Showing papers by "Martin Luther University of Halle-Wittenberg published in 1999"

Microbial heavy-metal resistance

Abstract: We are just beginning to understand the metabolism of heavy metals and to use their metabolic functions in biotechnology, although heavy metals comprise the major part of the elements in the periodic table. Because they can form complex compounds, some heavy metal ions are essential trace elements, but, essential or not, most heavy metals are toxic at higher concentrations. This review describes the workings of known metal-resistance systems in microorganisms. After an account of the basic principles of homoeostasis for all heavy-metal ions, the transport of the 17 most important (heavy metal) elements is compared.

The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism

Abstract: Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.

MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis.

Abstract: MAPKAP kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAP kinase. By introducing a targeted mutation into the mouse MK2 gene, we have determined the physiological function of MK2 in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is due to a reduction of approximately 90% in the production of tumor necrosis factor-alpha (TNF-alpha) and not to a change in signalling from the TNF receptor. The level and stability of TNF-alpha mRNA is not reduced and TNF-alpha secretion is not affected. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNF-alpha at a post-transcriptional level.

Adrenergic and Muscarinic Receptors in the Human Heart

Abstract: Cardiac function is controlled by the autonomic nervous system (i.e., the sympathetic and the parasympathetic nervous systems), which act via adrenoceptors and muscarinic acetylcholine receptors, respectively. At least nine adrenoceptor subtypes and five muscarinic receptor subtypes exist. In recent

The World is More Just for Me than Generally: About the Personal Belief in a Just World Scale's Validity

Abstract: Differences between personal and general belief in a just world were studied in four questionnaire studies and one experiment. Personal just world belief could reliably be differentiated from general just world belief, and subjects endorsed more strongly the personal compared to the general just world belief. Moreover, personal belief in a just world predicted subjective well-being and self-esteem, and this positive impact was independent of general just world belief and favorable self-perceptions. Finally, the more subjects were aware of their own unfairness, the more the personal belief in a just world showed a negative impact on self-esteem. Results give evidence to the just world beliefs' character as world views and as indicators of a personal contract between individual and social world.

Functional mammalian homologues of the Drosophila PEV‐modifier Su(var)3‐9 encode centromere‐associated proteins which complex with the heterochromatin component M31

Abstract: The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin.

Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly.

Abstract: Holoprosencephaly (HPE) is a common, severe malformation of the brain that involves separation of the central nervous system into left and right halves. Mild HPE can consist of signs such as a single central incisor, hypotelorism, microcephaly, or other craniofacial findings that can be present with or without associated brain malformations1,2,3. The aetiology of HPE is extremely heterogeneous, with the proposed participation of a minimum of 12 HPE-associated genetic loci as well as the causal involvement of specific teratogens acting at the earliest stages of neurulation4. The HPE2 locus was recently characterized as a 1-Mb interval on human chromosome 2p21 that contained a gene associated with HPE. A minimal critical region was defined by a set of six overlapping deletions and three clustered translocations in HPE patients5. We describe here the isolation and characterization of the human homeobox-containing SIX3 gene from the HPE2 minimal critical region (MCR). We show that at least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5´ end of SIX3. Mutational analysis has identified four different mutations in the homeodomain of SIX3 that are predicted to interfere with transcriptional activation and are associated with HPE. We propose that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eye in humans.

Optimal Grid-Clustering: Towards Breaking the Curse of Dimensionality in High-Dimensional Clustering

Abstract: Many applications require the clustering of large amounts of high-dimensional data. Most clustering algorithms, however, do not work e ectively and e ciently in highdimensional space, which is due to the so-called "curse of dimensionality". In addition, the high-dimensional data often contains a signi cant amount of noise which causes additional e ectiveness problems. In this paper, we review and compare the existing algorithms for clustering highdimensional data and show the impact of the curse of dimensionality on their e ectiveness and e ciency. The comparison reveals that condensation-based approaches (such as BIRCH or STING) are the most promising candidates for achieving the necessary e ciency, but it also shows that basically all condensation-based approaches have severe weaknesses with respect to their e ectiveness in highdimensional space. To overcome these problems, we develop a new clustering technique called OptiGrid which is based on constructing an optimal grid-partitioning of the data. The optimal grid-partitioning is determined by calculating the best partitioning hyperplanes for each dimension (if such a partitioning exists) using certain projections of the data. The advantages of our new approach are (1) it has a rm mathematical basis (2) it is by far more e ective than existing clustering algorithms for highdimensional data (3) it is very e cient even for large data sets of high dimensionality. To demonstrate the e ectiveness and e ciency of our new approach, we perform a series of experiments on a number of di erent data sets including real data sets from CAD and molecular biology. A comparison with one of the best known algorithms (BIRCH) shows the superiority of our new approach. Permission to copy without fee all or part of this material is granted provided that the copies are not made or distributed for direct commercial advantage, the VLDB copyright notice and the title of the publication and its date appear, and notice is given that copying is by permission of the Very Large Data Base Endowment. To copy otherwise, or to republish, requires a fee and/or special permission from the Endowment. Proceedings of the 25th VLDB Conference, Edinburgh, Scotland, 1999.

