Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.     

The potential of biotechnology for developed and developing countries

Summary The extraordinary developments occurring in biotechnology throughout the world will lead to significant changes in world commerce, as well as in human health and welfare. A few dramatic advances have already been made in medical and agricultural biotechnology. Production of vaccines employing recombinant DNA methods is one example of the potential for genetic engineering applications in the treatment of hereditable and communicable diseases. Lesser understood, but with equally dramatic promise, is marine biotechnology, an example where the excellent possibilities for industry applications in both developing and developed countries can be elucidated. Recent successes in the cloning of growth hormone genes in salmon and other important genes and gene complexes in fin fish and shellfish promise major changes in production of commercially important fish and shellfish. Pharmacologically active compounds from marine animals and plants can now be harvested effectively, using biotechnology. Specialty chemicals and enzymes with unusual properties, unique to the marine environment, with potential for industrial applications, are being discovered. Thus, new products and new markets appear on the horizon. What has changed the picture significantly is the ready access which is now available to these new materials and compounds by the application of the methods of genetic engineering. No longer is it necessary to depend on sustained harvest, which may be subject to vagaries of weather or climate. By means of genetic engineering, genes coding for economically desirable traits can be cloned and those systems established in the laboratory in controlled culture and production. The enormous natural resources of developing countries cannot be productively harnessed by the developing countries themselves, since the industrial resources needed for the technology and the trained intellectual potential are not in abundant supply. Partnerships between developed and developing countries should be established so that the benefits of biotechnology can be achieved by both. An important role for applied microbiology, thus, is described and a dramatic impact at the global level can be predicted if effective biotechnology partnerships between the developing and developed countries of the world are created.

---

Resumen El extraordinario desarrollo de la biotecnología en todo el mundo va a generar importantes cambios tanto en el comercio mundial como en la salud y bienestar de la humanidad. En las áreas de biotecnología médica y agrícola se han realizado importantes progresos. Un ejemplo de las posibilidades de la ingeniería genética en el tratamiento de enfermedades hereditarias y contagiosas es la produccion de vacunas utilizando técnicas de recombinación de ADN. La biotecnología marina es un área menos conocida pero no por ello con menos posibilidades de aplicaciones industriales tanto en países en desarrollo como en los desarrollados. Los éxitos obtenidos recientemente en la clonación de genes que codifican para las hormonas de crecimiento del salmón, así como de otros genes y conjuntos de genes de distintos peces y moluscos, suponen cambios importantes en la producción comercial de pescados y mariscos. Compuestos farmacológicamente activos procedentes de plantas y animales marinos pueden ahora cosechar se más eficientemente gracias a la biotecnología. Se estan descubiendo compuestos químicos y enzimas con propiedades especiales, unicas en el ambiente marino, cuya aplicación industrial tiene un futuro prometedor. Así pues, nuevos productos y nuevos mercados aparecen en el horizonte. La facilidad de acceso ahora disponible a estos nuevos materiales y compuestos, mediante la aplicación de los métodos de la ingeniería genética, ha cambiado significativamente el panorama. Ya no se ha de depender de cosechas sujetas a las variaciones del tiempo y del clima. Mediante la ingeniería genética los genes que codifican para características económicamente deseables pueden clonarse y los sistemas así obtenidos establecerse en laboratorio en condiciones de cultivo y producción controladas. La cantidad de recursos naturales que poseen los países en desarrollo no pueden ser utilizados productivamente por ellos mismos debido a la escasez de desarrollo tecnológico y potencial intelectual de los mismos. Deberián establecerse colaboraciones entre países en desarrollo y países desarrollados de manera que tanto unos como otros puedan beneficiarse de los progresos en biotecnología. Es este un papel importante que puede tener la microbiología aplicada y se puede predicir, a nivel global, un enorme impacto si se crean colaboraciones eficientes entre países en desarrollo y países desarrollados.

