KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis

Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade. In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p. This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively. We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family. The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S. cerevisiae. Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence. Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K. lactis. Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway. Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and in K. lactis. The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway.     

Standardized low-load repetitive work: evidence of different motor control strategies between experienced workers and a reference group

The purpose of this laboratory study was to evaluate the possible differences in motor strategies to a new standardized low-load repetitive work task in between healthy experienced workers and a reference group. Work task event duration, i.e. working rhythm, cutting forces, surface electromyographic (EMG) activity from four shoulder muscles, postural activity, and arm and trunk movements in 3D were recorded during low-load repetitive work simulation. The experienced group showed lower EMG activity and frequency contents (P<0.05), more abducted position of the upper arm and forward flexion of the trunk prior to work simulation (P<0.05), and increased arm and trunk range of motion (P<0.05) compared with the reference group. The results highlight that experienced butchers have a different motor strategy compared with a reference group, i.e. more variable form of coordination pattern. Furthermore, the initial implementation of a possible protective motor strategy by experienced workers might be a very important prognostic factor.     

On-line coupling of size exclusion chromatography with electrospray ionization-tandem mass spectrometry for the analysis of aquatic fulvic and humic acids

A method was developed for the analysis of humic and fulvic acids by size-exclusion chromatography-electrospray ionization-tandem mass spectrometry using a completely volatile eluent. Humic and fulvic acids were separated into three peaks. These fractions occupied different mass ranges and showed differences in the fine structure of their mass spectra. The low-molecular-weight (LMW) fraction of fulvic acids is most sensitively determined by ESI-MS, and it appears that previous results obtained by infusion-ESI-MS were primarily determined by this fulvic acid fraction. The average molecular weight of this fractions turned out to be lower than that reported from infusion-ESI-MS measurements. Its scan spectra and the product ion spectra of some of its molecular anions perfectly match those previously obtained from whole fulvic acid mixtures. Obviously, a class of well-defined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. With decreasing elution time and increasing molecular weight, detection by ESI-MS loses sensitivity as compared to the parallel UV recording, and the fine structure of the scan spectra becomes increasingly uniform for both fulvic and humic acids. The average molecular weight of the HMW fraction exceeds those values calculated from infusion experiments. Scan spectra and product ion spectra of the high-molecular-weight (HMW) fraction of both the humic and the fulvic acids suggest that the HMW fraction consists of several subunits that originate from the LMW fraction.     

High summer concentrations of mercury in big perch (Perca fluviatilis L) from the Tvärminne archipelago (SW Finland) and Nåtö (Aland Islands) Baltic sea

During high summer seasons in 1994-1999 84 big (20-42 cm total length) female perch (Perca fluviatilis L) were sampled from the waters around Tv?rminne Zoological Station on the Hanko-Hang? peninsula on the SW coast of Finland for mercury analysis. Additional sampling of 13 big female perch during the same season were carried out from the waters of N?t? Biological Station on the Aland Islands in 1997-1998. High summer is the most important season for perch fishing and consumption not only for private families during leisure time but also for professional fishery including trade and export, processing and preparations for restaurants. Fillets of big specimen of perch, s.c. "fillet perch" or "sea perch" from outer coastal waters were chosen for the study to find out the levels of mercury concentration in these desirable fish. The mean concentration of mercury in the dorsal muscle tissue (fillet) of big female "sea perch" from the coastal waters of Tv?rminne Zoological Station was calculated to 0.22 (SD 0.08), range 0.08-0.43 mg/kg, f wt and for big female perch from the waters of N?t? Biological Station to 0.18 (SD 0.07), range 0.07-0.30 mg/kg, f wt respectively. The results show a considerably lower mercury level in big "fillet perch" caught from the outer coastal waters in both Finland (and Estonia) compared with the levels recorded of big "fillet perch" from fresh water lakes and reservoirs. Thus, the common expression "sea perch" may also be used in the form "Baltic sea perch" meaning big healthy perch with low concentrations of mercury and caught from the outer coastal waters in S and SW Finland (and W Estonia) when marketing perch for human consumption.     

An Epoxy Photoresist Modified by Luminescent Nanocrystals for the Fabrication of 3D High‐Aspect‐Ratio Microstructures

An epoxy‐based negative‐tone photoresist, which is known as a suitable material for high‐aspect‐ratio surface micromachining, is functionalized with red‐light‐emitting CdSe@ZnS nanocrystals (NCs). The proper selection of a common solvent for the NCs and the resist is found to be critical for the efficient incorporation of the NCs in the epoxy matrix. The NC‐modified resist can be patterned by standard UV lithography down to micrometer‐scale resolution, and high‐aspect‐ratio structures have been successfully fabricated on a 100 mm scaled wafer. The “as‐fabricated”, 3D, epoxy‐based surface microstructures show the characteristic luminescent properties of the embedded NCs, as verified by fluorescence microscopy. This issue demonstrates that the NC emission properties can be conveniently conveyed into the polymer matrix without deteriorating the lithographic performance of the latter. The dimensions, the resolution, and the surface morphology of the NC‐modified‐epoxy microstructures exhibit only minor deviations with respect to that of the unmodified reference material, as examined by means of microscopic and metrologic investigations. The proposed approach of the incorporation of emitting and non‐bleachable NCs into a photoresist opens novel routes for surface patterning of integrated microsystems with inherent photonic functionality at the micro‐ and nanometer‐scale for light sensing and emitting applications.     

Interactive lectures: Effective teaching and learning in lectures using wireless networks

Among the severe instructional shortcomings of lectures is their lack of interactivity. To overcome these problems, the learning environment of the interactive lecture is introduced, in which students and lecturer interact by using mobile computers in a wireless network. In an experimental study, 44 students participated in interactive and in conventional lecture meetings. A quiz service that allowed question to be posted, students’ answers to be evaluated, and the results to be presented graphically, was implemented on mobile computers. The participants rated the interactive condition better, also reporting higher levels of attention, activity and perceived learning success for this condition. Objective measures indicated a slight but insignificant difference toward better learning results in the interactive condition.     

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined.     

Model for knowledge and legal expert systems

This paper presents a four layer model for working with legal knowledge in expert systems. It distinguishes five sources of knowledge. Four contain basic legal knowledge found in published and unpublished sources. The fifth consists of legal metaknowledge. In the model the four basic legal knowledge sources are placed at the lowest level. The metaknowledge is placed at levels above the other four knowledge sources. The assumption is that the knowledge is represented only once. The use of metaknowledge at various levels should make it possible to use the appropriate knowledge for the problem presented to the system. The knowledge has to be represented as closely to the original format as possible for this purpose. Suitable representation formalisms for the various types of knowledge in the five knowledge sources are discussed. It is not possible to indicate a best representation formalism for each knowledge source.