Light-activated deoxyguanosine: photochemical regulation of peroxidase activity

Photochemical activation of a deoxyribozyme with peroxidase activity was achieved by the synthesis and incorporation of a caged deoxyguanosine.     

Murine cytomegalovirus m38.5 protein inhibits Bax-mediated cell death

Many viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes to mitochondria and protects human HeLa cells and fibroblasts from apoptosis triggered by proteasome inhibitors but not from Fas-induced apoptosis. However, the ability of this protein to suppress the apoptosis of murine cells and its role during MCMV infection have not been investigated previously. Here we show that m38.5 is expressed at early time points during MCMV infection. Cells infected with MCMVs lacking m38.5 showed increased sensitivity to cell death induced by staurosporine, MG132, or the viral infection itself compared to the sensitivity of cells infected with wild-type MCMV. This defect was eliminated when an m38.5 or Bcl-X(L) gene was inserted into the genome of a deletion mutant. Using fibroblasts deficient in the proapoptotic Bcl-2 family proteins Bak and/or Bax, we further demonstrated that m38.5 protected from Bax- but not Bak-mediated apoptosis and interacted with Bax in infected cells. These results consolidate the role of m38.5 as a viral mitochondrion-localized inhibitor of apoptosis and its functional similarity to the human cytomegalovirus UL37x1 gene product. Although the m38.5 gene is not homologous to the UL37x1 gene at the sequence level, m38.5 is conserved among rodent cytomegaloviruses. Moreover, the fact that MCMV-infected cells are protected from both Bak- and Bax-mediated cell death suggests that MCMV possesses an additional, as-yet-unidentified mechanism to block Bak-mediated apoptosis.     

Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression

Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowett (nude) rats has been established as a representative animal model for human GBMs, with a tumor take rate close to 100%. In immunocompetent littermates however, primary human GBM tissue is invariably rejected. Here we show that after repeated passaging cycles in nude rats, human GBM spheroids are enabled to grow in the brain of immunocompetent rats. In case of engraftment, xenografts in immunocompetent rats grow progressively and host leukocytes fail to enter the tumor bed, similar to what is seen in nude animals. In contrast, rejection is associated with massive infiltration of the tumor bed by leukocytes, predominantly ED1+ microglia/macrophages, CD4+ T helper cells and CD8+ effector cells, and correlates with elevated serum levels of pro-inflammatory cytokines IL-1β, IL-18 and TNF-α. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo passaging cycles, characterized by striking attenuation of microglial infiltration. Furthermore, tumor-derived chemokines that promote leukocyte migration and their entry into the CNS such as CXCL-10 and CXCL-12 are down-regulated, and the levels of TGF-β2 increase. We propose that through serial in vivo passaging in nude rats, human GBM cells learn to avoid and or/ suppress host immunity. Such adapted GBM cells are in turn able to engraft in immunocompetent rats without signs of an inflammatory response.     

Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana

Thylakoid rhodanase‐like protein (TROL) is a nuclear‐encoded protein of thylakoid membranes required for tethering of ferredoxin:nicotinamide adenine dinucleotide phosphate (NADPH) oxydoreductase (FNR). It has been proposed that the dynamic interaction of TROL with flavoenzyme FNR, influenced by environmental light conditions, regulates the fate of photosynthetic electrons, directing them either to NADPH synthesis or to other acceptors, including reactive oxygen species detoxification pathways. Inside the chloroplasts, TROL has a dual localization: an inner membrane precursor form and a thylakoid membrane mature form, which has been confirmed by several large‐scale chloroplast proteomics studies, as well as protein import experiments. Unlike the localization, the topology of TROL in the membranes, which is a prerequisite for further studies of its properties and function, has not been experimentally confirmed yet. Thermolysin was proven to be a valuable protease to probe the surface of chloroplasts and membranes in general. By treating the total chloroplast membranes using increasing protease concentration, sequential degradation of TROL was observed, indicating protected polypeptides of TROL and possible domain orientation. To further substantiate the obtained results, TROL‐overexpressing Arabidopsis line (OX) and line in which the central rhodanase‐like domain (RHO) has been partially deleted (ΔRHO), were used as well. While OX line showed the same degradation pattern of TROL as the wild‐type, surprisingly, TROL from ΔRHO membranes was not detectable even at the lowest protease concentration applied, indicating the importance of this domain to the integrity of TROL. In conclusion, TROL is a polytopic protein with a stroma‐exposed C‐terminal FNR‐binding region, and the thylakoid lumen‐located RHO domain.     