---

Résumé Les extraordinaires développements de la biotechnologie dans le monde modifieront de façon significative le commerce mondial, aussi bien que la santé et le bien-être de l'homme. Des progrès considérables ont déjà été accomplis dans le domaine de la technologie médicale et agricole. La production de vaccins par les méthodes de l'ADN recombinant est un exemple du potentiel du génie génétique pour le traitement des maladies héréditaires et transmissibles. On connait moins le potentiel tout aussi considérable de la biotechnologie marine où on trouve d'excellents exemples d'applications industrielles réalisables aussi bien dans le pays développés que dans les pays en voie de développement. Les succès récents, obtenus par clonage des gènes d'hormones de croissance chez le saumon et par clonage d'autres gènes et groupes de gènes chez les poissons et les crustacés, laissent prévoir des changements majeurs dans le domaine de la production des poissons et crustacés d'intérêt commercial. Grâce à la biotechnologie, des composés pharmacologiquement actifs peuvent être obtenus à partir d'animaux et de végétaux marins. Des produits chimiques et des enzymes doués de propriétés nouvelles et spécifiques au milieu marin ont été découverts et sont suscéptibles d'applications industrielles. Ainsi, de nouveaux produits et de nouveaux marchés pointent à l'horizon. Ce qui a changé le tableau de façon décisive, c'est la facilité avec laquelle ces nouvelles substances peuvent être obtenues par les méthodes du génie génétique. Il n'est plus nécessaire d'avoir recours à des récoltes exposées aux aléas climatiques. Les gènes codant pour des caractères économiquement désirables peuvent être clonés au laboratoire et utilisés pour une production en cultures contrôlées. Faute de disposer des moyens technologiques et du potentiel scientifique nécessaires, les pays en voie de développement ne peuvent pas utiliser par eux-mêmes leurs énormes ressources naturelles. Des coopérations entre pays développés et moins développés doivent s'établir de façon à ce que la biotechnologie soit mutuellement profitable. La biotechnologie aura ainsi un rôle important et dont on peut attendre des effets considérables à l'échelle mondiale, à condition toutefois d'instituer une coopération effective entre les pays hautement industrialisés et le Tiers Monde.

---

---

Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16.8.1985. Opening Session     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Neuronal nets and nerve cell interactions in vitro in insect systems

Summary The development of a synthetic medium that supports growth and differentiation of insect embryonic tissues afforded the possibility of studying the interactions between nerve and other cell types in long term cultures. The mechanical dissociation of embryonic nerve tissues results in survival of nerve cells but not of glial cells. The dissociated glial-free neurons produce a dense fibrillar network in the presence, but not in the absence, of foregut explants or other tissues from same donors. Nerve fiber bundles outgrowing from dissociated neurons enter foregut segments and establish synaptic connections with muscle cells. Foregut explants undergo differentiation and become contractile in long term cultures when innervated by dissociated nerve cells. The progressive deterioration of similar foregut tissues cultured alone contrasts with the excellent condition of innervated explants and suggests that this is due to trophic factors released by nerve fibers. The same in vitro systems provided the opportunity of studying the interaction between nerve fibers produced by the autonomic ingluvial ganglion, which adheres to the surface of the alimentary tract, and muscle cells. Multiple esophagus explants from cockroach embryos become interconnected by fibers emerging from ingluvial ganglia, when the explants are combined in vitro at short distance from each other. Muscle cells migrating from the esophagi line up on axons branching out in the medium, or form contractile ribbons which, in turn, establish connections with nerve fibers. The thigmotropism of muscle cells and strong affinity for nerve fibers reveal a new aspect of muscle cells-to-fibers interaction, amenable to further analysis in vitro. This work was supported in part by United States Public Health Service grant NS-03777 and grant GB-16330 X from the National Science Foundation     

The Occurence of δ-Tocopherylquinone in Higher Plants and Its Relation to Senescence

δ-Tocopherylquinone (δTQ) content was determined in tobacco and yellow maple leaves, green ivy leaves and cactus tissues. It was found that the concentration of δ-TQ was highest in mature or senescent tissues, such as white tobacco leaves (0.02 μmole/g dry wt) while its detection was uncertain in young, green leaves from the apex of tobacco plants. Fractionation by centrifugation of senescent tobacco leaves showed that the osmiophilic globule fraction was enriched in δ-TQ. Electron microscope studies of young, mature and senescent tobacco tissues showed progressive changes in the size and number of osmiophilic globules. After chloroplast breakdown in senescent tobacco leaves, these globules became the predominant constituents of the organelle. δ-TQ which is associated with osmiophilic globules may play a role in the development of plants, particularly during senescence.     

