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Malignant glioma represents a fatal disease with a poor prognosis and development of resistance mechanisms against conventional therapeutic approaches. The distinct tumor zones of this heterogeneous neoplasm develop their own microenvironment, in which subpopulations of cancer cells communicate. Adaptation to hypoxia in the center of the expanding tumor mass leads to the glycolytic and angiogenic switch, accompanied by upregulation of different glycolytic enzymes, transporters, and other metabolites. These processes render the tumor microenvironment more acidic, remodel the extracellular matrix, and create energy gradients for the metabolic communication between different cancer cells in distinct tumor zones. Escape mechanisms from hypoxia-induced cell death and energy deprivation are the result. The functional consequences are more aggressive and malignant behavior with enhanced proliferation and survival, migration and invasiveness, and the induction of angiogenesis. In this review, we go from the biochemical principles of aerobic and anaerobic glycolysis over the glycolytic switch, regulated by the key transcription factor hypoxia-inducible factor (HIF)-1α, to other important metabolic players like the monocarboxylate transporters (MCTs)1 and 4. We discuss the metabolic symbiosis model via lactate shuttling in the acidic tumor microenvironment and highlight the functional consequences of the glycolytic switch on glioma malignancy. Furthermore, we illustrate regulation by micro ribonucleic acids (miRNAs) and the connection between isocitrate dehydrogenase (IDH) mutation status and glycolytic metabolism. Finally, we give an outlook about the diagnostic and therapeutic implications of the glycolytic switch and the relation to tumor immunity in malignant glioma.

Gliomas are brain-born tumors with devastating impact on their brain microenvironment. Novel approaches employ multiple combinations of chemical compounds in synthetic hybrid molecules to target malignant tumors. Here, we report on the chemical hybridization approach exemplified by artesunic acid (ARTA) and naturally occurring triterpene betulinic acid (BETA). Artemisinin derived semisynthetic compound artesunic acid (ARTA) and naturally occurring triterpene BETA were used to synthetically couple to the hybrid compound termed 212A. We investigated the impact of 212A and its parent compounds on glioma cells, astrocytes and neurons. ARTA and BETA showed cytotoxic effects on glioma cells at micromolar concentrations. ARTA was more effective on rodent glioma cells compared to BETA, whereas BETA exhibited higher toxic effects on human glioma cells compared to ARTA. We investigated these compounds on non-transformed glial cells and neurons as well. Noteworthy, ARTA showed almost no toxic effects on astrocytes and neurons, whereas BETA as well as 212A displayed neurotoxicity at higher concentrations. Hence we compared the efficacy of the hybrid 212A with the combinational treatment of its parent compounds ARTA and BETA. The hybrid 212A was efficient in killing glioma cells compared to single compound treatment strategies. Moreover, ARTA and the hybrid 212A displayed a significant cytotoxic impact on glioma cell migration. Taken together, these results demonstrate that both plant derived compounds ARTA and BETA operate gliomatoxic with minor neurotoxic side effects. Altogether, our proof-of-principle study demonstrates that the chemical hybrid synthesis is a valid approach for generating efficacious anti-cancer drugs out of virtually any given structure. Thus, synthetic hybrid therapeutics emerge as an innovative field for new chemotherapeutic developments with low neurotoxic profile.

Selenoproteins have been recognized as modulators of brain function and signaling. Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a unique member of the selenium-dependent glutathione peroxidases in mammals with a pivotal role in brain development and function. GPx4 exists as a cytosolic, mitochondrial, and nuclear isoform derived from a single gene. In mice, the GPx4 gene is located on chromosome 10 in close proximity to a functional retrotransposome that is expressed under the control of captured regulatory elements. Elucidation of crystallographic data uncovered structural peculiarities of GPx4 that provide the molecular basis for its unique enzymatic properties and substrate specificity. Monomeric GPx4 is multifunctional: it acts as a reducing enzyme of peroxidized phospholipids and thiols and as a structural protein. Transcriptional regulation of the different GPx4 isoforms requires several isoform-specific cis-regulatory sequences and trans-activating factors. Cytosolic and mitochondrial GPx4 are the major isoforms exclusively expressed by neurons in the developing brain. In stark contrast, following brain trauma, GPx4 is specifically upregulated in non-neuronal cells, i.e., reactive astrocytes. Molecular approaches to genetic modification in mice have revealed an essential and isoform-specific function for GPx4 in development and disease. Here we review recent findings on GPx4 with emphasis on its molecular structure and function and consider potential mechanisms that underlie neural development and neuropathological conditions.

In N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5Kβ is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5Kβ and Rac1 and generated a point mutant of PIP5Kβ that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5Kβ and thereby the localised synthesis of PtdIns(4,5)P2 required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5)P2 induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5)P2, attenuate PIP5K- and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5)P2 synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics.

