A patient with an interstitial deletion in Xp22.3 locates the gene for X-linked recessive chondrodysplasia punctata to within a one megabase interval

A male patient carrying an interstitial deletion in Xp22.3 and affected by Kallmann syndrome, X-linked ichthyosis and mental retardation, but without chondrodysplasia punctata or short stature, was investigated with molecular probes from the distal Xp22.3 region. By means of a novel probe, M115, from the relevant region, the distal deletion breakpoint was shown to be between 3.18 and 3.57 Mb from Xptel. As the patient is not affected by X-linked recessive chondrodysplasia punctata, the gene for this disease can therefore be located to within an interval of less than one megabase proximal to the pseudoautosomal boundary. If the chondrodysplasia punctata gene is associated with a CpG island, this leaves only two islands at 2760 and 3180 kb from the Xp telomere as the most promising candidate sites for this gene.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils

The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzyme.Abbreviations IL-8 interleukin-8 - fMLP fMet-Leu-Phe - MBP myelin basic protein - ERK extracellular signal regulated kinase - MAP2 microtubule-associated protein 2 - PK-A cAMP dependent protein kinase - PKI protein kinase inhibitor - PMSF phenyl-methanesulfonyl fluoride - PVDF poly-vinylidene difluoride - HBSF Hank's buffered salt solution - DAB 3,3-diaminobenzidine tetrahydrochloride - PNPP p-nitrophenyl-phosphate - HSA human serum albumin - EGTA [ethylenebis (oxyethylenenitrilo)]tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O₂ and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O₂ and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O₂ habitat, whereas pH varied from pH 6–9 with depth. Physicochemical gradients of O₂ and pH of the coral skeleton explained the β-diversity of the bacterial communities, and skeletal layers that showed O₂ peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.     

Gene-rich plastid genomes of two parasitic red algal species,Laurencia australis and L. verruciformis (Rhodomelaceae,Ceramiales), and a taxonomic revision of Janczewskia

Parasitic red algae are an interesting system for investigating the genetic changes that occur in parasites. These parasites have evolved independently multiple times within the red algae. The functional loss of plastid genomes can be investigated in these multiple independent examples, and fine-scale patterns may be discerned. The only plastid genomes from red algal parasites known so far are highly reduced and missing almost all photosynthetic genes. Our study assembled and annotated plastid genomes from the parasites Janczewskia tasmanica and its two Laurencia host species (Laurencia elata and one unidentified Laurencia sp. A25) from Australia and Janczewskia verruciformis, its host species (Laurencia catarinensis), and the closest known free-living relative (Laurencia obtusa) from the Canary Islands (Spain). For the first time we show parasitic red algal plastid genomes that are similar in size and gene content to free-living host species without any gene loss or genome reduction. The only exception was two pseudogenes (moeB and ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating potential for future loss of these genes. Further comparative analyses with the three highly reduced plastid genomes showed possible gene loss patterns, in which photosynthetic gene categories were lost followed by other gene categories. Phylogenetic analyses did not confirm monophyly of Janczewskia, and the genus was subsumed into Laurencia. Further investigations will determine if any convergent small-scale patterns of gene loss exist in parasitic red algae and how these are applicable to other parasitic systems.     

Localization of the enzymes involved in H2 and formate metabolism inSyntrophospora bryantii

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min^–1 mg^–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min^–1 mg^–1 protein). The K _M value and threshold value of the hydrogenase for H₂ were 0.21 mM and 18 µM, respectively, whereas the K _M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO₂ reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H₂ is formed intracellularly while formate is formed at the outside of the cell.     

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.     

The effect of sulfate and nitrate on methane formation in a freshwater sediment

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35–40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetion of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.     

A dimeric germacranolide and other sesquiterpene lactones from Mikania species

The investigation of two Mikania species, both previously placed in the genus Kanimia, afforded in addition to known compounds several new germ