysis is notable for its very high glycine content and indicates that the molecule may be related to those found in arthropod silk proteins.We wish to thank Mr. G. S. Bell and Mr. C. G. Ogden for their help with the amino acid analysis and electron microscopy respectively.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.     

The ultrastructure and ultracytochemistry of the basement membrane of the Galleria mellonella fat body

Summary The fat body lobes of Galleria mellonella are surrounded by basement membrane — a fine granular layer of connective tissue. This membrane has an affinity for ruthenium red. The results obtained after treatment of the fat body with neuraminidase, hyaluronidase, phospholipase C and proteolytic enzymes suggest that glycoproteins and phospholipoproteins are constituents of this basement membrane. The basement membrane also has the ability to bind concanavalin A-peroxidase, which is associated with the presence of mannoside residues.The preliminary results of these studies were presented at the IX Conference of Electron Microscopy, Gdask, Poland (Dutkowski, 1975)I am greatly indebted to Professor A. Przececka for her encouragement to undertake this study. The excellent technical assistance of Mrs. K. Mroziska, Mrs. Z. Kamiska and the engineering staff of the Laboratory is gratefully acknowledged     

The arrangement of neurosecretory and catecholamine fibres in relation to the pituitary intermedia cells of the skate,Raja radiata

Summary The innervation pattern of the intermediate lobe of the skate (Raja radiata) was studied with histological and fluorescence histochemical methods. Neurosecretory fibres, stained with i.a. pseudo-iso-cyanine, were found running in bundles in the central parts of the cell cords. They terminated partly around the perinuclear parts of the intermedia cells, partly around the apices of the cells close to the vascular walls.A catecholamine innervation of the intermedia was also established. Catecholaminecontaining fibres with the appearance of nerve terminals were found around the intermedia cell apices close to the vessels. In some specimens, catecholamine fibres also seemed to terminate at the perinuclear parts of the cells.Thus it is possible, judging solely from structural relations, that both the cell body (the synthesis pole) and the cell apex (the release pole) receive a dual innervation. Recent experimental evidence indicates that the release of MSH from the pars intermedia is controlled by catecholamine fibres, but as yet there is only structural evidence for a special control of hormone synthesis.This study was supported by grants from the Swedish Natural Science Research Council (No. 99-35 and 2126-2) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-712-05 and B70-14X-56-06).     

Anionic sites of the basement membrane of rat seminiferous tubules during ontogenesis

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as cryptorchidism, fetal irradiation, and ligation of the ductuli efferents lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.     

杜鹃花属映山红亚属植物叶表皮显微形态特征及其分类学意义

为澄清映山红亚属(Rhododendron subg. Tsutsusi)内的系统关系问题,该研究选择杜鹃花属映山红亚属37种植物作为对象,其中29种为首次报道,通过扫描电镜观察其叶表皮显微特征。结果表明:(1)气孔器均为无规则型且均在远轴面。(2)根据叶片微形态特征将映山红亚属的种类分为杜鹃型(R. simisii-type)、岭南杜鹃型(R. mariae-type)、皋月杜鹃型(R. indicum-type)、崖壁杜鹃型(R. saxatile-type)及丁香杜鹃型(R. farrerae-type)5种类型。(3)杜鹃型植物的气孔器周边无或具间断的条形突起。(4)岭南杜鹃型植物的气孔器周围有多层条形突起环绕,保卫细胞两极不具T型加厚。(5)皋月杜鹃型植物的叶表皮保卫细胞两极具有T型加厚,与叶状苞亚属(Subg. Therorhodion)叶状苞杜鹃(R. redowskianum)的气孔器特征相似,推测其与叶状苞亚属具有一定的亲缘关系。(6)崖壁杜鹃型植物的叶表皮毛单一,多呈卷曲状,与其他类型有所不同且未见腺体。(7)丁香杜鹃型植物的叶表皮仅有腺体。(8)依据叶表皮微形态特征讨论了一些近缘种类的关系,如倾向于将背绒杜鹃(R. hypoblematosum)和千针叶杜鹃(R. polyraphidoideum)处理为独立的种,支持保留紫薇春(R. naamkwanense var. cryptonerve)作为南昆杜鹃(R. naamkwanense)的变种地位,不支持将腺花杜鹃(R. adenanthum)并入细瘦杜鹃(R. tenue)作异名处理等。该研究结果表明叶表皮显微特征在映山红亚属内物种划分上具有重要价值。     

Growth of exocrine acinar cells on a reconstituted basement membrane gel

Summary Methods have been developed for culturing a dividing population of morphologically differentiated rat parotid, lacrimal, and pancreatic acinar cells in vitro. Isolated acinar cells were plated onto tissue culture dishes coated with a three-dimensional, reconstituted basement membrane gel. After attachment in Ham’s nutrient mixture F12, the cells were cultured at 35°C in F12 supplemented with 10% heat inactivated rat serum, epidermal growth factor, dexamethasone, insulin, transferrin, selenium, putrescine, reduced glutathione, ascorbate, penicillin, streptomycin, and the appropriate secretagogue. Under these conditions, the cells attached rapidly and DNA synthesis was initiated within 2 to 3 d. Although the cells flattened on the substratum, they continued to maintain their differentiated morphology. The cells contained secretory granules, and the secretory enzymes peroxidase and amylase could be detected. The use of a reconstituted basement membrane gel proved critical for the attachment and growth of exocrine acinar cells.