Development of the nervous system in the "head" of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> (Chelicerata: Xiphosura): morphological evidence for a correspondence between the segments of the chelicerae and of the (first) antennae of Mandibulata

We investigated brain development in the horseshoe crab Limulus polyphemus and several other arthropods via immunocytochemical methods, i.e. antibody stainings against acetylated alpha-tubulin and synapsin. According to the traditional view, the first appendage-bearing segment in chelicerates (the chelicerae) is not homologous to the first appendage-bearing segment of mandibulates (first antenna, deutocerebrum) but to the segment of the second antenna (tritocerebrum) or the intercalary segment in hexapods and myriapods. Accordingly, the segment of the deutocerebrum in chelicerates would be completely reduced. The main arguments for this view are: (1) the postoral origin of the cheliceral ganglion, (2) a poststomodaeal commissure, and (3) a connection of the cheliceral ganglion to the stomatogastric system. Our data show that these arguments are not convincing. During the development of horseshoe crabs there is no evidence for a former additional segment in front of the chelicerae. Instead, comparison of the brain structure (neuropil ring) between chelicerates, crustaceans and insects shows remarkable similarities. Furthermore, the cheliceral commissure in horseshoe crabs runs mainly praestomodaeal, which would be unique for a tritocerebral commissure. An unbiased view of the developing nervous system in the "head" of chelicerates, crustaceans and insects leads to a homologisation of the cheliceral segment and that of the (first) antenna (= deutocerebrum) of mandibulates that is also congruous to the interpretation of the Hox gene expression patterns. Thus, our data provide morphological evidence for the existence of a chelicerate deutocerebrum.     

Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum

 The aquaporin NtAQP1 from Nicotiana tabacum L. is insensitive to heavy-metal ions. In addition to water, the transport of urea or glycerol is facilitated by this plasma-membrane-located water channel. Northern hybridization and whole-mount in situ hybridization revealed a high steady-state level of NtAQP1-RNA in roots, a decreased content in shoots and a low content in leaves. By immunolocalization with an antibody targeted to the N-terminus of the aquaporin, the localization of NtAQP1-protein at sites of expected high water transport rates from and to the apoplast or symplast could be demonstrated. The specific pattern of NtAQP1 distribution in petioles strongly indicates a transcellular movement of water. Received: 12 August 1999 / Accepted: 27 December 1999     

Intracellular pathways and degradation of endosomal contents in basal epithelial cells of freshwater sponges (Porifera, Spongillidae)

 The digestive system expressed by basal epithelial cells of the freshwater sponges Spongilla lacustris and Ephydatia muelleri is mainly represented by a population of 30–50 preexisting lysosomes located in the close vicinity of the central nucleus. The strongly acidic vacuoles (pH 4–4.5) vary in size between 1 and 3 μm, and contain a set of different lysosomal enzymes. Immunocytochemical studies succeeded in the detection of β-hexosaminidase, cathepsin D, acid phosphatase, and α-glucosidase. Endosomes resulting from fluid-phase macropinocytosis, receptor-mediated endocytosis, or phagocytotic activity deliver their exogenous contents to the preexisting lysosomes for enzymatic degradation. Macropinosomes and phagosomes follow a rather reduced intracellular pathway by immediate fusion with the lysosomal compartment, whereas substances conveyed by coated vesicles pass through two additional vacuolar stages, namely early and late endosomes. Early endosomes serve as sorting organelles and segregate various constituents of complex ligands (BSA-AU_6, BSA-AU₁₂) by size into individual late endosomes, which then coalesce with preexisting lysosomes. As a whole, the intracellular pathways and hydrolytic processing of endosomal and phagosomal contents in freshwater sponge cells share certain similarities with the respective mechanisms in cells of higher eukaryotes. Accepted: 7 October 1997     

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.     

Visible light (>395 nm) causes micronuclei formation in mammalian cells without generation of cyclobutane pyrimidine dimers

Solar radiation gives rise to DNA damage in mammalian cells not only directly by excitation of DNA, which generates predominantly pyrimidine dimers, but also indirectly by the excitation of endogenous photosensitizers, which causes oxidative DNA modifications. The latter mechanism has a low quantum yield, but it is the only one proceeding in the visible range of the spectrum. To investigate its relevance for the genotoxicity of sunlight, we have analysed the generation of micronuclei associated with the induction of oxidative DNA damage by visible light in melanoma cells and primary human skin fibroblasts. Similar yields of light-induced oxidative DNA base modifications sensitive to the repair glycosylase Fpg (7,8-dihydro-8-oxoguanine and other oxidative purine modifications) were observed in the normal fibroblasts and the malignant melanoma cells of the same donor. When irradiations were carried out at intervals to compensate for a photodecomposition of the endogenous chromophore, a significant generation of micronuclei was observed in both cell types. Cyclobutane pyrimidine dimers could be excluded to be responsible for the micronuclei induction at wavelengths >395 nm. Experiments with a cut-off filter indicate that the ratio of pyrimidine dimers and Fpg-sensitive oxidative modifications in irradiated cells not only reflects the relative contributions of direct and indirect mechanisms, but is also similar to the ratio by which the two mechanisms contribute to the generation of the micronuclei. The results suggest that indirectly generated oxidative DNA modifications can contribute significantly to the adverse effects of sunlight.     

