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Biomedical subjects

K H Krause

Publications and source records attributed to K H Krause.

At least 19 recordsLinked to original sources
PubMed2004

[Infections in the elderly].

Demographic aging is now well established. Thus, infections in the elderly will continue to become an increasingly important public health issue. Infections are not only more frequent in the elderly, but they also have distinct properties. Infectious diseases of the elderly are distinct with respect to epidemiology, clinical presentation, treatment, and outcome. Theses specificities are explained through a variety of factors, including the ageing process itself, but also comorbidities, sociological and environmental factors. Geriatric infectious diseases is likely to develop as an increasingly important branch of geriatrics and infectious disease.

Adult↗
PubMed2001

Functional specialization of calreticulin domains.

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Animals↗
PubMed2001

A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes.

Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91(phox) subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca(2+) concentration it generated large amounts of superoxide. Upon Ca(2+) activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H(+) channel in a Ca(2+)-dependent manner. NOX5 is likely to be involved in Ca(2+)-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.

Amino Acid Sequence↗
PubMed2001

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase.

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.

Animals↗
PubMed2001

Alternative splice variants of hTrp4 differentially interact with the C-terminal portion of the inositol 1,4,5-trisphosphate receptors.

The molecular basis of capacitative (or store-operated) Ca2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store-operated Ca2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N-terminal region of the inositol 1,4,5-triphosphate receptors (InsP3R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as alpha-hTrp4 and beta-hTrp4. The shorter variant beta-hTrp4 is generated through alternative splicing and lacks the C-terminal amino acids G785-S868. Using a yeast two-hybrid assay and glutathione-S-transferase-pulldown experiments, we found that the C-terminus of alpha-hTrp4, but not of beta-hTrp4, associates in vitro with the C-terminal domain of the InsP(3) receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP3R and we provide evidence suggesting that the formation of hTrp4-InsP3R complexes may be regulated by alternative splicing.

Alternative Splicing↗
PubMed2001

Identification of BARD1 as mediator between proapoptotic stress and p53-dependent apoptosis.

The BRCA1-associated protein BARD1 is a putative tumor suppressor. We suggest that BARD1 is a mediator of apoptosis since (1) cell death in vivo (ischemic stroke) and in vitro is accompanied by increased levels of BARD1 protein and mRNA; (2) overexpression of BARD1 induces cell death with all features of apoptosis; and (3) BARD1-repressed cells are defective for the apoptotic response to genotoxic stress. The proapoptotic activity of BARD1 involves binding to and elevations of p53. BRCA1 is not required for but partially counteracts apoptosis induction by BARD1. A tumor-associated mutation Q564H of BARD1 is defective in apoptosis induction, thus suggesting a role of BARD1 in tumor suppression by mediating the signaling from proapoptotic stress toward induction of apoptosis.

Animals↗
PubMed2001

Effects of heat and cold application on turns and amplitude in surface EMG.

The purpose of this study was to investigate the effects of cooling and heating of muscles on parameters of surface EMG (SEMG) under various well defined grades of isometric muscle contraction. In 32 healthy volunteers, aged 20-30 years, turns and amplitude (RMS) analysis was done in SEMG from the hand extensors. Muscle strength was defined by a new developed hand dynamometer. Values for RMS and turns in case of maximal voluntary contraction (MVC) over periods of 20 sec were determined for each volunteer and subsequently RMS and turns were measured during isometric contractions of 10%, 30%, 50% and 80% of MVC, interrupted each by rest periods. This procedure was repeated after cooling and warming the forearm with thermo packs. For control purposes the same investigations with three measurement periods of SEMG were done following the same time schedule without thermic stimuli. With increasing isometric contractions turns increased after heat application and decreased after ice application, compared to the values achieved without thermic stimuli. The differences were significant from a level of 30% of MVC (p < 0.05). No corresponding changes were seen for RMS. In the control experiment values of turns and RMS did not differ in the three measurement periods. It is concluded, that the amplitude in SEMG tracings seems to be a reliable parameter of muscle force, whereas turns are sensitive to temperature. They might be useful markers for therapeutic approaches targeted at the muscle.

Adult↗
PubMed2000

Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils.

Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn-) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn- neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn- neutrophils was reduced ( approximately 50%) but not to the same extent as ingestion ( approximately 73%). This was not due to reduced surface expression of the Fcgamma-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn- neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton.

Actins↗
PubMed2000

Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum.

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.

Animals↗
PubMed2000

Increased striatal dopamine transporter in adult patients with attention deficit hyperactivity disorder: effects of methylphenidate as measured by single photon emission computed tomography.