Wirtskreis und Infektionscyclus eines neu isolierten Bdellovibrio bacteriovorus-Stammes

Zusammenfassung Der neu isolierte Stamm W von Bdellovibio bacteriovorus infiziert und lysiert Rhodospirillum rubrum F und alle anderen untersuchten Athiorhodaceae, nicht aber Pseudomonas aeruginosa und Spirillum serpens. Er befällt auch zahlreiche Enterobacteriaceae und von den grampositiven Bakterien Streptococcus faecalis und Lactobacillus plantarum.Nach dem Festheften an der Zellwand wird diese in 3–20 min durchdrungen. In 10–60 min ist Bdellovibrio vollständig in die Zelle eingedrungen und hat sich im Raum zwischen Zellwand und cytoplasmatischer Membran angesiedelt.In 3–5 Std wird der gesamte Zellinhalt bis auf die Membranen aufgelöst. In dieser Phase erfolgt die Vermehrung von Bdellovibrio. In den ghosts sind die Parasiten in lebhafter Bewegung. Die Geißel hat einen Gesamtdurchmesser von 29 m und eine Länge von etwa 3 . Sie ist von einer Geißelscheide umgeben, die in Verbindung zur Zellwand steht. Der Durchmesser der Geißel ohne Scheide beträgt etwa 18 m. Bdellovibrio kann oberhalb eines Sauerstoffpartialdruckes von 4–5 mm Hg infizieren und sich vermehren. Der Titer von Bdellovibrio nimmt bei Aufbewahrung in lysierten Kulturen in 36 Tagen von 10⁸ auf 10¹ pfu (plaque forming units) je ml ab. Bei Aufbewahrung in Nährkultur sinkt der Titer nur auf 10⁴ pfu/ml ab. Die Zahl der Plaques im Verhältnis zum Titer der Impfsuspension von Bdellovibrio schwankt in Abhängigkeit vom Wirtsstamm. Wenn man die Plaque-Bildungsrate bei R. rubrum gleich 1 setzt, beträgt sie bei Serratia marcescens 0,0001, bei Proteus vulgaris 10. Bd. bacteriovorus, Stamm W wächst nicht in synthetischer Nährlösung oder Lysaten. Ein geringes Wachstum ohne Zellteilung findet in Zellextrakten von R. rubrum statt. Der Stamm vermehrt sich jedoch in hitzeinaktiviertem R. rubrum. Die Plaque-Bildungsrate ist unter diesen Bedingungen aber sehr niedrig.In Lysaten treten encystierte Dauerformen von Bdellovibrio bacteriovorus auf.

---

The host range and the infectious cycle of a new isolated, on gram-positive and gram-negative bacteria parasiting Bdellovibrio bacteriovorus strain

Summary Rhodospirillum rubrum and all other investigated Athiorhodaceae are infected and lysed by the new isolated strain W of Bdellovibrio bacteriovorus. This strain W parasites on numerous Enterobacteriaceae and the gram-positive bacteria Streptococcus faecalis and Lactobacillus plantarum, but not on Pseudomonas aeruginosa and Spirillum serpens.After attachment of Bdellovibrio to the host, the cell wall is penetrated in 3 to 20 min. In 10 to 60 min Bdellovibrio has completely entered the host cell. He remains in the space between cell wall and cytoplasmic membrane of the host.The host cell is completely lysed within 3 to 5 hours. During this phase the size and cell number of Bdellovibrio are increased and a new flagellum is likely to be formed. In the ghosts of the host cell a strong movement is observed. The single polar flagellum of Bdellovibrio has a diameter of 29 m. The flagellum consists of an inner core ( 18 m) and an outer sheath which is continued into the cell wall. Bdellovibrio is able to grow and to infect only under aerobic or semiaerobic conditions (oxygen partial pressure 4 to 5 mm Hg and more). The titer of Bdellovibrio is gradually decreased from 10⁸ to 10¹ plaque forming units (pfu) per ml, when kept in the lysate for 36 days. In a synthetic medium there is a diminution of 10⁴ pfu/ml only. The plating efficiency is dependent of the host strain. If the plating efficiency of Bdellovibrio with Rhodospirillum rubrum is 1.0, the rate varies from 0.0001 with Serratia marcescens to 10 with Proteus vulgaris. Bdellovibrio bacteriovorus strain W does not grow in a synthetic medium. However, it grows but does not multiply in cell free extracts of Rhodospirillum rubrum. The parasite is also able to infect and lyse heat inactivated R. rubrum. But the plating efficiency in this case is very low.It has been observed that in lysed cells of R. rubrum certain amount of Bdellovibrio is encysted. The morphology and fine structure of these cells is quite different from the normal virulent type.

     

Effect of Temperature on the Potential and Current Thresholds of Axon Membrane

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 µa at 267deg;C to 1 µa at 7.5°C. Threshold potential values ranged from 50 mv at 26°C to 6 mv at 7.5°C. The mean Q₁₀ of threshold current was 2.3 (SD = 0.2), while the Q₁₀ for threshold potentials was 2.0 (SD = 0.1).