Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 °C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1–10 μg ml^–1 (EC₅₀) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.     

Effects of insulin therapy on liver fat content and hepatic insulin sensitivity in patients with type 2 diabetes

We determined whether insulin therapy changes liver fat content (LFAT) or hepatic insulin sensitivity in type 2 diabetes. Fourteen patients with type 2 diabetes (age 51+/-2 yr, body mass index 33.1+/-1.4 kg/m2) treated with metformin alone received additional basal insulin for 7 mo. Liver fat (proton magnetic resonance spectroscopy), fat distribution (MRI), fat-free and fat mass, and whole body and hepatic insulin sensitivity (6-h euglycemic hyperinsulinemic clamp combined with infusion of [3-(3)H]glucose) were measured. The insulin dose averaged 75+/-10 IU/day (0.69+/-0.08 IU/kg, range 24-132 IU/day). Glycosylated hemoglobin A1c (Hb A1c) decreased from 8.9+/-0.3 to 7.4+/-0.2% (P<0.001). Whole body insulin sensitivity increased from 2.21+/-0.38 to 3.08+/-0.40 mg/kg fat-free mass (FFM).min (P<0.05). This improvement could be attributed to enhanced suppression of hepatic glucose production (HGP) by insulin (HGP 1.04+/-0.28 vs. 0.21+/-0.19 mg/kg FFM.min, P<0.01). The percent suppression of HGP by insulin increased from 72+/-8 to 105+/-11% (P<0.01). LFAT decreased from 17+/-3 to 14+/-3% (P<0.05). The change in LFAT was significantly correlated with that in hepatic insulin sensitivity (r=0.56, P<0.05). Body weight increased by 3.0+/-1.1 kg (P<0.05). Of this, 83% was due to an increase in fat-free mass (P<0.01). Fat distribution and serum adiponectin concentrations remained unchanged while serum free fatty acids decreased significantly. Conclusions: insulin therapy improves hepatic insulin sensitivity and slightly but significantly reduces liver fat content, independent of serum adiponectin.     

Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems

Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.     

Transferrin and tooth morphogenesis: Retention of transferrin by mouse embryonic teeth in organ culture

Transferrin is the only serum protein that is required for the early morphogenesis of mouse embryonic teeth in organ culture. Transferrin is able to support tooth morphogenesis and dental cell differentiation by stimulating cell proliferation. Its role in this process is restricted exclusively to iron transport, which takes place by receptor-mediated endocytosis of iron-loaded transferrin. A lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), can replace transferrin and support tooth morphogenesis in organ culture. We studied the effects of these two iron transporters on cell proliferation in tooth germs during culture. We found that Fe-PIH and transferrin stimulate proliferation to a similar extent in early cap-stage teeth of 14-day mouse embryos, but have no effect on cell proliferation in bell-stage teeth of 16-day mouse embryos. Day-16 teeth undergo morphogenesis in unsupplemented chemically defined medium, whereas transferrin or Fe-PIH is needed for the morphogenesis of day-14 teeth. Although the need for exogenous iron-transport molecules is lost with advancing development, the level of mitotic activity is still fairly high in bell-stage teeth. The abundant binding of transferrin in areas of active cell proliferation in bell-stage teeth also suggests that transferrin is still needed and used for the transport of iron into proliferating cells. Transferrin is not degraded by the process of receptor-mediated endocytosis. After releasing iron into a cell, transferrin is returned to the extracellular space and is reused. We therefore studied whether the transferrin needed by bell-stage teeth could be adequately supplied by endogenous transferrin synthesized or stored in tissue explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems

In this work we describe the new concept of using fungal hydrophobins as efficient tags for purification of recombinant fusion proteins by aqueous two-phase separation. Hydrophobins are a group of small surface-active proteins produced by filamentous fungi. Some characteristics of hydrophobins are that they are relatively small (approximately 100 amino acids), they contain eight disulfide-forming Cys residues in a conserved pattern, and they self-assemble on interfaces. The aqueous two-phase systems studied were based on nonionic surfactants that phase-separate at certain temperatures. We show that the use of hydrophobins as tags has many advantages such as high selectivity and good yield and is technically very simple to perform. Fusion proteins with target proteins of different molecular size were compared to the corresponding free proteins using a set of different surfactants. This gave an understanding on which factors influence the separation and what rationale should be used for optimization. This unusually strong and specific interaction between polymeric surfactants and a soluble protein shows promise for new developments in interfacing proteins and nonbiological materials for other applications as well.     

