Temporal and Environmental Factors Driving Vibrio Vulnificus and V. Parahaemolyticus Populations and Their Associations With Harmful Algal Blooms in South Carolina Detention Ponds and Receiving Tidal Creeks.

Incidences of harmful algal blooms (HABs) and Vibrio infections have increased over recent decades. Numerous studies have tried to identify environmental factors driving HABs and pathogenic Vibrio populations separately. Few have considered the two simultaneously, though emerging evidence suggests that algal blooms enhance Vibrio growth and survival. This study examined various physical, nutrient, and temporal factors associated with incidences of HABs, V. vulnificus, and V. parahaemolyticus in South Carolina coastal stormwater detention ponds, managed systems where HABs often proliferate, and their receiving tidal creek waters. Five blooms occurred during the study (2008-2009): two during relatively warmer months (an August 2008 cyanobacteria bloom and a November 2008 dinoflagellate bloom) followed by increases in both Vibrio species and V. parahaemolyticus, respectively, and three during cooler months (December 2008 through February 2009) caused by dinoflagellates and euglenophytes that were not associated with marked changes in Vibrio abundances. Vibrio concentrations were positively and significantly associated with temperature and dissolved organic matter, dinoflagellate blooms, negatively and significantly associated with suspended solids, but not significantly correlated with chlorophyll or nitrogen. While more research involving longer time series is needed to increase robustness, findings herein suggest that certain HAB species may augment Vibrio occurrences during warmer months.

What is being measured, and by whom? Facilitation of communication on technical measures amongst competent authorities in the implementation of the European Union Broiler Directive (2007/43/EC).

The European Union (EU) Broiler Directive (2007/43/EC) is unique amongst current EU Directives, which address animal welfare, in that it uses outcome data collected at abattoirs and on farm to monitor on-farm broiler welfare and vary the maximum permitted stocking density on farm. In this study, we describe how, by bringing together personnel from the competent authorities in 22 member states (MSs) who have responsibility for implementing the Directive, and engaging in exchange of information and technical methods regarding the Broiler Directive, it has been possible to identify differences in approach with regard to 'what data is being collected, and by whom' across EU MSs. Online questionnaires and workshop exercises enabled us to identify priority areas for knowledge transfer and training. For example, foot pad dermatitis, hock burn, dead on arrival and total rejections (birds rejected as unfit for human consumption by the meat inspection staff at slaughter) were identified by the MSs as measures of medium-to-low priority in terms of knowledge transfer because there are assessment methods for these conditions that are already well accepted by competent authorities. On the other hand, breast lesions, cellulitis, emaciation, joint lesions, respiratory problems, scratches, wing fractures and a number of environmental measures were identified as having high priority in terms of knowledge transfer. The study identified that there is significant variability in the stage of implementation between MSs, and responses from the participating MSs indicated that sharing of guidance and technical information between MSs may be of value in the future set-up process for those MSs engaged in implementation of the Directive.

A spatial assessment of baseline nutrient and water quality values in the Ashepoo-Combahee-Edisto (ACE) Basin, South Carolina, USA.

The Ashepoo-Combahee-Edisto (ACE) Basin (South Carolina, USA) National Estuarine Research Reserve System (NERRS) encompasses some of the least developed USA coastline. Yet, periodic sampling showed that certain regions have higher nutrient, fecal coliform, and chlorophyll a levels, often with lower dissolved oxygen, than other South Carolina estuaries. To evaluate the spatial extent of these issues, a summer (2008) baseline study was conducted. Physical water quality, total nitrogen and phosphorus, chlorophyll a, dissolved organic carbon, and suspended solids were measured from surface waters of 67 stations (30 tidal creek, 37 open water). Nutrient and chlorophyll a levels were significantly (p<0.01) and negatively correlated with the extent of open water (% land cover), and chlorophyll a and nitrogen levels were, at times, elevated relative to concentrations typical of other estuaries in the state, reinforcing previous findings. This survey also identified several creeks not previously monitored that exhibited elevated nutrients.

[German translation and validation of the Stress Appraisal Measure (SAM)]. / Deutsche Übersetzung und Validierung des Stress Appraisal Measure (SAM).

