The air-blood barrier in the human lung

Summary The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3–7 superimposed ridges (fibrils) on the P face and complementary grooves on the E face, i.e., a structure corresponding to a tight junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2–4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a leaky type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.A portion of this work was presented in partial fulfillment for the degree of Dr. med., Hannover Medical School     

Histochemical and chemical studies on pre- and postnatal development of the different systems of “short” and “long” adrenergic neurons in peripheral organs of the rat

Summary The adrenergic innervation in the submaxillary gland, heart, kidney, small intestine, and accessory male genital organs and the development of the adrenal chromaffin cells and the sympathetic ganglia were studied in the rat from 15 days post coitum to 16 days post partum using the fluorescence histochemical method of Falck and Hillarp. The postnatal development of the noradrenaline concentrations in the heart and vas deferens was followed by fluorometric determinations.At about 15 days post coitum, the anlagen of the sympathetic chains were well visible in the form of two dorsal segmented columns of small branching sympathicoblasts exhibiting an intense catecholamine fluorescence. In the midline, ventrally to these two anlagen, another column of sympathicoblasts developed; this seemed to give rise to the prevertebral ganglia and to the short adrenergic neurons supplying the internal genital organs. At the level of the adrenal anlagen, small intensely fluorescent chromaffin cells were collected in two bilateral groups which became enclosed by adreno-cortical cells. This enclosure was, however, not complete even at two weeks post partum.Bundles of growing sympathetic nerves were visible in the periphery of the various organs studied at 19–21 days post coitum. A terminal innervation of the organs suggestive of a functional transmitter mechanism did not start to establish until at or immediately after birth. The final pattern of innervation was usually reached at about one week post partum, and the following development proceeded largely in the form of a quantitative increase in the number of nerves participating in the innervation apparatus. The adult level of noradrenaline in the heart and vas deferens was reached three to five weeks after birth. The small intestine was an exception in that the final pattern of innervation in the wall was attained immediately after birth.There was no overt difference in the rate of development of the terminal sympathetic innervation in organs supplied by short adrenergic neurons (accessory male genital organs) compared to the innervation of the submaxillary gland, heart and kidney, which receive classical long adrenergic neurons.The work was supported by a grant from the Association for the Aid of Crippled Children, New York, and was carried out within a research organization sponsored by the Swedish Medical Research Council (grants No. B71-14X-56-07A and B71-14X-712-06A).     

Direct arteriovenous connections and the intermediate circulation in dog spleen,studied by scanning electron microscopy of microcorrosion casts

Summary The exact nature of the circulatory pathways in dog spleen, particularly with reference to the intermediate circulation and the possible existence of direct arteriovenous connections, has been studied by scanning electron microscopy (SEM) of microcorrosion casts. A new casting procedure was developed in which minimal amounts of material were injected into contracted spleens, thus filling preferentially the faster channels for flow. Extensive filling of the red pulp was thereby avoided, leaving an open view of blood vessels and their connections. The depth of focus of the SEM, incomparably greater than those of transmission electron or light microscopes, enabled vascular pathways to be traced over considerable distances.Using this approach, we have obtained clear evidence for abundant connections between arterial capillaries and venous sinuses (i.e., closed circulation). Typically, the terminal arteriole bifurcates repeatedly, in quick succession, giving rise to as many as twelve short capillaries, each leading directly to at least one sinus. However, an open circulation also exists, inasmuch as the majority of all capillaries end in the marginal zone around lymphatic nodules. In the dilated spleen, direct connections to sinuses are rarely visible but endings in the red pulp are found, in addition to those going to the marginal zone.     

Energy Metabolism of Synaptosomal Subpopulations from Different Neuronal Systems of Rat Hippocampus: Effect of L-Acetylcarnitine Administration In Vivo

