Susceptibility and Tumor Size Changes During the Time Course of Standard Treatment in Recurrent Glioblastoma.

BACKGROUND AND PURPOSE: Susceptibility-weighted magnetic resonance imaging (SWI) yields information regarding tumor biology (e.g., hemorrhage) of growing gliomas. SWI changes can also be observed as a consequence of treatment, for example radiation therapy. The aim of our study was to investigate how susceptibility changes occur during the time course after completion of standard treatment in newly diagnosed glioblastoma (GBM). METHODS: Eighteen GBM patients were retrospectively analyzed. After completion of therapy, imaging was performed every 3 months. MRI was analyzed at the following time points: after the third and sixth cycle of adjuvant temozolomide chemotherapy, thereafter in 3 month intervals and at recurrence. The number of SWI positive tumor pixels was quantified and compared with progression as defined by the RANO criteria on T2- and contrast-enhanced T1-weighted MRI sequences (T1-CE). RESULTS: The MRI interval between completion of the sixth chemotherapy cycle and last MRI before progression was 390 ± 292 days. Between the last MRI before progression and at progression a significant increase in SWI positive tumor pixels was observed (P = .012), whereas tumor size remained unchanged (RANO T2: P = .385; RANO T1-CE: P = .165). The number of SWI positive pixels remained unchanged between last MRI before progression until progression (P = .149), whereas RANO T2 and T1-CE showed tumor progression (interval 128 ± 69 days). CONCLUSIONS: SWI positive pixel count increases significantly prior to changes in tumor size (RANO). Our findings may be explained by microbleeds compatible with stimulation of angiogenesis and possibly serve as an early biomarker of tumor progression.

Inhibiting 12/15-lipoxygenase to treat acute stroke in permanent and tPA induced thrombolysis models.

12/15-Lipoxygenase (12/15-LOX) contributes to the brain damage after middle cerebral artery occlusion (MCAO) in the acute phase of stroke. The aim of this study was to investigate the effects of a 12/15-LOX inhibitor, LOXBlock-1(LB1), in mice using a FeCl3-induced permanent distal MCAO model and FeCl3-induced ischemia/thrombolysis with tPA. In order to induce permanent distal MCAO, 30% FeCl3 was used in C57BL6 mice. LB1 or DMSO treatments were applied intraperitoneally 2 h following MCAO. For FeCl3-induced ischemia/thrombolysis experiments, 10% FeCl3 was preferred so as to obtain reperfusion with tPA in CD1 mice. 4 h following ischemia either LB1 or DMSO and iv tPA was administered. Outcomes were NSS, weight loss, infarct volume, hemorrhage area and reperfusion rate. FeCl3-induced distal MCAO caused an increase in 12/15-LOX signal in the ischemic cortex with an increase in MDA2 and AIF immunoreactivity. LB1 treatment, applied 2 h after ischemia, significantly decreased the infarct volume at 24 h of permanent distal MCAO. Weight loss was also significantly reduced in LB1 treated group. Distal MCAO and tPA application with LB1 or DMSO showed that treatment significantly decreased the infarct volume and the hemorrhage area. The reperfusion rate in the LB1-treated group was surprisingly higher than in the DMSO group and NSS results were significantly improved. These data suggest that LB1 can be used as an adjuvant agent to tPA. This study not only shows the effects of LB1 treatment in distal MCAO but also confirms that FeCl3-induced MCAO model can be a useful tool to screen novel treatment options in stroke.

GSK-3ß-dependent downregulation of γ-taxilin and αNAC merge to regulate ER stress responses.

The signaling pathway leading to the endoplasmic reticulum (ER) stress responses has not been fully elucidated. Here we showed that glycogen synthase kinase-3ß (GSK-3ß)-dependent downregulation of γ-taxilin and nascent polypeptide-associated complex α-subunit (αNAC) mediates hypoxia-induced unfolded protein responses (UPRs) and the subsequent apoptotic and autophagic pathways. The degradation of γ-taxilin or αNAC was sufficient to initiate UPRs in normoxic cells. However, the ER stress signaling pathways initiated by γ-taxilin or αNAC were distinct, triggering different ER stress sensors and activating different downstream pathways. Hypoxia caused GSK-3ß-dependent tau hyperphosphorylation and cleavage in neuronal cells, but γ-taxilin ablation induced tau hyperphosphorylation alone and αNAC ablation induced neither changes. Notably, downregulation of γ-taxilin and αNAC occurs in the brain of patients with Alzheimer's disease. These results suggest that GSK-3ß-dependent downregulation of γ-taxilin and αNAC, which differently activate the UPRs, merge to regulate hypoxia-induced ER stress responses and provide a new insight into the pathogenesis of neurodegenerative diseases.

