An alternative route for multistep tumorigenesis in a novel case of hereditary renal cell cancer and a t(2;3)(q35;q21) chromosome translocation.

Through allele-segregation and loss-of-heterozygosity analyses, we demonstrated loss of the translocation-derivative chromosome 3 in five independent renal cell tumors of the clear-cell type, obtained from three members of a family in which a constitutional t(2;3)(q35;q21) was encountered. In addition, analysis of the von Hippel-Lindau gene, VHL, revealed distinct insertion, deletion, and substitution mutations in four of the five tumors tested. On the basis of these results, we conclude that, in this familial case, an alternative route for renal cell carcinoma development is implied. In contrast to the first hit in the generally accepted two-hit tumor-suppressor model proposed by Knudson, the familial translocation in this case may act as a primary oncogenic event leading to (nondisjunctional) loss of the der(3) chromosome harboring the VHL tumor-suppressor gene. The risk of developing renal cell cancer may be correlated directly with the extent of somatic (kidney) mosaicism resulting from this loss.     

Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).     

Mobility of endogenous ecotropic murine leukemia viral genomes within mouse chromosomal DNA and integration of a mink cell focus-forming virus-type recombinant provirus in the germ line

Characterization of endogenous ecotropic Akv proviruses in DNA of low and high leukemic mouse strains revealed the presence of one to six copies of the Akv genome per haploid genome equivalent integrated in the germ line. Low leukemic strains analyzed so far contained only one complete copy of the Akv proviral DNA. The site of integration varied among strains, although genetically related strains often carried the Akv proviral gene in the same chromosomal site. The different substrains of the AKR mouse displayed the presence of variable numbers (two to six) of Akv genomes. In all substrains one Akv genome was present in an identical chromosomal site; this locus probably comprised the progenitor genome. Closely related substrains had several Akv proviral DNAs integrated in common sites. The accumulation of Akv genomes in the germ line of the AKR/FuRdA strain is likely the result of independent integration events, since backcross studies with the Akv-negative 129 strain showed random segregation of all six proviral loci. The AKR/Cnb strain carried a recombinant provirus in the germ line. This provirus resembled in structure the AKR mink cell focus-forming viruses, which are generated by somatic recombination during leukemogenesis. Therefore, the germ-line amplification of Akv proviral DNAs occurs most likely through infection of embryonic cells by circulating virus.     

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body.

MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.     

Thiophene Rings Improve the Device Performance of Conjugated Polymers in Polymer Solar Cells with Thick Active Layers

Developing novel materials that tolerate thickness variations of the active layer is critical to further enhance the efficiency of polymer solar cells and enable large‐scale manufacturing. Presently, only a few polymers afford high efficiencies at active layer thickness exceeding 200 nm and molecular design guidelines for developing successful materials are lacking. It is thus highly desirable to identify structural factors that determine the performance of semiconducting conjugated polymers in thick‐film polymer solar cells. Here, it is demonstrated that thiophene rings, introduced in the backbone of alternating donor–acceptor type conjugated polymers, enhance the fill factor and overall efficiency for thick (>200 nm) solar cells. For a series of fluorinated semiconducting polymers derived from electron‐rich benzo[1,2‐b:4,5‐b′]dithiophene units and electron‐deficient 5,6‐difluorobenzo[2,1,3]thiazole units a steady increase of the fill factor and power conversion efficiency is found when introducing thiophene rings between the donor and acceptor units. The increased performance is a synergistic result of an enhanced hole mobility and a suppressed bimolecular charge recombination, which is attributed to more favorable polymer chain packing and finer phase separation.     