OBJECTIVE: In the present study, the German-language version of the Stress Appraisal Measure (SAM) by Peacock and Wong was validated in a student population. SAM is a relatively short questionnaire (28 items) that evaluates a current, stress-triggering event. The theoretical background is provided by the stress model of Lazarus and Folkman. METHOD: 85 students (age: 23; 59 female, 26 male) were exposed to two stress scenarios in order to test whether they were suited to provoke stress. A factor analysis was performed and the internal consistency of the seven SAM scales was determined. In addition, the convergent validity of SAM with State and Trait Anxiety Inventory (STAI), Coping Inventory for Stressful Situations (CISS) and specific emotion scales was investigated via Pearson's product-moment correlation. RESULTS: The two stress scenarios were suited to evoke stress. The factor structure and the internal consistency of the individual scales, as well as the convergent validity of SAM were replicated with minor limitations in the present German version. Some items (especially from the fifth factor) were only replicated partially. CONCLUSION: SAM can also be employed in the German language version.

[Importance of the rearing period for laying hens in alternative systems]. / Bedeutung der Aufzucht der Legehennen für alternative Haltungsformen.

Feather pecking and cannibalism are still major problems in alternative systems for laying hens. Literature and practical experience indicate that unfavourable rearing conditions might be important risk factors for the occurrence of these behavioural disturbances during the laying period. Typical rearing conditions of laying hens from 50 rearing units in Germany and Austria are presented. Obvious risk factors during rearing for feather pecking and cannibalism during the laying period were found. Most flocks were kept under high stocking density (mean: 15 pullets per m' useable area) and some flocks had access to litter only after the second week of life or access to raised perches after the fourth week of life. Plumage condition of pullets and laying hens varied widely in non-beak-trimmed as well as in beak-trimmed flocks. The percentage of pullets with damaged plumage was higher in beak-trimmed than in non-beak-trimmed flocks (medians: 53 % versus 30 %, p = 0,022). In laying hens there was a higher percentage of hens with plumage damage in non-beak-trimmed flocks compared to beak-trimmed flocks (medians: 23 % versus 50 %, p = 0,007). Data analysis will be continued, especially with regard to particular risk factors.

Linkages between cellular biomarker responses and reproductive success in oysters--Crassostrea virginica.

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May-June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.

Vibrational dynamics of myoglobin determined by the phonon-assisted Mössbauer effect.

The phonon-assisted Mössbauer effect is used to determine the partial phonon density of states of the iron within the active center of deoxymyoglobin, carboxymyoglobin, and dry and wet metmyoglobin between 40 and 300 K. Between 0 and 1 meV the iron density of states increases quadratically with the energy, as in a Debye solid. Mean sound velocities are extracted from this slope. Between 1 and 3 meV a nearly quadratic "Debye-like" increase follows due to the similar strength of intermolecular and intramolecular forces. Above 3 meV, optical vibrations are characteristic for the iron-ligand conformation. The overall mean square displacements of the heme iron atom obtained from the density of states agree well with the values of Mössbauer absorption experiments below 180 K. In the physiological temperature regime the data confirm the existence of harmonic vibrations in addition to the protein specific dynamics measured by Mössbauer absorption. In the Debye energy regime the mean square displacement of the iron is in agreement with that of the hydrogens measured by incoherent neutron scattering demonstrating the global character of these modes. At higher energies the vibration of the heavy iron atom at 33 meV in metmyoglobin is as large as that of the lightweight hydrogens at that energy. A freeze dried, rehydrated (h=0.38 g H2O/g protein) metmyoglobin sample shows an excess of states above the Debye law between 1 and 3 meV, similar to neutron scattering experiments. The room temperature density of states below 3 meV exhibit an increase of the density compared to the low temperature data, which can be interpreted as mode softening.

Expression of P-glycoprotein in southeastern oysters, Crassostrea virginica.

These studies provide important fundamental information regarding the expression of P-glycoprotein (p-gp) in southeastern oysters (Crassostrea virginica). Using rhodamine transport studies, p-gp activity was detected in newly fertilized embryos. A monoclonal antibody (C219) was used to evaluate p-gp expression in oyster tissues. On the basis of laboratory studies, p-gp expression tended to be higher in gill tissues than mantle tissues, and was generally not related to salinity differences. Seasonal studies were conducted with oysters collected monthly for 1 year from Lighthouse Creek, an unpolluted site. There was a general pattern of higher p-gp expression in the warmer months and lower expression in the colder months. In contrast, total gill protein concentrations decreased during the warmer months and increased during the colder months. These studies indicate that there are seasonal patterns in p-gp expression which may represent an adaptive response to natural stressors associated with summer conditions.

