Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.     

Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)

Lecudina tuzetae is a parasitic protozoan (Gregarine, Apicomplexa) living in the intestine of a marine polychaete annelid, Nereis diversicolor. Using electron and fluorescence microscopy, we have characterized the dynamic changes in microtubule organization during the sexual phase of the life cycle. The gametocyst excreted from the host worm into seawater consists of two (one male and one female) gamonts in which cortical microtubule arrays are discernible. Each gamont undergoes multiple nuclear divisions without cytokinesis, resulting in the formation of large multinucleate haploid cells. After cellularization, approximately 1000 individual gametes are produced from each gamont within 24 h. Female gametes are spherical and contain interphase cytoplasmic microtubule arrays emanating from a gamma-tubulin-containing site. In male gametes, both interphase microtubules and a flagellum with "6 + 0" axonemal microtubules extend from the same microtubule-organizing site. At the beginning of spore formation, each zygote secretes a wall to form a sporocyst. Following meiotic and mitotic divisions, each sporocyst gives rise to eight haploid cells that ultimately differentiate into sporozoites. The ovoid shaped sporocyst is asymmetric and forms at least two distinctive microtubule arrays: spindle microtubules and microtubule bundles originating from the protruding apical end corresponding to the dehiscence pole of the sporocyst. Because antibodies raised against mammalian centrosome components, such as gamma-tubulin, pericentrin, Cep135, and mitosis-specific phosphoproteins, react strongly with the microtubule-nucleating sites of Lecudina, this protozoan is likely to share common centrosomal antigens with higher eukaryotes.     

G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex

We have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.     

Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling

Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues.     

Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams.     

Schistosoma mansoni: ferredoxin-NADP(H) oxidoreductase and the metabolism of reactive oxygen species

Mitochondrial-type ferredoxin-NADP(H) oxidoreductases (FNR) catalyze the electron transport between NADPH and substrates such as ferredoxins. Even though enzymes belonging to this family are present in several organisms, including prokaryotes, their biological function is not clearly understood. In a previous work, we reported the existence of a mitochondrial-type FNR in the trematode Schistosoma mansoni (SmFNR). This enzyme conferred tolerance to oxidative stress conditions when tested in an heterologous system. In this work, we demonstrate that the SmFNR can be imported to mitochondria in mammal cells and show that its expression is induced in parasite cultures by reactive oxygen species (ROS). The results reported herein give further support to the involvement of SmFNR in ROS metabolism.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.     

Specificity of the MAP kinase ERK2 for phosphorylation of tyrosine hydroxylase

Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2mol of phosphate into each subunit of tyrosine hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.     

Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-alpha induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to R-848

Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-alpha-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-alpha release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-alpha release correlated with divergent ligand-induced changes in monocyte TNF-alpha mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-alpha release on adult monocytes without any effect on R-848-induced TNF-alpha release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.