3′‐End processing of pre‐mRNA in eukaryotes

Abstract: 3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3'-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-gamma uses a different signaling pathway.

Abstract: STAT1 is an essential transcription factor for macrophage activation by IFN-γ and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-α occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-γ-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-γ-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-α caused activation of p38 MAPK whereas IFN-γ did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-α production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKα and β but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-γ-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Angiotensin II Induces LOX-1, the Human Endothelial Receptor for Oxidized Low-Density Lipoprotein

Abstract: Background—Oxidatively modified LDL (oxLDL) plays an important role in the development of atherosclerosis. OxLDL effects, eg, foam cell formation, are mediated in part by the classic scavenger receptor, whereas other effects may involve the recently cloned endothelial oxLDL receptor, LOX-1 (lectinlike oxLDL receptor-1), which is distinct from macrophage scavenger receptors. Because the regulation of LOX-1 must still be defined, we investigated whether LOX-1 is regulated by the potentially proatherosclerotic stimulant angiotensin II (Ang II). Methods and Results—Using competitive reverse transcription–polymerase chain reaction (RT-PCR), we quantified mRNA expression of LOX-1 in primary cultures of human umbilical vein endothelial cells (HUVECs). After treatment with Ang II for 3 hours (1 nmol/L to 1 μmol/L), LOX-1 mRNA was concentration-dependently induced (from 6.9±1.4 to 23.1±5.5 relative units [RU] by 1 μmol/L Ang II; P<0.05). The angiotensin II type 1 (AT1) receptor antagonist losartan prevented this i...

Influence of the hypoxic subvolume on the survival of patients with head and neck cancer.

Abstract: Purpose: Tumor hypoxia is regarded as an important factor influencing radiation response, disease-free, and overall survival of patients with squamous cell carcinoma of the head and neck (SCCHN). This study was performed to reevaluate the prognostic significance of the “classical oxygenation parameters” hypoxic fraction (percentage of pO2 values < 5 mmHg or < 2.5 mmHg, respectively) and median pO2, and to determine the influence of a new radiobiological factor. This factor was termed the “hypoxic subvolume” (HSV) and was defined as percentage of pO2-values below 5 mmHg multiplied by the total tumor volume. The rationale of this parameter was to quantify approximately the amount of hypoxic tissue which should be correlated to the number of hypoxic cells in the tumor. It is obvious that a tumor of 100 cm3 with a hypoxic fraction of 20% (HSV = 20 cm3) contains more hypoxic cells than a tumor of 1 cm3 with a hypoxic fraction of 50% (HSV = 0.5 cm3). Methods and Materials: Pretreatment pO2 was assessed in 59 patients with SCCHN with the Eppendorf histograph, and pretreatment volume was determined by ultrasonography (lymphnode metastases) and computer tomography (primaries). All patients were referred to our departments for radiotherapy (n = 27, median dose 70 Gy) or radiochemotherapy (n = 32; 5-FU, mitomycin C, median dose 70 Gy), respectively. All parameters were evaluated using the Kaplan-Meier analysis, and significance was assumed at a p-value of < 0.05 (log-rank test, Cox-Mantel). A multivariate analysis was performed to control for confounding factors. The median follow-up was 233 days. At the time of the evaluation, 34 of the 59 patients were dead. Results: In univariate analyses, the hypoxic fraction (pO2 < 5 mmHg, pO2 < 2.5 mmHg [p < 0.05]), the hemoglobin concentration (p < 0.05), and the hypoxic subvolume (p< 0.01) were of prognostic significance for overall survival. In multivariate analysis, the hemoglobin concentration and the hypoxic subvolume (p = 0.01) were significant prognosticators. We found no significant correlation between tumor volume or median pO2 and overall survival. No clear correlation was found between tumor volume and hypoxic fraction. Conclusion: These data suggest that the total amount of hypoxic tissue, as determined by the hypoxic subvolume, influences the prognosis of patients suffering from SCCHN. In addition, our data confirm the statements of previous studies that low pretherapy pO2-values indicate a worse prognosis.