The application of ultrasound in neuroendoscopic procedures: first results with the new tool "NECUP-2"

In this paper, our experience with originally constructed Neurosurgical Endoscopic Contact Ultrasound Probe "NECUP-2" in neuroendoscopy is reported. Between June 1997 and June 2007, 132 neuroendoscopic procedures have been performed: 102 endoscopic thrid ventriculostomies (ETV), 15 arachnoid cysts and 5 intraventricular tumours operations. The "NECUP-2" was applied effectively in all cases in which blunt perforation was not possible: 38/102 ETY, 10/10 septostomies, 15/15 arachnoid cysts. In five cases of intraventricular tumours, neuroendoscopic procedure was combined with open microsurgery for tumour removal with preservation of vascular structures. There were no "NECUP-2" related complications. Of postoperative complications, we had liquorrhea (9 patients), and symptoms of meningitis (6 patients). In the follow-up period (6 months to 6 years), we had a patency rate of 80% (50/63 patients). All patients improved in clinical status. According to the first results, it seems that ultrasonic contact probe NECUP-2 presents a new device in neurosurgical armamentarium that can be used in various fields of neurosurgery. With minimal and controlled lesion that is produced at the tip of the probe, it can be used in highly demanding operations such as third ventriculostomy and tumour resection.     

Chondromyxoid fibroma of the second metacarpal bone--a case report

This report describes a chondromyxoid fibroma of the second metacarpal bone in a 32-year-old female patient. Chondromyxoid fibroma is a rare, benign, slow-growing bone tumor of cartilaginous origin. Tumor has a high recurrance rate. Our aim was to show successful treatment of a metacarpal chondromyxoid fibroma with wide resection and implantation of finger join endoprosthesis.     

Differential Activity of NADPH-Producing Dehydrogenases Renders Rodents Unsuitable Models to Study IDH1R132 Mutation Effects in Human Glioblastoma

The somatic IDH1^R132 mutation in the isocitrate dehydrogenase 1 gene occurs in high frequency in glioma and in lower frequency in acute myeloid leukemia and thyroid cancer but not in other types of cancer. The mutation causes reduced NADPH production capacity in glioblastoma by 40% and is associated with prolonged patient survival. NADPH is a major reducing compound in cells that is essential for detoxification and may be involved in resistance of glioblastoma to treatment. IDH has never been considered important in NADPH production. Therefore, the authors investigated NADPH-producing dehydrogenases using in silico analysis of human cancer gene expression microarray data sets and metabolic mapping of human and rodent tissues to determine the role of IDH in total NADPH production. Expression of most NADPH-producing dehydrogenase genes was not elevated in 34 cancer data sets except for IDH1 in glioma and thyroid cancer, indicating an association with the IDH1 mutation. IDH activity was the main provider of NADPH in human normal brain and glioblastoma, but its role was modest in NADPH production in rodent brain and other tissues. It is concluded that rodents are a poor model to study consequences of the IDH1^R132 mutation in glioblastoma.     

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.     

Targeting the NG2/CSPG4 proteoglycan retards tumour growth and angiogenesis in preclinical models of GBM and melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy.     

Spinal anesthesia at the L2-3 and L3-4 levels: comparison of analgesia and hemodynamic response

Aim of this study was to evaluate level of analgesia and hemodynamic response to spinal anesthesia obtained by administering 15 mg 0.5% isobaric bupivacaine at L2-3 vs. L3-4 interspace for inguinal herniorrhaphy, since studies comparing analgesia and hemodynamic response at the L2-3 vs. L3-4 interspaces are lacking. In a prospective, randomized clinical study that encountered 72 patients undergoing elective inguinal herniorrhaphy randomly allocated in to two equal groups L2-3 (N = 36) and L3-4 (N = 36) according to lumbar interspace where intrathecal injection of bupivacaine was administered. Analgesia was evaluated by intraoperative "rescue" fentanyl requirements, the absence of pain and the maximal visual analogue scale (VAS) scores reached per patient during the operation. The severity of intraoperative pain was quantified by a 10 cm VAS scale (VAS 0: no pain to 10: worst pain imaginable) every 5 minutes after skin incision until the end of the operation. VAS > 3 was treated with intravenous fentanyl 25 microg. Hemodynamic response was monitored and evaluated, heart rate was continuously monitored as well as, baseline systolic, diastolic and mean arterial pressure prior to induction and every 5 minute after applying spinal anesthesia until surgical completion. Intraoperative fentanyl requirements were significantly higher in group L3-4 (L2-3 0%, 97.5% confidence interval [CI] 0.0-0.11 vs. L3-4 17%, 95% CI 0.07-0.32, p = 0.025). Absence of pain was significantly higher in L2-3 group at the beginning of the operation (L2-3 89%, 95% CI 0.74-0.96 vs. L3-4 67%, 95% CI 0.50-0.79, p = 0.047). The maximal VAS scores reached per patient during the operation in L2-3 group were lower then in L3-4 group (L2-3 median [M] 0, range [R] 0-3, L3-4 M 0, R 0-8, p = 0.014). There were no significant differences (p > 0.05) in the incidence of hypotension (L2-3 19%, 95% CI 0.09-0.35 vs. L3-4 17%, 95% CI 0.07-0.32) and bradycardia (L2-3 19%, 95% CI 0.09-0.35 vs. L3-4 8%, 95% CI 0.02-0.23). Spinal anesthesia with isobaric bupivacaine administered in L2-3 interspace for inguinal herniorrhaphy provides superior analgesia and equal hemodynamic stability as compared to neuroaxial anesthesia administered in the L3-4 interspace.