Mapping and genomic characterization of the gene encoding diacylglycerol kinase γ (DAGK3): assessment of its role in dominant optic atrophy (OPA1)

The family of diacylglycerol kinases (DAGKs) is known to play an important role in signal transduction linked to phospholipid turnover. In the fruitfly Drosophila melanogaster, a human DAGK ortholog, DGK2, was shown to underlie the phenotype of the visual mutant retinal degeneration A (rdgA). Previously, the gene encoding a novel member of the human DAGK family, termed DAGK3, was cloned and demonstrated to be abundantly expressed in the human retina. Based on these findings we reasoned that DAGK3 might be an excellent candidate gene for a human eye disease. In the present study, we report the genomic organization of the human DAGK3 gene, which spans over 30 kb of genomic DNA interrupted by 23 introns. In addition, we have mapped the gene locus by fluorescence in situ hybridization to 3q27–28, overlapping the chromosomal region known to contain the gene underlying dominant optic atrophy (OPA1), the most common form of hereditary atrophy of the optic nerve. Mutational analysis of the entire coding region of DAGK3 in 19 unrelated German OPA1 patients has not revealed any disease-causing mutations, therefore excluding DAGK3 as a major cause underlying OPA1. Received: 24 August 1998 / Accepted: 13 October 1998     

Arenavirus Dynamics in Experimentally and Naturally Infected Rodents

Infectious diseases of wildlife are typically studied using data on antibody and pathogen levels. In order to interpret these data, it is necessary to know the course of antibodies and pathogen levels after infection. Such data are typically collected using experimental infection studies in which host individuals are inoculated in the laboratory and sampled over an extended period, but because laboratory conditions are controlled and much less variable than natural conditions, the immune response and pathogen dynamics may differ. Here, we compared Morogoro arenavirus infection patterns between naturally and experimentally infected multimammate mice (Mastomys natalensis). Longitudinal samples were collected during three months of bi-weekly trapping in Morogoro, Tanzania, and antibody titer and viral RNA presence were determined. The time of infection was estimated from these data using a recently developed Bayesian approach, which allowed us to assess whether the natural temporal patterns match the previously observed patterns in the laboratory. A good match was found for 52% of naturally infected individuals, while most of the mismatches can be explained by the presence of chronically infected individuals (35%), maternal antibodies (10%), and an antibody detection limit (25%). These results suggest that while laboratory data are useful for interpreting field samples, there can still be differences due to conditions that were not tested in the laboratory.     

Effectiveness and safety of a long-acting,once-daily,two-phase release formulation of methylphenidate (Ritalin<Superscript>®</Superscript> LA) in school children under daily practice conditions

Long-acting (LA) preparations of methylphenidate allow for once-daily dosing; however, pharmacokinetics may vary and depend on food intake. The objective was to evaluate effectiveness of a two-phase release formulation (Ritalin^® LA) under daily practice conditions. This was a prospective, multicenter, observational study in Germany. Eligibility and dosing were determined by the physician based on the drug label. Outcomes included changes over 3 months of treatment in assessments of effect duration, clinical global impression (CGI), and quality of life (ILK). In 101 sites, 262 patients (197 boys, 63 girls, and two unknown) with a mean age of 10.9 years were enrolled; 50 were treated for the first time; 212 switched medication to Ritalin^® LA. After 3 months, CGI improved in 59.4 % of patients, and well-being overall was rated as good by 61.0 % of parents and 63.7 % of children. Based on parents’ assessment, the proportion of children suffering from strong disease burden decreased from 40.7 to 15.1 %. In 123 insufficient responders to previous ADHD medications, benefit from Ritalin^® LA was above average and effect duration was significantly prolonged as compared to pretreatment. Overall, 28 patients (10.7 %) had treatment-related adverse events with one case being serious; 23 patients (8.8 %) discontinued therapy, 7 (2.7 %) due to poor treatment response; and 212 patients (81 %) continued treatment beyond the study. In line with clinical trial data, Ritalin^® LA provides significant benefit also under routine practice conditions.     

Escherichia coli ghosts promote innate immune responses in human keratinocytes

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.