Optical Measurements of Invasive Forces Exerted by Appressoria of a Plant Pathogenic Fungus

Abstract: Many plant pathogenic fungi, such as the cereal pathogen Colletotrichum graminicola, differentiate highly specialized infection structures called appressoria, which send a penetration peg into the underlying plant cell. Appressoria have been shown to generate enormous turgor pressure, but direct evidence for mechanical infection of plants by fungi is lacking. A microscopic method was developed that uses elastic optical waveguides to visualize and measure forces locally exerted by single appressoria. By this method, the force exerted by appressoria of C. graminicola was found to be about 17 micronewtons.

The Armadillo Family of Structural Proteins

Abstract: The armadillo gene is a segment polarity gene of Drosophila involved in signal transduction through wingless. Since the mid-1980s, a growing number of related proteins have been identified based on sequence homologies. These proteins share a central domain that is composed of a series of imperfect 45 amino acid repeats. Armadillo family members reveal diverse cellular locations reflecting their diverse functions. A single protein exerts several functions through interactions of its armadillo repeat domain with diverse binding partners. The proteins combine structural roles as cell-contact and cytoskeleton-associated proteins and signaling functions by generating and transducing signals affecting gene expression. The study of armadillo family members has made it increasingly clear that a distinction between structural proteins on the one hand and signaling molecules on the other is rather artificial. Instead armadillo family members exert both functions by interacting with a number of distinct cellular-binding partners.

CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34.

Abstract: The Czc system of Ralstonia sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane α-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion of czcD in a Ralstonia sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural czcCBA genes, both in the absence and presence of inducers. The czcD deletion could be fully complemented in trans by CzcD and two other CDF proteins from Saccharomyces cerevisiae, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in Ralstonia, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.

Intestinal Transport of β-Lactam Antibiotics: Analysis of the Affinity at the H+/Peptide Symporter (PEPT1), the Uptake into Caco-2 Cell Monolayers and the Transepithelial Flux

Abstract: Purpose. This study on the intestinal transport of β-lactam antibiotics was undertaken to investigate the correlation between cellular transport parameters and the bioavailability.

Early specification of limb muscle precursor cells by the homeobox gene Lbx1h.

Abstract: During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses1,2. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs3,4,5. Regulatory molecules such as receptor tyrosine kinases like c-Met ( ref. 6) and those encoded by homeobox-containing genes, including c-Met (ref. 6), Tbx1 ( ref. 7), Mox2 (ref. 8), Six1 and Six2 (ref. 9), Pitx2, Pax3 (refs 10,11) and Lbx1h (refs 12,13), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium6,10,11. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevented an analysis of later functions of these genes during limb muscle development14. Based on quail-chick chimaera studies3,15, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Here we show that Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells12,13, led to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect was caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. Our results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles.

Release of carbon and nitrogen compounds by plant roots and their possible ecological importance

