The vocal expression of feeding motivation and frustration in the domestic laying hen, Gallus gallus domesticus

Thwarting of feeding behaviour in the laying hen results in an increase in stereotyped pacing, displacement preening, and the gakel-call. These behaviours therefore reflect the frustration arousal caused by the thwarting of feeding behaviour. This raises the question whether the level of frustration also varies with the intensity of the motivation to perform the thwarted behaviour. This study investigated the relationship between the intensity of the motivation and level of frustration on the one hand and the gakel-call on the other hand. In Experiment 1, the strength of the motivation to feed was varied by thwarting hens in their feeding behaviour in an operant procedure after different durations of food deprivation (0, 8, 23 and 47 h). Trend analysis showed that with increasing hunger state, an increasing number of gakel-calls was given. No effect of treatments on temporal characteristics of the gakel-call was found. In Experiment 2, the level of frustration was varied by reducing or increasing the duration of access to food for food-deprived hens compared to the duration of access during training. It was assumed that a shorter duration of access to food compared to training would elicit frustration, which in turn would affect the performance of behaviours indicative of thwarting. However, we found neither a relation between the number of gakel-calls nor the temporal features of the gakel-call and the duration of access to food. Possibly, the differences between treatments were not large enough to induce differences in frustration level. Also, other factors that might have influenced the motivation are discussed.     

The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors

Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-gamma) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-gamma receptor alpha chain (IFN-gammaRalpha) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-gammaR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-gamma-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-gamma hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1(-/-) mice were resistant to IFN-gamma. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT(-/-) mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).     

The ins and outs of EBV infection

Epstein-Barr virus (EBV) infects almost the entire adult population of the world. The success of this virus appears to be based on its ability to infect the B cell, rather than any other cell type. We review EBV B-cell tropism, and discuss the mechanisms by which the virus may gain access to, and egress from, B cells in the normal host.     

A selective peroxisome proliferator-activated receptor-gamma (PPARgamma) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists such as the thiazolidinediones are insulin sensitizers used in the treatment of type 2 diabetes. These compounds induce adipogenesis in cell culture models and increase weight gain in rodents and humans. We have identified a novel PPARgamma ligand, LG100641, that does not activate PPARgamma but selectively and competitively blocks thiazolidinedione-induced PPARgamma activation and adipocyte conversion. It also antagonizes target gene activation as well as repression in agonist-treated 3T3-L1 adipocytes. This novel PPARgamma antagonist does not block adipocyte differentiation induced by a ligand for the retinoid X receptor (RXR), the heterodimeric partner for PPARgamma, or by a differentiation cocktail containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Surprisingly, LG100641, like the PPARgamma agonist rosiglitazone, increases glucose uptake in 3T3-L1 adipocytes. Such selective PPARgamma antagonists may help determine whether insulin sensitization by thiazolidinediones is mediated solely through PPARgamma activation, and whether there are PPARgamma-ligand-independent pathways for adipocyte differentiation. If selective PPARgamma modulators block adipogenesis in vivo, they may prevent obesity, lower insulin resistance, and delay the onset of type 2 diabetes.     

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Background

Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background.

Results

Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type.

Conclusions

The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.

     

X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management

Background

Pompe disease (Glycogen storage disease type II, GSD II, acid alpha-glucosidase deficiency, acid maltase deficiency, OMIM # 232300) is an autosomal-recessive lysosomal storage disorder due to a deficiency of acid alpha-glucosidase (GAA, acid maltase, EC 3.2.1.20, Swiss-Prot P10253). Clinical manifestations are dominated by progressive weakness of skeletal muscle throughout the clinical spectrum. In addition, the classic infantile form is characterised by hypertrophic cardiomyopathy.

Methods

In a cross-sectional single-centre study we clinically assessed 3 patients with classic infantile Pompe disease and 39 patients with non-classic presentations, measured their acid alpha-glucosidase activities and analysed their GAA genes.

Results

Classic infantile patients had nearly absent residual enzyme activities and a typical clinical course with hypertrophic cardiomyopathy until the beginning of therapy. The disease manifestations in non-classic patients were heterogeneous. There was a broad variability in the decline of locomotive and respiratory function. The age of onset ranged from birth to late adulthood and correlated with enzyme activities. Molecular analysis revealed as many as 33 different mutations, 14 of which are novel. All classic infantile patients had two severe mutations. The most common mutation in the non-classic group was c.-32-13?T?>?G. It was associated with a milder course in this subgroup.

Conclusions

Disease manifestation strongly correlates with the nature of the GAA mutations, while the variable progression in non-classic Pompe disease is likely to be explained by yet unknown modifying factors. This study provides the first comprehensive dataset on the clinical course and the mutational spectrum of Pompe disease in Germany.     

