Genomic structure of the human ezrin gene

The ERM proteins, ezrin, radixin, and moesin, act as linkers between the plasma membrane and actin cytoskeleton. They are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures, and are highly homologous, both in protein sequence and in functional activity, with merlin/schwannomin, a neurofibromatosis-2-associated tumor-suppressor protein. We report here the genomic structure and intron junction sequences of the human ezrin gene. Ezrin consists of 13 exons and spans approximately 24 kb genomic DNA. The coding parts of the exons range in size from 12 bp to 275 bp and the introns from 182 bp to 7 kb. The genomic structures of ezrin and moesin are highly conserved, suggesting their recent divergence. Radiation hybrid mapping has refined the location of ezrin to the interval between D6S442 and D6S281. Received: 1 June 1998 / Accepted: 25 August 1998     

Dimer-tetramer transition between solution and crystalline states of streptavidin and avidin mutants

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.     

Leukoregulin, a T-cell derived cytokine, upregulates stromelysin-1 gene expression in human dermal fibroblasts: evidence for the role of AP-1 in transcriptional activation.

Leukoregulin (LR), a product of activated T-cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455-1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin-1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose- and time-dependent elevation of stromelysin-1 mRNA levels, the maximum enhancement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 1.3 kb of 5' flanking DNA of the human stromelysin-1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This enhancement was abolished by a single base substitution in the AP-1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP-1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin-1 gene expression by LR is mediated by AP-1 regulatory elements which are necessary, but not sufficient, for gene response.     

Effects of Break Crops on Yield and Grain Protein Concentration of Barley in a Boreal Climate

Rotation with dicotyledonous crops to break cereal monoculture has proven to be beneficial to successive cereals. In two fields where the soil had been subjected to prolonged, continuous cereal production, two 3-year rotation trials were established. In the first year, faba bean, turnip rape and barley were grown, as first crops, in large blocks and their residues tilled into the soil after harvest. In the following year, barley, buckwheat, caraway, faba bean, hemp and white lupin were sown, as second crops, in each block and incorporated either at flowering stage (except barley) or after harvest. In the third year, barley was grown in all plots and its yield and grain protein concentration were determined. Mineral N in the plough layer was determined two months after incorporation of crops and again before sowing barley in the following year. The effect of faba bean and turnip rape on improving barley yields and grain protein concentration was still detectable two years after they were grown. The yield response of barley was not sensitive to the growth stage of second crops when they were incorporated, but was to different second crops, showing clear benefits averaging 6-7% after white lupin, faba bean and hemp but no benefit from caraway or buckwheat. The effect of increased N in the plough layer derived from rotation crops on barley yields was minor. Incorporation of plants at flowering stage slightly increased third-year barley grain protein concentration but posed a great potential for N loss compared with incorporation of crop residues after harvest, showing the value of either delayed incorporation or using catch crops.     

Diagnosis of flavobacteriosis by direct amplification of rRNA genes

A broad-range bacterial PCR method with universal 16S rDNA targeting primers and bacterial cultivation was used to identify the putative pathogen in flavobacterial outbreaks. Restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the partial 16S rDNA PCR products of 10 skin samples and 10 representative isolates derived from the same fish specimens revealed differences between direct molecular and cultivation-based analysis. Flavobacterium columnare-like sequences dominated in the direct molecular analysis in most cases, whereas most of the isolates belonged to a phylogenetically heterogeneous group of flavobacteria clustering with F. hibernum. F. columnare was isolated in only 1 outbreak. The possible explanations for the different results may be attributable to difficulties in the plate cultivation procedure of external flavobacterial samples. During plate cultivation, the dominating Flavobacterium species can be masked by saprophytic species of the same genus or other genera, or the growth of flavobacteria can be completely inhibited by antagonistic bacteria such as Pseudomonas. Direct analysis of the prevailing 16S rDNA sequences avoids the problems with cultivation and may thus be preferable for the diagnosis of flavobacterial diseases. When isolating flavobacteria from external samples, serial dilution of the sample before plating can improve the results.     

Rho‐kinase inhibitor Y‐27632 increases cellular proliferation and migration in human foreskin fibroblast cells

The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho‐kinase inhibitor Y‐27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short‐term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 μM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell‐IQ time‐lapse imaging analysis. Rho‐kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin‐associated focal adhesions were present in the ROCKi‐treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte‐specific genes, such as procollagen α₁(II) and aggrecan.     

Complex metacommunity structure for benthic invertebrates in a low‐diversity coastal system

The majority of studies in metacommunity ecology have focused on systems other than marine benthic ecosystems, thereby providing an impetus to broaden the focus of metacommunity research to comprise marine systems. These systems are more open than many other systems and may thus exhibit relatively less discrete patterns in community structure across space. Metacommunity structure of soft‐sediment benthic invertebrates was examined using a fine‐grained (285 sites) data set collected during one summer across a large spatial extent (1700 km²). We applied the elements of metacommunity structure (EMS) approach, allowing multiple hypothesis of variation in community structure to be tested. We demonstrated several patterns associated with environmental variation and associated processes that could simultaneously assemble species to occur at the sites. A quasi‐Clementsian pattern was observed frequently, suggesting interdependent ecological relationships among species or similar response to an underlying environmental gradient across sites. A quasi‐nested clumped species loss pattern was also observed, which suggests nested habitat specialization. Species richness declined with depth (from 0.5 to 44.8 m). We argue that sensitive species may survive in shallower water, which are more stable with regard to oxygen conditions and present greater habitat complexity, in contrast to deeper waters, which may experience periodic disturbance due to hypoxia. Future studies should better integrate disturbance in terms of temporal dynamics and dispersal rates in the EMS approach. We highlight that shallow water sites may act as sources of recruitment to deeper water sites that are relatively more prone to periodic disturbances due to hypoxia. However, these shallow sites are not currently monitored and should be better prioritized in future conservation strategies in marine systems.     

Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.