Vitis pollen dispersal in and from organic vineyards: I. Pollen trap and soil pollen data

The recent discovery of Roman vineyards in Britain [Antiquity 75 (2001) 745] based on both archaeological and palynological data has raised questions concerning the palynological record of viticulture in NW Europe. This paper presents data collected from 30 pollen traps and 29 soil pollen samples from within, and around, three organic vineyards in England. The soil trap data were collected over one flowering season. Within-vineyard pollen accumulation is compared with off-site accumulation and the soil pollen data. Although there is extremely high intravineyard variation in Vitis accumulation, the data confirm the role of stemflow transport and the exponential decrease in Vitis pollen accumulation with distance away from the vineyard. Pollen trap values of over 2% total pollen (TP) Vitis and soils with over 0.2% TP (based on 1000+ pollen sum) are likely to indicate the on-site presence of a vineyard. This is in accordance with the values recorded from known Roman vineyards in the Nene Valley. A compilation of occasional finds of Vitis pollen in England reveals typical values of 0.2% or less; although higher values are recorded, they are generally an artefact of a low total pollen count. However, the clustering of these occasional finds into the Roman and high Medieval periods suggests that long-distance Vitis influx increased during periods of widespread viticulture in NW Europe including England. The monitoring of Vitis pollen dispersal suggests that where encountered as part of routine pollen counting, the total pollen sum should be increased to ca. 1000 TP in order to establish a better estimate of the frequency of occurrence. This will allow palynology to play a less ambiguous part in the mapping of ancient viticulture.     

Influence of diet on pre-ingestive particle processing in bivalves: I: Transport velocities on the ctenidium

Suspension-feeding bivalve molluscs can assume a large ecological role by linking benthic and pelagic ecosystems. Therefore, a knowledge of the factors that influence feeding rates and processes at the level of the individual is important in understanding bivalve-dominated environments. We examined the roles of diet quality and concentration on particle processing by the ctenidia of four species of bivalves: the mussels Mytilus edulis L. and Mytilus trossulus Gould, and oysters Crassostrea virginica (Gmelin) and Crassostrea gigas (Thunberg). Bivalves were delivered diets of three different qualities at three different concentrations (1-2×10³, 10⁴, 10⁵ particles ml⁻¹). The high-quality diet consisted of the cryptophyte Rhodomonas lens Pascher et Ruttner; the low-quality diet consisted of particles prepared from ground Spartina sp. detritus; the medium-quality diet consisted of a 50:50 mixture (by particle number) of both particle types. Particle transport velocities on the ventral groove and dorsal tracts were then measured by means of video endoscopy. Ventral-groove transport velocities of M. edulis were the most responsive, demonstrating a significant increase with increasing diet quality, and a significant decrease with increasing diet concentration. Transport velocities in the ventral groove and dorsal tracts of C. virginica were not significantly affected by changes in diet quality, but significantly increased with increasing diet concentration. Transport velocities of M. trossulus and C. gigas demonstrated little change with diet quality or concentration, indicating that responses are species specific. Our data suggest that differential control of particle transport on the bivalve ctenidium is one of the underlying mechanisms that contribute to compensatory feeding responses exhibited by the entire organism.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp.: Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, α-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Quadruplex real-time quantitative PCR assay for the detection of pathogens related to late-onset ventilator-associated pneumonia: A preliminary report

A quadruplex real-time (RT) qPCR assay for the detection and quantification in 4 h of Staphylococcusaureus, Pseudomonasaeruginosa, Acinetobacterbaumannii and Stenotrophomonasmaltophilia directly from bronchoalveolar lavage specimens was developed. The specificity of the assay was 100% for all four species.     

