Electron microscopy of the tracheal ciliated mucosa in rat

Summary The structure of the tracheal epithelial cells from rat has been studied by electron microscopy on approximately 200 Å thick sections with a resolution of better than 30 Å.The epithelium is found to be of a simple columnar type composed of ciliated cells, mucus producing (goblet) cells, basal cells and a fourth kind of cell, here called brush cell. A great number of non-ciliated cells has also been encountered. It has been proved that these represent goblet cells in different stages of intracellular synthesis of mucous granules. The ciliated cells have approximately 8–9 cilia per square micron and there are about 270 cilia on each cell, the calculated surface area being 33 square microns. They are covered by a 70 Å thick membrane. The ciliary filaments are arranged in a pattern of 2 separate ones in the center and a ring of 9 peripheral ones, each divided into 2 subfilaments by a wall with same thickness as the filamentous wall itself, this being 60 Å. The peripheral filaments are continuous with the basal corpuscles. The structure of the corpuscles as compared with earlier findings is discussed. A number of 0.05 micron thick and 1 micron long filiform projections emerge from the cell surface. No cuticle is present.The cell membrane facing adjacent cells is 90 Å and separated from their cell membrane by a 105 Å wide space, this space, being expanded towards a level corresponding to the proximal parts of the cell. A structure that represents terminal bar has been encountered. The cytoplasm is loose and composed of 160 Å thick granules. Spaces enclosed by 50 Å thick membranes with attached 160 Å thick granules (-cytomembranes) are rare. The Golgi zone is analyzed and its regular composition of -cytomembranes, granules and vacuoles is confirmed. The mitochondria with a mean width of 0.23 micron differ to their inner structure from the common type in that the triple layered membranes are highly interconnected. Large opaque granules are encountered in the cytoplasm. Ring-shaped, 850 Å wide, structures are present in the nuclear membrane. The goblet cells are not as abundant as the ciliated cells, the ratio being 14. Small filiform projections covered by a 95 Å thick membrane protrude from the cell surface. This membrane is continuous with the cell membrane, the latter with the same dimensions as in the ciliated cells. Terminal bars are present. The cytoplasm is very opaque due to a dense packing of the 165 Å opaque granules, many in clusters of 4–6. The -cytomembranes have the same dimensions as mentioned above for those present in the ciliated cells. The Golgi zone is of regular composition. There is a suggestion that the Golgi vacuoles and the -cytomembranes are involved in the formation of mucus. In the stage of cellular activity with but few mucous granules, there is a great number of large opaque granules, the size varying from 0.4–1 micron. The mitochondria with a mean width of 0.23 micron have an outer triple layered membrane with a total thickness of 180 Å. The central less opaque layer is 70 Å and the opaque layer on either side is 55 Å. The inner membranes are arranged parallel to each other and have a triple layered composition where the central less opaque layer is 65 Å and the opaque layers each 60 Å. The brush cells belong to the non-ciliated cells. They are encountered singly, surrounded by goblet cells. The surface structures are shaped like brushes or clumsy protrusions which emerge from the distal end of the cell, and are covered by a 95 Å thick membrane. There have been no suggestions of the brushes being cilia in a stage of growth, nor is it probable that they represent stereocilia. They most nearly resemble the intestinal brush border extensions and thus might serve as a resorbing structure.The cytoplasm of the brush cells appears of medium opacity between the ciliated cells and goblet cells and is composed of 155 Å opaque granules. The -cytomembranes are very rare. The Golgi zone is diminutive though of regular composition. The mitochondria are abundant and small with a mean width of 0.14 micron. The outer and inner membranes are triple layered with approximately the same dimensions as reported for the mitochondria of the ciliated and goblet cells. The inner membranes are very few, often only one or two are present. Some of the large opaque granules have inside a very regular arrangement of small 60 Å thick opaque granules arranged in a crystallinic pattern. In the cytoplasm 0.5–1 micron long bundles of 30–40 Å wide fibrils are encountered. The nucleolus shows a characteristic structure of concentrically arranged thin membranes. The basal cells are believed to represent lymphocytes or white blood cells. They sometimes rest on the basement membrane, sometimes are encountered in the distal part of the intercellular spaces. They are bordered by a 110 Å thick cell membrane and have a rather opaque cytoplasm characterized by 160 Å thick opaque granules. A very small Golgi zone is present. The mitochondria, the mean width being 0.14 micron, have triple layered outer and inner membranes, where the less opaque central layer is 65–70 Å and the opaque layers 45–50 Å each. The basement membrane has a thickness of 600 Å. No inner structure has been resolved. The basement membrane is separated from adjacent parts of the ciliated, goblet, brush, and basal cells by a 250 Å wide less opaque space. Below the basement membrane is the lamina propria of the trachea, which is composed of collagen and elastin fibers together with fibroblasts, white blood cells and lymphocytes. The relationship between different types of tracheal epithelial cells in rat has been analyzed. There has been found no indication of a transformation of any type of cells observed into a different type of cell. The development of basal cells via supporting cells or intermediate cells to goblet cells or ciliated cells has not been noticed. On the contrary, all cells that in light microscopy could have been considered to be supporting or intermediate cells, we have been able to recognize as brush cells or as goblet cells to a varying degree filled with mucous granules. If the cells did not seem to reach the cell surface it has been found to be due to a diagonal direction of the sectioning. In this connection it should be emphasized that this relationship is valid only in rat where it is known that the epithelium is of a simple columnar type as distinct from the conditions in man, that epithelium being of a pseudostratified columnar type.This paper is based on a report given at the meeting of Deutsche Gesellschaft für Elektronenmikroskopie in Münster, March 28–31, 1955 and at the Scandinavian Electron Microscope Society Meeting in Stockholm, May 13, 1955.     