Abstract: The root-borne C- and N-flux in the plant/soil system was studied by determining the 14C- or 15N-balances in pot trials with soil as a substrate (14CO2- or 15NH3-application to the shoots, comparison of sterile and nonsterile treatments for quantification of root-borne substances). The following results were obtained: 1. The amount of (primary) root-borne carbon compounds released into soil was (besides root respiration) 11—20% of net-CO2-assimilation or 13—32% of the 14C incorporated into the plants (= 1 t C · ha—1). 5—6% of 15N assimilated by the plants were released as root-borne N compounds (= 15 kg N · ha—1). 2. A considerable portion of the root-borne C (about 6% = 600 kg C · ha—1) was found in the rooted soil zone at the end of the experiments (rhizodeposition). 3. (Primary) root-borne C and N compounds found in immediate vicinity of the roots (about 60—80%) were mainly water soluble, whereas most of the C and N compounds found in a greater distance were water insoluble. The water soluble exudates consisted mainly of neutral (carbohydrates) and acid fractions (organic acids). The basic fraction (amino acids) made up a small portion only. 4. The root-borne C and N compounds influenced the nutrient balance of soil and plant directly and/or indirectly via microbes (depending on species, variety and nutritional status of plants). 5. Microbes stimulated the release of C- and N-compounds, but rapidly respired 65—85% of the root-borne C-compounds, thereby putting a burden on the C-budget of the “host” plant. 6. It could be shown by the example of hup+Rhizobium meliloti strains (tested by 3H2-incorporation) and the wheat-Serratia-association, that energy efficient microbenplant systems can improve plant performance. Freisetzung von C- und N-Verbindungen durch Pflanzenwurzeln und ihre mogliche okologische Bedeutung Mit Hilfe von 14C- bzw. 15N-Bilanzierungsmethoden (14CO2- bzw. 15NH3-Sprosbegasung, Steril/Nichtsterilvergleich zur direkten Quantifizierung wurzelburtiger C-Verbindungen) wurde in Gefasversuchen mit Boden die C- und N-Verteilung im System Pflanze/Boden untersucht. Folgende Resultate sind erzielt worden: 1. Die Menge der freigesetzten (primar) wurzelbartigen C-Verbindungen betrug (ohne die Wurzelatmung) 11—20% der Netto-CO2-Assimilation bzw. 13—32% des 14C-Einbaus in die Pflanze (= 1 t C · ha—1), die der (primar) wurzelburtigen N-Verbindungen 5—6% des 15N-Einbaus in die Pflanze (= 15 kg N · ha—1). 2. Betrachtliche Anteile des wurzelburtigen C (ca. 6% = 600 kg C · ha—1) konnten am Ende der Versuchszeit in der Wurzelumgebung aufgefunden werden (Rhizodeposition). 3. Die in Wurzelnahe vorliegenden (primar) wurzelburtigen C- und N-Verbindungen (etwa 60—80%) lagen vornehmlich in H2O-loslicher Form vor. Die wurzelfernen C-Verbindungen waren im wesentlichen wasserunloslich. Die wasserloslichen Exsudate enthielten vor allem neutrale (Zucker) und saure Fraktionen (organische Sauren). Die basischen Fraktionen (Aminosauren) hatten nur einen geringen Mengenanteil. 4. Die wurzelburtigen C- und N-Verbindungen beeinflusten direkt oder indirekt (uber Mikroben) den Nahrstoffhaushalt von Boden und Pflanze (Abhangigkeit von Art, Sorte und Ernahrungsstatus). 5. Mikroben regten die C- und N-Freisetzung der Pflanzen an, veratmeten aber sehr schnell 65—85% der wurzelburtigen C-Verbindungen und „belasteten” somit den C-Haushalt der „Wirts-pflanze”. 6. Am Beispiel hup-positiver Rhizobium-meliloti-Stamme (Nachweis durch 3H2-Einbau) und der Weizen-Serratia-Assoziation lies sich nachweisen, das energieeffektive Mikroben-Pflanzen-Systeme zur Leistungssteigerung von Pflanzen beitragen konnen.

Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide

Abstract: Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a messenger molecule with multiple clinical implications. Understanding the activation of sGC is an important step for establishing new therapeutic principles. We have now overexpressed sGC in a baculovirus/Sf9 system optimized for high protein yields to facilitate spectral and kinetic studies of the activation mechanisms of this enzyme. It was expressed in a batch fermenter using a defined mixture of viruses encoding the α1 and β1 subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosolic fraction by anion exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography. By use of this new method 2.5 l culture yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. The enzyme did not contain stoichiometric amounts of copper. The basal activities of the purified enzyme were 153 and 1259 nmol min–1 mg–1 in the presence of Mg2+ and Mn2+, respectively. The nitric oxide releasing agent 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fold with Mg2+, whereas the NO-independent activator 3-(5’-hydroxymethyl-2’-furyl)-1- benzylindazole (YC-1) induced an increase in the activity of 101-fold at a concentration of 300 µM. The combination of DEA/NO (10 µM) and YC-1 (100 µM) elicited a dose-dependent synergistic stimulation with a maximum of a 792-fold increase over the basal activity in the presence of Mg2+, resulting in a specific activity of 121 µmol min–1 mg–1. The synergistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 µM) by 94%. In a different experimental setup a saturated carbon monoxide solution in the absence of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme spectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 424 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme spectra were not affected by YC-1 in the absence or in the presence of DEA/NO or of carbon monoxide, which reflects the fact that YC-1 does not interact directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The described method should help to facilitate the investigation of the new therapeutic principle of NO-independent guanylyl cyclase activators.