The synthesis of two sets of analogues of 2-5A trimer containing 9-(3-fluoro-3-deoxy-β-D-xylo-furanosyl)adenine (AF) or 3′-fluoro-3′-deoxyadenosine (AF) at different positions of the chain is described, along with the preparation of the corresponding 5′-monophosphates and 5′-diphosphorylated (core) trimers. The ability of each ribo and xylo isomeric pair of fluorodeoxy analogues of 2-5A (i) to compete with p3(A2′p)3A3′[32P]pC3′p for binding to RNase L in L929 cell extracts, and (ii) to activate the partially purified RNase L from L929 cell extracts to hydrolyze poly(U)[3H], was compared to that of the related 3′-deoxy analogue [Torrence et al., J. Biol. Chem. 263, 1131 (1988)] and the parent trimer, p3A3, using radiobinding and RNase L-(2′,5′)pentaadenylate(core)-agarose assays, respectively. Evidence is presented to show that the stereochemistry of the trimers plays an important role, specifically in the second process. The most striking observation is that, compared to 2-5A, p3A(AF)A was found to be nine times more effective an activator of RNase L, whereas isomeric p3A(AF)A is 30 times less effective.

Although dietary selenium (Se) deficiency results in phenotypes associated with selenoprotein depletion in various organs, the brain is protected from Se loss. To address the basis for the critical role of Se in brain function, we carried out comparative gene expression analyses for the complete selenoproteome and associated biosynthetic factors. Using the Allen Brain Atlas, we evaluated 159 regions of adult mouse brain and provided experimental analyses of selected selenoproteins. All 24 selenoprotein mRNAs were expressed in the mouse brain. Most strikingly, neurons in olfactory bulb, hippocampus, cerebral cortex, and cerebellar cortex were exceptionally rich in selenoprotein gene expression, in particular in GPx4, SelK, SelM, SelW, and Sep15. Over half of the selenoprotein genes were also expressed in the choroid plexus. A unique expression pattern was observed for one of the highly expressed selenoprotein genes, SelP, which we suggest to provide neurons with Se. Cluster analysis of the expression data linked certain selenoproteins and selenocysteine machinery genes and suggested functional linkages among selenoproteins, such as that between SelM and Sep15. Overall, this study suggests that the main functions of selenium in mammals are confined to certain neurons in the brain.

Fully modified homopyrimidine 2'‐deoxy‐xylo nucleic acid (dXNA) form triple helixes with complementary DNA/RNA with thermal stabilities comparable to those of the corresponding DNA:DNA and DNA:RNA duplexes. However, a single or few insertions of dXNA monomers in a DNA strand significantly lower duplex stabilities. The dXNA monomers are known to adopt predominantly an N‐type furanose conformation in solution. With a desire to increase the binding affinity, seven sugar‐modified XNA monomers (H, F, N, M, K, P and Q) have been synthesised and their effect on hybridization towards DNA and RNA complements studied. The introduction of 2'‐fluoro and 2'‐hydroxy substituents was expected to induce conformational restriction towards C3'‐endo‐type furanose conformation of monomer F derived from 1‐(2'‐deoxy‐2'‐fluoro‐β‐D‐xylofuranosyl)thymine and monomer H derived from 1‐(β‐D‐xylofuranosyl)thymine. The presence of functionalites facing the minor groove as in 1‐(2'‐amino‐2'‐deoxy‐2'‐N,4'‐C‐methylene‐β‐D‐xylofuranosyl)thymine (monomer N), 1‐[4‐C‐(N‐methylpiperazinyl)methyl‐β‐D‐xylofuranosyl]thymine (monomer P), 1‐(4‐C‐piperazinylmethyl‐β‐D‐xylofuranosyl)thymine (monomer Q), 1‐(4‐C‐hydroxymethyl‐β‐D‐xylofuranosyl)thymine (monomer M) and 9‐(4‐C‐hydroxymethyl‐β‐D‐xylofuranosyl)adenine (monomer K) was studied, with monomer N being locked in an N‐type furanose conformation. Besides, an efficient synthesis of known xylo‐LNA phosphoramidite 19, required for the incorporation of 1‐(2'‐O,4'‐C‐methylene‐β‐D‐xylofuranosyl)thymine (monomer L) is described. For comparison, hydridization data of various XNAs reported in the literature are included in the discussion section. The thermal denaturation studies show that XNAs containing conformationally locked monomers (N and L) display improved binding affinity, and that partially modified DNA/XNA chimera, or fully modified XNA display preferential hybridization towards RNA complements. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

A sperm nucleus glutathione peroxidase (snGPx), which is closely related to the phospholipid hydroperoxide glutathione peroxidase (phGPx), was recently discovered in late spermatids. Both GPx isoforms originate from a joint ph/snGPx gene, but their N-terminal peptides are encoded by alternative first exons. The expression of the two enzymes is differentially regulated in various cells, but little is known about the regulatory mechanisms. To explore the tissue-specific regulation of expression of the two isoenzymes, we first investigated their tissue distribution. Whereas phGPx is expressed at low levels in many organs, snGPx was only detected in testis, kidney, and in the human embryonic kidney cell line HEK293. Subcellular fractionation studies and immunoelectron microscopy revealed a cytosolic localization. To explore the mechanistic reasons for the differential expression pattern, we first tested the activity of the putative phGPx and snGPx promoters. The 5′-flanking region of the joint ph/snGPx gene exhibits strong promoter activity. In contrast, the putative snGPx promoter, which comprises 334 bp of intronic sequences, lacks major promoter activity. However, it strongly suppresses the activity of the ph/snGPx promoter. These data suggest negative regulatory elements in the first intron of the ph/snGPx gene, and DNase protection assays revealed the existence of several protein-binding sites. The corresponding trans-regulatory proteins (SP1, ERG1, GATA1, SREBP1, USF1, and CREBP1) were identified, and in vivo binding of EGR1 and SREBP1 was shown by chromatin immunoprecipitation. These data indicate for the first time somatic expression of the snGPx and provide evidence for the existence of intronic negative cis-regulatory elements in the ph/snGPx gene. Our failure to detect an alternative snGPx promoter suggests that transcription of the ph/snGPx gene may be regulated by a joint basic promoter. The decision, which GPx isoform is expressed in a given cell, appears to be made by alternative splicing of a joint primary transcript.

