Band 382, Heft 12

A new control of mitochondrial membrane potential ΔΨm and formation of reactive oxygen species (ROS) is presented, based on allosteric ATPinhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100 120 mV), the ATP/ADP ratio will also be maximal at this ΔΨm (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADPratio keeps ΔΨm low. In contrast, the known respiratory control leads to an inhibition of respiration only at high ΔΨm values (150 200 mV) which cause ROS formation. ATPinhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that stress hormones which increase intracellular [Ca 2+ ] also increase ΔΨm and ROS formation, which promote degenerative diseases and accelerate aging.

A clone was isolated from a cDNA library from early embryos of Xenopus laevis that codes for a highly charged protein containing 339 amino acids. Two putative nuclear localization signals could be identified in its sequence, but no other known motifs or domains. Closely related ORFs are present in the genomes of man, C. elegans, yeast and Arabidopsis. A fusion protein with GFP expressed in HeLa cells or Xenopus oocytes was found to be localized in the nucleolus and coiled (Cajal) bodies. Moreover, immunoprecipitation experiments demonstrated that the new Xenopus protein interacts with 5S, 5.8S and 28S RNAs of large ribosomal subunits. The name Brix (biogenesis of ribosomes in Xenopus) is proposed for this protein and the corresponding gene. In Saccharomyces cerevisiae, the essential gene YOL077c, now named BRX1, codes for the Brix homolog, which is also localized in the nucleolus. Depletion of Brx1p in a conditional yeast mutant leads to defects in rRNA processing, and a block in the assembly of large ribosomal subunits.

In contrast to signal generation and transmission, the mechanisms and molecules that negatively regulate receptor tyrosine kinase (RTK) signaling are poorly understood. Here we characterize Mig-6 as a novel negative feedback regulator of the epidermal growth factor receptor (EGFR) and potential tumor suppressor. Mig-6 was identified in a yeast twohybrid screen with the kinase active domain of the EGFR as bait. Upon EGF stimulation Mig-6 binds to the EGFR involving a highly acidic region between amino acids 985 995. This interaction is kinase activitydependent, but independent of tyrosine 992. Mig-6 overexpression results in reduced activation of the mitogenactivated protein kinase ERK2 in response to EGF, but not FGF or PDGF, stimulation and in enhanced receptor internalization without affecting the rate of degradation. The induction of Mig-6 mRNA expression in response to EGF, but not FGF, indicates the existence of a negative regulatory feedback loop. Consistent with these findings, a possible role as tumor suppressor is indicated by Mig-6-mediated inhibition of EGFR overexpressioninduced transformation of Rat1 cells.

Epidemiological studies suggest that βcarotene is able to modulate the risk of cancer. A number of in vitro studies reported that βcarotene inhibits the growth of cancer cells; however, so far little is known about the molecular mechanisms of the antiproliferative effect of βcarotene. Here we have investigated the effects of two βcarotene preparations, (i) βcarotene dissolved in tetrahydrofuran (final concentration in cell culture medium: 0.5%) and (ii) βcarotene incorporated in a water dispersible bead form, on cultured human colon carcinoma cells HT29. The treatment of cells with βcarotene up to 30 M for 72 h led to a significant increase in the cellular βcarotene concentration and formation of retinol. βCarotene showed only low cytotoxicity for confluent cells tested up to 30 M, but at dietary relevant concentrations for the intestinal tract (10, 30 M) βcarotene was strongly cytotoxic for growing cells and induced apoptosis in HT29 cells as assessed by the AnnexinV assay (the maximal effect was observed 15 h after treatment with βcarotene). Exposure of cells to retinol at concentrations yielding cellular retinol levels similar to those observed by βcarotene treatment had no antiproliferative or cytotoxic effect. Furthermore, βcarotene did not affect the activation of the extracellular signalregulated kinases (ERK1 and ERK2) that are essential for cellular growth. In summary, βcarotene can inhibit growth of human colon carcinoma cells in vitro by induction of apoptosis in proliferating cells.

