The Pseudomonas fluorescens AlgG protein,but not its mannuronan C-5-epimerase activity,is needed for alginate polymer formation

Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.     

Concentrations of Cd, Co, Cu, Fe, Mn, Rb, V, and Zn in formalin-fixed brain tissue in amyotrophic lateral sclerosis and parkinsonism-dementia complex of guam determined by high-resolution ICP-MS

Amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) are neurodegenerative disorders that occurred with extremely high frequency among the native population on Guam, especially in the 1950s and 1960s, but have substantially declined over the last half-century. The etiology of these diseases is unknown, but the most plausible hypothesis centers on imbalances in essential and toxic metals. We have determined the concentrations of Cd, Co, Cu, Fe, Mn, Rb, V, and Zn in formalin-fixed brain tissue collected during the period 1979–1983 from eight Guamanian patients with ALS, four with PDC, and five control subjects using high-resolution inductively coupled plasma-mass spectrometry. The concentrations of Cd are markedly and significantly elevated both in gray and white matter in ALS, but not in PDC patients. The concentrations of Zn are elevated for both patient groups, in both gray and white matter, but only the difference in gray matter for PDC is significant. For the other metals, no significant differences are found.     

Tumor classification and marker gene prediction by feature selection and fuzzy c-means clustering using microarray data

Background  

Using DNA microarrays, we have developed two novel models for tumor classification and target gene prediction. First, gene expression profiles are summarized by optimally selected Self-Organizing Maps (SOMs), followed by tumor sample classification by Fuzzy C-means clustering. Then, the prediction of marker genes is accomplished by either manual feature selection (visualizing the weighted/mean SOM component plane) or automatic feature selection (by pair-wise Fisher's linear discriminant).     

Spooneromyces, a new genus to accommodate Peziza laeticolor and the new species S. helveticus

A new genus Spooneromyces is proposed for Peziza laeticolor (syn. Melastiza asperula ). A second species of the genus is described here as S. helveticus sp. nov. Spooneromyces has the following combination of distinctive generic marks: superficial, stiff, brownish hairs; orange to reddish carotenoid-containing hymenium, and heavily sculptured ascospore walls. It is referred to tribe Scutellinieae of the Pyrone-metaceae.     

Phylogenetic and structural studies in the thelebolaceae (Ascomycota)

Several minute dung-inhabiting discomycetes have been classified in the family Thelebolaceae, which has traditionally been included in the order Pezizales. The non-operculate type-genusThelebolus has recently been excluded from the Pezizales. The phylogenetic distribution of other genera associated with Thelebolaceae is still obscure. We have analysed ca. 580 bp from a variable part of the nuclear SSU rRNA gene fromAscozonus, Caccobius, Lasiobolus andThecotheus, and compared these with ca. 1700 bp sequences fromThelebolus, Pleospora, Pezizales, leotiales and Leotiales-related taxa. In the resulting tree,Ascozonus andCaccobius group withThelebolus and the inoperculate discomycetes;Lasiobolus groups withAscodesmis, andThecotheus withAscobolus within Pezizales. SEM pictures of fruit-bodies and ascus apices ofAscozonus, and ascospores fromThecotheus are presented to illustrate characteristic features of these taxa. Submitting author: FAX: +47-22 85 46 64, TEL: +47-22 85 46 61.     

Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of ¹²⁵I-asialofetuin (¹²⁵I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of ¹²⁵I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.     

Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography

In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.     

Molecular phylogenetics suggest a North American link between the anthropogenic dry rot fungus Serpula lacrymans and its wild relative S. himantioides

The dry rot fungus Serpula lacrymans causes damages in wooden buildings and constructions in temperate regions worldwide. In this study, the global phylogeography of S. lacrymans and its wild relative S. himantioides has been investigated to clarify genealogical relationships and determine the origin and spread of the building strains. Internal transcribed spacer (ITS) nrDNA and parts of the beta-tubulin (tub) and the translation elongation factor (efa) 1a genes were sequenced, and phylogenetic relationships inferred. Some analyses suggest that S. lacrymans may have originated from an ancient S. himantioides lineage, but most results support that S. lacrymans and S. himantioides are monophyletic sister species. Phylogenetic analysis of the ITS data revealed two subgroups within S. lacrymans corresponding to two earlier described varieties; one group occurring frequently in houses worldwide ('Domesticus'), and one group represented by individuals from forests in Northern California ('Shastensis'). A few collections from nature were included in the Domesticus group as well, among other specimens from two newly discovered localities in Far East Russia and Siberia. In the Domesticus group little sequence variation occurs, suggesting a recent worldwide dispersal, possibly linked to human activity. Phylogenetic analyses indicate that the Domesticus group may have originated from an ancient lineage related closely to the Shastensis group. A remarkable shift in morphology and habitat preferences has occurred during the evolution of the Domesticus lineage, linked to the transition from nature to human-made habitats.     

Ecological significance of endosymbionts in a mixotrophic ciliate--an experimental test of a simple model of growth coordination between host and symbiont

Ciliates with endosymbiotic algae (green ciliates) have oftenbeen found to be more viable than aposymbiotic (without endosymbionts)counterparts during periods of starvation. However, the possiblebenefit of algal endosymbionts to the growth of ciliate hostshas rarely been quantified. Growth coordination between hostand symbionts is essential to maintain the symbiosis, but themechanism behind this is also uncertain. Our hypothesis is thatthe growth rate of the symbionts is always close to its maximum,irrespective of the nutritional status of the host. To testthis hypothesis we built a model based on a constant symbiontgrowth rate, and performed an experiment where we observed thegrowth rate of aposymbiotic and green Coleps cells under differentlight conditions and food concentrations. The results were ingood agreement with the model, and showed that at low food concentrationthe growth rate of green Coleps was clearly higher than thatof aposymbiotic Coleps, while there were no significant differenceswhen food was abundant. Our results indicate that algal grossgrowth rate is always close to maximum, and that growth coordinationbetween host and symbiont is obtained by a variable degree ofleakage of photosynthetic products from the symbionts to theciliate host.