Malignant gliomas belong to the most threatening tumor entities and are hallmarked by rapid proliferation, hypervascularization and an invasive growth pattern. The primary obstacle in surgical treatment lies in differentiation between healthy and pathological tissue at the tumor margins, where current visualization methods reach their limits. Here, we report on a novel technique (vascular dual intraoperative visualization approach - vDIVA) enabling visualization of different tumor zones (TZ I–III) on the basis of angiogenic hotspots. We investigated glioblastoma patients who underwent 5-ALA fluorescence-guided surgery with simultaneous intraoperative ICG fluorescence angiography. This vDIVA technique revealed hypervascularized areas which were further histologically investigated. Neuropathological assessments revealed tissue areas at the resection margins corresponding to TZ II and postoperative CD34- and Map2 immunostaining confirmed these angiogenic hotspots to be occupied by glioma cells. Hence, the vascular architecture in this transitional zone could be well differentiated from both primary tumor bulk and healthy brain parenchyma. These data demonstrate that ICG fluorescence angiography improves state-of-the-art glioma surgery techniques and facilitates the future characterization of polyclonal attributes of malignant gliomas.

Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules. These findings support the hypothesis of a temporal accessibility of membrane-bound factors governing the layer-specific sprouting of remaining axons following perforant path lesion in vivo.

2-Deoxy-2-sulfenyl- arabino -α-nucleosides and 2-deoxy-2-sulfenyl- ribo -β-nucleosides were obtained with excellent stereoselectivity from 1-thio- ribo - and 1-thio- arabino -glycosides under Mitsunobu conditions.

Cell recognition molecules of the immunoglobulin superfamily are involved in the formation, establishment, and plasticity of neural circuits in the central nervous system (CNS). We used a polymerase chain reaction-based approach to specifically amplify molecules with conserved sequence elements of immunoglobulin-like domains. This approach enabled us to isolate Kilon, a novel immunoglobulin that has been described by Funatsu et al. (J Biol Chem 1999;274:8224–8230) from the hippocampus. The sequence of Kilon shows a high degree of homology to that of the chicken protein neurotractin, a molecule involved in neurite outgrowth and capable of interacting with LAMP. In situ hybridization analysis was performed to analyze the Kilon mRNA distribution in the developing and adult rat brain and to compare it to that of LAMP mRNA. Kilon mRNA was found to be specifically expressed in the dentate gyrus (DG) of the adult rat, whereas LAMP transcripts were present in all regions of the hippocampal formation. These results were corroborated by RT-PCR semiquantification of gene expression in microdissected tissue prepared from the DG and the CA1 region of the hippocampus. We also performed mRNA expression analysis of both genes following hippocampal deafferentation and seizure, but neither Kilon nor LAMP gene expression showed significant alterations after lesioning on the in situ hybridization level. Our results show that the expression patterns of Kilon and LAMP during development and in the mature hippocampus are clearly distinguishable from one another, which suggests different roles for these related molecules in the hippocampus. Hippocampus 2000;10:632–644. © 2000 Wiley-Liss, Inc.

We analysed the effects of semaphorin D on axons from the developing rat entorhinal-hippocampal formation. Explants from superficial layers of the entorhinal cortex and of the hippocampus anlage were obtained from various developmental stages and co-cultured with cell aggregates expressing semaphorin D. Neurites extending from entorhinal explants that had been isolated from early embryonic stages (E16 and E17) were not affected by semaphorin D, but were repelled at later stages (E20 and E21). Axons from hippocampal neurons explanted at E21 were also repelled by semaphorin D. In situ hybridization studies revealed expression of the semaphorin D receptor neuropilin-1 in the entorhinal cortex from stage E17 to stage P7, and in the dentate gyrus and CA1-3 regions between E17 and adulthood. These data suggest that semaphorin D is involved in the formation of the perforant pathway and acts, via the neuropilin-1 receptor, as a repulsive signal that prevents entorhinal fibres from growing into the granular layer of the dentate gyrus. These data also suggest a role for semaphorin D in the development of intrahippocampal connections.

Synthesis of 9-(2,3-dideoxy-3-fluoro-beta-D-ribofuranosyl)-2-chloroadenine (7b) and -2-chloro-6-methoxypurine (9b), as well as the alpha-D-anomer 7a of the former and its N-7 isomer 10a is reported. Among the compounds synthesized, only the beta-D-anomer 7b displays moderate cytotoxic activity.