Joins and intersections

Abstract: In this chapter, we develop the theme of estimating dimensions of joins and sets of tangencies, often under assumptions of connectedness, using results from Chapter 3. In Section 4.1 we study the v-cycle again and show as an application of the connectedness theorem that it has no gaps, and this is interpreted in terms of normal cones. We apply this to linear projections and describe in some cases the structure of such a projection if its image has minimal dimension.

Deviations from the Area Law for Supersymmetric Black Holes

Abstract: We review modifications of the Bekenstein-Hawking area law for black hole entropy in the presence of higher-derivative interactions. In four-dimensional N=2 compactifications of string theory or M-theory these modifications are crucial for finding agreement between the macroscopic entropy obtained from supergravity and the microscopic entropy obtained by counting states in string or M-theory. Our discussion is based on the effective Wilsonian action, which in the context of N=2 supersymmetric theories is defined in terms of holomorphic quantities. At the end we briefly indicate how to incorporate non-holomorphic corrections.

Myocardial Gene Expression of Regulators of Myocyte Apoptosis and Myocyte Calcium Homeostasis During Hemodynamic Unloading by Ventricular Assist Devices in Patients With End-Stage Heart Failure

Abstract: Background —In patients with end-stage heart failure, characterized by an increased susceptibility to cardiomyocyte apoptosis and a labile cardiomyocyte calcium homeostasis, a ventricular assist device (VAD) is implanted for bridging to cardiac transplantation and results in myocardial unloading. Although phenotype changes in the failing heart are assumed to result from hemodynamic overload, the reversibility of these changes under unloading is unknown. Methods and Results —By use of quantitative reverse-transcription polymerase chain reaction, mRNA expression analyses were performed on left ventricular specimens obtained from 10 nonfailing donor hearts (from 8 patients with dilated cardiomyopathy and 2 patients with coronary heart disease) at the time of VAD implantation and 36 to 169 days later during VAD removal with subsequent cardiac transplantation. In terminally failing hearts before VAD support, left ventricular mRNA analyses revealed increased Pro-ANP, reduced antiapoptotic Bcl-x L and antiapoptotic Fas isoform FasExo6Del, and a decreased ratio of sarcoplasmic reticulum Ca 2+ -ATPase per sarcolemmal Na + -Ca 2+ exchanger in comparison with nonfailing ventricles. After VAD unloading, ventricular transcription of Pro-ANP was immediately normalized, and apoptotic DNA fragmentation was attenuated. In patients with dilated cardiomyopathy, mRNAs of Bcl-x L and FasExo6Del/Fas were enhanced depending on time on VAD. The Bcl-x L mRNA level correlated positively with that of the Bcl-x L protein. Transcription of sarcoplasmic reticulum Ca 2+ -ATPase/Na + -Ca 2+ exchanger demonstrated recovery in only 4 of 10 patients. Conclusions —Mechanical support of the failing heart induces a time-dependent change in myocardial gene expression compatible with a decreased susceptibility to apoptosis.

Poly(vinyl alcohol)/poly(acrylic acid) hydrogels: FT-IR spectroscopic characterization of crosslinking reaction and work at transition point

Abstract: The crosslinking reaction of pure poly(acrylic acid) and its blend with poly(vinyl alcohol) was studied by FT-IR spectroscopy. It is demonstrated that also in blends the anhydride formation characteristic for pure poly(acrylic acid) is the predominant crosslinking reaction upon heating. But the ester formation between poly(vinyl alcohol) and poly(acrylic acid) is detectable due to the ester C=O vibrations and C—O—C vibrations, respectively. The degree of swelling and the Young's modulus of the crosslinked blend in deionized water depend on the time and temperature of the heat treatment. In dependence of the pH-value of the swelling agent the blends swell or shrink. The working energy at the shrinking or swelling process induced by a change of the pH-value of differently treated blends was measured. The values are in a range of technical interests and comparable with other microactuators.