Photobiology of microorganisms: how photosensors catch a photon to initialize signalling

The afa-3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea-associated Escherichia coli strains. It contains six genes ( afaA–afaF  ), among which the afaE3 gene is known to code for the structural AfaE-III adhesin (previously designated AFA-III), whereas no role has yet been identified for the afaD gene product. The afa-3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE-III adhesin; however, unlike the AfaE-III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa-3 and daa operons. Analyses of the phenotypes conferred by these afa-3 / daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE-III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE-III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE-III, AfaD was found to be a surface-exposed protein as well as an adhesin; both AfaD and AfaE-III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.     

Enhancement of the light-triggered electrical response in plant cells following their de-energization with uncouplers

Light-triggered membrane potential changes in cells of a liverwort Anthoceros are greatly enhanced by the ionophorous uncouplers nigericin and monesin. Stimulation of the light-triggered electrical response (LTER) by nigericin occurred concomitantly with inhibition of a slow decline in the chlorophyll fluorescence, which suggests that the transmembrane pH gradient in thylakoids is not essential for generation of LTER at the plasma membrane. The extent of monensin-stimulated LTER remained high under a diminished driving force for the ionophore-induced proton-cation exchange across the plasma membrane (elevation of the external Na⁺ concentration from 1 to 50 m M ), which indicates that energy uncoupling in chloroplasts is more related to the electric response enhancement than the induction of the H⁺/K⁺(Na⁺) exchange at the plasma membrane. Enhancement of LTER by ionophores occurs in parallel with stimulation of light-triggered pH changes (alkalinization) in the vicinity of the cell surface, which suggests an association of trans-membrane H⁺ fluxes with LTER. The results are consistent with the hypothesis that illumination produces a temporary inhibition of the plasma membrane H⁺ pump with a subsequent activation of gated channels and transient rapid depolarization of the cell.     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.     

Utilization of limiting concentrations of ortho-phosphate and production of extracellular organic phosphates in cultures of marine diatoms

The utilization of ortho-phosphate by two coastal marine diatomspecies, Nitzschia closterium and Cyclotella cryptica, was studiedin batch cultures. The hypothesis was tested that thresholdconcentrations in the phosphate uptake determine the lower limitof environmental phosphate, permitting the existence of species.The turn-over time of residual medium phosphate in culturesis {small tilde}10 min, indicating a rapid equilibration ofconcentration dependent on uptake with leakage of ortho-phosphate.Increasing phosphate starvation in cultures diminished the residualortho-phosphate in the range of {small tilde}60–<2nmol l^–1, as measured radiochemically after elution onSephadex® G-10 gel. These concentrations encompass the rangeof limiting phosphate concentration in continuous cultures ofthe few microalgae, for which these concentrations are actuallymeasured. The diatoms excreted {small tilde}20–100 nmolI^–1 of organic phosphate. One dominating compound, probablyan unusual nucleotide, was incompletely or not resorbed underphosphate starvation. In contrast, Nitzschia closterium excretedunder ample phosphate supply a series of three related compounds,probably phospholipids, that were resorbed under depletion.The association of the organic phosphates with macromolecularexudates is interpreted, along with the other observations,as an indication for a hardly explored periplasmatic phosphatemetabolism in these algae. ³Dedicated to Prof. Dr. H.-A. von Stosch in honour of his 75thbirthday. ⁴This study was conducted at the University of Marburg undersupport of the Humboldt Foundation Publication no. 64 of theproject "Biological Research of the Eems-Dollard Estuary".     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

Behavioural responses of two small ermine moth species (Lepidoptera: Yponomeutidae) to plant constituents

On the basis of electrophysiological screening of lateral and medial styloconic sensilla of caterpillars of Yponomeuta species, several hypotheses concerning behavioural effects of plant constituents can be posed (Van Drongelen, 1979). Feeding experiments have been carried out with intact larvae of Y. cagnagellus (Hb.) and Y. evonymellus (L.). Both species display neural responses to dulcitol, phloridzin and prunasin. Leaf discs of the host plants of these species were modified using these three constituents and effects were studied in choice and non-choice situations. Dulcitol appears to stimulate food intake of both species, whereas phloridzin acts as a deterrent exclusively in a choice situation. Prunasin inhibits feeding of Y. cagnagellus in a choice situation, but Y. evonymellus does not show a reaction to this compound at the concentration applied. Neural mechanisms probably underlying these behaviours are discussed.

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Zusammenfassung Aufgrund elektrophysiologischer Versuche mit lateralen und medialen Sensilla styloconica von Yponomeuta-Arten können mehrere Hypothesen über Verhaltenseffekte von Pflanzenbestandteilen aufgestellt werden (Van Drongelen, 1979). Y. cagnagellus und Y. evonymellus zeigen neurale Reaktionen auf Dulcitol, Phlorizin und Prunasin.Es wurden Frassexperimente mit intakten Raupen beider Arten durchgeführt. Blattscheiben der Wirtspflanzen wurden mit den genannten Stoffen modifiziert, und die Effekte wurden in Wahl- und Nichtwahlversuchen studiert. Dulcitol scheint die Futteraufnahme beider Arten anzuregen, während Phlorizin als Abhaltestoff wirkt und zwar nur in einer Wahlsituation. Prunasin verhindert Fressen bei Y. cagnanellus in einer Wahlsituation, während Y. evonymellus auf diesen Stoff in der geprüften Konzentration nicht reagiert. Neurale Mechanismen, welche wahrscheinlich diesem Verhalten zugrunde liegen, werden diskutiert.

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Supported by the Foundation for Fundamental Biological Research (BION), subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).