Heart morphology in wild and farmed Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss

The normal shape of the salmonid ventricle is a triangular pyramid with the apex pointing caudoventrally. A strong positive correlation has been established between this shape and optimum cardiac output and function. Domesticated salmonids appear to have developed a more rounded ventricle with misaligned bulbus arteriosus. Several reports from fish health veterinarians indicate that fish with abnormal heart morphology have a high mortality rate during stress-inducing situations like grading, transportation and bath treatments. The present paper compares and describes the ventricle morphology of wild vs. farmed Atlantic salmon, and wild steelhead (anadromous rainbow trout) vs. farmed rainbow trout. Several parameters were measured to provide numerical measurement of the differences in shape, i.e. height:width ratio and the angle between the longitudinal ventricular axis and the axis of the bulbus arteriosus. We conclude that the hearts of farmed fish are rounder than those in corresponding wild fish, and that the angle between the ventricular axis and the axis of the bulbus arteriosus is more acute in wild fish than in their farmed counterparts. Further studies are necessary to reveal the prevalence, functional significance and possible causes of these abnormal hearts.     

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.     

Rapidly Evolving Genes Are Key Players in Host Specialization and Virulence of the Fungal Wheat Pathogen Zymoseptoria tritici (Mycosphaerella graminicola)

The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola). In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in Z. tritici. We deleted the four genes in Z. tritici and confirm a virulence-related role of three of the four genes ΔZt80707, ΔZt89160 and ΔZt103264. The two mutants ΔZt80707 and ΔZt103264 show a significant reduction in virulence during infection of wheat; the ΔZt89160 mutant causes a hypervirulent phenotype in wheat. Mutant phenotypes of ΔZt80707, ΔZt89160 and ΔZt103264 can be restored by insertion of the wild-type genes. However, the insertion of the Zt80707 and Zt89160 orthologs from Z. pseudotritici and Z. ardabiliae do not restore wild-type levels of virulence, suggesting that positively selected substitutions in Z. tritici may relate to divergent host specialization. Interestingly, the gene Zt80707 encodes also a secretion signal that targets the protein for cell secretion. This secretion signal is however only transcribed in Z. tritici, suggesting that Z. tritici-specific substitutions relate to a new function of the protein in the extracellular space of the wheat-Z. tritici interaction. Together, the results presented here highlight that Zt80707, Zt103264 and Zt89160 represent key genes involved in virulence and host-specific disease development of Z. tritici. Our findings illustrate that evolutionary predictions provide a powerful tool for the identification of novel traits crucial for host adaptation and pathogen evolution.     

Electron microscopy of thin protein crystal sections.

In continuation of an earlier publication (Hoppe et al., 1968), further experiments are described here on the preparation of thin film sections of embedded protein crystals for investigation by electron microscopy and electron diffraction. Several embedding media were compared, the best being Aquon. Periodicities were observed in electron micrographs as well as in electron diffraction patterns. In diffraction experiments the best resolution observed was approximately 10 to 11 Å.     

Wild arctic char Salvelinus alpinus and trout Salmo trutta: hosts and reservoir of the salmonid pathogen Spironucleus salmonicida (Diplomonadida; Hexamitidae)

Spironucleus salmonicida is a diplomonad flagellate known to cause systemic infections in farmed salmonids. In northern Norway, outbreaks of spironucleosis in farmed Atlantic salmon Salmo salar have been a recurring problem. Common to all these outbreaks was the origin of smolts: all came from the same farm. In the present study, wild Arctic char Salvelinus alpinus and brown trout Salmo trutta were sampled from the lakes used as a water source for the smolt supplier. In addition, smolt and three-spined sticklebacks Gasterosteus aculeatus were sampled from the smolt farm. Bile and intestinal contents from the sampled fish were examined by light microscopy and PCR. Spironucleus salmonicida was identified in both wild Arctic char and brown trout from the lakes used as water sources by the smolt farm, suggesting that the farmed fish were exposed to this pathogen before transfer to the sea. Spironucleus barkhanus and Spironucleus salmonis were also identified in the sampled fish. The present study also demonstrated that infections with multiple Spironucleus species are present in wild salmonids. No indications of disease related to diplomonad infections were observed in the wild fish, suggesting that wild salmonids are reservoir hosts of Spironucleus salmonicida.     

Protected hinge in the immunoglobulin G2‐A2 disulfide isoform

Human IgG2 consists of disulfide‐mediated structural isoforms, classified by the number of Fab arms disulfide‐linked to the heavy chain hinge. In the IgG2‐B isoform, both Fab arms are linked to the hinge region, and in IgG2‐A, neither Fab arm are linked to the hinge. IgG2‐A/B is a hybrid between these two forms, with only one Fab arm disulfide‐linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide‐linkage differences. Here we explored the structural basis for the A₁ and A₂ isoform subtypes. Whereas A₁ isoform converts into the A/B and B isoforms under mild redox conditions, A₂ does not. Characterization of the disulfide connectivities of A₂ isoform revealed a similar structure to A₁ isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A₂ isoform were resistant to reduction under conditions where A₁ isoform hinge disulfides became reduced and they required thermal treatment (>55°C) to obtain thiol‐dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A₂ isoform behavior. ¹H NMR studies showed that the A₁ isoform Fc glycan was more dynamic than that on A₂ isoform and showed some other conformational differences. Results point to an IgG2‐A₂ upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.     

Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His₆-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.     

LASP1, a Newly Identified Melanocytic Protein with a Possible Role in Melanin Release,but Not in Melanoma Progression

The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release.