Fasciola hepatica: Screening of chemical compounds on immature stages in the mouse: I. Pathology and method of investigation

Examinations in albino mice under rigorously standardized conditions demonstrated that the most marked reactions resulting from fascioliasis are: increase of the weight of the liver and spleen, lowering of the hemoglobin content and of the hematocrit. The effect of the extent of infection on these parameters was investigated. In addition, the effect of the intensity of infection with regard to the average number and the length of the parasites was examined, as well as the mortality in the host and the occurrence of blood in the abdominal cavity.     

Effects of temperature on two Mediterranean populations of Dinophilus gyrociliatus (Polychaeta: Dinophilidae): I. Effects on life history and sex ratio

The effects of temperature on the life history characteristics of two populations of the polychaete Dinophilus gyrociliatus, one from Ravenna (northern Adriatic Sea) and the other from Genoa (Ligurian Sea), were investigated. The temperatures tested (6, 12, 18, 24 and 30 °C) cover a wider range than those prevailing in the natural environment. In the populations studied there are broad differences in timing of development and reproduction. At 6 °C, the adults of both populations survive for a long time but they are unable to reproduce. At 12 °C, only the animals from Ravenna manage to reproduce. At the higher temperatures (18, 24 and 30 °C), the development of the animals belonging to the Genoa strain is faster than that of the Ravenna strain. The duration of the various phases of the biological cycle is very similar in both populations, but that from Ravenna exhibits greater tolerance of low temperatures, slower development rate and lower development threshold temperature than does the Genoa population. Temperature and geographical origin also have strong effects on reproductive characteristics. The highest fecundity values were observed at 12 °C in the Ravenna strain, the lowest at 30 °C in both groups. At 18 °C, the Genoa population is more fecund than the Ravenna one, while the situation is reversed at 12 °C. The smallest ovigerous capsules are produced at 30 °C, the biggest at 12 °C, and the Genoa females produce larger capsules than do the females from Ravenna, except at 12 °C. The size of both male and female eggs varies in relation to temperature, the smallest female eggs generally being laid at the higher temperatures. At all the temperatures tested, the sex ratio of the Ravenna population is higher than that of the Genoa population. In the Ravenna strain, temperature has no effect on the sex ratio, while in the Genoa strain the sex ratio at 24 °C is lower than at 18 and 30 °C. Comparison of the two populations at the same temperature reveals considerable differences in the characteristics of their respective life histories and sex ratios. It is very likely that the extreme selectivity of the harbor environments has favored the fragmentation of the species into differentiated populations that have adapted to the conditions prevailing in the different localities.     

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions: 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (αβ)₃ resulted first in monomerization (αβ), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

Effects of temperature on two Mediterranean population of Dinophilus gyrociliatus (Polychaeta: Dinophilidae): II. Effects on demographic parameters

The effects of temperature on demographic characteristics of two populations from Ravenna and Genoa of the polychaete Dinophilus gyrociliatus were investigated. Temperature affects age-specific survival and fecundity and all the demographic parameters often to a different degree in the two populations. Individuals from Ravenna survive longer than those from Genoa. The most evident differences in the age-specific fecundity curves of the experimental groups are related to age at maturity and the duration of the reproductive period that are in inverse proportion to temperature. In both populations of D. gyrociliatus, the maximum daily fecundity is observed at intermediate temperatures. In all cases, the Genoa females mature earlier, attain their maximum fecundity more quickly and have a shorter reproductive period than their Ravenna counterparts.Age at maturity, fecundity during the first reproductive events and juvenile survival are by far the most important characteristics in determining the fitness of the two populations at the tested temperatures. Even though the greatest net growth rates and highest expectation of life were recorded at 12 °C in the Ravenna population, the delay in the attainment of sexual maturity means that, at this temperature, the population growth rate is lowest. The higher juvenile survivorship and the greater fecundity observed at 24 °C is counter-balanced by the early attainment of sexual maturity induced at 30 °C. The comparison of the population growth rate calculated in laboratory with field data suggests that temperature is one of the main environmental parameters determining the fitness of D. gyrociliatus.     

