Regulatory interactions between two actin nucleators, Spire and Cappuccino

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila melanogaster oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. We find that the interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming.     

Agonistic anti-CD40 antibody profoundly suppresses the immune response to infection with lymphocytic choriomeningitis virus

Previous work has shown that agonistic Abs to CD40 (anti-CD40) can boost weak CD8 T cell responses as well as substitute for CD4 T cell function during chronic gammaherpes virus infection. Agonistic anti-CD40 treatment has, therefore, been suggested as a potential therapeutic strategy in immunocompromised patients. In this study, we investigated whether agonistic anti-CD40 could substitute for CD4 T cell help in generating a sustained CD8 T cell response and prevent viral recrudescence following infection with lymphocytic choriomeningitis virus (LCMV). Contrary to expectations, we found that anti-CD40 treatment of MHC class II-deficient mice infected with a moderate dose of LCMV resulted in severe suppression of the antiviral CD8 T cell response and uncontrolled virus spread, rather than improved CD8 T cell immune surveillance. In Ab-treated wild-type mice, the antiviral CD8 T cell response also collapsed prematurely, and virus clearance was delayed. Additional analysis revealed that, following anti-CD40 treatment, the virus-specific CD8 T cells initially proliferated normally, but an increased cell loss compared with that in untreated mice was observed. The anti-CD40-induced abortion of virus-specific CD8 T cells during LCMV infection was IL-12 independent, but depended partly on Fas expression. Notably, similar anti-CD40 treatment of vesicular stomatitis virus-infected mice resulted in an improved antiviral CD8 T cell response, demonstrating that the effect of anti-CD40 treatment varies with the virus infection studied. For this reason, we recommend further evaluation of the safety of this regimen before being applied to human patients.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Nucleic acids potentiate Factor VII-activating protease (FSAP)-mediated cleavage of platelet-derived growth factor-BB and inhibition of vascular smooth muscle cell proliferation

FSAP (Factor VII-activating protease) can cleave and inactivate PDGF-BB (platelet-derived growth factor-BB) and thereby inhibits VSMC (vascular smooth-muscle cell) proliferation. The auto-activation of FSAP is facilitated by negatively charged polyanions such as heparin, dextransulfate or extracellular ribonucleic acids. Since auto-activation is essential for the anti-proliferative function of FSAP, the influence of nucleic acids as cofactors for the FSAP-mediated inhibition of PDGF-BB was investigated. Natural or artificial RNA was an effective cofactor for FSAP mediated PDGF-BB degradation, whereas the effect of DNA was weak. RNA-induced cleavage of PDGF-BB was inhibited by serine protease inhibitors. The pattern of PDGF-BB cleavage was identical with either heparin or RNA as a cofactor. One of the cleavage sites in PDGF-BB was at the positions 160-162 (R160KK162), which is an important region for receptor binding and activation. In VSMCs, PDGF-BB-stimulated DNA synthesis was inhibited by FSAP in the presence of RNA. RNA was more effective than DNA and the cofactor activity of RNA was neutralized after pretreatment with RNase. FSAP binding to RNA protected the nucleic acid from degradation by RNase. These data are relevant to situations where extracellular nucleic acids released from necrotic or apoptotic cells could activate local FSAP, leading to inhibition of PDGF-BB.     

Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo

The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.     

Skp2B stimulates mammary gland development by inhibiting REA, the repressor of the estrogen receptor

Skp2B, an F-box protein of unknown function, is frequently overexpressed in breast cancer. In order to determine the function of Skp2B and whether it has a role in breast cancer, we performed a two-hybrid screen and established transgenic mice expressing Skp2B in the mammary glands. We found that Skp2B interacts with the repressor of estrogen receptor activity (REA) and that overexpression of Skp2B leads to a reduction in REA levels. In the mammary glands of MMTV-Skp2B mice, REA levels are also low. Our results show that in virgin transgenic females, Skp2B induces lobuloalveolar development and differentiation of the mammary glands normally observed during pregnancy. As this phenotype is identical to what was observed for REA heterozygote mice, our observations suggest that the Skp2B-REA interaction is physiologically relevant. However, in contrast to REA(+/-) mice, MMTV-Skp2B mice develop mammary tumors, suggesting that Skp2B affects additional proteins. These results indicate that the observed expression of Skp2B in breast cancer does contribute to tumorigenesis at least in part by modulating the activity of the estrogen receptor.     

