The fluorescent properties of acridines in the presence of chloroplasts or liposomes. On the quantitative relationship between the fluorescence quenching and the transmembrane proton gradient

The fluorescent properties of 9-aminoacridine were studied in chloroplasts and phospholipid liposomes.

In energized chloroplasts it was found that the percentage of fluorescence quenching was dependent on both the 9-aminoacridine concentration and the chlorophyll concentration. On the other hand, it was independent of the osmolarity of the medium.

In phospholipid liposomes the dependence of the fluorescence quenching on the concentration of 9-aminoacridine was similar to that in chloroplasts. Moreover, the fluorescence quenching depended on the presence of charged compounds in the membrane being larger in negatively charged than in positively charged liposomes.

The fluorescence of both the monoamine 9-amino-6-chloro-2-methoxyacridine and the diamine atebrin is quenched more extensively than that of 9-aminoacridine. Although the percentage of fluorescence quenching of both atebrin and 9-aminoacridine is dependent on the outside pH, the relationship between the fluorescence quenching of the two probes under similar conditions is not pH-dependent.

It is concluded that calculation of ΔpH from the percentage of fluorescence quenching of fluorescent amines is not meaningful, that the osmotic volume of chloroplasts is not involved in the quenching process and, consequently, that the interaction between the acridines and energized membranes is more likely to occur at the level of the membrane proper.     

Lactic acid production by aggregated continuous cultures of Lactobacillus acidophilus under aerobic conditions

Summary The ability of Lactobacillus acidophilus to aggregate, to produce lactic acid for a long term continuous fermentation process and to exist as aggregate cell cultures in a gas-lift reactor under aerobic conditions was studied. The main product of fermentation was lactic acid and only the traces of other end-products were determined. The highest fermentation efficiency of lactic acid was 98.6% and the highest productivity was 9.6 g.l^–1.h^–1 of lactic acid.     

Planktonic Food Web Structure along the Sau Reservoir (Spain) in Summer 1997

We studied the planktonic food web in eutrophic Sau Reservoir (Catalonia, NE Spain). Along the longitudinal axis from the Ter River downstream to the dam, we characterized a microbial succession of food web dominance of bacteria‐HNF‐ciliates. The Ter River transports a large load of organic material into the reservoir, with a bacterial density of ∼9 · 10⁶ large cells per ml. While at the first lacustrine station of the Reservoir HNF were the dominant bacterial consumers, at the others, an oligotrich ciliate, Halteria grandinella, was the main protozoan bacterivore. Most of the bacterial production in the reservoir epilimnion was consumed by grazing. The spatial succession of the reservoir microbial food webs was followed downstream by maximum densities of their potential predators among zoo‐plankters – rotifers, and early developmental stages of copepods.     

Foraging behaviour of nest predators at open-cup nests of woodland passerines

Nest predation patterns and processes cannot be understood without studying the behaviour of predators. I videotaped the behaviour of 22 species of predators at 171 depredated nests of 13 passerine species, in woodland in the Czech Republic. About 32% (60/187) of all events occurred during the night; mammals accounted for 95% (57/60) and 22% (28/127) of nocturnal and diurnal predation, respectively. About 67% (57/85) of mammalian predation, but only 3% (3/102) of avian predation, occurred during night. Multiple predations by the same species were detected in at least 7% (6/82) and 42% (37/88) of nests depredated by mammals and birds, respectively. Martens Martes martes/foina took nest content mostly all at once; birds (mainly Jay Garrulus glandarius) revisited partially depredated nest during 1–4 consecutive days. Martens stayed at the depredated nest about five times longer than Jays. Martens spent similar time at nests with eggs and nestling, while Jays stayed about twice longer at nests with eggs. Mammals consumed eggs always at the nest (23/23), but took nestlings away in at least 48% (31/64) cases. Birds took the eggs and nestling away in at least 31% (18/58) and 76% (71/94) cases, respectively. Predator visits to active nests without taking the content, repeated partial predation and revisitation of previously depredated nests suggest an effect of memory on predator’s foraging behaviour.     

Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.     