We have previously demonstrated that hamster embryo fibroblasts (HEFs) transformed by Rous Sarcoma virus, Schmidt-Ruppin strain (RSV-SR) are highly resistant to damage by H 2 O 2 (H 2 O 2 R ), (in contrast to HEFs transformed spontaneously, or by bovine adenovirus and SV40), while N-ras transfection of RSV-SR transformants leads to suppression of pp60 v-src and of H 2 O 2 R . In this study we have examined (1) mechanisms of antioxidant activity (AOA) of HEFs transformed by these agents and (2) the possible role of the v-src gene in unusually high AOA of RSV-SR transformants and of activated ras oncogenes in its suppression. All transformants exhibit increased catalase and glutathione peroxidase (GP) activities, while SOD, glutathione and glutathione reductase (GR) were reduced. As compared with other transformants, the significantly higher catalase and the low SOD activities were characteristic of RSV-SR-transformants, while an increase in GP was observed in all types of transformants. Correspondingly, RSV-SR-transformants showed an extremely high H 2 O 2 -catabolizing activity (H 2 O 2 CA ) and no lipid peroxidation chain reaction (LPCR). N-ras-induced suppression of pp60 v-src of RSV-SR-transformed HEFs coincided with the suppression of catalase, GP, H 2 O 2 R and H 2 O 2 CA . However, suppression of catalase and GP was also observed in N-ras- and Ha-ras-transfected, spontaneously transformed HEFs. Thus, extremely high catalase activity and suppression of LPCR are apparently the main mechanisms of the unusually high H 2 O 2 R of RSV-SR transformants, while its suppression by activated ras oncogenes may also take place in some transformants, free of v-src activity.

The synthesis of some 3'-C-branched-3'-deoxy adenine nucleosides is described. Starting from the known 3-deoxy-3-C-(hydroxymethyl)-1,2;5,6-di-O-isopropylidene-alpha-D-allof uranose (1), a versatile glycosylating agent, namely 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-C-(mesyloxymethyl)-beta-D-ri bofuranose (6), was prepared in three steps. Condensation of the latter with bis(trimethysilylated) N6-benzoyladenine in the presence of tin(IV) chloride gave the N9-beta-nucleoside 7. Compound 7 was converted into (i) 9-[3-C-(azidomethyl)-3-deoxy-beta-D-ribofuranosyl]adenine (10), (ii) 9-[3-C-(azidomethyl)-2,3-dideoxy-beta-D-glycero-pent-2-enofuran osyl]adenine (14) and 9-[2-azido-3-C-(azidomethyl)-2,3-dideoxy-beta-D-arabinofuranosyl]a denine (15), and (iii) 9-[3-deoxy-2-O,3-C-(methylene)-beta-D-ribofuranosyl]adenine (16). None of the tested nucleosides showed marked cytostatic or antiviral activity in vitro.

A large number of papers have been published on the magnetic field effect (MFE) in various kinds of chemical which were detected mainly by the transient absorption meth0d~9~ and product analy~is.~,~ The radical pair model of CIDNP9$Io has been proposed for the mechanism of this magnetic field dependence and was confirmed by several techniques. For the photoreduction of quinones in sodium dodecyl sulfate (SDS) micellar solutions, we found that the ESR transitions of both the SDS radical and the semiquinone radical affect the yield of a product that is produced only from the SDS radical (i.e. spin adduct).” This is direct evidence of the RP model for this kind of magnetic field dependence. As another evidence of the RP model, Closs et a1.I2 and McLauchlan et al.I3 have explained a new-type of CIDEP found by Sakaguchi et al.I4 for a similar system with the interaction in the RP. Because with our method the ESR spectrum of the RP is traced by the change of the final-product yield, we call this method product-yield-detected ESR,l5%l6 or PYESR in short. Since these spin-dependent phenomena are deeply concerned with the relation between the chemical bond and the spin correlation of the two unpaired electrons from which a new bond is created,I7 these are undoubtedly an important theme of the basic chemistry. In the PYESR technique, we usually use the spin trapping meth~d’~.’~ to separate the escape product as a spin adduct from the cage product, and also to determine the yield by ESR with which in-situ observation is easy. Since PYESR is also possible by using the HPLC method,20 other techniques such as NMR should be potentially applicable. As a similar technique to detect the ESR of a spin pair,21 the optically-detected ESR technique has been well-known in solid-state and subsequently applied to solution chemi~try.~~-~~ These studies have been successful in elucidating the structure of ionic species in the irradiated sy~tems,~~,~~,~~ the reaction mechanism of the photosynthetic system,27 and the dynamics of an ion pair in a photolytically ionized system.28 The main difference with our method is that we observe the yield of a final product and the

Three pulse sequences have been employed for laser and microwave-pulse irradiation in the pulse mode product-yield-detected ESR technique to detect the dynamical feature of transient radical pair. In this method, the decrease in spin adduct yield as functions of delay times for irradiation/termination of the microwave pulse after the laser pulse are simulated with the Runge-Kutta method for a detailed reaction model to obtain the rate constants of cage product formation, escape of the radical pair, as well as the hydrogen abstraction reaction. As the representative reaction systems, photoreduction of anthraquinone, anthraquinone-2-sulfonate, and benzophenone in SDS micellar solution were employed. Effects of cyclohexene, added as a hydrogen donor, NaCl and Ethanol, added to change the microviscosity of micelle, on these reaction rate constants have been tested.