Ablation of connexin43 in smooth muscle cells of the mouse intestine: functional insights into physiology and morphology

Connexin43 (Cx43) gap-junction channels are highly abundant in intestinal smooth muscle but their functional impact has not been studied so far. Here, we have aimed to elucidate the functional role of Cx43 in the tunica muscularis of the mouse intestine in vivo. Transgenic mice with conditional deletion of Cx43 in smooth muscle cells (SMC) were generated. Histological investigations by immunofluorescence analyses and organ-bath recordings to assess the contractility of intestinal tissue strips were carried out. Measurements of gastrointestinal transit and of the visceromotor response by utilizing a standardized colorectal distension model to quantify alterations of visceral sensory function were also performed in SMC-specific Cx43 null mice and control littermates. Histologically, we found thickening of the tunica muscularis and a 13-fold increase of neutrophil infiltration of the gastrointestinal wall of SMC-specific Cx43 null mice. These animals also exhibited a decrease of 29% in gastrointestinal transit time. In contrast, the visceromotor response to a standardized colorectal distension was elevated, as was the contractility in SMC-specific Cx43 null mice, compared with controls. Thus, SMC-specific ablation of Cx43 in mice leads to morphological and functional alterations of the intestinal tunica muscularis, to gastrointestinal motor dysfunction and to altered visceral sensory function. This study was supported by a grant from the German Research Association (Wi 270/25-1,2) to K.W. and in part by the IFORES program of the University Hospital, Essen, Germany.     

Interclonal variation in diel horizontal migration behaviour of the water flea <Emphasis Type="Italic">Daphnia magna</Emphasis>—searching for a signature of adaptive evolution

In shallow temperate lakes, zooplankton populations may exhibit diel horizontal migration (DHM) and move towards macrophytes during the day to avoid fish. Using a natural Daphnia magna population, we undertook an experimental investigation aimed to describe the genetic variation for DHM and to study whether an adaptive micro-evolutionary response occurred to changes in macrophyte coverage and fish predation pressure through time. Twenty-seven D. magna clones were hatched from ephippia in the sediment of shallow Lake Ring, Denmark. This lake was eutrophied during the 20th century and was subject to restoration measures in the 1970s. The DHM behaviour of the clones was observed both in the presence and absence of fish kairomone. Significant interclonal variation in DHM behaviour occurred in both treatments. To study the micro-evolutionary response of the Lake Ring D. magna population, two approaches were used. First, we compared the DHM behaviour of clones derived from ephippia collected at different depths. A comparison was conducted between clones resurrected from the period of eutrophication (1960–1980) and from the period of recovery (1986–2000). A significant treatment (presence and absence of fish kairomone) × period interaction effect was identified, suggesting a significant micro-evolutionary response for DHM behaviour. The D. magna clones exhibited a significantly stronger horizontal migration response during the period of eutrophication than in the recovery phase. Second, clonal means, representing the influence of the genotype on the trait, were correlated with environmental conditions (macrophyte cover, fish predation pressure and Secchi depth). The results of this analysis also suggest that a micro-evolutionary response by Daphnia has occurred in reaction to changes in fish predation pressure. In periods with high fish predation pressure, Daphnia migrated more strongly towards the plants. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

The autotransporter esterase EstA of Pseudomonas aeruginosa is required for rhamnolipid production, cell motility, and biofilm formation

Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.     

Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.