Biomedical subjects

Anja Hoffmann

Publications and source records attributed to Anja Hoffmann.

15 recordsLinked to original sources

PubMed2006

The lantibiotic mersacidin is an autoinducing peptide.

The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.

Anti-Bacterial Agents↗

PubMed2006

The C-terminal domain of Escherichia coli trigger factor represents the central module of its chaperone activity.

In bacteria, ribosome-bound Trigger Factor assists the folding of newly synthesized proteins. The N-terminal domain (N) of Trigger Factor mediates ribosome binding, whereas the middle domain (P) harbors peptidyl-prolyl isomerase activity. The function of the C-terminal domain (C) has remained enigmatic due to structural instability in isolation. Here, we have characterized a stabilized version of the C domain (C(S)), designed on the basis of the recently solved atomic structure of Trigger Factor. Strikingly, only the isolated C(S) domain or domain combinations thereof (NC(S), PC(S)) revealed substantial chaperone activity in vitro and in vivo. Furthermore, to disrupt the C domain without affecting the overall Trigger Factor structure, we generated a mutant (Delta53) by deletion of the C-terminal 53 amino acid residues. This truncation caused the complete loss of the chaperone activity of Trigger Factor in vitro and severely impaired its function in vivo. Therefore, we conclude that the chaperone activity of Trigger Factor critically depends on its C-terminal domain as the central structural chaperone module. Intriguingly, a structurally similar module is found in the periplasmic chaperone SurA and in MPN555, a protein of unknown function. We speculate that this conserved module can exist solely or in combination with additional domains to fulfill diverse chaperone functions in the cell.

Binding Sites↗

PubMed2006

Trigger factor forms a protective shield for nascent polypeptides at the ribosome.

In prokaryotes, the ribosome-associated Trigger Factor is the first chaperone newly synthesized polypeptides encounter when they emerge from the ribosomal exit tunnel. The effects that Trigger Factor exerts on nascent polypeptides, however, remain unclear. Here we analyzed the potential of the Trigger Factor to shield nascent polypeptides at the ribosome. A set of arrested nascent polypeptides differing in origin, size, and folding status were synthesized in an Escherichia coli-based in vitro transcription/translation system and tested for sensitivity to degradation by the unspecific protease proteinase K. In the absence of Trigger Factor, nascent polypeptides exposed outside the ribosomal exit tunnel were rapidly degraded unless they were folded into a compact domain. The presence of Trigger Factor, as well as a Trigger Factor fragment lacking its peptidyl-prolyl isomerase domain, counteracted degradation of all unfolded nascent polypeptides tested. This protective function was specific for ribosome-tethered Trigger Factor, since neither non-ribosomal Trigger Factor nor the DnaK system, which cooperates with Trigger Factor in the folding process in vivo, revealed a comparable efficiency in protection. Furthermore, shielding by Trigger Factor was not restricted to short stretches of nascent chains but was evident for large, non-native nascent polypeptides exposing up to 41 kDa outside the ribosome. We suggest that Trigger Factor supports productive de novo folding by shielding nascent polypeptides on the ribosome thereby preventing untimely degradation or aggregation processes. This protected environment provided by Trigger Factor might be particularly important for large multidomain proteins to fold productively into their native states.

Escherichia coli↗

PubMed2006

Calcium ions are involved in the delay of plant cell cycle progression by abiotic stresses.

Higher plants respond to environmental stresses by a sequence of reactions which include the reduction of growth by affecting cell division. It has been shown that calcium ions plays a role as a second messenger in mediating various defence responses under environmental stresses. In this study, the role of calcium ions on cell cycle progression under abiotic stresses has been examined in tobacco BY-2 suspension culture cells. Using synchronized BY-2 cells expressing the endogenous calcium sensor aequorin as experimental system, we could show that oxidative and hypoosmotic stress both induce an increase of intracellular calcium and cause a delay of the cell cycle. The inhibitory effect of these abiotic stress stimuli on cell cycle progression could be mimicked by increasing the intracellular calcium concentration via application of an external electrical field. Likewise, depletion of calcium ions in the culture medium suppressed the effect of the stimuli tested. These results demonstrate that calcium signalling is involved in the regulation of cell cycle progression in response to abiotic stress.

Calcium↗

PubMed2006

Evaluation of patient satisfaction after therapy of unilateral clefts of lip, alveolus and palate.