We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligandmediated protection and phagemediated selection. The effectiveness of this new approach was demonstrated by displaying the T7tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7tag antibody was used as protective ligand for T7tagdisplaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10 000 cells displaying the T7tag and approximately 10exp8 cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibodydependent survival of T7tag displaying cells was observed, yielding an enrichment factor of up to 10exp7-fold. The CISTEM technology was used to select sequences from a T7tagbased, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display platform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of proteinligand interactions.

The thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, Nterminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL ). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E' 0 ) for the active thiol/ disulfide was determined to be 0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active site cysteines and the structure of the reduced form.

Some cocoas and chocolates are rich in ()epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dosedependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC 50 of 0.8 M. Among the monomeric flavanols, epigallocatechin gallate (IC 50 = 4 M) and epicatechin gallate (5 M) were more potent than ()epicatechin (IC50 = 60 M). ()Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxyeicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocytetype 12- lipoxygenase (another representative of the 12/15- lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 M) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2 O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2 O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione Stransferase, glutathione peroxidase) were studied. Exposure to H2 O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2 O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2 O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2 O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.

The Nterminal heparin/fibrin binding domain of human plasma fibronectin (pFN) contains a cryptic proteinase. The enzyme could be generated and activated in the presence of Ca 2+ from the purified 70 kDa pFN fragment produced by cathepsin D digestion of pFN. In this work we cloned and expressed the serine proteinase, designated fibronectinase (Fnase), in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, subjected to folding and autocatalytic degradation in the presence of Ca 2+ , and yielded an active enzyme capable of digesting fibronectin. Cleavage of pFN and the synthetic peptides AcIEGKpNA and BzIEGRpNA demonstrated identical specificity of the recombinant and the isolated fibronectinase. Further investigations of the substrate specificity revealed for the first time the muscle proteins actin and myosin as being substrates of fibronectinase. The enzyme can be inhibited by α1-proteinase inhibitor. In the context of induced cathepsin D release, e. g. from granulocytes under inflammatory conditions, these results indicate an increase in specific proteolytic potential against muscular proteins in dystrophic diseases by the release of cryptic fibronectinase.

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280 000 and 290 000 by nondenaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDSPAGE in the absence and presence of βME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km , Vmax , kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 M, 3.7 molper mg and min, 33.1 per s and 4.87 10,000 per s and M for AlaAlaPheMCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl 2 , and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide ArgValTyr from angiotensin III more rapidly than from AlaAlaPheMCA, and also released GlyAsnLeu from neuromedin B with the same velocity as from AlaAlaPheMCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as λRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > / = liver > heart > brain > lung > spleen >> skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.

Methods to assess metabolism are important analytical tools in neuroscience. The fluorophore nicotinamide adenine dinucleotide (NADH) is a parameter of cellular metabolism. NADH fluorescence was measured using a laserbased fluorescence detector with spectral and temporal filters. Distribution and intensity of NADH fluorescence were investigated in frozen brain sections. In sections containing hippocampus the intensity of NADH fluorescence was correlated to brain structures. In order to investigate the consequences of neurotoxic lesions, 5,7-dihydroxytryptamine was injected into the dorsal raphe nucleus 4 to 240 days prior to the measurement. NADH fluorescence decreased in the affected region by 50%, indicating that no recovery in metabolic activity had occurred.

Heterodimeric calpain, consisting of the large (80 kDa) and the small (30 kDa) subunit, was isolated and purified from human erythrocytes by a highly reproducible fourstep purification procedure. Obtained material is more than 95% pure and has a specific activity of 6 7 mU/mg. Presence of contaminating proteins could not be detected by HPLC and sequence analysis. During storage at 80 C the enzyme remains fully activatable by Ca 2+ , although the small subunit is partially processed to a 22 kDa fragment. This novel autolysis product of the small subunit starts with the sequence 60 RILG and is further processed to the known 18 kDa fragment. Active forms and typical transient and stable autolysis products of the large subunit were identified by protein sequencing. In caseinzymograms only the activatable forms 80 kDa+30 kDa, 80 kDa+22 kDa and 80 kDa+18 kDa displayed caseinolysis.