Akt and mTOR mediate programmed necrosis in neurons.

Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3ß, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.

Mitochondrial distribution of neuroglobin and its response to oxygen-glucose deprivation in primary-cultured mouse cortical neurons.

Neuroglobin (Ngb) is a new member of the globin family and a novel endogenous neuroprotective molecule, but its neuroprotective mechanisms remain largely undefined. Previous studies suggest Ngb is both physically and functionally related to mitochondria, however without direct evidence. Our recent discovery has shown that Ngb can physically interact with a number of mitochondrial proteins. In this study we aimed to define the physical interaction between Ngb and mitochondria by determining whether there is a mitochondrial distribution of Ngb under both physiological-resting and pathological oxygen-glucose deprivation (OGD) conditions. Western blot for the first time revealed a small portion of Ngb was physically localized in mitochondria, and the relative mitochondrial Ngb level was significantly increased after OGD in primary-cultured mouse cortical neurons, indicating a translocation of Ngb into mitochondria. Complementary approaches including confocal imaging and immuno-electron microscopy confirmed Ngb distribution in mitochondria under both basal-resting condition and OGD. Inhibitors of mitochondria permeability transition pore (mPTP) and Voltage-Dependent Anion Channel (VDAC) blocked OGD-induced increase of mitochondrial Ngb level, demonstrating a possible role of mPTP in Ngb's mitochondrial translocation. We further found that Ngb overexpression-conferred neuroprotection was correlated with increased mitochondrial Ngb level, suggesting the mitochondria distribution of Ngb is clearly associated with and may contribute to Ngb's neuroprotection.

Brain tumor imaging with transcranial sonography: state of the art and review of the literature.

Transcranial sonography (TCS) is a widely used non-invasive bedside method to evaluate the brain, its vessels, perfusion and pathologies. Transcranial brain tumor sonography emerged in the early nineties and while B-mode imaging and Color-Doppler have acquired widespread use, especially for intraoperative imaging, other ultrasound modalities such as Perfusion Imaging are applied more in the research field. The aim of this review is to give an overview of the different ultrasound modalities and their respective application in sonographic brain tumor imaging.

alphaNAC depletion as an initiator of ER stress-induced apoptosis in hypoxia.

Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3beta (GSK-3beta) and that the activated GSK-3beta destabilizes alphaNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of alphaNAC and ER stress responses involving PERK, ATF4, gamma-taxilin, elF2alpha, Bip, and CHOP. Depletion of alphaNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3beta, and that GSK-3beta inhibition prevented alphaNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, alphaNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that alphaNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3beta may participate upstream in this mechanism.

[Fluorescence guided resection of malignant brain tumors - breakthrough in the surgery of brain tumors]. / Fluoreszenzgestützte Resektion bösartiger Hirntumoren - Durchbruch bei der Hirntumor-Operation.

Malignant gliomas, among others the glioblastoma multiforme, are the most frequent brain tumors. The glioblastoma itself represents the most unfavorable tumor. In Switzerland, about 450 humans contract a glioblastoma each year. The median survival time is under 12 months, thus the prognosis is dismal despite a combination of surgery, radiotherapy and chemotherapy. Surgical resection presents the major constituent in the management of patients with a glioblastoma. Postoperative radio- and chemotherapy have beneficial effects on the survival time and quality of life. Surgical removal of glioblastomas is challenging due to their infiltrative growth pattern. Therefore, the target extent of resection can only be achieved partially. For some time now, a new in Germany developed method is used in the Neurosurgical Clinic of the Cantonal Hospital in St. Gallen: The 5-ALA-guided microsurgical resection method allows a targeted and secure surgical resection of the tumor. A preoperative administered substance colors the tumor and makes it better visible for the neurosurgeon. Consequently, the healthy brain tissue can be better distinguished from the tumor. This permits not only a larger complete surgical resection of the tumor but also minimizes the resection of healthy tissue.

15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet.

Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of approximately 81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the alpha- and beta-type. The 15-LOX and various components of the ubiquitin-proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins.

Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner.

Inhibition of 15-lipoxygenase leads to delayed organelle degradation in the reticulocyte.