Effect of diet and mating status on ovarian development and oviposition in the polyphagous predator Macrolophus caliginosus (Heteroptera: Miridae)

The mirid bug Macrolophus caliginosus is commercially reared on eggs of Ephestia kuehniella, constituting an effective but expensive factitious food. Artificial diets can decrease the rearing costs of this natural enemy, but developing and evaluating an artificial diet is a very time-consuming activity. In the current study, development and reproduction of M. caliginosus on two artificial diets based on egg yolk were investigated. The artificial diets resulted in longer development and lower adult weights, but survival was comparable with that of control insects fed E. kuehniella eggs. Reproductive potential of the predator reared on factitious and artificial foods was assessed using a dissection method. The influence of nymphal food on fecundity was less important than that of adult food. Adults fed E. kuehniella eggs had a preoviposition period of about 4 days, whereas adults offered only plant material started laying eggs about 7 days after emergence. Ovarian scores at day 7 were higher for females fed E. kuehniella eggs than for those given access only to a tobacco leaf. Ovarian scores were not significantly affected by mating status. In a final test, a parallel comparison of two methods for assessing reproductive response to diet was made. Here, adult couples were offered one of four diets: E. kuehniella eggs, one of two artificial diets or no food. Half of the females were dissected and the other half was held for determining lifetime oviposition. Females fed E. kuehniella eggs had superior ovarian scores and laid more eggs than those fed either artificial diet or those given no extra food. A good correlation (r = 0.97) was obtained between ovarian scores and oviposition data, indicating that dissecting females after 1 week provides a reliable estimate of fecundity as affected by diet quality. Rapid reproductive assessments as used in the current study will help to increase the rate of development of artificial diets and may contribute to more cost effective production methods for augmentative biological control agents.     

Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[N-(2-hydroxypropyl)methacrylamide

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) containing 4-nitrophenyl ester (ONp) or thiazolidine-2-thione (TT) reactive groups in side chains and telechelic/semitelechelic pHPMA with TT groups were designed as highly hydrophilic biocompatible polymers suitable for chemical coating of polyelectrolyte-based DNA-containing nanoparticles bearing amino groups on the surface. The course of the coating reaction carried out in aqueous solution was evaluated on model self-assembling polyelectrolyte DNA/poly(L-lysine) (DNA/PLL) complexes either by monitoring the amount of residual polymer reactive groups by UV spectroscopy or by monitoring changes in the weight-average molecular weight and hydrodynamic size of the complexes using light scattering methods. Physicochemical stability of the coated complexes in buffered saline solution was also investigated. Contrary to uncoated particles, the coated complexes showed remarkable stability to aggregate in 0.15 M NaCl. Coating with pHPMA had practically no effect on the size distribution of the most stable complexes prepared by complexation of DNA with high-molecular-weight PLL (M(w) = 134 000) as shown by dynamic light scattering. The coating reaction was faster and more efficient with multivalent HPMA copolymers containing TT reactive groups than that with HPMA copolymers containing ONp groups.     

Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.     

Kinetic models for detection of toxicity in a microbial fuel cell based biosensor

Currently available models describing microbial fuel cell (MFC) polarization curves, do not describe the effect of the presence of toxic components. A bioelectrochemical model combined with enzyme inhibition kinetics, that describes the polarization curve of an MFC-based biosensor, was modified to describe four types of toxicity. To get a stable and sensitive sensor, the overpotential has to be controlled. Simulations with the four modified models were performed to predict the overpotential that gives the most sensitive sensor. These simulations were based on data and parameter values from experimental results under non-toxic conditions. Given the parameter values from experimental results, controlling the overpotential at 250 mV leads to a sensor that is most sensitive to components that influence the whole bacterial metabolism or that influence the substrate affinity constant (Km). Controlling the overpotential at 105 mV is the most sensitive setting for components influencing the ratio of biochemical over electrochemical reaction rate constants (K1), while an overpotential of 76 mV gives the most sensitive setting for components that influence the ratio of the forward over backward biochemical rate constants (K2). The sensitivity of the biosensor was also analyzed for robustness against changes in the model parameters other than toxicity. As an example, the tradeoff between sensitivity and robustness for the model describing changes on K1 (IK1) is presented. The biosensor is sensitive for toxic components and robust for changes in model parameter K2 when overpotential is controlled between 118 and 140 mV under the simulated conditions.