Showing papers by "Heidelberg University published in 1997"

Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR/O, the TetR/O and AraC/I1-I2 Regulatory Elements

Abstract: Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

The Heidelberg classification of renal cell tumours

Abstract: This paper presents the conclusions of a workshop entitled 'Impact of Molecular Genetics on the Classification of Renal Cell Tumours', which was held in Heidelberg in October 1996. The focus on 'renal cell tumours' excludes any discussion of Wilms' tumour and its variants, or of tumours metastatic to the kidneys. The proposed classification subdivides renal cell tumours into benign and malignant parenchymal neoplasms and, where possible, limits each subcategory to the most commonly documented genetic abnormalities. Benign tumours are subclassified into metanephric adenoma and adenofibroma, papillary renal cell adenoma, and renal oncocytoma. Malignant tumours are subclassified into common or conventional renal cell carcinoma; papillary renal cell carcinoma; chromophobe renal cell carcinoma; collecting duct carcinoma, with medullary carcinoma of the kidney; and renal cell carcinoma, unclassified. This classification is based on current genetic knowledge, correlates with recognizable histological findings, and is applicable to routine diagnostic practice.

Matrix‐based comparative genomic hybridization: Biochips to screen for genomic imbalances

Abstract: Comparative genomic hybridization (CGH) to metaphase chromosomes has been widely used for the genome-wide screening of genomic imbalances in tumor cells. Substitution of the chromosome targets by a matrix consisting of an ordered set of defined nucleic acid target sequences would greatly enhance the resolution and simplify the analysis procedure, both of which are prerequisites for a broad application of CGH as a diagnostic tool. However, hybridization of whole genomic human DNA to immobilized single-copy DNA fragments with complexities below the megabase pair level has been hampered by the low probability of specific binding because of the high probe complexity. We developed a protocol that allows CGH to chips consisting of glass slides with immobilized target DNAs arrayed in small spots. High-copy-number amplifications contained in tumor cells were rapidly scored by use of target DNAs as small as a cosmid. Low-copy-number gains and losses were identified reliably by their ratios by use of chromosome-specific DNA libraries or genomic fragments as small as 75 kb cloned in PI or PAC vectors as targets, thus greatly improving the resolution achievable by chromosomal CGH. The ratios obtained for the same chromosomal imbalance by matrix CGH and by chromosomal CGH corresponded very well. The new matrix CGH protocol provides a basis for the development of automated diagnostic procedures with biochips designed to meet clinical needs.

Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

Abstract: Nano-electrospray tandem mass spectrometry allows qualitative and quantitative analysis of complex membrane lipid mixtures at the subpicomole level. We have exploited this technique to selectively detect individual classes of phospholipids from unprocessed total cellular lipid extracts by either precursor ion or neutral loss scanning. This way phosphatidylcholine, sphingomyelin, phosphatidylinositol and -phosphates, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and their plasmalogen analogues can be detected. The optimized ionization and fragmentation conditions described together with the principle of internal standardization by nonnatural analogues allow the rapid and quantitative determination of membrane lipid compositions down to sample amounts of 1000 cells.

Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome

Abstract: Growth retardation resulting in short stature is a major concern for parents and due to its great variety of causes, a complex diagnostic challenge for clinicians. A major locus involved in linear growth has been implicated within the pseudoautosomal region (PAR1) of the human sex chromosomes. We have determined an interval of 170 kb of DNA within PAR1 which was deleted in 36 individuals with short stature and different rearrangements on Xp22 or Yp11.3. This deletion was not detected in any of the relatives with normal stature or in a further 30 individuals with rearrangements on Xp22 or Yp11.3 with normal height. We have isolated a homeobox-containing gene (SHOX) from this region, which has at least two alternatively spliced forms, encoding proteins with different patterns of expression. We also identified one functionally significant SHOX mutation by screening 91 individuals with idiopathic short stature. Our data suggest an involvement of SHOX in idiopathic growth retardation and in the short stature phenotype of Turner syndrome patients.

Substrate specificity of the DnaK chaperone determined by screening cellulose‐bound peptide libraries

Abstract: Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.

Abstract: Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells. We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells. At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2). Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53-/- (Hep3B). In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells. Microinjection of wt p53 cDNA into HepG2 cells had the same effect. In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells. Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression. Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction. These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system. The same applies to other anti-cancer drugs such as cisplatin and methotrexate. These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance.

Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.

Abstract: The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.