Quantitative assessment of peptide-lipid interactions.: Ubiquitous fluorescence methodologies

Peptide-membrane interactions have been gaining increased relevance, mainly in biomedical investigation, as the potential of the natural, nature-based and synthetic peptides as new drugs or drug candidates also expands. These peptides must face the cell membrane when they interfere with or participate in intracellular processes. Additionally, several peptide drugs and drug leads actions occur at the membrane level (e.g., antimicrobial peptides, cell-penetrating peptides and enveloped viruses membrane fusion inhibitors). Here we explore fluorescence spectroscopy methods that can be used to monitor such interactions. Two main approaches are considered, centered either on the peptide or on the membrane. On the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine. Regarding membrane-centric approaches, we review methods based on lipophilic probes sensitive to membrane potentials. The use of fluorescence constitutes a simple and sensitive method to measure these events. Unraveling the molecular mechanisms that govern these interactions can unlock the key to understand specific biological processes involving natural peptides or to optimize the action of a peptide drug.     

Genetic diversification of the chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Germany

Chestnut blight in south-western Germany was first reported in 1992 and is since expanding in distribution. Here we investigated the invasion history of Cryphonectria parasitica and its associated hypovirus. For this, we characterized 284 isolates collected between 1992 and 2012 for hypovirulence, vegetative compatibility (vc), mating type, and microsatellite haplotype. A total of 27 haplotypes and 15 vc types were observed, although the C. parasitica population analyzed is currently dominated to 50 % by one haplotype and to 64 % by the vc type EU-2. Structure analysis indicated two divergent genetic pools. Over 66 % of the haplotypes belonged to a pool probably originating from northern Italy. Further diversification is expected due to ongoing sexual recombination, but also to new migration and additional introductions. Cryphonectria hypovirus 1 (CHV-1) was found in four of five C. parasitica populations from Baden-Württemberg. Genetic analysis of the 35 CHV-1 isolates obtained revealed that they all belong to the German subtype, although they have clearly diverged from the first German hypovirus isolated in 1992. Our study suggests that C. parasitica has been introduced into Germany several times from two different gene pools, whereas the hypovirus most probably has a single origin.     

Horseradish peroxidase-catalyzed oxidation of chlorophyll a with hydrogen peroxide: Characterization of the products and mechanism of the reaction

Horseradish peroxidase was verified to catalyze, without any phenol, the hydrogen peroxide oxidation of chlorophyll a (Chl a), solubilized with Triton X-100. The 13²(S) and 13²(R) diastereomers of 13²-hydroxyChl a were characterized as major oxidation products (ca. 60%) by TLC on sucrose, UV-vis, ¹H, and ¹³C NMR spectra, as well as fast-atom bombardment MS. A minor amount of the 15²-methyl, 17³-phytyl ester of Mg-unstable chlorin was identified on the basis of its UV-vis spectrum and reactivity with diazomethane, which converted it to the 13¹,15²-dimethyl, 17³-phytyl ester of Mg-purpurin 7. The side products (ca. 10%) were suggested to include the 17³-phytyl ester of Mg-purpurin 18, which is known to form easily from the Mg-unstable chlorin. The side products also included two red components with UV-vis spectral features resembling those of pure Chl a enolate anion. Hence, the two red components were assigned to the enolate anions of Chl a and pheophytin a or, alternatively, two different complexes of the Chl a enolate ion with Triton X-100. All the above products characterized by us are included in our published free-radical allomerization mechanism of Chl a, i.e. oxidation by ground-state dioxygen. The HRP clearly accelerated the allomerization process, but it did not produce bilins, that is, open-chain tetrapyrroles, the formation of which would require oxygenolysis of the chlorin macrocycle. In this regard, our results are in discrepancy with the claim by several researchers that ‘bilirubin-like compounds’ are formed in the HRP-catalyzed oxidation of Chl a. Inspection of the likely reactions that occurred on the distal side of the heme in the active centre of HRP provided a reasonable explanation for the observed catalytic effect of the HRP on the allomerization of Chl. In the active centre of HRP, the imidazole nitrogen of His-42 was considered to play a crucial role in the C-13² deprotonation of Chl a, which resulted in the Chl a enolate ion resonance hybrid. The Chl enolate was then oxidized to the Chl 13²-radical while the HRP Compound I was reduced to Compound II. The same reactive Chl derivatives, i.e. the Chl enolate anion and the Chl 13²-radical, which are produced twice in the HRP reaction cycle, happen to be the crucial intermediates in the initial stages of the Chl allomerization mechanism.     