Excitotoxic brain lesions, such as stroke and epilepsy, lead to increasing destruction of neurons hours after the insult. The deadly cascade of events involves detrimental actions by free radicals and the activation of proapoptotic transcription factors, which finally result in neuronal destruction. Here, we provide direct evidence that the nutritionally essential trace element selenium has a pivotal role in neuronal susceptibility to excitotoxic lesions. First, we observed in neuronal cell cultures that addition of selenium in the form of selenite within the physiological range protects against excitotoxic insults and even attenuates primary damage. The neuroprotective effect of selenium is not directly mediated via antioxidative effects of selenite but requires de novo protein synthesis. Gel shift analysis demonstrates that this effect is connected to the inhibition of glutamate-induced NF-kappaB and AP-1 activation. Furthermore, we provide evidence that selenium deficiency in vivo results in a massive increase in susceptibility to kainate-induced seizures and cell loss. These findings indicate the importance of selenium for prevention and therapy of excitotoxic brain damage.

A convenient preparation of α- and β-anomers of 1,5-di-O-benzoyl-2,3-dideoxy-3-fluoro-D-erythro-pentofuranose is described. Each anomer is studied as the glycosylating agent in the reaction with bis-trimethylsilylated thymine in the presence of trimethylsilyl triflate. Under optimum conditions, 2',3'-dideoxy-3'-fluorothymidine (FLT) and its α-anomer are isolated in 64 and 25% yields, respectively

A series of 3'-deoxy-3'-fluoro- and 2'-azido-2',3'-dideoxy-3'-fluoro-D-ribofuranosides of natural heterocyclic bases have been synthesized with the use of universal carbohydrate precursors, viz., 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-fluoro-D-ribofuranose and methyl 2-azido-5-O-benzoyl-2,3-dideoxy-3-fluoro-beta-D-ribofuranoside, respectively. The cytostatic and antiviral activity of the compounds was evaluated against a variety of tumor cell lines and DNA/RNA viruses, respectively. As the most active compound, from both a cytostatic and antiviral activity viewpoint, emerged 3'-deoxy-3'-fluoroadenosine. It inhibited the proliferation of some tumor cell lines (i.e. murine leukemia L1210 and human T-lymphocyte MT-4) at a concentration of 0.2-2 micrograms/mL, and proved inhibitory to the replication of positive-stranded RNA viruses (i.e. polio, Coxsackie, Sindbis, Semliki forest), double-stranded RNA viruses (i.e. reo), and some DNA viruses (i.e. vaccinia) at a concentration of 1-4 micrograms/mL, which is well below the cytotoxicity threshold (40 micrograms/mL).

A large number of papers have been published on the magnetic field effect (MFE) in various kinds of chemical which were detected mainly by the transient absorption meth0d~9~ and product analy~is.~,~ The radical pair model of CIDNP9$Io has been proposed for the mechanism of this magnetic field dependence and was confirmed by several techniques. For the photoreduction of quinones in sodium dodecyl sulfate (SDS) micellar solutions, we found that the ESR transitions of both the SDS radical and the semiquinone radical affect the yield of a product that is produced only from the SDS radical (i.e. spin adduct).” This is direct evidence of the RP model for this kind of magnetic field dependence. As another evidence of the RP model, Closs et a1.I2 and McLauchlan et al.I3 have explained a new-type of CIDEP found by Sakaguchi et al.I4 for a similar system with the interaction in the RP. Because with our method the ESR spectrum of the RP is traced by the change of the final-product yield, we call this method product-yield-detected ESR,l5%l6 or PYESR in short. Since these spin-dependent phenomena are deeply concerned with the relation between the chemical bond and the spin correlation of the two unpaired electrons from which a new bond is created,I7 these are undoubtedly an important theme of the basic chemistry. In the PYESR technique, we usually use the spin trapping meth~d’~.’~ to separate the escape product as a spin adduct from the cage product, and also to determine the yield by ESR with which in-situ observation is easy. Since PYESR is also possible by using the HPLC method,20 other techniques such as NMR should be potentially applicable. As a similar technique to detect the ESR of a spin pair,21 the optically-detected ESR technique has been well-known in solid-state and subsequently applied to solution chemi~try.~~-~~ These studies have been successful in elucidating the structure of ionic species in the irradiated sy~tems,~~,~~,~~ the reaction mechanism of the photosynthetic system,27 and the dynamics of an ion pair in a photolytically ionized system.28 The main difference with our method is that we observe the yield of a final product and the

The oxidation of methyl 5-O- benzyl-3(2)-deoxy-3(2)-fluoro- α- d - pentofuranosides with dimethyl sulfoxide-acetic (trifluoroacetic) anhydride was accompanied by epimerization at the carbon atom bearing a fluoro function, resulting in the formation of the corresponding 2- or 3-keto derivatives as mixtures of two epimers in high combined yield. Reduction of a mixture of the erythro / threo epimeric 2-keto sugars (isolated as stable hydrates) with sodium borohydride in benzene-ethanol proceeded stereoselectively leading to the formation of 3-deoxy-3-fluoro ribo and lyxo-furanosides, respectively. In the case of the ribo and arabino epimers of the 3-keto sugar (isolated as free ketones), reduction stereoselectivity of the former was > 95% for the 2-deoxy-2-fluoro ribo sugar, whereas a ca. 3:1 lyxo / arabino ratio of products was obtained for the latter. Treatment of a mixture of the 2-epimeric 3-keto sugars with triethylamine in carbon tetrachloride at room temperature for 3–5 h afforded the 2-deoxy-2-fluoro ribo ketone (ca. 90%). The synthesis of 1-O- acetyl-2,5-di -O- benzoyl-3-deoxy-3-fluoro- α,β- d - lyxofuranose (8) and 1-O- acetyl-3,5-di -O- benzoyl-2-deoxy-2-fluoro -β- d - ribofuranose (16) and their use as glycosylating agents for bis-trimethylsilylated N6-benzoyladenine is described.

