Effect of fluvastatin on apoptosis in human CD4+ T cells

Statins are lipid-lowering agents with pleiotropic effects. We investigated the apoptotic effects of fluvastatin on peripheral CD4+ T cells from healthy subjects. Fluvastatin induced apoptosis in resting CD4+ T cells but not in CD4+ T cells strongly activated with a high concentration of PMA plus ionomycin (PMA/I) analyzed with annexin V and propidium iodide staining. However, CD4+ T cells activated with a low concentration of PMA/I or with anti-CD3 antibodies were apoptotic after treatment with fluvastatin. Activities of caspases-8, -9, and -3 were increased in resting CD4+ T cells treated with fluvastatin (10 microM). In strongly activated CD4+ T cells, fluvastatin inhibited the activation of caspase-8 induced by PMA/I and increased caspase-9 activity. The caspase-3 activity did not differ between untreated and fluvastatin-treated strongly activated CD4+ T cells. Treatment with fluvastatin (10 microM) enhanced cytochrome c release and increased the Bax/Bcl-2 ratio in both resting and strongly activated CD4+ T cells. Although the in vitro concentration of fluvastatin used in this study is higher than in vivo, other factors may sensitize apoptotic cell death of CD4+ T cells in vivo. In conclusion, fluvastatin induces apoptosis in resting T cells but not in strongly activated T cells, a difference that might be due to the interaction between caspase-8 and caspase-9.     

Overexpression of cathepsin B in gastric cancer identified by proteome analysis

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.     

Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide

A method for the measurement of proton T(1)(rho) relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90 degrees excitation in a HOHAHA pulse sequence. The experiment is applied to alpha-bungarotoxin (alpha-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) alpha-subunit. A good correlation between high T(1)(rho) values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the alpha-BTX complex with an AChR peptide and the model for alpha-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor alpha-subunit is exhibited, while residues implicated in binding gamma- and delta-subunits retain their mobility. In addition, the quantitative T(1)(rho) measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.     

Roles for common cytokine receptor gamma-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo

NK cells differentiate in adult mice from bone marrow hemopoietic progenitors. Cytokines, including those that signal via receptors using the common cytokine receptor gamma-chain (gamma(c)), have been implicated at various stages of NK cell development. We have previously described committed NK cell precursors (NKPs), which have the capacity to generate NK cells, but not B, T, erythroid, or myeloid cells, after in vitro culture or transfer to a fetal thymic microenvironment. NKPs express the CD122 Ag (beta chain of the receptors for IL-2/IL-15), but lack other mature NK markers, including NK1.1, CD49b (DX5), or members of the Ly49 gene family. In this report, we have analyzed the roles for gamma(c)-dependent cytokines in the generation of bone marrow NKP and in their subsequent differentiation to mature NK cells in vivo. Normal numbers of NKPs are found in gamma(c)-deficient mice, suggesting that NK cell commitment is not dependent on IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. Although IL-2, IL-4, and IL-7 have been reported to influence NK cell differentiation, we find that mice deficient in any or all of these cytokines have normal NK cell numbers, phenotype, and effector functions. In contrast, IL-15 plays a dominant role in early NK cell differentiation by maintaining normal numbers of immature and mature NK cells in the bone marrow and spleen. Surprisingly, the few residual NK cells generated in absence of IL-15 appear relatively mature, expressing a variety of Ly49 receptors and demonstrating lytic and cytokine production capacity.     

Involvement of phosphoinositide 3-kinase gamma, Rac, and PAK signaling in chemokine-induced macrophage migration

In macrophages, chemotactic stimuli cause the activation of Rac and PAK, but little is known about the signaling pathways involved and their role in chemotactic gradient sensing. Herein, we report that in macrophages, the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 activates the small GTPase Rac and its downstream target PAK2 within seconds. This response depends on Gi activation and largely on the subsequent triggering of phosphoinositide 3-kinase gamma (PI3Kgamma) and Rac. Retroviral transduction of tagged Rac1 and -2 indicates that RANTES/CCL5-mediated activation of PI3Kgamma triggers Rac1 but not Rac2. In agreement, silencing of Rac1 by shRNA blocks PAK2 activity and inhibits RANTES/CCL5-induced macrophage polarization and directional migration. On the other hand, the tyrosine kinase receptor agonist CSF-1 activates PAK2 independently of PI3Kgamma and Rac. Our results thus demonstrate a chemokine-specific signaling pathway in which Gi and PI3Kgamma coordinate to drive Rac1 and PAK2 activation that eventually controls the chemotactic response.     

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.     

Potato tuber proteomics: comparison of two complementary extraction methods designed for 2-DE of acidic proteins

Two protein extraction procedures were tested in order to remove interfering compounds prior to 2-DE of potato tubers. These methods using SDS lysis buffer and phenol-phase extraction were compared regarding the quality of the resulting 2-D gel. While the resolution of SDS extracts on semipreparative gels seems better, both methods lead to similar extraction yields and total number of spots. The procedures are complementary regarding the Mr range of preferentially extracted proteins.