Specific, nongenomic actions of steroid hormones

Abstract: Traditionally, steroid hormone action has been described as the modulation of nuclear transcription, thus triggering genomic events that are responsible for physiological effects. Despite early observations of rapid steroid effects that were incompatible with this theory, nongenomic steroid action has been widely recognized only recently. Evidence for these rapid effects is available for steroids of all clones and for a multitude of species and tissues. Examples of nongenomic steroid action include rapid aldosterone effects in lymphocytes and vascular smooth muscle cells, vitamin D3 effects in epithelial cells, progesterone action in human sperm, neurosteroid effects on neuronal function, and vascular effects of estrogens. Mechanisms of action are being studied with regard to signal perception and transduction, and researchers have developed a patchy sketch of a membrane receptor-second messenger cascade similar to those involved in catecholamine and peptide hormone action. Many of these effects appear to involve phospholipase C, phosphoinositide turnover, intracellular pH and calcium, protein kinase C, and tyrosine kinases. The physiological and pathophysiological relevance of these effects is unclear, but rapid steroid effects on cardiovascular, central nervous, and reproductive functions may occur in vivo. The cloning of the cDNA for the first membrane receptor for steroids should be achieved in the near future, and the physiological and clinical relevance of these rapid steroid effects can then be established.

Aggregation, blowup, and collapse: the abc's of taxis in reinforced random walks

Abstract: In many biological systems, movement of an organism occurs in response to a diffusible or otherwise transported signal, and in its simplest form this can be modeled by diffusion equations with advection terms of the form first derived by Patlak [Bull. of Math. Biophys., 15 (1953), pp. 311--338]. However, other systems are more accurately modeled by random walkers that deposit a nondiffusible signal that modifies the local environment for succeeding passages. In these systems, one example of which is the myxobacteria, the question arises as to whether aggregation is possible under suitable hypotheses on the transition rules and the production of a control species that modulates the transition rates. Davis [Probab. Theory Related Fields, 84 (1990), pp. 203--229] has studied this question for a certain class of random walks, and here we extend this analysis to the continuum limit of such walks. We first derive several general classes of partial differential equations that depend on how the movement rules are...

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus

Abstract: The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.

NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex

Abstract: NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells

Abstract: Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco.

Abstract: Nia30(145) transformants with very low nitrate reductase activity provide an in vivo screen to identify processes that are regulated by nitrate. Nia30(145) resembles nitrate-limited wild-type plants with respect to growth rate and protein and amino acid content but accumulates large amounts of nitrate when it is grown on high nitrate. The transcripts for nitrate reductase (NR), nitrite reductase, cytosolic glutamine synthetase, and glutamate synthase increased; NR and nitrite reductase activity increased in leaves and roots; and glutamine synthetase activity increased in roots. The transcripts for phosphoenolpyruvate carboxylase, cytosolic pyruvate kinase, citrate synthase, and NADP-isocitrate dehydrogenase increased; phosphoenolpyruvate carboxylase activity increased; and malate, citrate, isocitrate, and [alpha]-oxoglutarate accumulated in leaves and roots. There was a decrease of the ADP-glucose pyrophosphorylase transcript and activity, and starch decreased in the leaves and roots. After adding 12 mM nitrate to nitrate-limited Nia30(145), the transcripts for NR and phosphoenolpyruvate carboxylase increased, and the transcripts for ADP-glucose pyrophosphorylase decreased within 2 and 4 hr, respectively. Starch was remobilized at almost the same rate as in wild-type plants, even though growth was not stimulated in Nia30(145). It is proposed that nitrate acts as a signal to initiate coordinated changes in carbon and nitrogen metabolism.

Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

Abstract: Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated M r of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

Acute stroke: usefulness of early CT findings before thrombolytic therapy.

Abstract: PURPOSE: To determine whether the extent of subtle parenchymal hypoattenuation detected on computed tomographic (CT) scans obtained within 6 hours of ischemic stroke is a factor in predicting patients' response to thrombolytic treatment. MATERIALS AND METHODS: The baseline CT scans of 620 patients, who received either recombinant tissue plasminogen activator (rt-PA) or a placebo, in a double-blind, randomized multicenter trial were prospectively evaluated and assigned to one of three categories according to the extent of parenchymal hypoattenuation: none, 33% or less (small), or more than 33% (large) of the middle cerebral artery territory. The association between the extent of hypoattenuation on the baseline CT scans and the clinical outcome in the placebo-treated and the rt-PA-treated groups after 3 months was analyzed. RESULTS: In 215 patients with a small hypoattenuating area, treatment increased the chance of good outcome. In 336 patients with a normal CT scan and in 52 patients with a large hypoatte...

Giant Intermolecular Decay and Fragmentation of Clusters

Abstract: In sharp contrast to molecules, electronic states of clusters with an excited intermediate-shell electron can efficiently decay via an intermolecular Coulombic mechanism. Explicit examples are presented using large scale {ital ab initio} propagator calculations. The mechanism is illustrated and its generality is stressed. {copyright} {ital 1997} {ital The American Physical Society}

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A) + RNA and nuclear pores

Abstract: An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.

Role of NFkappaB in the mortality of sepsis.