Differential effect of phosphatidylethanolamine depletion on raft proteins: Further evidence for diversity of rafts in Saccharomyces cerevisiae

A considerable amount of evidence supports the idea that lipid rafts are involved in many cellular processes, including protein sorting and trafficking. We show that, in this process, also a non-raft lipid, phosphatidylethanolamine (PE), has an indispensable function. The depletion of this phospholipid results in an accumulation of a typical raft-resident, the arginine transporter Can1p, in the membranes of Golgi, while the trafficking of another plasma membrane transporter, Pma1p, is interrupted at the level of the ER. Both these transporters associate with a Triton (TX-100) resistant membrane fraction before their intracellular transport is arrested in the respective organelles. The Can1p undelivered to the plasma membrane is fully active when reconstituted to a PE-containing vesicle system in vitro. We further demonstrate that, in addition to the TX-100 resistance at 4 °C, Can1p and Pma1pa exhibit different accessibility to nonyl glucoside (NG), which points to distinct intimate lipid surroundings of these two proteins. Also, at 20 °C, these two proteins are extracted by TX-100 differentially. The features above suggest that Pma1p and Can1p are associated with different compartments. This is independently supported by the observations made by confocal microscopy. In addition we show that PE is involved in the stability of Can1p-raft association.     

Influence of diet on pre-ingestive particle processing in bivalves: II. Residence time in the pallial cavity and handling time on the labial palps

Previous studies have demonstrated that bivalve molluscs respond to changing food conditions through processes such as preferential selection and ingestion of particulate matter. Little is known, however, about the underlying mechanisms accountable for these responses. To further explain feeding processes at the level of the pallial organs, we determined pallial cavity residence times, or the amount of time it took particles to travel from the inhalant aperture to the stomach, in two species of bivalves, Crassostrea virginica and Mytilus edulis, under conditions of differing particle quality, particle concentration, and temperature. From these residence times, particle-handling times on the labial palps were determined. Diets of three different qualities were tested, including Rhodomonas lens cells, particles prepared from ground Spartina sp. detritus, and a 50/50 mixture of both. Bivalves were delivered one of the three diets along with 10-μm fluorescent polystyrene beads (tracer), removed from feeding chambers at intervals from 30 s up to 20 min, and placed in liquid nitrogen to halt particle transport. Digestive systems of bivalves were then dissected and examined for the presence of tracer beads. Particle-residence times in the pallial cavity and handling times on the labial palps of C. virginica were significantly affected by changes in diet type. Particle-handling times on the palps decreased with increasing diet quality and ranged from 2.2 min (100% R. lens) to 22.8 min (100% ground Spartina sp.), accounting for 88% and 99%, respectively, of the total time particles spent in the pallial cavity. In contrast, diet quality had little effect on particle-residence times in the pallial cavity of M. edulis. However, residence times were affected by temperature and diet concentration. Temperature significantly affected residence times at particle concentrations of both 20 and 100 particles μl⁻¹, whereas particle concentration affected residence times at 20 °C, but not at 5 °C. Particle-handling times on the labial palps ranged from less than 1 to 5.5 min, depending on temperature and concentration, accounting for 50% to 82%, respectively, of the total time particles spent in the pallial cavity. We suggest that (1) observed interspecific differences in particle handling on the labial palps may be due to differences in palp morphology and function, and (2) particle sorting and selection on the labial palps is a rate-limiting step of pre-ingestive feeding processes in by bivalves.     