Expression of P-glycoprotein in the gills of oysters, Crassostrea virginica: seasonal and pollutant related effects.

The expression of p-glycoprotein (p-gp) in aquatic organisms has been proposed as a biomarker of pollution exposure. Previous research has provided evidence that p-gp is inducible by organic xenobiotics and that p-gp is overexpressed in mussels from degraded areas. However, seasonal changes in expression at polluted sites has not been described previously. The purposes of these studies were to evaluate the expression of p-gp in polluted and unpolluted sites in oysters (Crassostrea virginica) and to determine if there were seasonal differences. P-gp expression and total protein concentrations were measured seasonally in the gills of southeastern oysters, Crassostrea virginica, at degraded and undegraded sites in Charleston Harbor, South Carolina, USA. At all sites, p-gp expression was generally higher during the warmer months and lower in the colder months. Polluted sites tended to show a decrease in p-gp expression in June and September, suggesting that p-gp inhibition may have occurred. P-gp expression was not significantly related to specific classes of sediment contaminants or to overall sediment contaminant loading. Total gill protein concentrations at all sites were lower during the warmer months and higher during the colder months. In general, all sites tended to show decreased total gill protein concentrations when compared to the control site. Total protein concentrations were significantly related to water temperature, specific classes of sediment contaminants and overall sediment contaminant loads, but there was no relationship with salinity.

Nuclear forward scattering of synchrotron radiation by deoxymyoglobin.

Nuclear forward scattering of synchrotron radiation is used to determine the quadrupole splitting and the mean square displacement of the iron atom in deoxymyoglobin in the temperature range between 50 K and 243 K. Above 200 K an abnormally fast decay of the forward scattered intensity at short times after the synchrotron flash is observed, which is caused by protein-specific motions. The results strongly support the picture that protein dynamics seen at the position of the iron can be understood by harmonic motions in the low temperature regime while in the physiological regime diffusive motions in limited space are present. The shape of the resonance broadening function is investigated. An inhomogeneous broadening with a Lorentzian distribution indicating dipole interactions results in a better agreement with the experimental data than the common Gaussian distribution.

Biomarker studies with juvenile oysters (Crassostrea virginica) deployed in-situ.

Hatchery-reared juvenile oysters (Crassostrea virginica) were deployed in situ for approximately 1 month from mid-May to mid-June of 1996 at sites that were classified as reference, agricultural, suburban, or urban/industrial. Cellular responses (lysosomal destabilization, glutathione concentrations, lipid peroxidation, heat shock proteins, metallothioneins, and multi-xenobiotic resistance proteins) were analysed, and their efficacy as biomarkers of stress was evaluated. Increased lysosomal destabilization, glutathione depletion, increased lipid peroxidation, and induction of heat shock proteins and metallothioneins were observed at many of the polluted sites, but increases in multixenobiotic resistance proteins were not. Significant correlations between sediment contaminants and lysosomal destabilization or glutathione concentrations were observed. Similarly, there were significant correlations between sediment cadmium and copper levels and metallothioneins. Although elevated lipid peroxidation products and heat shock proteins were observed at some of the contaminated sites, there were no significant correlations with contaminants. These studies suggest that lysosomal destabilization and glutathione depletion are sensitive, robust indicators of contaminant stress. Although lipid peroxidation and heat shock protein responses were not correlated with contaminants, they are still regarded as valuable indicators of stress. These studies demonstrate the value of using a suite of cellular biomarkers to identify and characterize stress responses related to anthropogenic perturbations.

Cytoplasmic carbonic anhydrase II of rat coagulating gland is secreted via the apocrine export mode.