2-Deoxy-2-sulfenyl- arabino -α-nucleosides and 2-deoxy-2-sulfenyl- ribo -β-nucleosides were obtained with excellent stereoselectivity from 1-thio- ribo - and 1-thio- arabino -glycosides under Mitsunobu conditions.

Oxidative brain damage, such as excitotoxicity and stroke, leads to primary neuronal destruction. The primary damage is further potentiated by macrophages and microglial cells, which are attracted and invade into the zone of damage resulting in secondary neuronal death. Since the essential trace element selenium has anti-inflammatory properties, we analyzed the effects of selenium on these inflammatory cells. Here, we show that the essential trace element selenium abrogates the stress-induced migration of microglial cells. Thus, the antimigratory effects of selenium may attenuate the secondary cell death cascade by preventing microglial invasion.

Autotaxin is a secreted cell motility-stimulating exo-phosphodiesterase with lysophospholipase D activity that generates bioactive lysophosphatidic acid. Lysophosphatidic acid has been implicated in various neural cell functions such as neurite remodeling, demyelination, survival and inhibition of axon growth. Here, we report on the in vivo expression of autotaxin in the brain during development and following neurotrauma. We found that autotaxin is expressed in the proliferating subventricular and choroid plexus epithelium during embryonic development. After birth, autotaxin is mainly found in white matter areas in the central nervous system. In the adult brain, autotaxin is solely expressed in leptomeningeal cells and oligodendrocyte precursor cells. Following neurotrauma, autotaxin is strongly up-regulated in reactive astrocytes adjacent to the lesion. The present study revealed the cellular distribution of autotaxin in the developing and lesioned brain and implies a function of autotaxin in oligodendrocyte precursor cells and brain injuries.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

Molecular biology has recently contributed significantly to the recognition of selenium (Se)2 and Se-dependent enzymes as modulators of brain function. Increased oxidative stress has been proposed as a pathomechanism in neurodegenerative diseases including, among others, Parkinson's disease, stroke, and epilepsy. Glutathione peroxidases (GPx), thioredoxin reductases, and one methionine-sulfoxide-reductase are selenium-dependent enzymes involved in antioxidant defense and intracellular redox regulation and modulation. Selenium depletion in animals is associated with decreased activities of Se-dependent enzymes and leads to enhanced cell loss in models of neurodegenerative disease. Genetic inactivation of cellular GPx increases the sensitivity towards neurotoxins and brain ischemia. Conversely, increased GPx activity as a result of increased Se supply or overexpression ameliorates the outcome in the same models of disease. Genetic inactivation of selenoprotein P leads to a marked reduction of brain Se content, which has not been achieved by dietary Se depletion, and to a movement disorder and spontaneous seizures. Here we review the role of Se for the brain under physiological as well as pathophysiological conditions and highlight recent findings which open new vistas on an old essential trace element.

In addition to acting as an essential nutrient for the immune system and overall body function, it is apparent that selenium also plays a critical role in the operation of the nervous system. Selenium itself is a constituent of selenoproteins, which are primarily involved in antioxidant function and redox status. However, apart from its covalent incorporation into these proteins, selenium also performs neuroprotective actions independent of translational processes. Furthermore, low selenium intake has detrimental effects on proper brain function, such as epileptic episodes and neuronal cell death, which have, in turn, been shown to be mitigated by higher selenium levels. Understanding the mechanisms of selenium action will be crucial to determining its potential as a preventive and therapeutic agent against excitatory brain damage.

Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as ligands that bind to specific receptors. Here, we report a new membrane-associated lipid phosphate phosphatase that we have named plasticity-related gene 1 (PRG-1), which facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ecto-enzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, we propose a novel mechanism by which axons are able to control phospholipid-mediated signaling and overcome the growth-inhibiting, phospholipid-rich environment of the extracellular space.

We report synthesis and high-affinity hybridization of fully modified home-thymine 2′-deoxy and 2′-deoxy-2′-fluoro xylo nucleic acids.

Xylo-Configured oligonucleotides (XNA) containing a novel conformationally restricted 2′-deoxy-2′-fluoro-β- d -xylofuranosyl nucleotide monomer, a novel conformationally locked 2′-amino-2′-deoxy-2′-N,4′-C-methylene-β- d -xylofuranosyl nucleotide monomer, and a known 2′-deoxy-β- d -xylofuranosyl nucleotide monomer (XNA monomers) have been synthesized and their hybridization towards DNA and RNA complements studied. Thermal denaturation studies of nine-mer mixed-base sequences composed of a mixture of XNA monomers and DNA monomers revealed preferential hybridization towards RNA complements relative to DNA complements. For 14-mer homo-thymine XNAs containing thirteen XNA monomers, stable complexes towards single-stranded DNA and RNA were formed at pH 7. Gel-shift experiments revealed these complexes to involve at least two XNA strands per DNA or RNA target strand.