Die Aktivitätsperiodik von Nagern im künstlichen 24-Stunden-Tag mit 6–20 Stunden lichtzeit

Zusammenfassung Die Tagesperiodik der lokomotorischen Aktivität von weißen Ratten und Mäusen ist nicht einfach 24-Std-periodisch. Man beobachtet auch im künstlichen Licht-Dunkel-Wechsel 2 Maxima der Aktivität, die den beiden Umkehrpunkten der Umweltperiode: Licht-an und Licht-aus zugeordnet sind. Veränderungen des Verhältnisses von Lichtzeit zu Dunkelzeit (bei unveränderter Dauer der Periode mit 24 Std) führt zu entsprechenden Verformungen der tierischen Periodik: die Maxima folgen mehr oder weniger streng den Verschiebungen der Umkehrpunkte, wie das auch von den jahreszeitlichen Änderungen der Vogelperiodik unter natürlichen Bedingungen bekannt ist.Wird die Zahl der Lichtstunden im 24-Std-Kunsttag von normal 12 Std um 6 Std herauf- oder herabgesetzt, so folgen die Maxima den Umkehrpunkten nicht in gleichem Ausmaß. Bei der Maus beträgt der Abstand zwischen Morgen- und Abendmaximum im Kunsttag mit 12 Std Licht rund 15,5 Std. Im Kunsttag mit 6 oder 18 Std Licht wird dieser Abstand nur um jeweils 1,5 Std verkleinert oder vergrößert. Das gilt auch für Tiere, die bereits 6 Wochen an das entsprechende Licht-Dunkel-Verhältnis angepaßt wurden. Die endogene Komponente der Tagesperiodik läßt Verformungen durch den Zeitgeber nur im begrenzten Umfang zu.Das Verhältnis der Lichtstundenzahl zur Dunkelstundenzahl übt einen starken Einfluß auf die insgesamt vom Tier entwickelte Aktivität aus. Bei schrittweiser Vergrößerung der Lichtstundenzahl von 12 über 14 auf 16 Std/die Licht versuchen dunkelaktive Tiere durch Steigerung der stündlichen Aktivitätsleistung zumal in der Dunkelzeit die Verkürzung der von ihnen bevorzugten Zeitspanne auszugleichen; sie erreichen im allgemeinen im Kunsttag mit rund 14–16 Std/die Licht die größte Gesamtaktivität je 24 Std. Im Kunsttag mit 18 Std Licht und mehr bricht diese Regulation zusammen — die Gesamtaktivität nimmt stark ab. Dasselbe gilt bei Verkürzung der Lichtstundenzahl auf 6: sowohl in der Lichtzeit wie in der Dunkelzeit wird unter diesen Umständen die je Stunde entwickelte Aktivität auf weniger als die Hälfte der Werte herabgedrückt, die für den Kunsttag mit mittlerer Lichtstundenzahl gelten.Die Ergebnisse legen den Schluß nahe, daß je nach Tierart bestimmte Verhältnisse von Licht zu Dunkel eine optimale Umwelt darstellen und daß ganz allgemein nicht nur die Durchschnittswerte der wichtigsten Umweltgrößen sondern auch deren periodische Änderungen entscheidend die Lebensäußerungen der Tierwelt beeinflussen.     

