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Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures that have been classified into 12 immunotypes. A feature of meningococcal LPS is the reversible, high-frequency switching of expression (phase variation) of terminal LPS structures. A number of studies are strongly suggestive of a key role for these terminal structures, and their phase-variable expression, in pathogenesis. In a previous study, a locus of three LPS biosynthetic genes, lgtABE, involved in the biosynthesis of one of these terminal structures, lacto-N-neotetraose, was described. The molecular mechanism of phase-variable expression of this structure is by high-frequency mutation in a homopolymeric tract of G residues in the lgtA gene. To investigate the genetic basis of the structural differences between the immunotypes, and the potential for strains to express alternative immunotypes, this locus was examined in all of the immunotype strains. Initially, the lgt locus of strain 126E, an L1 immunotype strain, was cloned and sequenced, revealing two active genes, lgtC and lgtE. The remnants of the lgtA and lgtB genes and an inactive lgtD gene were also present, indicating that the locus may have once contained five active genes, similar to a locus previously reported in Neisseria gonorrhoeae strain F62. Probes based on each of the lgt genes (ABCDE), and the recently reported lgtG gene, were used to determine the presence or absence of lgt genes within individual strains, allowing the prediction of the phase variation repertoire of these strains. Sequencing to determine the nature of homopolymeric tract regions within the lgt genes was carried out to establish the potential for LPS switching. In general, the set of strains examined could be sorted into two distinct groups : one group which phase-vary the α-chain extension via lgtA or lgtC but cannot make β-chain; the second group phase-vary the β-chain extension via lgtG but do not vary α-chain (lacto-N-neotetraose).

Caboxamycin, a new benzoxazole antibiotic, was detected by HPLC-diode array screening in extracts of the marine strain Streptomyces sp. NTK 937, which was isolated from deep-sea sediment collected in the Canary Basin. The structure of caboxamycin was determined by mass spectrometry, NMR experiments and X-ray analysis. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and the enzyme phosphodiesterase.

SUMMARYNeuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression. Activation of Alk signaling, in particular overexpression of ALK wt and NB ALK mutants resulted in the upregulation of NMyc and trkB, which may contribute to continued proliferation and NB predisposition. MATERIALS AND METHODS Expression plasmidsPcDNA3.1 expression plasmids for human ALK wt , ALK F1174L and ALK R1275Q, and pCAGGS expression plasmids for Hand2, Phox2b wt and Phox2b K155X have been described previously (Janoueix-Lerosey et al., 2008;Reiff et al., 2010). shRNA constructsThe shRNA against Gallus gallus Alk and Mk were designed using BLOCK-iT RNAi Designer (Invitrogen, Karlsruhe, Germany) and target the following sequences: shAlk, 5Ј-AAU GGU UUC UCU CUA UGU CCA ACU C-3Ј; shMk, 5Ј-GAG CUG ACU GCA AGU ACA AGU UUG A-3Ј. Scrambled shRNAs (sc-shRNA, Invitrogen, Karlsruhe, Germany) served as controls. In situ hybridizationNon-radioactive in situ hybridization on cryosections and preparation of digoxigenin labeled probes for chick Phox2b was carried out as described previously (Ernsberger et al., 1997;Stanke et al., 1999). qPCR analysisEqual amounts of RNA were used to synthesize cDNA with Oligo(dT)-primers and Superscript-III-reverse-transcriptase according to manufacturer's instructions (Invitrogen, Karlsruhe, Germany). The PCR was carried out using Abgene's Absolute Blue SYBR-Green qPCR Mix (Abgene, Epsom, UK) in a Stratagene Mx3000p Light Cycler (Stratagene, Waldbronn, Germany). All primers (MWG Biotech AG, Ebersberg, Germany) were designed to anneal optimally at 58°C with PerlPrimer (Marshall, 2004) (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, repeated for 50 cycles and a subsequent dissociation curve). The primer pairs (see Table S1 in the supplementary material) were ...