Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.     

Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility

Two tropomyosin isoforms, human Tm5(NM1) and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5(NM1) cells, we observed large stress fibres which showed predominant co-localisation of beta-actin and low-molecular-weight gamma-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the beta- and gamma-actin isoforms, predominately populated by high-molecular-weight alpha- and beta-tropomyosin isoforms. The stress fibres observed in Tm5(NM1) cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5(NM1) isoform with a human-specific Tm5(NM1) siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.     

IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions

A tight spatiotemporal coordination of the machineries controlling membrane bending and trafficking, and actin dynamics is crucial for the generation of cellular protrusions. Proteins that are simultaneously capable of regulating actin dynamics and sensing or inducing membrane curvature are predicted to have a prominent role. A prototypical example of this type of proteins is the insulin receptor tyrosine kinase substrate of 53kDa, the founding member of a recently discovered family of proteins, including missing-in-metastasis and ABBA (actin-bundling protein with BAIAP2 homology). Structural, biochemical and cell biological experiments support the unique role of this family as transducers of signalling, linking the protruding membrane to the underlying actin cytoskeleton.     

Filopodia: molecular architecture and cellular functions

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.     

Ultrasound Transmission Technique as a Potential Tool for Physical Evaluation of Monolithic Matrix Tablets

The aim of this study was to investigate the effects of tablet porosity and particle size fraction of compacted Starch acetate powders, with and without model drug caffeine, on acoustic properties of tablets. The ultrasound velocity was determined from the transmission measurements. Tablets of starch acetate (SA DS 2.7) powder with two particle size fractions of 0–53 and 0–710 μm were compressed with a compaction simulator. Porosities of tablets varied in the range from 12% to 43% for both particle size fractions. Strong associations were found between the ultrasound velocity and physical properties of the tablets such as porosity and particle size fraction. Interestingly, ultrasound velocity was practically insensitive to inclusion of the model drug caffeine with the concentrations used. Based on this study ultrasound transmission method is a potential non-destructive tool for studying structural changes of tablets and other solid dosage forms.     

Ant predation ofEriocrania miners in a polluted area

We tested the hypothesis that air pollution may affect population densities of birch-feeding leafminers via changes in ant predation. Foraging activity of three ant species (Formica rufa, F. fusca andF. lemani), predation rates and population densities of both solitary and gregariousEriocrania (Lepidoptera: Eriocraniidae) miners were investigated at 13 sites around the Harjavalta copper-nickel smelter, SW Finland. Ant species differed in their distribution patterns relative to pollution. However, the total percentage of birch trees foraged by ants (all species combined) showed no correlation with the distance from the factory complex. As a result, no clear trends in predation rates were apparent in relation to the distance from the pollution source for either solitary or gregariousEriocrania species. Densities of the solitaryEriocrania species tended to increase with the distance from the pollution source whereas densities of the gregariousE. haworthi peaked close to the factory complex. No corresponding differences in predation rates between solitary and gregarious miners were found. Ant predation, thus, did not explain density patterns ofEriocrania miners in the polluted area.     

Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling "old" actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.     

Scorer and modality agreement for the detection of intervertebral disc calcification in Dachshunds

Background

The Dachshund is a chondrodystrophic breed of dog predisposed to premature degeneration and calcification, and subsequent herniation, of intervertebral discs (IVDs). This condition is heritable in Dachshunds and breeding candidates are screened for radiographically detectable intervertebral disc calcification (RDIDC), a feature of advanced disc degeneration and a prognostic factor for clinical disease. RDIDC scoring has been previously shown to be consistent within scorers; however, strong scorer effect (subjectivity) was also reported. The aim of this study was to estimate the within- and between-scorer agreement (repeatability and reproducibility, respectively) of computed tomography (CT) scanning and magnetic resonance imaging (MRI) for scoring IVD calcification, and to compare these modalities with radiographic scoring.

Results

Twenty-one Dachshund dogs were screened for IVD calcification using the three imaging modalities. Three scorers scored each case twice, independently. Repeatability was highest for radiography (95.4%), and significantly higher than for CT (90.4%) but not MRI (93.8%). Reproducibility was also highest for radiography (92.9%), but not significantly higher than for CT or MRI (89.4% and 86.4%, respectively). Overall, CT scored IVDs differently than radiography and MRI (64.8% and 62.7% agreement, respectively), while radiography and MRI scored more similarly (85.7% agreement).

Conclusions

Despite high precision for radiography, previous evidence of scorer subjectivity was confirmed, which was not generally observed with CT and MRI. The increased consistency of radiography may be related to prior scorer experience with the modality and RDIDC scoring. This study does not support replacing radiography with CT or MRI to screen for heritable IVD calcification in breeding Dachshunds; however, evaluation of dog-level precision and the accuracy of each modality is recommended.

     

Spatial distribution of spawning sites of pikeperch [Sander lucioperca (L.)] in a highly eutrophic clay-turbid lake – implications for management

The spawning sites of pikeperch were mapped using artificial spawning nests in the clay‐turbid Lake Tuusulanjärvi in southern Finland to protect the most important spawning areas. Altogether 272 artificial spawning nests were set before actual spawning on potential spawning grounds. The first observations of eggs were made on 26 May at a surface water temperature of 12°C. No new eggs were found after 10 June at water temperature of 16°C. The nests were removed on 18 June when the larvae had already hatched. The study demonstrated that artificial spawning nests could be used to evaluate both duration and spatial dimensions of spawning of pikeperch.     

Effects of abiotic factors on movements of pikeperch during pre-spawning and spawning season in a Baltic archipelago

The effects of temperature, water level and moon phase on the movements of pikeperch (Sander lucioperca) were studied during pre-spawning and spawning season from April to June. Trends in catches and temperature were removed with linear regression. Cross-correlation analysis showed that temperature change one and two days earlier had the strongest effect on catches. Close to the new moon, pikeperch catches increased, whereas changes in water level had no effects on movements. Handling editor: J. A. Cambray