Territorial Behaviour by a Clan of Spotted Hyaenas Crocuta crocuta

Territorial behaviour of a clan of spotted hyaenas, Crocuta crocuta, was investigated in the Kruger National Park over a period of 27 months. These hyaenas were highly territorial, spending ? of their total activities on territory patrol by scent-marking intensively and monitoring 64 marking posts, particularly in border regions. Females, the more philopatric sex, were most active in territory defence. Local intensity of territorial activities by residents within their 130-km² territory was directly proportional to intrusion pressure by neighbours. When clan size was reduced, the ability to defend disputed land declined and larger neighbouring clans appropriated parts of the territory. We propose a relationship between resource distribution, intrusion pressure and territory defence.     

Kinetics of cooperative ligand–lattice binding: Fast Monte Carlo integration

A fast Monte Carlo integration algorithm with varying time step is described for cooperative binding of ligands of arbitrary length to a one-dimensional lattice. This algorithm is particularly suitable for strongly cooperative or anticooperative systems, i.e., when the time scales for different kinetic events are very different. As an application, the kinetics of a bimodal two-ligand system are briefly discussed.     

Mechanisms responsible for the positive diversity–productivity relationship in Minnesota grasslands

Species’ extinctions have spurred debate on whether interactions among few or among many species cause a positive diversity–productivity relationship in experimentally assembled grasslands. We addressed this question by quantifying the productivity of 14 species across an experimental diversity gradient in Minnesota. We found that interspecific interactions leading to coexistence and competitive displacement both determine which species overyield; i.e. are more productive at high diversity. Overyielding species were either superior N competitors (C4 grasses) or N fixers (legumes). Surprisingly, these species were not most productive in monoculture, thus, the ‘selection’ of productive species in diverse plots did not cause the positive diversity–productivity relationship. Both positive (with legumes) and negative interspecific interactions (with C4 grasses) determined whether individual species overyielded. Foliar pathogens did not cause overyielding, although other natural enemies may be responsible. Overyielding species are not displacing underyielding species over time, implying that other diversity‐promoting interactions also operate in this experiment.     

Homeotic duplication of the pelvic body segment in regenerating tadpole tails induced by retinoic acid

 Homeosis, the ectopic formation of a body part, is one of the key phenomena that prompted the identification of the essential selector genes controlling body organization. Shared elements of such homeotic genes exist in all studied animal classes, but homeotic transformations of the same order of magnitude as in insects, such as the duplication of the thorax in Drosophila mutants, have not been described in vertebrates. Here we investigate the capacity of retinoic acid to modify tail regeneration in amphibians. We show that retinoic acid causes the formation of an additional body segment in regenerating tails of Rana temporaria tadpoles. A second pelvic section, including vertebral elements, pelvic girdle elements and limb buds, forms at the mid-tail level. This is the first report of a homeotic duplication of a whole body segment in vertebrate axial regeneration. Received: 16 August 1996 / Accepted: 20 September 1996     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific α-satellite DNA

CENP-B, a highly conserved centromere-associated protein, binds to -satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENP-B box. By fluorescence in situ hybridization (FISH) with an oligomer specific for the CENP-B box sequence, we have demonstrated the abundance of CENP-B boxes on all chromosomes (except the Y) of humans, chimpanzee, pygmy chimpanzee, gorilla, and orangutan. This sequence motif was not detected in the genomes of other primates, including gibbons, Old and New World monkeys, and prosimians. Our results indicate that the CENP-B box containing subtype of -satellite DNA may have emerged recently in the evolution of the large-bodied hominoids, after divergence of the phylogenetic lines leading to gibbons and apes; the box is thus on the order of 15–25 million years of age. The rapid process of dispersal and fixation of the CENP-B box sequence throughout the human and great ape genomes is thought to be a consequence of concerted evolution of -satellite subsets on both homologous and nonhomologous chromosomes.Correspondence to: T. Haaf     

Slow conformational exchange in DNA minihairpin loops: A conformationalstudy of the circular dumbbell d〈pCGC-TT-GCG-TT〉

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson–Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decanter 5′-d〈pCGC- TT-GCG- TT〉 -3′ ( I ) is studied in solution by means of nmr spectroscope. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson–Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K. The L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop with only one remaining solvent-accessible hydrogen bond between NH_α of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5′-CTTG-3′ loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5′-3′ loop-stem junction in the 5′-GTTC-3′ loop. The two conformers. L2L2 and L2L4, occur in slow equilibrium (2–20 s^?1). Moderate line broadening of specific ¹H, ¹³C, and ³¹P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T₁ relaxation measurements, chemical-shift considerations, and complete hand-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational slates in the anti–syn equilibrium. © 1995 John Wiley & Sons, Inc.     

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.