Two different pathways for protein secretion are described for epithelial cells of rat coagulating gland and dorsal prostate: the classical merocrine and the alternative apocrine release mode. Apocrine-secreted proteins are synthesized on cytoplasmic polyribosomes and are subsequently exported in protrusions on the apical cell surface (aposomes). In this article we report the identification and purification to homogeneity of a 29-kD protein from the secretion of rat coagulating gland. N-terminal amino acid sequence analyses revealed 100% identity to rat brain carbonic anhydrase II (CAH II). In addition, the 29-kD protein showed CAH enzyme activity. On Western blot analysis, a polyclonal anti-CAH II antibody raised in rabbit reacted specifically with the rat and human but not bovine CAH II isoforms. Immunohistochemical studies on rat coagulating gland showed strong labeling for CAH II protein in aposomes. Immunoelectron microscopy confined CAH II protein to the cytoplasm and aposomes, whereas no staining was visible in the compartments of the classical merocrine route, the endoplasmic reticulum and Golgi apparatus. The resident cytoplasmic protein lactate dehydrogenase, however, was not found in the secretion. Taken together, the morphological and biochemical data clearly indicate that cytoplasmic CAH II from rat coagulating gland is specifically selected and then secreted via the apocrine pathway.

The course and morphology of efferent nerve fibres in the papilla basilaris of the pigeon (Columba livia).

This paper describes the course and morphology of efferent fibres in an avian cochlea. Horseradish peroxidase stained efferent fibres in the pigeon papilla basilaris were identified by Nomarski optics and camera lucida drawings. There are at least two types of efferent fibres: Large thick fibres take mainly a transversal course and contact short and intermediate hair cells over the free basilar membrane as well as hyaline cells. Large efferent fibres contact both hair cells and hyaline cells. Small thin fibres contact short or intermediate hair cells over the free basilar membrane or tall hair cells over the neural limbus. A physiological consequence of the findings is that efferent activity will concomitantly lead to a contraction of hyaline cells and a hyperpolarization of hair cells.

SVS II--an androgen-dependent actin-binding glycoprotein in rat semen.

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.

A new staining method for the detection of activities of H2O2-producing oxidases on gels and blots using cerium and 3,3'-diaminobenzidine.

Cerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown-black precipitate by the subsequent reaction with 3,3'-diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecificity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms.

Purification and characterization of transglutaminases from the genital tract of the male rat.

In the genital tract of the male rat two different forms of the enzyme transglutaminase (TGase) could be identified and characterized. The coagulating gland and the dorsal prostate secrete a glycosylated and acylated TGase with a molecular weight of 65,000 dalton and pI value of 8.7. This secretory form was purified to homogeneity using preparative isoelectric focusing and gel filtration on a Superdex 200 column. Running fast protein liquid chromatographic gel filtration on a Superose 12 column in the presence of calcium ions, high-molecular-weight aggregates were physically formed which could only be eluted using drastic conditions (0.1 M sodium hydroxide). In the presence of 10 mM EDTA this tendency to aggregate was greatly diminished. Utilizing a Superdex 200 column for gel filtration, the secretory TGase was even eluted as a monomeric protein. Testicular TGase was isolated by ion-exchange fast protein liquid chromatography on a Mono Q and by gel filtration on a Superdex 200 column. This enzyme represents a tissue-type TGase with a molecular weight of 82,000 dalton and pI value of 5.25. Hydrophobic interaction chromatography on a phenyl-Superose column showed no further enrichment of the GTP-binding form of transglutaminase.

Purification and molecular characterization of a secretory transglutaminase from coagulating gland of the rat.

A transglutaminase (TGase, EC 2.3.2.13) was isolated from the secretion of rat coagulating gland (CGS-TGase). The protein consists of a single polypeptide chain and has a molecular mass of 65 kDa. During purification the net charge changes from pI 7.6 in the crude extract to pI 8.5-8.7 for the purified protein. Nearly equal numbers of glutamyl- and lysyl-residues were detected by amino acid analysis. The protein therefore represents an appropriate substrate of autocatalytic crosslinking. The total number of cysteine residues is 18-19, six of which being present in free form. One of the thiol groups is essential for the enzymic activity. The protein core is glycosylated with mannosyl residues and in addition substituted with saturated acyl residues and phosphoinositol. The phosphoinositol anchor was demonstrated by use of a specific antibody. Removal of the acyl- and glycosyl-residues or of the total anchor group results in autoaggregation and decrease of enzymic activity. In contrast to tissue-type TGases, Ca2+ dependent enzymic activity of CGS-TGase is not inhibited by GTP. The secretory TGase shows no immunological cross-reactivity to tissue-type enzyme or blood factor XIII.