Testing for temporal coherence across spatial extents: the roles of climate and local factors in regulating stream macroinvertebrate community dynamics

Temporal coherence or spatial synchrony refers to the tendency of population, community or ecosystem dynamics to behave similarly among locations through time as a result of spatially‐correlated environmental stochasticity (Moran effect), dispersal or trophic interactions. While terrestrial studies have treated synchrony mainly as a population‐level concept, the majority of freshwater studies have focused on community‐level patterns, particularly in lake planktonic communities. We used spatially and temporally hierarchical data on benthic stream invertebrates across six years, with three seasonal samples a year, in 11 boreal streams to assess temporal coherence at three spatial extents: 1) among regions (watersheds), 2) among streams within a region, and 3) among riffles within a stream, using the average of correlation coefficients for stream/riffle pairs across years. Our results revealed the primacy of strongly synchronized climatic factors (precipitation, air temperature) in inducing temporal coherence of macroinvertebrate assemblages across geographically distinct sites (i.e. Moran effect). Coherence tended to decrease with increasing spatial extent, but positive coherence was detected for most biological variables even at the largest extent (about 350 km). The generally high level of coherence reflected the strong seasonality of boreal freshwater communities. A hydrologically exceptional year enhanced the synchrony of biological variables, particularly total macroinvertebrate abundance. Regionally low precipitation in that year led to a substantial decrease in benthic densities across a broad spatial extent, followed by a rapid post‐drought recovery. Coherence at the among‐riffle (within‐stream) extent was lower than expected, implying that local‐scale habitat filters determine community dynamics at smaller spatial extents. Thus, temporal coherence of stream benthic communities appears to be controlled by partly different processes at different spatial scales.     

Species–environment relationships of fish and map‐based variables in small boreal streams: Linkages with climate change and bioassessment

Species–environment relationships were studied between the occurrence of 13 fish and lamprey species and 9 mainly map‐based environmental variables of Finnish boreal small streams. A self‐organizing map (SOM) analysis showed strong relationships between the fish species and environmental variables in a single model (explained variance 55.9%). Besides basic environmental variables such as altitude, catchment size, and mean temperature, land cover variables were also explored. A logistic regression analysis indicated that the occurrence probability of brown trout, Salmo trutta L., decreased with an increasing percentage of peatland ditch drainage in the upper catchment. Ninespine stickleback, Pungitius pungitius (L.), and three‐spined stickleback, Gasterosteus aculeatus L., seemed to benefit from urban areas in the upper catchment. Discovered relationships between fish species occurrence and land‐use attributes are encouraging for the development of fish‐based bioassessment for small streams. The presented ordination of the fish species in the mean temperature gradient will help in predicting fish community responses to climate change.     

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.     

Intra- and Interspecific Competition in Hatchery Landlocked Salmon and Brown Trout in Semi-Natural Streams

I studied inter- and intraspecific competition in two hatchery stocks: landlocked salmon with long-hatchery background and a heterogenic brown trout stock. These species are potential competitors in the natural environment when landlocked salmon is being restored to wild by stocking hatchery juveniles. Behavioural responses were studied in four indoor laboratory flumes (400 cm long and 37 cm wide) and habitat use in six semi-natural outdoor streams (26 m long and 1.5 m long). Video recordings were used to monitor fish behaviour and electrofishing for fish positioning in the outdoor channels. The study design included five treatments: two densities of brown trout and salmon in solitary and both species together. The results of the study demonstrated that juvenile brown trout changed their behaviour in laboratory streams in response to presence of the landlocked salmon and the density of the conspecifics also tended to alter the habitat use by brown trout in semi-natural streams. Landlocked salmon juveniles showed no response to treatments. I conclude that possible poor adaptive ability to conditions outside hatchery by the hatchery salmon together and more competitive brown trout stocks may limit the success of management action in restoring landlocked salmon back to their natural streams of stocking.