INTRODUCTION: Cleft lip, alveolus and palate (CLAP) is a craniofacial abnormality and is one of the most frequent human developmental anomalies. Therapy of clefts does not only comprise surgical closure of the cleft, but rather aims at an aesthetically and functionally optimal result at adult age. MATERIAL AND METHODS: Thirty-three cleft patients with total clefts of lip, alveolus and palate were enrolled in this study. Osseous bridging of the alveolar cleft (osteoplasty) was performed in all patients followed by different types of subsequent treatment. All patients answered a questionnaire to assess their satisfaction with the treatment result and their facial appearance. Patient satisfaction was correlated to the type of alveolar cleft repair. RESULTS: The returned questionnaires revealed varying patient satisfaction with their appearance, occlusal conditions, and dental aesthetics depending on the type of dental treatment in the alveolar cleft area. Questionnaire analysis by gender revealed clear gender-dependent differences in self-rated satisfaction. CONCLUSION: Aesthetics gain increasing importance for self-perception. Therefore, patient satisfaction with her facial appearance should move even more into focus of therapy of clefts.

Adolescent↗

PubMed2005

Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing.

Small Rho GTPases are key regulators of the cytoskeleton in a great variety of cells. Rho function mediates morphological changes as well as locomotor activity. Using astrocyte cultures established from neonatal mice we investigated the role of Rho in process formation during astrocyte stellation. Using a scratch-wound model, we examined the impact of Rho on a variety of morphological and functional variables such as stellation and migratory activity during wound healing. C3 proteins are widely used to study cellular Rho functions. In addition, C3 derived from Clostridium botulinum (C3bot) is considered selectively to promote neuronal regeneration. Because the latter requires a balanced activity of neurones and glial cells, the effects of C3 protein on glial cells such as astrocytes have to be considered carefully. Low nanomolar concentrations of C3 proteins significantly promoted process outgrowth and increased process branching. Besides enzymatic inactivation of Rho by ADP-ribosylation, changes in protein levels of the various Rho GTPases may also contribute to the observed effects. Furthermore, incubation of scratch-wounded astrocyte cultures with C3bot accelerated wound healing. By inhibiting the Rho downstream effector ROCK with the selective inhibitor Y27632 we were able to demonstrate that the accelerated wound closure resulted from both enhanced polarized process formation and increased migratory activity of astrocytes into the lesion site. These results suggest that Rho negatively regulates astrocytic process growth and migratory responses after injury and that its inactivation by C3bot in nanomolar concentrations promotes astrocyte migration.

Amides↗

PubMed2005

Zinc ions are endogenous modulators of neurotransmitter-stimulated capacitative Ca2+ entry in both cultured and in situ mouse astrocytes.

Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.

Animals↗

PubMed2004

Dynamic rearrangements of transvacuolar strands in BY-2 cells imply a role of myosin in remodeling the plant actin cytoskeleton.

Plant cells typically contain a large central vacuole that confines the cytoplasm and organelles to the periphery of the cell and the vicinity of the nucleus. These two domains are often connected by transvacuolar strands (TVS), thin tubular structures that traverse the vacuole. The TVS are thought to act as important transport routes for the distribution of organelles and metabolites, and also to play a role in the positioning of the nucleus. Most TVS depend on internal actin filaments for their existence, and rearrangements of TVS can therefore indicate modifications in the actin cytoskeleton. In this study we describe time-lapse observations of tobacco BY-2 suspension-cultured cells that document the dynamic behavior of TVS. The TVS formed, branched, and collapsed, and their attachment points in the nuclear or cortical cytoplasm, as well as on other TVS, moved around. These dynamic rearrangements were inhibited within 5 min by the myosin inhibitor 2,3-butanedione monoxime (BDM). In particular, the movements of TVS attachment points and the variations in TVS length were significantly reduced in the presence of the drug. Similarly, movements of the nucleus were reduced by two thirds in BDM-treated cells. The number of TVS, together with the number of attachment and branch points, also dropped during BDM treatment. All effects of BDM on TVS dynamics were reversible upon removal of the drug. These results suggest a role for myosin motors in the rearrangement of TVS, which is likely to occur through their interaction with actin filaments.

Actin Cytoskeleton↗

PubMed2004

ZnT-1 expression in astroglial cells protects against zinc toxicity and slows the accumulation of intracellular zinc.

Zinc ions are emerging as an important factor in the etiology of neurodegenerative disorders and in brain damage resulting from ischemia or seizure activity. High intracellular levels of zinc are toxic not only to neurons but also to astrocytes, the major population of glial cells in the brain. In the present study, the role of ZnT-1 in reducing zinc-dependent cell damage in astrocytes was assessed. Zinc-dependent cell damage was apparent within 2 h of exposure to zinc, and occurred within a narrow range of approximately 200 microM. Pretreatment with sublethal concentrations of zinc rendered astrocytes less sensitive to toxic zinc levels, indicating that preconditioning protects astrocytes from zinc toxicity. Fluorescence cell imaging revealed a steep reduction in intracellular zinc accumulation for the zinc-pretreated cells mediated by L-type calcium channels. Heterologous expression of ZnT-1 had similar effects; intracellular zinc accumulation was slowed down and the sensitivity of astrocytes to toxic zinc levels was reduced, indicating that this is specifically mediated by ZnT-1 expression. Immunohistochemical analysis demonstrated endogenous ZnT-1 expression in cultured astroglia, microglia, and oligodendrocytes. Pretreatment with zinc induced a 4-fold increase in the expression of the putative zinc transporter ZnT-1 in astroglia as shown by immunoblot analysis. The elevated ZnT-1 expression following zinc priming or after heterologous expression of ZnT-1 may explain the reduced zinc accumulation and the subsequent reduction in sensitivity toward toxic zinc levels. Induction of ZnT-1 may play a protective role when mild episodes of stroke or seizures are followed by a massive brain insult.

