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.     

Chemical and physiological changes in fungi during autolysis

A comparative study was done on some of the chemical changes occurring during autolysis of cultures ofAspergillus flavus in both physiologically acid and alkaline media. The mycelium ofA. flavus lost during autolysis 44 % of its maximum dry weight in the physiologically alkaline medium, whereas this loss was apparently nil in the physiologically acid medium. Nitrogen containing compounds seemed not to be affected by autolysis either in the physiologically acid or alkaline media. The disappearance of P-containing compounds in mycelium ofA. flavus autolysed in both conditions (NO ₃ ^– and NH ₄ ⁺ as N source) amounted to 64 % in the alkaline autolysis and to nearly 77 % in the acid autolysis. The results we have obtained for the acid autolysis strongly suggest that very little activity is shown by autolytic enzymes in the interval 10–133 days of incubation, when measuring autolysis by the loss in mycelial dry weight.

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Zusammenfassung Eine vergleichende Untersuchung war unternommen an einigen der chemischen Veränderungen, die während der Autolyse der Kulturen vonAspergillus flavus in physiologischen sauren und alkalischen Medien vorkommen. Die Myzelien vonA. flavus haben während der Autolyse 44 % ihres größten Trockengewichtes in physiologisch alkalischem Medium verloren, während dieser Verlust in physiologisch saurem Medium anscheinend Null gewesen ist. Stickstoff enthaltende Substanzen erschienen während der Autolyse weder in physiologisch saueren noch in alkalischen Medien beeinflußt zu sein. Das Verschwinden von P-enthaltenden Substanzen in Myzelien vonA. flavus in Autolyse unter beiden Bedingungen (NO ₃ ^– und NH ₄ ⁺ als Stickstoffquelle) erreichte 64 % in alkalischer Autolyse und beinahe 77 % in der saueren Autolyse. Die Ergebnisse, die wir in der saueren Autolyse erhalten haben legen es sehr nahe, daß autolytische Enzyme eine sehr geringe Aktivität in der Zeitspanne von 10–133 Tagen der Inkubazion zeigen, wenn die Autolyse an dem Verlust des mycelialen Trockengewichtes gemessen wird.

     

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.

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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).     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.