The Pif1 Helicase,a Negative Regulator of Telomerase,Acts Preferentially at Long Telomeres

Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.     

Impact of Acetazolamide and CPAP on Cortical Activity in Obstructive Sleep Apnea Patients

Study Objectives

1) To investigate the impact of acetazolamide, a drug commonly prescribed for altitude sickness, on cortical oscillations in patients with obstructive sleep apnea syndrome (OSAS). 2) To examine alterations in the sleep EEG after short-term discontinuation of continuous positive airway pressure (CPAP) therapy.

Design

Data from two double-blind, placebo-controlled randomized cross-over design studies were analyzed.

Setting

Polysomnographic recordings in sleep laboratory at 490 m and at moderate altitudes in the Swiss Alps: 1630 or 1860 m and 2590 m.

Patients

Study 1: 39 OSAS patients. Study 2: 41 OSAS patients.

Interventions

Study 1: OSAS patients withdrawn from treatment with CPAP. Study 2: OSAS patients treated with autoCPAP. Treatment with acetazolamide (500–750 mg) or placebo at moderate altitudes.

Measurements and Results

An evening dose of 500 mg acetazolamide reduced slow-wave activity (SWA; approximately 10%) and increased spindle activity (approximately 10%) during non-REM sleep. In addition, alpha activity during wake after lights out was increased. An evening dose of 250 mg did not affect these cortical oscillations. Discontinuation of CPAP therapy revealed a reduction in SWA (5–10%) and increase in beta activity (approximately 25%).

Conclusions

The higher evening dose of 500 mg acetazolamide showed the “spectral fingerprint” of Benzodiazepines, while 250 mg acetazolamide had no impact on cortical oscillations. However, both doses had beneficial effects on oxygen saturation and sleep quality.     

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

Background

30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).

Results

With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.

Conclusion

Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.     

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.     

TagFinder for the quantitative analysis of gas chromatography--mass spectrometry (GC-MS)-based metabolite profiling experiments

MOTIVATION: Typical GC-MS-based metabolite profiling experiments may comprise hundreds of chromatogram files, which each contain up to 1000 mass spectral tags (MSTs). MSTs are the characteristic patterns of approximately 25-250 fragment ions and respective isotopomers, which are generated after gas chromatography (GC) by electron impact ionization (EI) of the separated chemical molecules. These fragment ions are subsequently detected by time-of-flight (TOF) mass spectrometry (MS). MSTs of profiling experiments are typically reported as a list of ions, which are characterized by mass, chromatographic retention index (RI) or retention time (RT), and arbitrary abundance. The first two parameters allow the identification, the later the quantification of the represented chemical compounds. Many software tools have been reported for the pre-processing, the so-called curve resolution and deconvolution, of GC-(EI-TOF)-MS files. Pre-processing tools generate numerical data matrices, which contain all aligned MSTs and samples of an experiment. This process, however, is error prone mainly due to (i) the imprecise RI or RT alignment of MSTs and (ii) the high complexity of biological samples. This complexity causes co-elution of compounds and as a consequence non-selective, in other words impure MSTs. The selection and validation of optimal fragment ions for the specific and selective quantification of simultaneously eluting compounds is, therefore, mandatory. Currently validation is performed in most laboratories under human supervision. So far no software tool supports the non-targeted and user-independent quality assessment of the data matrices prior to statistical analysis. TagFinder may fill this gap. Strategy: TagFinder facilitates the analysis of all fragment ions, which are observed in GC-(EI-TOF)-MS profiling experiments. The non-targeted approach allows the discovery of novel and unexpected compounds. In addition, mass isotopomer resolution is maintained by TagFinder processing. This feature is essential for metabolic flux analyses and highly useful, but not required for metabolite profiling. Whenever possible, TagFinder gives precedence to chemical means of standardization, for example, the use of internal reference compounds for retention time calibration or quantitative standardization. In addition, external standardization is supported for both compound identification and calibration. The workflow of TagFinder comprises, (i) the import of fragment ion data, namely mass, time and arbitrary abundance (intensity), from a chromatography file interchange format or from peak lists provided by other chromatogram pre-processing software, (ii) the annotation of sample information and grouping of samples into classes, (iii) the RI calculation, (iv) the binning of observed fragment ions of equal mass from different chromatograms into RI windows, (v) the combination of these bins, so-called mass tags, into time groups of co-eluting fragment ions, (vi) the test of time groups for intensity correlated mass tags, (vii) the data matrix generation and (viii) the extraction of selective mass tags supported by compound identification. Thus, TagFinder supports both non-targeted fingerprinting analyses and metabolite targeted profiling. AVAILABILITY: Exemplary TagFinder workspaces and test data sets are made available upon request to the contact authors. TagFinder is made freely available for academic use from http://www-en.mpimp-golm.mpg.de/03-research/researchGroups/01-dept1/Root_Metabolism/smp/TagFinder/index.html.     

Blockade of CTLA-4 decreases the generation of multifunctional memory CD4+ T cells in vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.     

Probing the role of a secondary structure element at the 5'- and 3'-splice sites in group I intron self-splicing: the tetrahymena L-16 ScaI ribozyme reveals a new role of the G.U pair in self-splicing

Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.     

Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells

Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, H?hn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.