Animals↗

PubMed2004

Microglia express GABA(B) receptors to modulate interleukin release.

gamma-Aminobutyric acid (GABA) can act as a neuroprotective agent besides its well-established role as the main inhibitory neurotransmitter in the CNS. Here we report that microglial cells express GABA(B) receptors indicating that these prominent immunocompetent cells in the brain are a target for GABA. Agonists of GABA(B) receptors triggered the induction of K(+) conductance in microglial cells from acute brain slices and in culture. Both subunits of GABA(B) receptors were identified in cultured microglia by Western blot analysis and immunocytochemistry, and were detected on a subpopulation of microglia in situ by immunohistochemistry. In response to facial nerve axotomy, we observed an increase in GABA(B) receptor expressing microglial cells in the facial nucleus. We activated microglial cells in culture with lipopolysaccharide (LPS) to induce the release of interleukin-6 and interleukin-12p40. This release activity was attenuated by simultaneous activation of the GABA(B) receptors indicating that GABA can modulate the microglial immune response.

Animals↗

PubMed2004

The tyrosine kinase inhibitor AG126 restores receptor signaling and blocks release functions in activated microglia (brain macrophages) by preventing a chronic rise in the intracellular calcium level.

We recently reported that lasting activation of mouse microglial cells with bacterial lipopolysaccharide (LPS) chronically elevated the basal intracellular calcium concentration ([Ca2+]i). This correlated to an attenuated calcium signaling of complement (C5a) and purinergic (UTP) receptors as well as to the capacity for effective production of cytokines-chemokines. Here, we demonstrate that these adjustments in the [Ca2+]i regulation require a critical protein tyrosine kinase (PTK) function--even in varying stimulation scenarios. Changes in basal [Ca2+]i and calcium signaling are not restricted to Gram-negative bacterial confrontation. Pneumococcal cell wall (PCW) modelling Gram-positive infection causes virtually the same effects. Moreover, decreases in calcium signaling efficacy are neither associated with altered receptor expression, nor mediated by autocrine loops. Administration of microglial release products, transfer of conditioned supernatant or presence of a radical scavenger during LPS or PCW treatments have no consequence. However, both the elevation in basal [Ca2+]i as well as the suppression of C5a- and UTP-evoked calcium signals are selectively and dose-dependently reversed by tyrphostin AG126, a PTK inhibitor that, moreover, blocks inducible nitric oxide and cytokine-chemokine release. The findings suggest that the AG126-sensitive PTK critically controls both sensory and executive features of the microglial activation process via sustained up-regulation of basal [Ca2+]i.

Animals↗

PubMed2004

Localization and functional analysis of PepI, the immunity peptide of Pep5-producing Staphylococcus epidermidis strain 5.

Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.

Amino Acid Sequence↗

PubMed2003

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia.

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H2O2), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1-5 mM H2O2 for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H2O2 and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H2O2 in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia.

Adenosine Diphosphate Ribose↗

PubMed2003

Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release function.

Microglia-brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia.

Animals↗

PubMed2002

Combination of antibiotic mechanisms in lantibiotics.

Recent studies on the mode of action have revealed exciting features of multiple activities of nisin and related lantibiotics making these peptides interesting model systems for the design of new antibiotics (Molec. Microbiol. 30 (1998) 317; Science 286 (1999) 2361; J. Biol. Chem. 276 (2001) 1772.). In contrast to other groups of antibiotic peptides, the lantibiotics display a substantial degree of specificity for particular components of bacterial membranes. Mersacidin and actagardine were shown to bind with high affinity to the lipid coupled peptidoglycan precursor, the so-called lipid II, which prevents the polymerisation of the cell wall monomers into a functional murein sacculus. The lantibiotics nisin and epidermin also bind tightly to this cell wall precursor; however, for these lantibiotics the binding of lipid II has two consequences. Like with mersacidin blocking of lipid II inhibits peptidoglycan biosynthesis; in addition, lipid II is used as a specific docking molecule for the formation of pores. This combination of lethal effects explains the potency of these peptides, which are active in nanomolar concentration. Other type-A lantibiotics are believed to also use docking molecules for pore formation, although identification of such membrane components has not yet been achieved.

Amino Acid Sequence↗