Ten previously untreated adults with attention deficit hyperactivity disorder (ADHD) were investigated before and after 4 weeks of treatment with a dose of 3x5 mg methylphenidate/d by single photon emission computed tomography (SPECT) with [Tc-99m]TRODAT-1, the first Tc-99m labelled SPECT ligand specifically binding to the dopamine transporter (DAT). For semiquantitative evaluation of the DAT, specific binding ([STR-BKG]/BKG) was calculated in the striatum (STR) with the cerebellum used as background (BKG). The patients with ADHD presented with increased specific binding of Tc-99m-TRODAT-1 to the DAT as compared with age and sex matched controls ([STR-BKG]/BKG 1.43+/-0.18 vs. 1.22+/-0.05, P<0.001). After treatment with methylphenidate specific binding decreased in all patients ([STR-BKG]/BKG 1.02+/-0.23, P<0.001). Thus, for the first time it could be demonstrated using SPECT that methylphenidate lowers increased striatal DAT availability in adults suffering from ADHD.

Adult↗
PubMed2000

Professional phagocytes: predators and prey of microorganisms.

Phagocytosis is an ancient cellular function. However, professional phagocytes have evolved only in higher organisms, where they play an important role in host defence. Professional phagocytes are capable of engulfing relatively large microorganisms and killing them with a combination of various microbicidal systems. Crucial killing mechanisms of phagocytes include superoxide generation by phagocyte NADPH oxidase and release of microbicidal proteins through exocytosis of performed granules. Phagocytes are also able to interfere with microbial growth through alteration of the phagosomal ionic environment (acidification, iron depletion). While the microbicidal mechanisms of phagocytes are extremely efficient and capable of killing most microorganisms, pathogenic microorganisms have developed mechanisms to resist phagocytes. Microorganisms capable of surviving within phagocytes are rare, but represent very successful pathogens, such as Mycobacterium tuberculosis. Other pathogens, such as S. aureus, have developed strategies to evade phagocytosis. How microorganisms are phagocytosed and killed, and why certain pathogens resist these mechanisms, are crucial questions for an understanding of the pathogenesis of infectious diseases and the development of innovative treatment approaches.

Humans↗
PubMed2000

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).

Alternative Splicing↗
PubMed2000

Attention deficit hyperactivity disorder: binding of [99mTc]TRODAT-1 to the dopamine transporter before and after methylphenidate treatment.

Involvement of the dopaminergic system has been suggested in patients suffering from attention deficit hyperactivity disorder (ADHD) since the symptoms can be successfully treated with methylphenidate, a potent blocker of the dopamine transporter (DAT). This study reports the findings on the status of the DAT in adults with ADHD before and after commencement of treatment with methylphenidate, as measured using [99mTc]TRODAT-1. Seventeen patients (seven males, ten females, aged 21-64 years, mean 38 years) were examined before and after the initiation of methylphenidate treatment (3x5 mg/day). All subjects were injected with 800 MBq [99mTc]TRODAT-1 and imaged 3 h p.i. Single-photon emission tomography (SPET) scans were acquired using a triple-headed gamma camera. For semiquantitative evaluation of the DAT, transverse slices corrected for attenuation were used to calculate specific binding in the striatum, with the cerebellum used as background [(STR-BKG)/BKG]. Data were compared with an age-matched control group. It was found that untreated patients presented with a significantly increased specific binding of [99mTc]TRODAT-1 to the DAT as compared with normal controls [(STR-BKG)/BKG: 1.43+/-0.18 vs 1.22+/-0.06, P<0.001]. Under treatment with methylphenidate, specific binding decreased significantly in all patients [(STR-BKG)/BKG: 1.00+/-0.14, P<0.001]. Our findings suggest that the number of DAT binding sites is higher in drug-naive patients suffering from ADHD than in normal controls. The decrease in available DAT binding sites under treatment with methylphenidate correlates well with the improvement in clinical symptoms. The data of this study help to elucidate the complex dysregulation of the dopaminergic neurotransmitter system in patients suffering from ADHD and the effect of treatment with psychoactive drugs.

Adult↗
PubMed2000

Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells.

Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell. Microbiol. 1:101-117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.

Adhesins, Bacterial↗
PubMed1999

Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells.

Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN(347-361) (the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (tau = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (tau = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN(347-361), demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions.

Cells, Cultured↗
PubMed1999

The HIV Nef protein alters Ca(2+) signaling in myelomonocytic cells through SH3-mediated protein-protein interactions.

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca(2+) signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef((PXXP)4(-))). In differentiated HL60 cells, Nef markedly altered cellular Ca(2+) signaling; the amount of intracellularly stored Ca(2+) was increased, and as a consequence, store-operated Ca(2+)-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca(2+) signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59(hck) co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca(2+) stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca(2+) stores through SH3-mediated interactions in myelomonocytic cells.

Calcitriol↗