The maximum rate (V_max) of some enzyme activities related to glycolysis, Krebs' cycle, acetylcholine catabolism and amino acid metabolism were evaluated in different types of synaptosomes obtained from rat hippocampus. The enzyme characterization was performed on two synaptosomal populations defined as large and small synaptosomes, supposed to originate mainly from the granule cell glutamatergic mossy fiber endings and small cholinergic nerve endings mainly arising from septohippocampal fiber synapses, involved with cognitive processes. Thus, this is an unique model of pharmacological significance to study the selective action of drugs on energy metabolism of hippocampus and the sub-chronic i.p. treatement with L-acetylcarnitine at two different dose levels (30 and 60 mg · kg^–1, 5 day a week, for 4 weeks) was performed. In control animals, the results indicate that these two hippocampal synaptosomal populations differ for the potential catalytic activities of enzymes of the main metabolic pathways related to energy metabolism. This energetic micro-heterogeneity may cause their different behaviour during both physiopathological events and pharmacological treatment, because of different sensitivity of neurons. Therefore, the micro-heterogeneity of brain synaptosomes must be considered when the effect of a pharmacological treatment is to be evaluated. In fact, the in vivo administration of L-acetylcarnitine affects some specific enzyme activities, suggesting a specific molecular trigger mode of action on citrate synthase (Krebs' cycle) and glutamate-pyruvate-transaminase (glutamate metabolism), but mainly of small synaptosomal populations, suggesting a specific synaptic trigger site of action. These observations on various types of hippocampal synaptosomes confirm their different metabolic machinery and their different sensitivity to pharmacological treatment.     

Colchicine effect on the chromosome number of barley embryos culturedin vitro

Summary The effect of colchicine upon the embryos of barley (Hordeum vulgare L.) culturedin vitro has been studied. Different concentrations of the drug as well as different times of culture in its presence have been tested, in order to ascertain the optimal conditions for the induction of non diploid cells. Tetraploid cells were always infrequent after the treatments, reaching the higher values during the first days of culture; aneuploid cells being frequent in some cases.     

“Gomori-positive” neurosecretion in the rat after deafferentation of the medial basal hypothalamus

A portion of the "Gomori-positive" peptidergic neurosecretory (NS) cells in the paraventricular and especially in the supraoptic and postoptic nuclei degenerate three weeks after deafferentation of the medial basal hypothalamus. Most of the remaining NS cells show signs of high activity. Regenerating NS fibres form "muffs" around the blood vessels laterally from the lesion; some of them enter the "isolated" area or persist there if a thin layer of the brain tissue is left somewhere untouched under the basal end of the cut. The regenerating NS fibres are also found outside the nervous tissue: within the scar tissue, in the proliferating connective tissue of the brain sheet below the basal end of the cut and in the mantel plexus area. The NS fibres make close contact with blood vessels invading or penetrating the vascular wall. It is suggested that peptide neurohormones discharged from the "Gomori-positive" NS terminals enter the general blood circulation as well as the portal blood at the site of these newly formed axovasal contacts. It is supposed that under these conditions monoaminergic terminals do not discharge monoamines because no stimulation of monoamine-producing NS cells occurs with deafferentation.     

Expression and inheritance of kanamycin resistance in a large number of transgenic petunias generated by Agrobacterium-mediated transformation

One hundred and four kanamycin-resistant Petunia Mitchell plants were regenerated from leaf discs cocultivated with Agrobacterium tumefaciens strain LBA4404 containing a binary vector pCGN200. Selection for kanamycin resistance was applied during plant regeneration at the initiation of both shoots and roots. The regenerated plants were analysed for expression and inheritance of their kanamycin resistance phenotype. Approximately half of the plants showed normal Mendelian inheritance for one or two kanamycin resistance genes. In one case, the two copies were inserted at closely linked sites on homologous chromosomes, and gave <0.05% kanamycin-sensitive progeny on backcrosses. Six plants had inheritance patterns suggesting that the kanamycin gene had inserted into an essential region of DNA. Forty-five plants showed lower than expected transmission of kanamycin resistance, which was associated with low expression of the resistance phenotype in most cases. Ten plants produced segregation ratios that are not readily interpreted by Mendelian inheritance.     

Melatonin enhances Th2 cell mediated immune responses: Lack of sensitivity to reversal by naltrexone or benzodiazepine receptor antagonists

Chronic administration of melatonin for 5 days to antigen-primed mice increased the production of pro-inflammatory cytokine IL10 but decreased the secretion of antiinflammatory cytokine TNF-. These results further confirm that melatonin activates Th2like immune response. Whether melatoninmediated Th2 response is dependent on opioid or central and peripheral benzodiazepine receptors was also examined. Hence, melatonin was administered to antigen-sensitised mice with either naltrexone (a opioid receptor antagonist) or flumazenil (a central benzodiazepine receptor antagonist) or PK11195 (a peripheral benzoidiazepine receptor antagonist). No significant difference in melatonin-induced Th2 cell response was observed by naltrexone, flumazenil or PK11195 treatment. These findings suggest that the Th2 cell response induced by melatonin in antigen sensitised mice neither dependent on endogenous opioid system nor is modulated through the central or peripheral benzodiazepine receptors.     

Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts

Summary Efficient delivery of genomic DNA fragments to maize protoplasts was obtained by new methods using the polycation Polybrene or Lipofectin cationic liposomes. Stable kanamycin-resistant secondary transformants were recovered after transfection with genomic DNA from a maize cell line that had previously been tagged with the bacterial gene neomycin phosphotransferase (nptII) in a first-round transformation. The frequency of secondary transformants with nptII-homologous DNA sequences was 3% or 6% of all randomly picked microcalli after Polybrene or Lipofectin-mediated transfection, respectively. Transformation with genomic DNA by these methods may allow easy transfer of uncloned genes encoding desirable characteristics to crop species that can be regenerated from protoplasts.     

Reproduction of,and ultrastructural changes induced by,Spiroplasma citri in sieve tube of citrus leaves

Summary Electron microscope examination of ultrathin sections of leaf veins of stubborn—affected citrus seedlings revealed three morphotypes ofSpiroplasma citri free in the cytoplasm of mature sieve elements. In addition to these, inclusions believed to beSpiroplasma citri, some in various stages of degeneration, were occasionally found inside spherical, ovoid, or angular membraneous structures (= packets) which occurred in sieve elements devoid of any recognizable organelles. These packets which varied in size from 1.0 to 1.8 m wide an 1.9 to 3.5 m long were bounded by unit membrane ca. 9 to 10 nm thick. Spiroplasmas and packets were apparently absent in sieve elements of leaf veins of healthy citrus seedlings. Three types of packets were recognized based on the size of spiroplasmas contained: type I packets contained large, intermediate, and small spiroplasmas, but small forms predominated; type II packets contained a mixture of large and intermediate forms, while type III packets contained essentially tightly—packed large forms. Results of the study suggested that the spiroplasma-containing packets are either definite reproductive structures peculiar toSpiroplasma citri or are sieve-tube cells in various stages of plasmolysis. Evidence is presented indicating that within a given packet small spiroplasmas were produced from large spiroplasmas by some process of cell constriction followed by fission, or by budding. Since these spiroplasma—containing packets were infrequently observed in infected tissues we suggest that cell division by budding, of by constriction followed by fission into unequal daughter cells may be the principal mode of reproduction inSpiroplasma citri.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

Present-Day State of Coral Reefs of Nha Trang Bay (Southern Vietnam) and Possible Reasons for the Disturbance of Habitats of Scleractinian Corals

Our investigations were conducted from 1990 to 2002. Sampling of bottom sediments and biological objects, as well as photo and video shooting, were performed during scuba diving. The state of the environment and coral reef communities was assessed using the chemical–analytical, fluorometric, and luminometric methods, as well as the Ames test and the transect technique. The research results suggest that the spectrum and distribution pattern of persistent congeners of PCDD/Fs (dioxins) in bottom sediments are similar to those of the defoliant Agent Orange and that the bottom sediments are toxic and display photo inhibition and a mutagenic effect. The bottom of the bay is heavily silted throughout its depth. Many large dead colonies of corals without mechanical damage were observed everywhere. The total coverage by live corals in all sites investigated does not exceed 30%. Although, without a doubt, many factors contributed much to the disturbance of the bay ecosystems, the actual trigger for the degradation of the coral reefs seems to be the input of dioxin-containing chemicals used as defoliants during the American–Vietnamese war (Vietnam War).     

Zur Feinstruktur der Spezialzellen von normalernährten und hungernden Aeolidiern (Gastr. Nudibranchia)

Zusammenfassung Die Spezialzellen im Bindegewebe der Kolben von Eubranchus farrani und Trinchesia granosa (Aeolidoidea) bilden Gruppen, die stets von einer Basallamina umschlossen werden. Die Zellen werden nicht durch desmosomale Bildungen untereinander verbunden. In den oft geweiteten Interzellularlakunen findet sich feinstflockiges Material. Spezialzellen normal ernährter Schnecken besitzen ein außerordentlich reiches, geordnetes, rauhes endoplasmatisches Retikulum und einen großen Kern mit großem Nucleolus. Golgi- und Vakuolenapparat der Zellen bleiben klein. Mit zunehmender Dauer des Fastens wird das rauhe ER durch ungeordnetes, glattes endoplasmatisches Retikulum ersetzt, wächst der Golgiapparat und nimmt die Zahl der Autolysosomen zu. Die feinstrukturellen Unterschiede zwischen Spezialzellen, Blasenzellen und Nervenzellen werden beschrieben, die Funktion der Spezialzellen abschließend diskutiert.

