Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance

The suitability of proteome-based strategies for the targeting of tumor-associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F-actin capping protein, gamma-actin, stathmin, tubulin-alpha, tubulin-beta and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor-associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.     

The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality

AIMS: In this study we determined the extent to which lactic acid bacteria (LAB) occurred in brandy base wines, their ability to catalyse the malolactic fermentation (MLF) and the effect of MLF on the quality of the base wine and the brandy distillate. METHODS AND RESULTS: Lactic acid bacteria were isolated and enumerated from grape juice, experimental and commercially produced brandy base wines. Spontaneous MLF occurred in approximately 50% of the commercial base wines. The occurrence of MLF had an influence on the quality of the base wines and the resulting distillates. In samples where MLF occurred there was a loss of fruitiness and in the intensity of aroma. Volatile compounds like iso-amyl acetate, ethyl acetate, ethyl caproate, 2-phenethyl acetate and hexyl acetate decreased in samples having undergone MLF, while ethyl lactate, acetic acid and diethyl succinate increased in the same samples. CONCLUSIONS: Spontaneous malolactic fermentation does occur in commercial brandy base wines and it has an influence on base wine and brandy quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that MLF influences the quality of the base wine and the resulting distillate and with this in mind commercial base wine producers should be able to produce brandy of higher quality.     

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79–87]. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLD_cab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLD_cab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLD_cab is N-terminally acetylated.     

Effects of hydrogen peroxide on bovine leukemia virus expression

Several activators of bovine leukemia virus (BLV) expression, including lipopolysaccharides, phorbol esters and calcium ionophores, are known to generate reactive oxygen species (ROS). Therefore the influence of H2O2 on BLV expression in two BLV producing cell lines was investigated. The effect of H2O2 on BLV expression is apparently dose-dependent. Incubation of FLK/BLV cells with low concentrations of H2O2 (2.5 to 10 microM) induced a marked enhancement of BLV p24 synthesis and an activation of the long terminal repeat (LTR). Higher concentrations resulted in a decrease of proliferation, induction of apoptosis and in a decrease of BLV synthesis. Furthermore, in both cell lines H2O2 treatment led to the activation of NF-kappaB. Pretreatment of cells with antioxidants abrogated the H2O2-induced BLV expression. Taken together, our findings suggest that oxidative stress stimulates BLV expression via activation of NF-kappaB, raising the possibility that biological sources of H2O2, such as stimulated phagocytes, may influence BLV expression.     

The role of the polo kinase Cdc5 in controlling Cdc14 localization

In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.     

Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cells<Emphasis Type="Italic">in vitro</Emphasis>

Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells invitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 μM) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikertet al.,FEBS Lett 2001, 506:131–134) on aspects of validation procedures as well as limitations and pitfalls of this method. Published: June 2, 2003     

Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.     

Complementary neuronal and glial expression of two high-affinity glutamate transporter GLT1/EAAT2 forms in rat cerebral cortex

The glutamate transporter GLT1 is essential in limiting transmitter signaling and restricting harmful receptor overstimulation. It has been shown recently that GLT1 exists in two forms, the generic GLT1 and a 3'-end-spliced variant of GLT1 (GLT1v), both with similar transport characteristics. To differentiate clearly the cellular distribution of both GLT1 forms in the cortex, specific cRNA probes for non-radioactive in situ hybridization were generated and applied to adult rat brain sections. The results were complemented by western and northern blot analyses and by immunocytochemical investigations using specific peptide antibodies against both GLT1 forms. The study confirmed that generic GLT1 mRNA was expressed predominantly in astrocytes and, to a small extent, in neurons, whereas GLT1 protein was detected only in cell membranes of astrocytes. On the other hand, GLT1v mRNA and protein were demonstrated predominantly in neurons and in non-astrocytic glial cells irrespective of the cortical areas studied. A cytoplasmic granular staining of neurons and astrocytes predominated in the demonstration of GLT1v protein. It is concluded that the cellular expression of the two GLT1 forms is complementary. The cytoplasmic vesicular distribution of GLT1v may represent an endogenous protective mechanism to limit glutamate-induced excitotoxicity.