The properties of two non-conventional substrates of the calf-spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3-(beta-D-ribofuranosyl)adenine (RibfAde) and 3-(beta-D-ribofuranosyl)hypoxanthine (RibfHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo-first-order rate constants (Vmax/K(m)) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the corresponding values are 22% and 30%, respectively. The Michaelis constants (K(m)) for RibfAde were 800 microM (calf PNP) and 150 microM (E. coli PNP). For RibfHyp, the corresponding K(m) values were 220 microM and 260 microM. Two well-characterized inhibitors of calf spleen PNP [9-(2-fluoro-3,4-dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3-beta-nucleosides occurs at the same active site as for the natural substrate Ino. In particular, the substrate properties of both 3-beta-nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9-beta-nucleosides in the conformation anti to the pyrimidine ring. The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.

A series of 3'-deoxy-3'-fluoro- and 2'-azido-2',3'-dideoxy-3'-fluoro-D-ribofuranosides of natural heterocyclic bases have been synthesized with the use of universal carbohydrate precursors, viz., 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-fluoro-D-ribofuranose and methyl 2-azido-5-O-benzoyl-2,3-dideoxy-3-fluoro-beta-D-ribofuranoside, respectively. The cytostatic and antiviral activity of the compounds was evaluated against a variety of tumor cell lines and DNA/RNA viruses, respectively. As the most active compound, from both a cytostatic and antiviral activity viewpoint, emerged 3'-deoxy-3'-fluoroadenosine. It inhibited the proliferation of some tumor cell lines (i.e. murine leukemia L1210 and human T-lymphocyte MT-4) at a concentration of 0.2-2 micrograms/mL, and proved inhibitory to the replication of positive-stranded RNA viruses (i.e. polio, Coxsackie, Sindbis, Semliki forest), double-stranded RNA viruses (i.e. reo), and some DNA viruses (i.e. vaccinia) at a concentration of 1-4 micrograms/mL, which is well below the cytotoxicity threshold (40 micrograms/mL).

Synthesis of 2′,3′-dideoxy-3′-C-(dihydroxyphosphinylmethyl)-adenosine and -thymidine 5, as well as of 2′-deoxy-2′-C-(dihydroxyphosphinylmethyl)-adenosine and -thymidine 9 was accomplished with the ...

The oxidation of methyl 5-O- benzyl-3(2)-deoxy-3(2)-fluoro- α- d - pentofuranosides with dimethyl sulfoxide-acetic (trifluoroacetic) anhydride was accompanied by epimerization at the carbon atom bearing a fluoro function, resulting in the formation of the corresponding 2- or 3-keto derivatives as mixtures of two epimers in high combined yield. Reduction of a mixture of the erythro / threo epimeric 2-keto sugars (isolated as stable hydrates) with sodium borohydride in benzene-ethanol proceeded stereoselectively leading to the formation of 3-deoxy-3-fluoro ribo and lyxo-furanosides, respectively. In the case of the ribo and arabino epimers of the 3-keto sugar (isolated as free ketones), reduction stereoselectivity of the former was > 95% for the 2-deoxy-2-fluoro ribo sugar, whereas a ca. 3:1 lyxo / arabino ratio of products was obtained for the latter. Treatment of a mixture of the 2-epimeric 3-keto sugars with triethylamine in carbon tetrachloride at room temperature for 3–5 h afforded the 2-deoxy-2-fluoro ribo ketone (ca. 90%). The synthesis of 1-O- acetyl-2,5-di -O- benzoyl-3-deoxy-3-fluoro- α,β- d - lyxofuranose (8) and 1-O- acetyl-3,5-di -O- benzoyl-2-deoxy-2-fluoro -β- d - ribofuranose (16) and their use as glycosylating agents for bis-trimethylsilylated N6-benzoyladenine is described.

Oxidation of 1 with DMSO-acetic anhydride resulted in the formation of a mixture of epimeric ketones 2 and 3 in the ratio of ≍3:1 in high combined yield. Acetolysis of methyl glycoside 5 afforded 1-O-acetyl-3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-ribofuranoside (6)(83%). The latter was reacted with silylated N6-benzoyladenine to give α- and β-ribosides (1:3.7; 61%, combined).

The brain represents a privileged organ with respect to selenium (Se) supply and retention. It contains high amounts of this essential trace element, which is efficiently retained even in conditions of Se deficiency. Accordingly, no severe neurological phenotype has been reported for animals exposed to Se-depleted diets. They are, however, more susceptible to neuropathological challenges. Recently, gene disruption experiments supported a pivotal role for different selenoproteins in brain function. Using these and other transgenic models, longstanding questions concerning the preferential supply of Se to the brain and the hierarchy among the different selenoproteins are readdressed. Given that genes for at least 25 selenoproteins have been identified in the human genome, and most of these are expressed in the brain, their specific roles for normal brain function and neurological diseases remain to be elucidated.

