Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1.

Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma, after which microglial cells migrate towards the sites of injury. At these sites, the cells produce large quantities of oxygen radicals and cause secondary damage that accounts for most of the loss of brain function. Here we show that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, regulated by the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) through the formation of a nuclear PARP-NF-kappaB-protein complex. Downregulation of PARP or CD11a by transfection with antisense DNA abrogated microglial migration almost completely and prevented neurons from secondary damage.

Rab11 regulates recycling through the pericentriolar recycling endosome.

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

Cells´ Flow and Immune Cell Priming under alternating g-forces in Parabolic Flight.

Gravitational stress in general and microgravity (µg) in particular are regarded as major stress factors responsible for immune system dysfunction in space. To assess the effects of alternating µg and hypergravity (hyper-g) on immune cells, the attachment of peripheral blood mononuclear cells (PBMCs) to adhesion molecules under flow conditions and the antigen-induced immune activation in whole blood were investigated in parabolic flight (PF). In contrast to hyper-g (1.8 g) and control conditions (1 g), flow and rolling speed of PBMCs were moderately accelerated during µg-periods which were accompanied by a clear reduction in rolling rate. Whole blood analyses revealed a "primed" state of monocytes after PF with potentiated antigen-induced pro-inflammatory cytokine responses. At the same time, concentrations of anti-inflammatory cytokines were increased and monocytes displayed a surface molecule pattern that indicated immunosuppression. The results suggest an immunologic counterbalance to avoid disproportionate immune responses. Understanding the interrelation of immune system impairing and enhancing effects under different gravitational conditions may support the design of countermeasures to mitigate immune deficiencies in space.

Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor.

Cholesterol entering cells in low-density lipoproteins (LDL) via receptor-mediated endocytosis is transported to organelles of the late endocytic pathway for degradation of the lipoprotein particles. The fate of the free cholesterol released remains poorly understood, however. Recent observations suggest that late-endosomal cholesterol sequestration is regulated by the dynamics of lysobisphosphatidic acid (LBPA)-rich membranes [1]. Genetic studies have pinpointed a protein, Niemann-Pick C-1 (NPC-1), that is required for the mobilization of late-endosomal/lysosomal cholesterol by an unknown mechanism [2]. Here, we report the removal of accumulated cholesterol by overexpression of the NPC-1 protein in NPC-1-deficient fibroblasts from patients with Niemann-Pick disease, and in normal fibroblasts upon release of a progesterone-induced block of cholesterol transport. We show that late-endosomal/lysosomal cholesterol mobilization is specifically inhibited by microinjection of Rab GDP-dissociation inhibitor (Rab-GDI). Moreover, clearance of the cholesterol deposits by NPC-1 in patients' fibroblasts is accompanied by the redistribution of LBPA and of a lysosomal hydrolase that utilizes the mannose-6-phosphate receptor. Our results reveal, for the first time, the involvement of a specific molecular component of the membrane-trafficking machinery in cholesterol transport and the coupling of late-endosomal cholesterol egress to the trafficking of other lipid and protein cargo.

Proteasomal degradation of oxidatively damaged endogenous histones in K562 human leukemic cells.

A number of antitumor drugs act via the oxidation of nuclear material in the tumor cell. It is therefore important to know if tumor cells can effectively and precisely cope not only with oxidatively induced DNA damage, but also with nuclear protein oxidation. In this study, we investigated the endogenous degradation of oxidatively damaged histones in K562 human leukemic cells after oxidative challenge and demonstrated a link to the overall cellular stress response pathways by poly-ADP-ribose-polymerase (PARP). After an oxidative challenge, endogenous nuclear protein degradation, as well as histone degradation, was enhanced. Among the histone fractions, histone H1 revealed the highest degradation rate, and more than 85% of the total degraded H1 disappeared in the first 30 min after oxidative challenge. Short-term degradation of histones up to 30 min, as well as long-term degradation up to 48 h after oxidative challenge, was significantly reduced in the presence of the PARP inhibitor 3-aminobenzamide, and nearly completely abrogated by the selective proteasome inhibitor lactacystin. Immunoprecipitation experiments indicated that the proteasome specifically degraded oxidized histones. Thus, we show that the nuclear proteosome system in tumor cells is capable of preventing the accumulation of oxidized proteins in this compartment and may suggest further treatment strategies to effectively interfere with the protein "repair" and replacement strategies of tumor cells.