2-D DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells

As part of the innate immune system, natural killer (NK) cells detect and lyse tumor and virus-infected cells without prior antigen-dependent recognition and expansion. To this end, they utilize dual-function organelles that combine properties of conventional lysosomes and exocytotic vesicles. Upon stimulation, these secretory lysosomes (SLs) release their cytotoxic molecules into the immunological synapse. In addition, several molecules associated with secretory vesicles become exposed on the plasma membrane. Recent studies often took advantage of the few established NK cell lines, for instance to analyze the exocytotic machinery associated with NK cell vesicles. NK cell lines and primary NK cells differ, however, substantially in the expression of "typical" surface receptors and their requirements to induce target cell lysis. Here, we directly compared the lysosomal compartments of different NK cell populations. We enriched SLs of two leukemic cell lines (YTS and NKL) and IL-2-expanded NK cells by subcellular fractionation and characterized their proteome by 2-D difference gel electrophoresis and MS. Although the overall protein composition of the lysosomal preparations was very similar and more than 90% of the proteins were present at comparable levels, we define a cell line-specific setup of functionally relevant proteins involved in antigen presentation and cytotoxic effector function.     

Biotic ligand model development predicting Zn toxicity to the alga Pseudokirchneriella subcapitata: possibilities and limitations

Biotic ligand models have been developed for various metals (e.g. Cu, Ag, Zn) and different aquatic species. These models incorporate the effect of physico-chemical water characteristics (major cations, pH, dissolved organic carbon) on the bioavailability and toxicity of the metal. In this study, the individual effects of calcium, magnesium, potassium, sodium and pH on zinc toxicity to the green alga Pseudokirchneriella subcapitata (formerly and better known as Selenastrum capricornutum and Raphidocelis subcapitata) were investigated. Stability constants for binding to algal cells (K(BL)) were derived for those cations affecting zinc toxicity, using the mathematical approach proposed by De Schamphelaere and Janssen [Environ. Sci. Technol. 63, (2002) 48-54]. Potassium proved to be the only cation tested that did not alter zinc toxicity to algae significantly. Log (K(BL)) values for Ca, Mg and Na, derived at pH 7.5, were 3.2, 3.9 and 2.8, respectively. Toxicity tests performed at different pH values (5.5-8.0) indicated that competition between H(+) and Zn(2+) reduces zinc toxicity. However, the observed relationship between (H(+)) and the 72h-EbC(50) [expressed as microM (Zn(2+))] is not linear and suggests that pH affects the physiology of the biotic ligand. Although, in general, our findings seem to suggest that zinc toxicity to algae can be modelled as a function of key water characteristics, the results also demonstrate that the part of the conventional BLM-hypothesis-i.e. that the binding characteristics of the biotic ligand are independent of the test medium characteristics-is not valid for algae. The observed pH-dependent change of stability constants should therefore be further investigated and incorporated in future BL-modelling efforts with algae.     

Surface modifications created by using engineered hydrophobins

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.     

Effects of anesthetics on systemic hemodynamics in mice

The aim of this study was to compare the systemic hemodynamic effects of four commonly used anesthetic regimens in mice that were chronically instrumented for direct and continuous measurements of cardiac output (CO). Mice (CD-1, Swiss, and C57BL6 strains) were instrumented with a transit-time flow probe placed around the ascending aorta for CO measurement. An arterial catheter was inserted into the aorta 4 or 5 days later for blood pressure measurements. After full recovery, hemodynamic parameters including stroke volume, heart rate, CO, mean arterial pressure (MAP), and total peripheral resistance were measured with animals in the conscious state. General anesthesia was then induced in these mice using isoflurane (Iso), urethane, pentobarbital sodium, or ketamine-xylazine (K-X). The doses and routes of administration of these agents were given as required for general surgical procedures in these animals. Compared with the values obtained for animals in the conscious resting state, MAP and CO decreased during all anesthetic interventions, and hemodynamic effects were smallest for Iso (MAP, -24 +/- 3%; CO, -5 +/- 7%; n = 15 mice) and greatest for K-X (MAP, -51 +/- 6%; CO, -37 +/- 9%; n = 8 mice), respectively. The hemodynamic effects of K-X were fully antagonized by administration of the alpha(2)-receptor antagonist atipamezole (n = 8 mice). These results indicate that the anesthetic Iso has fewer systemic hemodynamic effects in mice than the nonvolatile anesthetics.