Solubilization of Trichoplusia ni granulosis virus proteinic crystal: I. Kinetics

Methods for the solubilization of an insect granulosis virus capsule from Trichoplusia ni were studied using turbidity measurements. The solubilization in the solvents employed was most strongly influenced by pH. Various alcohols and hydrogen bond cleaving agents also enhanced solubilization. The most effective solvent tested at pH′s close to neutrality was 60% n-propanol saturated with guanidine. HCl. The effectiveness of this solvent was minimal at pH 5.2. It was shown that the time courses of the solubilization were generally composed of three phases. A plot of the third phase levels versus pH indicated a normal distribution of those capsules or capsular fragments in resisting solubilization. Validity of the calculation of rate constants for the dissociation reaction in the second phase was discussed. Logarithm of the rate constant showed a linear relationship with the reciprocal of absolute temperature. The activation energy for the dissociation calculated from this relationship using the Arrhenius equation indicated that hydrophobic and hydrogen binding forces play the major roles for the stability of the crystalline structure of the capsular protein. Inic binding forces were estimated to be of minor importance.     

Immobilization antigens of Tetrahymena pyriformis : I. Assay and extraction

A method using immunodiffusion has been established to assay the two mutually exclusive temperature dependent immobilization antigens, H and T, of Tetrahymena pyriformis. Specific antiserum was obtained by exploitation of allelic or temperature induced variations among inbred strains for absorption of antisera prepared against whole cells. The antigens were extracted both from isolated cilia and from whole cell bodies. Mild detergent extraction was found to be more efficient than mechanical disruption of the cells by freeze-thawing. The sedimentation behavior in sucrose density gradients of active H antigen was the same, whether freeze-thaw or detergent extracted; similarly, the sedimentation behavior of T was the same following the two extraction methods. Extraction with acetic acid, as reported by others, solubilized the same material as the detergent, but the acid denatured the antigen. An estimate of the molecular weight of the antigen of 29 000 for H and 23 000 for T was made.     

The pre-transmembrane region of the HCV E1 envelope glycoprotein: Interaction with model membranes

The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein-protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309-340, peptide E1_PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1_PTM strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.     

Hydration pressure and phase transitions of phospholipids: I. Piezotropic approach

Dehydration reduces the main phase transition pressure of phospholipids. An analysis based on the Gibbs-Duhem equation shows how the shift of the transition pressure is correlated to the hydration pressure.By using Fourier transform infrared (FT-IR) spectroscopy we determined the hydration-dependent phase transition pressure. The application of our new approach gives hydration pressure values which agree with the values obtained with the osmotic stress method.     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 2. Multi-frequency phase and modulation analysis evidences a loosely connected PSII pigment-protein complex

We report the first direct decomposition of the fluorescence lifetime heterogeneity during multiphasic fluorescence induction in dark-adapted leaves by multi-frequency phase and modulation fluorometry (PMF). A very fast component, assigned to photosystem I (PSI), was found to be constant in lifetime and yield, whereas the two slow components, which are strongly affected by the closure of the reaction centers by light, were assigned to PSII. Based on a modified “reversible radical pair” kinetic model with three compartments, we showed that a loosely connected pigment complex, which is assumed to be the CP47 complex, plays a specific role with respect to the structure and function of the PSII: (i) it explains the heterogeneity of PSII fluorescence lifetime as a compartmentation of excitation energy in the antenna, (ii) it is the site of a conformational change in the first second of illumination, and (iii) it is involved in the mechanisms of nonphotochemical quenching (NPQ). On the basis of the multi-frequency PMF analysis, we reconciled two apparently antagonistic aspects of chlorophyll a fluorescence in vivo: it is heterogeneous with respect to the kinetic structure (several lifetime components) and homogeneous with respect to average quantities (quasi-linear mean τ-Φ relationship).