Proteasome activation by poly-ADP-ribose-polymerase in human myelomonocytic cells after oxidative stress.

Cytotoxic action of a variety of antitumor drugs generate oxidatively modified proteins that are predominantly metabolized via the proteasome. In the present study, a differentiation-retrodifferentiation cell system was exposed to oxidative stress by hydrogen peroxide treatment. Thus, the activity of the nuclear proteasome in proliferating human U937 leukemic cells increased by 2.5-fold after hydrogen peroxide treatment. In contrast, growth-arrested differentiated U937 cells demonstrated 40% less constitutive proteasomal activity, which was not inducible after hydrogen peroxide exposure. After a retrodifferentiation process, however, in which differentiated U937 cells resume autonomous growth again, the proteasomal activity was indistinguishable from that in U937 control cells, both constitutively and after induction of oxidative stress. Moreover, cells of TUR, a differentiation-resistant U937 subclone, expressed an elevated constitutive proteasomal activity that increased by 2.5-fold after oxidative stress. Immunoblot analysis revealed that these differences in proteasomal activities did not correlate with proteasome protein expression but with protein levels of the nuclear enzyme poly-ADP-ribose-polymerase (PARP). Further studies using specific PARP inhibitors revealed that the noninducible proteasome activity in differentiated U937 cells was PARP independent, whereas the increased activity level in oxidatively stressed TUR cells was downregulated upon PARP inhibition. Immunoprecipitation experiments demonstrated a protein-protein interaction of the functional active PARP with the proteasome in correlation with the proteasome activity. Similar results were obtained by analyzing protein carbonyls after oxidative stress. Taken together, these data suggest that proliferating, rather than growth-arrested, cells metabolize oxidatively damaged nuclear proteins via the proteasome by expressing high levels of PARP.

Intracellular metabolism of 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.

The intracellular metabolism of 4-hydroxynonenal (HNE), a secondary product of lipid peroxidation and mediator of inflammation, which was found in the joints of patients with rheumatoid arthritis, was investigated in primary cultures of rabbit synovial fibroblasts. A consumption rate of 27.3 nmol/min x 10(6) cells was measured for the cultivated fibroblasts. It could be shown, that 4-hydroxynonenal enters the synovial fibroblasts and is metabolized mainly oxidatively to 4-hydroxynonenoic acid, intermediates of the tricarboxylic acid cycle and water and by formation of the glutathione-HNE adduct. The share of protein-bound HNE was about up to 8% of the total added HNE after 10 min of incubation. All metabolites accumulates intracellularly within the incubation time except of 4-hydroxynonenal itself. An increase of 4-hydroxynonenoic acid could be detected also extracellularly during the intracellular metabolism of 4-hydroxynonenal. Therefore, an involvement of synovial fibroblasts in the secondary antioxidant defense system of the joints during conditions of higher HNE concentrations like rheumatoid arthritis is suggested.

Degradation of hypochlorite-damaged glucose-6-phosphate dehydrogenase by the 20S proteasome.

Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by mitochondria isolated from rat kidney cortex.

The cytosolic lipid peroxidation product 4-hydroxynonenal (HNE) is rapidly metabolized in mitochondria isolated from rat kidney cortex. About 80% of HNE was degraded within 3 min of incubation. Main products of HNE which were identified in mitochondria were the hydroxynonenoic acid, the 1,4-dihydroxynonene and the glutathione-HNE-conjugate. Furthermore, formation of metabolites of the tricarboxylic acid cycle from HNE is suggested. The quantitative share of HNE binding to proteins was high with about 8% of total HNE consumption after 3 min of incubation. Therefore, rapid degradation of HNE by mitochondria might be involved in an intracellular antioxidative defense system.