Abstract: Binding activity for nuclear factor kappa B (NFkappaB) consensus probes was studied in nuclear extracts from peripheral blood mononuclear cells of 15 septic patients (10 surviving and 5 not surviving). Nonsurvivors could be distinguished from survivors by an increase in NFkappaB binding activity during the observation period (P < 0.001). The increase in NFkappaB binding activity was comparable to the APACHE-II score as a predictor of outcome. Intravenous somatic gene transfer with an expression plasmid coding for IkappaBalpha was used to investigate the role of members of the NFkappaB family in a mouse model of endotoxemia. In this model, increased NFkappaB binding activity was present after injection of LPS. Intravenous somatic gene transfer with IkappaBalpha given before LPS attenuated renal NFkappaB binding activity and increased survival. Endothelial cells and monocytes/macrophages were the major target cells for somatic gene transfer, transfected with an average transfection efficiency of 20-35%. Tissue factor, a gene under regulatory control of NFkappaB, was induced by LPS. Somatic gene transfer with a reporter plasmid containing the functional tissue factor promoter demonstrated NFkappaB-dependent stimulation by LPS. Intravenous somatic gene transfer with IkappaBalpha reduced LPS-induced renal tissue factor expression, activation of the plasmatic coagulation system (decrease of thrombin-antithrombin III complexes) and renal fibrin/fibrinogen deposition. Somatic gene transfer with an expression plasmid with tissue factor cDNA in the antisense direction (in contrast to sense or vector alone) also increased survival. Furthermore, antisense tissue factor decreased renal tissue factor expression and the activation of the plasmatic coagulation system.

Accumulation of nitrate in the shoot acts as a signal to regulate shoot‐root allocation in tobacco†

Abstract: Mutants and transformants of tobacco (Nicotiania tabacum L. cv Gatersleben 1) with decreased expression of nitrate reductase have been used to investigate whether nitrate accumulation in the shoot acts as a signal to alter allocation between shoot and root growth. (a) Transformants with very low (1–3% of wild-type levels) nitrate reductase activity had growth rates, and protein, amino acid and glutamine levels similar to or slightly lower than a nitrate-limited wild-type, but accumulated large amounts of nitrate. These plants should resemble a nitrate-limited wild-type, except in responses where nitrate acts as a signal. (b) Whereas the shoot:root ratio decreases from about 3.5 in a well-fertilized wild-type to about 2 in a nitrate-limited wild-type, the transformants had a very high shoot:root ratio (8–10) when they were grown on high nitrate. When they were grown on lower nitrate concentrations their shoot:root ratio declined progressively to a value similar to that in nitrate-limited wild-types. Mutants with a moderate (30–50%) decrease of nitrate reductase also had a small but highly significant increase of their shoot:root ratio, compared to the wild-type. The increased shoot:root ratio in the mutants and transformants was due to a stimulation of shoot growth and an inhibition of root growth. (c) There was a highly significant correlation between leaf nitrate content and the shoot:root ratio for eight genotypes growing at a wide range of nitrate supply. (d) A similar increase of the shoot:root ratio in nitrate reductase-deficient plants, and correlation between leaf nitrate content and the shoot:root ratio, was found in plants growing on ammonium nitrate. (f) Split-root experiments, in which the transformants were grown with part of their root system in high nitrate and the other part in low nitrate, showed that root growth is inhibited by the accumulation of nitrate in the shoot. High concentrations of nitrate in the rooting medium actually stimulate local root growth. (g) The inhibition of root growth in the transformants was relieved when the transformants were grown on limiting phosphate, even though the nitrate content of the root remained high. This shows that the nitrate-dependent changes in allocation can be overridden by other signals that increase allocation to root growth. (h) The reasons for the changed allocation were investigated in transformants growing normally, and in split-root culture. Accumulation of nitrate in the shoot did not lead to decreased levels of amino acids or protein in the roots. However, it did lead to a strong inhibition of starch synthesis and turnover in the leaves, and to decreased levels of sugars in the root. The rate of root growth was correlated with the root sugar content. It is concluded that these changes of carbon allocation could contribute to the changes in shoot and root growth.

GrpE‐like regulation of the Hsc70 chaperone by the anti‐apoptotic protein BAG‐1

Abstract: The BAG‐1 protein appears to inhibit cell death by binding to Bcl‐2, the Raf‐1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG‐1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG‐1 is a regulator of the Hsc70 chaperone. BAG‐1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady‐state ATP hydrolysis activity ∼40‐fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG‐1 accelerates the release of ADP from Hsc70. Thus, BAG‐1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70‐interacting protein Hip, which stabilizes the ADP‐bound state. Intriguingly, BAG‐1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti‐apoptotic function of BAG‐1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.

Interaction of Hsp70 chaperones with substrates

Abstract: Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing Hsp70 interaction with polypeptide chains. DnaK recognizes extended peptide strands composed of up to five consecutive hydrophobic residues within and positively charged residues outside the substrate binding cavity.

Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.

Abstract: Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

Abstract: Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.