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An electron-microscope study of cellules spéciales in normally nourished and starved aeolids (Gastr. nudibranchia)

Summary Cellules spéciales of Aelidoidea have been investigated in the cerata of Eubranchus farrani and Trinchesia granosa. All groups of cellules spéciales are limited by a basal lamina. Opposing membranes are not connected by specialized structures as desmosomes etc. The intercellular cleft often is widened and contains a fine floccular substance. In normally nourished animals the cellules spéciales show a rich, highly organized, rough surfaced reticulum, while there are only a few vacuoles and a small Golgi Complex. In starved animals mainly smooth reticulum is found, but the Golgi Apparatus has enlarged and autolysosomes are abundant. The fine structural differences of cellules spéciales, globular cells and nerve cells are described, the function of the cellules spéciales is discussed.

Mit dankenswerter Unterstützung durch den Schweizerischen Nationalfond zur Förderung der Wissenschaften und die Deutsche Forschungsgemeinschaft. Ich danke ferner für die guten Arbeitsmöglichkeiten in der Zoologischen Abteilung der Stazione Zoologica Napoli und Frau A. Merkelbach (Tübingen) für technische Assistenz.     

Biosynthesis and Molecular Genetics of Clavulanic Acid

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic -lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modfication as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the -lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack -lactamase inhibitory activity.     

Structural analyses of lipid A from lipopolysaccharides of nodulating and non-nodulating Rhizobium trifolii

Structural studies have been performed on lipid A preparations derived from lipopolysaccharides of nodulating (nod⁺) Rhizobium trifolii wild type strains and non-nodulating (nod^-) mutants deprived of the medium size Nod plasmid.All preparations contain a lipid A backbone composed of a 1,6-linked d-glucosamine disaccharide (GlcN II-1,6-GlcN I) which is bis-phosphorylated at positions 1 and 4. The phosphate group at position 4 (GlcN II) is nonstoichiometrically substituted probably by a neutral glycosyl residue. Another substituent (probably also a neutral sugar) substitutes partly position 4 (GlcN I) of the disaccharide. The hydroxyl groups at positions 3 and 3 are likely to be esterified by fatty acyl residue. In lipid A of nod⁺ strains, 3-hydroxyhexa- and octadecanoic acid are linked to the amino group of GlcN I, and 3-hydroxyhexa- and tetradecanoic acid to the amino group of GlcN II. In lipid A of nod^- strains, mainly 3-hydroxyhexadecanoic acid is linked to the amino group of GlcN I, and 3-hydroxytetra- and hexadecanoic acid to that of GlcN II.The results show that rhizobial lipid A expresses many similarities to the lipid A of Enterobacteria. The structure shows, however, also peculiarities, especially regarding substituents of the lipid A backbone.This paper is dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

The relation between the levels of extractable and diffusible IAA in almond fruits and their “June drop”

The relationship between the June drop of almond fruits(cv. Truoito) and the levels of extractable and diffusible IAA of the fruit wasstudied. June drop started almost simultaneously with thedecreasein the concentration of extractable free and ester IAA from seeds and theamountof diffusible free IAA from persisting fruits. The drop lasted for a period ofabout 17 days, during which time the auxin decreased constantly. In a secondexperiment, the relationship between fruit size, fruit drop and level of auxinwas examined. The fruits were classified according to size into threecategories, large, intermediate and small. Throughout the experiment thepercentage of small fruits that abscised (70%) was five times higher than thatof the other categories of fruits. The small persisting fruits had lower levelsof extractable free and diffusible free IAA than fruits of intermediate andlarge size. When the process of abscission advanced and the diffusion of theauxin had been interrupted, an increase in the concentration of extractablefreeand peptidic IAA in the seeds and pericarps of the abscising fruits wasobservedcompared with the persisting fruits.     

Process of apoptosis induced by TNF-α in murine fibroblast Ltk-cells: Continuous observation with video enhanced contrast microscopy

Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF- were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF- exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF- exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.