Dose- and wavelength-dependent oxidation of crystallins by UV light--selective recognition and degradation by the 20S proteasome.

The lens of the human eye is a suitable model for age-related alterations at the molecular level. Age-related cataract formation is closely related to the accumulation of oxidatively altered proteins. In this study the influence of UV-A, UV-B, and UV-C irradiation on the proteolytic susceptibility of alpha-, betaL-, and betaH-crystallins by the isolated 20S proteasome was investigated. The proteins were irradiated with 280, 300, and 350 nm monochromatic light. Changes of the physical properties of the crystallins were characterized by absorbance measurements at 280 nm, fluorescence spectra, and SDS-PAGE-electrophoresis. The proteolytic susceptibility of crystallins was maximal after irradiation at 280 nm and three- to fivefold lower at 300 nm. Irradiation at 350 nm was not able to initiate proteolysis, probably due to protein-aggregate formation of higher molecular weight, as shown by SDS-PAGE. The damage of crystallins by UV-C light might be a signal for its proteolytic degradation by the 20S proteasome, whereas UV-B and UV-A do not increase the proteolytic susceptibility to the same extent but promote the formation of crosslinked proteins. Therefore, irradiation with UV, which is not followed by an increase in the proteolytic susceptibility, is accompanied by the formation of crosslinked proteins. It was concluded, that also long UV-B and UV-A may be involved in age-related alterations of the human lens and cataract formation.

The effect of the lipid peroxidation product 4-hydroxynonenal and of its metabolite 4-hydroxynonenoic acid on respiration of rat kidney cortex mitochondria.

In rat kidney cortex mitochondria, 4-hydroxynonenal inhibits state 3 respiration as well as uncoupled respiration at micromolar concentrations. The inhibition is more distinct for NAD-linked than for FAD-linked respiration. 4-Hydroxynonenal increases the state 4 respiration. It is assumed that 4-hydroxynonenal behaves like a decoupling agent. 4-Hydroxynonenal augments the inhibitory effect of 2,4-dinitrophenol observed at superoptimal concentrations. 4-Hydroxynonenal is metabolised by renal mitochondria, and 4-hydroxynonenoic acid is one of the metabolites generated. This metabolite is without effect on respiration at concentrations up to 50 microM. Therefore, the effect of 4-hydroxynonenal on respiration is not mediated by this fatty acid derivative formed during respiratory measurements.

Dynamic and three-dimensional transcranial ultrasonography of an arachnoid cyst in the cerebral convexity. Technical note.

Structural imaging of the brain, such as cerebral computerized tomography (CT) and magnetic resonance (MR) imaging, is state-of-the-art. Dynamic transcranial (dTC) ultrasonography and three-dimensional (3D) transcranial color-coded duplex (TCC) ultrasonography are complementary, noninvasive procedures with the capacity for real-time imaging, which may aid in the temporary management of space-occupying lesions. A 16-year-old woman presented with recurrent tension-type headaches. A space-occupying arachnoid cyst in the cerebral convexity was demonstrated on MR images. The patient underwent an examination for raised intracranial pressure. which was performed using a standard color-coded duplex ultrasonography system attached to a personal computer-based system for 3D data acquisition. Transcranial ultrasonography was used to identify the outer arachnoid membrane of the cyst, which undulated freely in response to rotation of the patient's head (headshake maneuver). Three-dimensional data sets were acquired and, using a multiplanar reformatting reconstruction algorithm, the authors obtained high-resolution images that corresponded to the initial MR image and a follow-up cranial CT scan. No detectable differences were observed on dTC or 3D TC ultrasonograms obtained at follow-up examinations performed 9 and 28 months later. Three-dimensional TCC and dTC ultrasonography may complement conventional diagnostic procedures such as MR and CT imaging. This report represents evidence of the high resolution and good reproducibility of 3D TC methods. Ultrasonography is a mobile and inexpensive tool and may be used to improve management and therapeutic strategies for patients with space-occupying brain lesions in selected cases.

