Helicascolide C,a new lactone from an Indonesian marine algicolous strain of Daldinia eschscholzii (Xylariaceae,Ascomycota)

From an endophytic Daldinia eschscholzii strain isolated from the agar-producing red alga Gracilaria sp. SGR-1, collected from the coast of South Sulawesi, Indonesia, a new lactone helicascolide C (1) was obtained as colourless crystals from the ethyl acetate extract together with the related structurally known compound helicascolide A (2). The structure of the new compound 1 reveals a carbonyl group replacing an alcohol group of compound 2. The structure of 1 was elucidated by X-ray diffraction and spectral analyses. Compound 1 showed fungistatic activity against the phytopathogenic fungus Cladosporium cucumerinum.     

Synthesis, hydrolytic activation and cytotoxicity of etoposide prodrugs

Two 4'-propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cytotoxicity of the prodrugs of up to three orders of magnitude.     

Comparison of different IMAC techniques used for enrichment of phosphorylated peptides.

Four commercially available immobilized metal ion affinity chromatography (IMAC) methods for phosphopeptide enrichment were compared using small volumes and concentrations of phosphopeptide mixtures with or without extra-added bovine serum albumin (BSA) nonphosphorylated peptides. Addition of abundant tryptic BSA peptides to the phosphopeptide mixture increases the demand for selective IMAC capture. While SwellGel gallium Discs, IPAC Metal Chelating Resin, and ZipTipMC Pipette Tips allow for the possibility of enriching phosphopeptides, the Gyrolab MALDI IMAC1 also presents the possibility of verifying existing phosphopeptides after a dephosphorylation step. Phosphate-containing peptides are identified through a mass shift between phosphorylated and dephosphorylated spectra of 80 Da (or multiples of 80 Da). This verification is useful if the degree of phosphorylation is low in the sample or if the ionization is unfavorable, which often is the case for phosphopeptides. A peptide mixture in which phosphorylated serine, threonine, and tyrosine were represented was diluted in steps and thereafter enriched using the four different IMAC methods prior to analyses with matrix assisted laser desorption/ionization mass spectrometry. The enrichment of phosphopeptides using SwellGel Gallium Discs or Gyrolab MALDI IMAC1 was not significantly affected by the addition of abundant BSA peptides added to the sample mixture, and the achieved detection limits using these techniques were also the lowest. All four of the included phosphopeptides were detected by MALDI-MS only after enrichment using the Gyrolab MALDI IMAC1 compact disc (CD) and detection down to low femtomole levels was possible. Furthermore, selectivity, reproducibility, and detection for a number of other phosphopeptides using the IMAC CD are reported herein. For example, two phosphopeptides sent out in a worldwide survey performed by the Proteomics Research Group (PRG03) of the Association of Biomolecular Resource Facilities (ABRF) were detected and verified by means of the 80 Da mass shift achieved by on-column dephosphorylation.     

How Does Intracellular Ca2+ Oscillate: By Chance or by the Clock?

Ca²⁺ oscillations have been considered to obey deterministic dynamics for almost two decades. We show for four cell types that Ca²⁺ oscillations are instead a sequence of random spikes. The standard deviation of the interspike intervals (ISIs) of individual spike trains is similar to the average ISI; it increases approximately linearly with the average ISI; and consecutive ISIs are uncorrelated. Decreasing the effective diffusion coefficient of free Ca²⁺ using Ca²⁺ buffers increases the average ISI and the standard deviation in agreement with the idea that individual spikes are caused by random wave nucleation. Array-enhanced coherence resonance leads to regular Ca²⁺ oscillations with small standard deviation of ISIs.     

Transcutaneous anti-influenza vaccination promotes both CD4 and CD8 T cell immune responses in humans

Induction of T cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. The use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells in CD8 cell cross-priming and suggests that vaccination via the transcutaneous (TC) route may be relevant in the induction of cellular immune responses. We have previously shown that TC application of nanoparticles, on human skin explants, allows targeting of epidermal dendritic cells, possibly via hair follicles. In this study, we have investigated cellular immune responses against an influenza protein-based vaccine by TC vaccination, compared with i.m. vaccination in humans. In this study on 11 healthy volunteers, we found that a newly developed protocol based on cyanoacrylate skin surface stripping induced a significant increase in IFN-gamma-producing T cells specific for influenza vaccine by ELISPOT assays. Interestingly, TC vaccination induced both effector CD4 and CD8 T cell responses, whereas i.m. injection induced strong effector CD4 in the absence of CD8 T cells, as assessed by intracellular cytokine staining and tetramer analyses. This study proposes new perspectives for the development of vaccination strategies that trigger T cell immune responses in humans.     

Protein kinase C-theta critically regulates the proliferation and survival of pathogen-specific T cells in murine listeriosis

Protein kinase C-theta (PKC-theta) is essential for the activation of T cells in autoimmune disorders, but not in viral infections. To study the role of PKC-theta in bacterial infections, PKC-theta(-/-) and wild-type mice were infected with Listeria monocytogenes (LM). In primary and secondary listeriosis, the numbers of LM-specific CD8 and CD4 T cells were drastically reduced in PKC-theta(-/-) mice, resulting in increased CFUs in spleen and liver of both PKC-theta(-/-) C57BL/6 and BALB/c mice. Furthermore, immunization with peptide-loaded wild-type dendritic cells induced LM-specific CD4 and CD8 T cells in wild-type but not in PKC-theta(-/-) mice. In listeriosis, transfer of wild-type T cells into PKC-theta(-/-) mice resulted in a normal control of Listeria, and, additionally, a selective expression of PKC-theta in LM-specific T cells was sufficient to drive a normal proliferation and survival of these T cells in LM-infected PKC-theta(-/-) recipients, illustrating a cell-autonomous function of PKC-theta in LM-specific T cells. Conversely, adoptively transferred PKC-theta(-/-) T cells were partially rescued from cell death and proliferated in LM-infected wild-type recipients, demonstrating that a PKC-theta deficiency of LM-specific T cells can be partially compensated for by a wild-type environment. Additionally, in vitro experiments showed that only the addition of IL-2, but not an inhibition of caspase-3, induced proliferation and prevented death of PKC-theta(-/-) T cells stimulated with LM-infected wild-type dendritic cells, further demonstrating that the impaired proliferation and survival of PKC-theta(-/-) T cells in listeriosis is not intrinsically fixed and can be experimentally improved.