Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence.

BACKGROUND: Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.     

ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS : Effects on Mitochondria In Vivo

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a₃ show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.     

Strategies for antiviral resistance in transgenic plants

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.     

Mitochondrial energetic metabolism: A simplified model of TCA cycle with ATP production

Mitochondria play a central role in cellular energetic metabolism. The essential parts of this metabolism are the tricarboxylic acid (TCA) cycle, the respiratory chain and the adenosine triphosphate (ATP) synthesis machinery. Here a simplified model of these three metabolic components with a limited set of differential equations is presented. The existence of a steady state is demonstrated and results of numerical simulations are presented. The relevance of a simple model to represent actual in vivo behavior is discussed.     

��-Helical Domains Promote Translocation of Intrinsically Disordered Polypeptides into the Endoplasmic Reticulum

Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of α-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58^IPK promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.To ensure cellular homeostasis and to preclude toxic effects of aberrant protein conformers quality control mechanisms have evolved to recognize and degrade non-functional and misfolded proteins. In the cytosol the ubiquitin-proteasome system is the main pathway for regulated protein turnover (for reviews, see Refs. 1–3). Moreover the proteasome is part of a quality control system designated endoplasmic reticulum (ER)⁵-associated degradation (ERAD), which mediates post-translational degradation of non-native proteins generated in the ER. ERAD is the primary response to eliminate non-native ER proteins. It involves recognition of non-native polypeptides by ER-resident proteins and retrograde transport to the cytosol where proteasomal degradation occurs (for reviews, see Refs. 4–6). In case ERAD substrates accumulate in the ER lumen intracellular signaling pathways are induced that are collectively called the unfolded protein response (for reviews, see Refs. 7 and 8). Recently a preemptive, co-translocational quality control pathway was described that operates before translocation into the ER is completed (9, 10). Regulated translocation could act as an early quality control step to prevent an overload of the ER with non-native proteins. This regulation relies on an interplay between intrinsic features of the polypeptides and accessory factors able to modulate the translocation efficiency (for a review, see Ref. 11). Although numerous factors are known to be involved in ERAD less is known about mediators of the preemptive, co-translocational quality control pathway. In one study p58^IPK was identified as a key regulator (9), whereas in another it was shown that ER translocation during conditions of acute ER stress is controlled by the signal peptide (10). Indeed the signal peptide is an important intrinsic determinant. Although an exceptionally diverse set of sequences can target proteins for ER import it has been demonstrated that the translocation efficiency is modulated in a signal peptide sequence-specific manner (for reviews, see Refs. 12–14). Another attractive candidate for an intrinsic factor to regulate translocation is the folding state of the nascent polypeptide chain. Formation of secondary structure occurs already in the ribosome exit tunnel (15–18). Moreover it was shown that the polypeptide structure within the ribosomal exit tunnel can modulate translocation of distal parts of the nascent chain (19).The mammalian prion protein (PrP) is a suitable model protein to study whether formation of secondary structure could modulate translocation efficiency because it has an intriguing modular composition: the N-terminal domain spanning 120 amino acids is flexibly disordered followed by a highly structured C-terminal domain of ∼110 amino acids. This autonomously folding domain contains three α-helical regions and a short, two-stranded β-sheet (20–22). Notably folding of the C-terminal domain is one of the most rapid folding reactions measured in vitro (23). Interestingly previous studies indicated that the C-terminal folded domain of PrP is necessary and sufficient to promote ER import. In cultured cells and neurons of transgenic animals PrP-S230X (also known as PrPΔGPI or GPI⁻PrP), a mutant devoid of the C-terminal glycosylphosphatidylinositol (GPI) anchor signal peptide, is efficiently imported into the ER and secreted (24–27). However, by deleting parts of the α-helical domains located in the C-terminal domain a fraction is directed to proteasomal degradation in the cytosol (28).We have now analyzed the underlying mechanisms of this impaired ER import and show that ER-targeted proteins require a certain amount of structured domains to be imported into the ER. In addition, our study indicates that extended unstructured domains are signals for a preemptive/co-translocational degradation pathway.     

Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer

RNA interference is a powerful tool for the functional analysis of proteins by specific gene knockdown. In this study, we devised a rapid and efficient way to screen suitable siRNA sequences and subsequently employ them for specific gene knockdown in usually hard-to-transfect lymphoid cell lines, using a self-inactivating lentiviral vector. Two proteins with different half-lives were chosen, cyclin D1 and STAT3. A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences. Only siRNA molecules with more than 85% of knockdown efficiency were selected for the generation of lentiviral transfer vectors. Transduction rates of 75–99% were achieved in the lymphoma cell lines Granta 519 (mantle cell lymphoma), Karpas 299, and SUDHL-1 (anaplastic large T cell lymphoma), as demonstrated by green fluorescent protein expression in fluorescence-activated cell sorting analysis. The high level of transduction efficiency allows RNA interference studies to be performed on transduced cells without further manipulation, such as cell sorting or cloning. The LacZ reporter system together with the lentivirus technology is a very important tool in the hematology field, which enables experiments in lymphoid cells that were not possible before.

Electronic supplementary material

The online version of this article (doi:10.1007/s12308-008-0020-x) contains supplementary material, which is available to authorized users.     

Common and specific properties of herpesvirus UL34/UL31 protein family members revealed by protein complementation assay

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 beta-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.     

Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.