Deep hypothermia and circulatory arrest for surgery of complex intracranial aneurysms.

OBJECTIVE: Some intracranial aneurysms may not be operable by conventional neurosurgery due to their location or morphology. Cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest renders surgery of these complex aneurysms possible. Brain temperatures can be measured directly in this setting. METHODS: Eight patients with complex intracranial aneurysms were operated on with the aid of CPB. Femoro-femoral bypass with heparin-coated circuit components was used in all cases. Venous drainage was augmented by a centrifugal pump in six patients and by a newly developed vacuum technique in two patients. Temperatures were monitored by probes in brain, tympanum, nasopharynx, bladder, rectum, arterial and venous blood. These measurements were recorded on-line together with those of cerebral oxygen saturation, AP, CVP and PAP. Blood gas analyses and an EEG were also performed continuously. RESULTS: Outcome was excellent in seven patients, in one patient moderate neurological disability occurred. Mean time on cardiopulmonary bypass was 160 (117-215) min, for cooling to a brain temperature of 18 degrees C 33 (20-47) min, and for total circulatory arrest 27 (15-45) min. Additionally, terminal brain arteries were clamped for up to 68 min in four patients. No cardiac complications were observed. Actual brain temperatures were best reflected by the tympanum probes (max. deviation 2 degrees C), whereas temperatures measured in bladder or rectum exhibited deviations of up to 10 degrees C. EEG activities were arrested between brain temperatures of 19 and 26 degrees C. CONCLUSIONS: Complex intracranial aneurysms can be treated successfully using deep hypothermic circulatory arrest. Extensive monitoring adds to the speed and safety of the procedure. The resulting comparative measurements of temperatures at different body sites including brain, EEG, and other variables may be of general relevance for operations employing deep hypothermia and circulatory arrest.

Sonographic parenchymal and brain perfusion imaging: preliminary results in four patients following decompressive surgery for malignant middle cerebral artery infarct.

To investigate new methods of diagnostic transcranial sonography for brain parenchymal, vascular and perfusion imaging, we performed 3-D native tissue harmonic transcranial sonography (3D-nthTCS), 3-D transcranial color-coded duplex sonography (3D-TCCS), and "loss-of-correlation" imaging (LOC-TCCS) in four patients following early hemicraniectomy due to space-occupying "malignant" middle cerebral artery infarction (MMCAI). Three-dimensional datasets, utilizing 3D-nthTCS and 3D-TCCS, were created and up to 10 axial 2-D B-mode image planes, similar to CCT, reconstructed in each patient. Three-dimensional reconstructions of the circle of Willis documented one persistent carotid-T occlusion and three recanalizations of the MCA. LOC-TCCS, based on stimulated acoustic emission from an ultrasound (US) contrast agent, demonstrated a perfusion deficit in 2 of 3 patients, with regard to their infarcts. Concluding, 3D-nthTCS, 3D-TCCS and LOC-TCCS are promising tools for bedside monitoring, early prognosis and treatment evaluation for MMCAI in the postoperative period. Further studies should be performed to standardize these new methods and evaluate their applications through the intact calvarina.

The Bonebridge: preclinical evaluation of a new transcutaneously-activated bone anchored hearing device.