Mammalian cells are characterized by an endomembrane system. Nevertheless, some cells lose these membranes during their terminal differentiation, e.g. red blood cells and lens fiber cells of the eye. 15-Lipoxygenase is believed to be critical for this membrane degradation. Here we use cultivated rabbit reticulocytes in the presence or absence of a lipoxygenase inhibitor to provide further evidence for the importance of 15-lipoxygenase for the in vivo degradation of mitochondria. We find that inhibitor treatment retarded mitochondrial degradation, as shown by persistence of marker proteins and by direct visualization of mitochondria by electron microscopy.

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain.

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans -stereoconfiguration in the beta-position are described. The water-soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc-P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water-soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.

A function for lipoxygenase in programmed organelle degradation.

Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells of the eye lens, and keratinocytes. The degradation of these organelles must be tightly regulated with respect to both the time of activation and the specificity of membrane degradation. The expression of 15-lipoxygenase (15-LOX) peaks in reticulocytes immediately before organelle degradation. Here we show that 15-LOX integrates into the membranes of various organelles, allowing release of proteins from the organelle lumen and access of proteases to both lumenal and integral membrane proteins. In addition, by sparing the plasma membrane, 15-LOX shows the required specificity for organellar membranes. Thus, the action of 15-LOX provides a mechanism by which the natural degradation process can be explained. This conclusion is supported by our finding that lipoxygenase expression in the eye lens is restricted to the region at which organelle degradation occurs.

Limitations for three-dimensional ultrasound imaging through a bore-hole trepanation.

The intraoperative shift of neuroanatomical landmarks that serve as reference points is an unsolved problem in current neuronavigation. Monitoring the position of these landmarks requires repeated intraoperative imaging. We analyzed the effectiveness of a 3-D ultrasound system for imaging through a bore-hole trepanation. A tissue-mimicking ultrasound phantom and plastic pads with bore-holes were utilized for in vitro tests of the system. Reducing the diameter of the simulated bore-hole decreased the image quality and reduced the field of view. The multiple plane mode of the 3-D ultrasound system allows reconstruction of images in arbitrary imaging planes on the basis of intraoperatively acquired 3-D datasets. Selecting planes that are coplanar with preoperative MRI scans, we were able to identify neuroanatomical landmarks in the reconstructed ultrasound images. Repeated 3-D ultrasound during a procedure might, therefore, allow recognition of a shift of these landmarks.

Glycotripeptides are released by yeast but not by mammalian microsomes.

Glycotripeptides generated in vivo in the endoplasmic reticulum (ER) have been used as markers to assess the rate of vesicular bulk flow from the ER via the Golgi apparatus to the plasma membrane in mammalian cells. The applicability of such glycotripeptides as markers for bulk flow along this pathway has been questioned by a report on non-vesicular release of glycotripeptides from yeast semi-intact spheroplasts. We have therefore investigated direct release of glycotripeptides from yeast and from mammalian microsomes and report here that such release is specific to the yeast system and cannot be detected in mammalian microsomes.

Complete Golgi passage of glycotripeptides generated in the endoplasmic reticulum of mammalian cells.

The tripeptide, N-octanoyl-Asn-[125I]Tyr-Thr-NH2, which contains the acceptor sequence for N-glycosylation, is readily taken up by cell culture cells, glycosylated in the endoplasmic reticulum (ER), and secreted into the medium. Therefore such glycosylated tripeptides have been used as markers for the vesicular flow from the endoplasmic reticulum to the plasma membrane [(1987) Cell 50, 289-300; (1990) J. Biol. Chem. 265, 20027-20032]. We have now studied the pathway taken by the glycotripeptides in mammals in more detail. In the perfused rat liver, the glycotripeptides secreted to the medium were analyzed by digestion with exoglycosidases, and a significant fraction was found to contain the terminating sequence -Gal-Sial, which is generated by processing enzymes that reside in the late Golgi apparatus. Thus we conclude that these glycotripeptides have passed through the complete Golgi complex on their way from the ER to the cell surface.

Transcranial Doppler sonography and intracranial pressure monitoring in children and juveniles with acute brain injuries or hydrocephalus.

Transcranial Doppler sonography is a noninvasive method of obtaining information about changes in cerebral hemodynamics and intracranial pressure. After severe head injuries the development of brain swelling and brain edema can be assessed and the efficacy of treatment monitored. Development of severe brain edema accompanied by a rapid increase in intracranial pressure can be recognized by a decrease in blood flow velocity and rise in the pulsatility index. In hydrocephalic children the behavior of the cerebral blood flow velocity and the pulsatility index will warn of an increase of the ventricular fluid pressure or a shunt insufficiency.

Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors.

Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.