Cell cycle population effects in perturbation studies

Growth condition perturbation or gene function disruption are commonly used strategies to study cellular systems. Although it is widely appreciated that such experiments may involve indirect effects, these frequently remain uncharacterized. Here, analysis of functionally unrelated Saccharyomyces cerevisiae deletion strains reveals a common gene expression signature. One property shared by these strains is slower growth, with increased presence of the signature in more slowly growing strains. The slow growth signature is highly similar to the environmental stress response (ESR), an expression response common to diverse environmental perturbations. Both environmental and genetic perturbations result in growth rate changes. These are accompanied by a change in the distribution of cells over different cell cycle phases. Rather than representing a direct expression response in single cells, both the slow growth signature and ESR mainly reflect a redistribution of cells over different cell cycle phases, primarily characterized by an increase in the G1 population. The findings have implications for any study of perturbation that is accompanied by growth rate changes. Strategies to counter these effects are presented and discussed.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae

Background

Microorganisms that are exposed to pollutants in the environment, such as metals/metalloids, have a remarkable ability to fight the metal stress by various mechanisms. These metal-microbe interactions have already found an important role in biotechnological applications. It is only recently that microorganisms have been explored as potential biofactories for synthesis of metal/metalloid nanoparticles. Biosynthesis of selenium (Se⁰) nanospheres in aerobic conditions by a bacterial strain isolated from the coalmine soil is reported in the present study.

Results

The strain CM100B, identified as Bacillus cereus by morphological, biochemical and 16S rRNA gene sequencing [GenBank:GU551935.1] was studied for its ability to generate selenium nanoparticles (SNs) by transformation of toxic selenite (SeO₃ ^2-) anions into red elemental selenium (Se⁰) under aerobic conditions. Also, the ability of the strain to tolerate high levels of toxic selenite ions was studied by challenging the microbe with different concentrations of sodium selenite (0.5 mM-10 mM). ESEM, AFM and SEM studies revealed the spherical Se⁰ nanospheres adhering to bacterial biomass as well as present as free particles. The TEM microscopy showed the accumulation of spherical nanostructures as intracellular and extracellular deposits. The deposits were identified as element selenium by EDX analysis. This is also indicated by the red coloration of the culture broth that starts within 2-3 h of exposure to selenite oxyions. Selenium nanoparticles (SNs) were further characterized by UV-Visible spectroscopy, TEM and zeta potential measurement. The size of nanospheres was in the range of 150-200 nm with high negative charge of -46.86 mV.

Conclusions

This bacterial isolate has the potential to be used as a bionanofactory for the synthesis of stable, nearly monodisperse Se⁰ nanoparticles as well as for detoxification of the toxic selenite anions in the environment. A hypothetical mechanism for the biogenesis of selenium nanoparticles (SNs) involving membrane associated reductase enzyme(s) that reduces selenite (SeO₃ ^2-) to Se⁰ through electron shuttle enzymatic metal reduction process has been proposed.     

A Class III PDZ Binding Motif in the Myotilin and FATZ Families Binds Enigma Family Proteins: a Common Link for Z-Disc Myopathies

Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.The sarcomere of striated muscle consists of strictly organized subunits, myosin-containing thick filaments and actin-containing thin filaments. The thin filaments are aligned and cross-linked at the Z-discs by a molecular complex in which α-actinin is one of the core structures. Since the contractile force is transduced via the Z-discs, this structure has special requirements. It must provide extensive stability and yet undergo modulation in response to external signals. The Z-discs also serve as important sensors of extracellular cues and mediators of cellular signals that result in various adaptive responses (37). Muscle cells are able to sense changes in their workload and adapt accordingly via complex signaling pathways, some involving calcium, since its level in muscle cells alters in response to nerve pulses and muscle contraction. Of special importance is calcineurin, a sarcomeric calcium/calmodulin-dependent phosphatase that can act as a sensor of change. It is involved in the regulation of genes affecting muscle differentiation and fiber-type specification (12, 13).The special role of the Z-discs is indicated by the fact that mutations in several Z-disc proteins can result in neuromuscular disorders and cardiomyopathies. For instance, myofibrillar myopathy (desmin-related myopathy), a disease characterized by sarcomere disintegration and accumulation of thin filament material, is caused by dominantly inherited missense mutations in Z-disc proteins: myotilin, filamin-C, and Z-band alternatively spliced PDZ motif-containing protein (ZASP, also named LIM domain-binding factor 3, Cypher, or Oracle) (42, 43, 52). Missense mutations in myotilin can also result in limb-girdle muscular dystrophy 1A and spheroid body myositis (10, 18), while mutations in ZASP/Cypher (8, 57), myopalladin or FATZ-2 (calsarcin-1/myozenin-2) have been found to be associated with dominant familial dilated (7, 50) or hypertrophic cardiomyopathy (33). ZASP/Cypher knockout mice display a severe form of congenital myopathy and die postnatally (58), whereas myotilin knockout mice are virtually normal (31), suggesting redundancy between the myotilin family members and indicating that dysfunctional myotilin is more harmful to muscle cells than loss of the protein.Myotilin (40), palladin (32, 34), and myopalladin (3) are homologous Z-disc proteins that form a novel family of immunoglobulin-domain-containing actin-binding proteins. Biochemical studies on the best-characterized family member, myotilin, have demonstrated an association with important components of the sarcomere: α-actinin (40), which is a core structural component of the Z-disc; filamins (15, 49); the proteins of the FATZ (calsarcin/myozenin) family (15); and actin (51). Myotilin is linked to signaling networks by binding to the ubiquitin ligases Murf-1 and Murf-2 (54) and indirectly via FATZ (calsarcin/myozenin). Experiments using myotilin fragments with dominant-negative effect have shown its critical involvement in sarcomere organization. Myotilin bundles and stabilizes actin effectively, which suggests a role for myotilin in the organization and maintenance of Z-disc integrity.The FATZ (calsarcin/myozenin) proteins form another Z-disc family with structural and signaling functions. The three homologous members—FATZ-1 (calsarcin-2/myozenin-1), FATZ-2 (calsarcin-1/myozenin-2), and FATZ-3 (calsarcin-3/myozenin-3)—are localized in the Z-disc binding not only to myotilin but also to filamins A, B, and C (15), telethonin (T-cap), α-actinin, ZASP/Cypher, and calcineurin (9, 11, 12, 47). The three FATZ (calsarcin/myozenin) proteins share high homology at their N and the C terminals and, in fact, the binding sites for a variety of proteins occur in these regions. It has been suggested that the FATZ (calsarcin/myozenin) family may play a role in contributing to the formation and maintenance of the Z-disc, as well as in cell signaling, since its members bind calcineurin. FATZ-1 (calsarcin-2/myozenin-1) and FATZ-3 (calsarcin-3/myozenin-3) are highly expressed in skeletal muscle fast-twitch fibers, whereas FATZ-2 (calsarcin-1/myozenin-2) is highly expressed in cardiac muscle slow-twitch fibers. Mice lacking FATZ-2 (calsarcin-1/myozenin-2) showed an increase in the level of calcineurin, as well as a concurrent increase in the percentage of slow-twitch fibers (13). A recent report shows that FATZ-1 (calsarcin-2/myozenin-1) knockout mice have reduced body weight and fast-twitch muscle mass without exhibiting muscle atrophy (14). It is noteworthy that they also have the ability to run longer distances than control mice, thus exhibiting endurance to exercise. In fact, thus far only actinin-3 knockout mice have displayed this phenotype of endurance to exercise. FATZ-1 (calsarcin-2/myozenin-1)-deficient mice show an increase in oxidative muscle fibers and a switch from fast-twitch to slow-twitch fibers due to an increase in NFAT activity, as well as the regulator of calcineurin 1-4 (RCAN1-4), resulting in the concomitant increase in calcineurin signaling. Both FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-2) can regulate calcineurin/NFAT activity, thus influencing the fiber type composition of skeletal muscle (14).ZASP/Cypher (Oracle) (8, 35, 57) belongs to the Enigma family of proteins, the members of which all have a N-terminal PDZ domain and one or more LIM domains at the C-terminal (48). To date, there are six isoforms of ZASP/Cypher, all of which contain an N-terminal PDZ domain and none or three C-terminal LIM domains. ZASP/Cypher also contains a third domain known as the ZM motif which can also be found in ALP and CLP-36 (23, 24). It interacts with two different regions of α-actinin-2; its PDZ domain binds to the C-terminal EH-hand region of α-actinin-2, whereas its ZM motif binds to the rod region of α-actinin-2 (2, 23). ZASP/Cypher colocalizes with α-actinin-2 in the Z-disc, whereas the LIM domains interact with and are phosphorylated by all six isoforms of protein kinase C (PKC α, β1, γ, ζ, δ, and ɛ). ZASP/Cypher is important for the stability of the Z-disc; in fact, ZASP/Cypher knockout mice die in the first 24 h after birth as a result of functional failure of striated muscles caused by disruption of the Z-disc during muscle contraction (58). The PDZ of ZASP/Cypher is a classical type I PDZ domain that binds to the C-terminal of α-actinin-2.To better understand the biology of the Z-disc and pathogenesis of muscle disorders, it is important to unravel the dynamic interplay of Z-disc components. In the present study, we demonstrate a novel PDZ domain-binding motif common to the myotilin and FATZ (calsarcin/myozenin) protein families. This domain mediates interaction with ZASP/Cypher in a phosphorylation-dependent manner and is also involved in targeting ZASP/Cypher.