OBJECTIVES: To assess the functional performance of the Bonebridge (BB, MED-EL), a newly-designed transcutaneous bone conduction implant that allows the skin to remain intact and to compare it with the current clinical model of choice, a percutaneous bone conduction implant (BAHA BP100, Cochlear Bone Anchored Solutions AG). MATERIALS AND METHODS: The devices were compared using two methods: (1) Measurements of cochlear promontory acceleration in five cadaver heads: Accelerations of the cochlear promontories on both ipsilateral and contralateral sides were measured using a Laser Doppler system, with free-field sound stimuli of 90 dB SPL in the frequency range of 0.3-10 kHz (2) Measurements of pure-tone sound field thresholds in 5 normally hearing human adult subjects under a condition of simulated hearing loss. For the latter measurements, the devices were applied to the head using a Softband, and measurements were performed in the frequency range of 0.25-8 kHz. Within investigation comparisons (i.e., in cadavers or listeners) and a cross-comparison analysis of the cadaver and human results were done. RESULTS: Results from the cadaver heads showed that the cochlear promontory acceleration with the BB was higher within 10 dB on the ipsilateral side and lower within 5 dB on the contralateral side than the acceleration with the BAHA, in the frequency range of 0.7-10 kHz. The transcranial attenuation of the acceleration for the BB was greater than for the BAHA within 20 dB. For the sound-field threshold assessments with human subjects, the BB and BAHA showed similar threshold improvements of more than 10 dB HL for the ipsilateral side. For the contralateral side, the threshold improvement with the BB was less than with the BAHA, indicating better separation between ipsilateral and contralateral sides. CONCLUSIONS: Preclinical results imply that the BB has functional performance similar to the BAHA and could be beneficial to patients suffering with conductive and mixed hearing losses as well as for those with unilateral impairment. Based on these preliminary results, a carefully designed clinical trial with conservative inclusion criteria can be recommended. This article is part of a special issue entitled "MEMRO 2012".

Murine adult neural progenitor cells alter their proliferative behavior and gene expression after the activation of Toll-like-receptor 3.

Viral infections during pregnancy significantly increase the risk for psychological pathologies like schizophrenia in the offspring. One of the main morphological hallmarks of schizophrenia is a reduced size of the hippocampus. Since new neurons are produced in this particular brain compartment throughout life, it might be possible that low neurogenesis levels triggered by a maternal viral infection contribute to developmental deficits of the hippocampus. We injected polyinosinic:polycytidylic acid (Poly I:C) in pregnant C57Bl/6 mice to stimulate an anti-viral response through TLR3 and examined gene expressions in the neuronal progenitor cells (NPCs) of the offspring at different ages. Additionally, we treated adult NPC lines with Poly I:C to investigate its direct effect. We could show for the first time that TLR3 and its downstream effector molecule IRF3 are expressed in adult NPCs. Poly I:C treatment in vitro and in vivo led to the regulation of proliferation and genes involved in antiviral response, migration, and survival. These findings indicate that NPCs of the fetus are able to react towards an in utero immune response, and thus, changes in the neuronal stem cell pool can contribute to the development of neurological diseases like schizophrenia.

Accuracy of linear intraoral measurements using cone beam CT and multidetector CT: a tale of two CTs.

OBJECTIVES: The aim was to compare the accuracy of linear bone measurements of cone beam CT (CBCT) with multidetector CT (MDCT) and validate intraoral soft-tissue measurements in CBCT. METHODS: Comparable views of CBCT and MDCT were obtained from eight intact cadaveric heads. The anatomical positions of the gingival margin and the buccal alveolar bone ridge were determined. Image measurements (CBCT/MDCT) were performed upon multiplanar reformatted data sets and compared with the anatomical measurements; the number of non-assessable sites (NASs) was evaluated. RESULTS: Radiological measurements were accurate with a mean difference from anatomical measurements of 0.14 mm (CBCT) and 0.23 mm (MDCT). These differences were statistically not significant, but the limits of agreement for bone measurements were broader in MDCT (-1.35 mm; 1.82 mm) than in CBCT (-0.93 mm; 1.21 mm). The limits of agreement for soft-tissue measurements in CBCT were smaller (-0.77 mm; 1.07 mm), indicating a slightly higher accuracy. More NASs occurred in MDCT (14.5%) than in CBCT (8.3%). CONCLUSIONS: CBCT is slightly more reliable for linear measurements than MDCT and less affected by metal artefacts. CBCT accuracy of linear intraoral soft-tissue measurements is similar to the accuracy of bone measurements.