Purification of an EH domain-binding protein from rat brain that modulates the gating of the rat ether-à-go-go channel

Mutations in the gene encoding ether-à-go-go (EAG) potassium channel impair the function of several classes of potassium currents, synaptic transmission, and learning in Drosophila. Absence of EAG abolishes the modulation of a broad group of potassium currents. EAG has been proposed to be a regulatory subunit of different potassium channels. To further explore this regulatory role we searched for signaling molecules that associate with EAG protein. We have purified a approximately 95-kDa protein from rat brain membranes that binds to EAG. When co-expressed in mammalian cells this protein coimmunoprecipites with EAG and alters the gating of EAG channels. Expression of this protein is regulated during neuronal differentiation. The protein is identical to the recently reported rat protein epsin, which is an EH domain-binding protein similar to the Xenopus mitotic phosphoprotein MP90. These results show that proteins of the epsin family are modulators of channel activity that may link signaling pathways, or the cell cycle, to EAG and thus to various potassium channel functions.     

Development of a transient large strain contact method for biological heart valve simulations

A new 2D method to implement transient contact using Comsol Multiphysics (finite element analysis software that enables multiphysics simulations) is described, which is based on Hertzian contact. This method is compared to the existing (default) contact method that does not enable real transient simulations, but instead performs steady-state solutions where time is a constant. The two types of contact modelling have been applied to simple 2D biological heart valve models, undergoing strains in the region of 10% under 20 kPa pressure (applied over 0.3 s). Both the methods predicted comparable stress patterns, locations of peak stresses, deformations and directions of principal stress. The default contact method predicted slightly greater contact stresses, but spreads over a shorter surface length than the new contact method. The default contact method is useful for contact systems with little transient dependency, due to ease of use. However, where transient conditions are important the new contact method is preferred.     

Identification of land predators of African Penguins Spheniscus demersus through post-mortem examination

The African Penguin Spheniscus demersus is an endangered seabird endemic to southern Africa, and killing sprees by terrestrial predators have been one of the main threats for its mainland colonies. The methods employed to manage predators may differ depending on the species involved, therefore the implementation of strategies to limit the impacts of predation relies on the correct identification of the culprit predator. We report and quantify the lesions seen in African Penguins killed by four species of terrestrial predators: Caracal Caracal caracal (52 kills), Leopard Panthera pardus (27 kills), Domestic Dog Canis lupus familiaris (10 kills), and Cape Grey Mongoose Galerella pulverulenta (4 kills). We discuss patterns of necropsy findings that can be used to identify the predator species involved. Traditional forensic methods are useful tools to direct species-specific management actions for the conservation of the African Penguin and other seabirds so that effective mitigating measures can be deployed quickly to prevent further losses. It should be borne in mind, however, that the age, size and previous hunting experience of the predator are likely to influence the pattern of lesions that will be observed, and not all carcasses will have hallmark lesions or recognisable bite marks.     

Characterization of zebra mussel (Dreissena polymorpha) sperm motility: duration of movement, effects of cations, pH and gossypol

We have examined effects of time after activation, pH, sodium and potassium, and gossypol concentrations on sperm motility of zebra mussel (Dreissena polymorpha). Zebra mussel spermatozoa appeared to have remarkable viability in the fresh water in comparison with freshwater fish sperm. Duration of sperm motility in fresh water is possibly one of the longest among freshwater animals, since it was not significantly changed 3 h after incubation at room temperature (20 °C) or 24 h of incubation at ±0 °C. High osmotic pressure suppresses sperm motility and effects of sodium and potassium are similar. Spermatozoa were inactive at acid pH and became gradually motile when exposed to pH 6.0–9.0. Gossypol appeared to be a very potent spermicidal agent and inhibited motility. This compound also inhibited fertilization. We observed some differences in gossypol effects on spermatozoa between North American and European zebra mussels. These data on zebra mussel sperm biology may be useful for better handling of gametes under laboratory conditions.     

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production. Journal of Industrial Microbiology & Biotechnology (2001) 27, 322–328. Received 12 September 2000/ Accepted in revised form 21 July 2001     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

The effect of juglone on cholinergic mechanisms in rat brain synaptosomes

Juglone 5-hydroxy-1,4-napthoquinone exerts three activities on cortical synaptosomal preparations. It inhibits the release of acetylcholine and is an even more potent inhibitor of high-affinity choline transport into synaptosomes. In addition, as has already been shown by others using brain homogenates, juglone inhibits choline acetyltransferase.     

The influence of water level on macrophyte growth and trophic interactions in eutrophic Mediterranean shallow lakes: a mesocosm experiment with and without fish

1. Water‐level fluctuations are typical of lakes located in the semi‐arid Mediterranean region, which is characterised by warm rainy winters and hot dry summers. Ongoing climate change may exacerbate fluctuations and lead to more severe episodes of drought, so information on the effects of water level on the functioning of lake ecosystems in such regions is crucial. 2. In eutrophic Lake Eymir, Turkey, we conducted a 4‐month (summer) field experiment using cylindrical 0.8‐m‐ (low‐water‐level) and 1.6‐m‐deep (high‐water‐level) mesocosms (kept open to the sediment and atmosphere). Fish (tench, Tinca tinca, and bleak, Alburnus escherichii) were added to half of the mesocosms, while the rest were kept fishless. Ten shoots of Potamogeton pectinatus were transplanted to each mesocosm. 3. Sampling for physicochemical variables, chlorophyll a (chl‐a), zooplankton and per cent plant volume inhabited (PVI%) by macrophytes was conducted weekly during the first 5 weeks, and subsequently biweekly. Macrophytes were harvested on the last sampling date. During the course of the experiment, the water level decreased by 0.41 ± 0.06 m. 4. Throughout the experiment, fish affected zooplankton abundance (?), nutrient concentrations (+), chl‐a (+) and water clarity (?) most strongly in the low‐water‐level mesocosms and the zooplankton community shifted towards dominance of small‐sized forms. The fishless mesocosms had a higher zooplankton/phytoplankton ratio, suggesting higher grazing. 5. Greatest macrophyte growth was observed in the low‐water‐level fishless mesocosms. However, despite high nutrient concentrations and low water clarity, macrophytes were also abundant in the fish mesocosms and particularly increased following a water‐level decrease from midsummer onwards. Macrophyte growth was poor in the high‐water‐level mesocosms, even in the fishless ones with high water clarity. This was ascribed to extensive periphyton development reducing light availability for the macrophytes. 6. Our results indicate that a reduction in water level during summer may help maintain the growth of macrophytes in Mediterranean eutrophic shallow lakes, despite a strong negative effect of fish predation on water clarity. It is therefore probable that an expected negative effect of global climate change on water clarity because of eutrophication and enhanced top‐down control of fish may be, at least partly, counteracted by reduced water level, provided that physical disturbance is not severe.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

Functional analysis of cloned opioid receptors in transfected cell lines

Opioids modulate numerous central and peripheral processes including pain perception, neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (μ, δ, κ). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein α subunits and/or βγ subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K⁺ channels and reduces adenylyl cyclase and Ca²⁺ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca²⁺ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca²⁺ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH₃ cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.