Background The clinical impact of the positivity of the Deauville scale (DS) of positron emission tomography (PET) performed at the end of doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) in patients with advanced Hodgkin lymphoma (HL), in terms of providing rationale to shift poor responders onto a more intensive regimen, remain to be validated by histopathology. Patients and methods This prospective trial involved patients with stage IIB/IV HL who after six ABVD cycles underwent PET (PET6) and core-needle cutting biopsy (CNCB) of 2-deoxy-2[F-18] fluoro- d -glucose (FDG)-avid lymph nodes. Patients received high-dose chemotherapy/autologous haematopoietic stem cell rescue (HDCT/AHSCR) if CNCB was positive for HL, alternatively, if CNCB or PET was negative, received observation or consolidation radiotherapy (cRT) on residual nodal masses, as initially planned. The end-point was 5-year progression-free survival (PFS). Results In all, 43 of the 169 (25%) evaluable patients were PET6 positive (DS 4, 32; DS 5, 11). Among them, histology showed malignancy (HL) in 100% of DS 5 scores and in 12.5% of DS 4 scores. Fifteen patients with positive biopsy received HDCT/AHSCR, whereas 28 patients with negative biopsy, as well as 126 patients with negative PET6, continued the original plan (cRT, 78 patients; observation, 76 patients). The 5-year PFS in the negative PET6 group, negative biopsy group and positive biopsy group was 95.4%, 100% and 52.5%, respectively. Conclusion DS positivity of end-of-ABVD PET in advanced HL carried a certain number of CNCB-proven non-malignant FDG-uptakes. The DS 4 scores which were found to have negative histology appeared to benefit from continuing the original non-intensive therapeutic plane as indicated by the successful outcome in more than 95% of them by obtaining similar 5-year PFS to the PET6-negative group. By contrast, the DS 5 score had consistently positive histology and was associated with unsuccessful conventional therapy, promptly requiring treatment intensification or innovative therapeutic approaches.

Little is known about how CO2 affects neural processing of taste. We used functional magnetic resonance imaging to investigate the effects of carbonation on brain processing of sweet stimuli, which has relevance to studies of food selection and satiety. The presence of carbonation produced an overall decrease in the neural processing of sweetness-related signals, especially from sucrose. CO2 reduced the neural processing of sucrose more than that of artificial sweeteners. These findings might be relevant to dietary interventions that include noncaloric beverages, whereas the combination of CO2 and sucrose might increase consumption of sucrose.

To clarify the clinical significance of Tl-201 reverse redistribution (RR), 33 patients with chronic coronary artery disease (CAD) underwent stress-redistribution Tl-201 cardiac imaging with rest reinjection, coronary arteriography, and 2D-echocardiography. Rest Tc-99m MIBI scintigraphy was also performed in 27 of the 33 patients. A total of 495 segments were analyzed for Tl-201 scintigraphy (405 for Tc-99m MIBI). Each segment was assigned to one of the major coronary artery territories. Two patterns of RR were identified; 1) pattern A (RR-A) showed normal Tl-201 uptake on stress images and lower than normal on redistribution images, and 2) pattern B (RR-B) showed lower than normal Tl-201 uptake on stress images with further decrease on redistribution images. The RR phenomenon was found in 46 (9% of the total) segments; 25 with RR-A and 21 with RR-B. Reverse redistribution pattern A segments had lower Tc-99m MIBI uptake (84 +/- 9% versus 92 +/- 10%, P < 0.0001) and a higher percentage of stenosed coronary arteries (80% versus 49%, P < 0.05) compared to normal segments (n = 204, 41% of the total). No difference in wall motion was observed between RR-A and normal segments. Of the 25 segments with RR-A, 14 showed enhanced Tl-201 uptake after reinjection (Re+) and 11 remained unchanged after reinjection (Re-). Segments that were Re- showed significantly (P < 0.05) lower Tc-99m MIBI uptake (79 +/- 9%) compared to Re+ segments (87 +/- 8%) and normal segments (92 +/- 10%).(ABSTRACT TRUNCATED AT 250 WORDS)

In patients with chronic heart failure (CHF), the addition of coenzyme Q10 to conventional therapy reduces the hospitalization rate for worsening of heart failure and the incidence of serious cardiovascular complications. The present study was planned to assess the hemodynamic mechanisms underlying this phenomenon. Cardiac hemodynamics was evaluated continuously using an ambulatory radionuclide detector (VEST) which allows a noninvasive monitoring of left ventricular function. Six patients wit CHF (mean ejection fraction (EF): 29%) clinically documented were studied. This study was organized as a randomized double-blind, placebo controlled, cross-over trial. The enrolled patients, after a washout period, underwent the first hemodynamic evaluation with VEST. Subsequently they were randomized to receive placebo or coenzyme Q10 for 4 weeks. At the end of this period they underwent the second VEST study. The third VEST study was performed after a further 4-week period with inverted treatment. Cardiac hemodynamics were evaluated during bicycle exercise. The EF in control conditions (CC) changed from 27 +/- 11%, at rest, to 24 +/- 8%, at peak exercise. During coenzyme Q10 treatment EF showed a significant increase both at rest (33 +/- 13%, P < 0.05 vs CC) and at peak exercise (30 +/- 12%, P < 0.05 vs CC). The same trends were recorded for the stroke volume and the cardiac output. Our results demonstrate that coenzyme Q10 improves cardiac hemodynamic response to exercise in patients with CHF and suggest that noninvasive monitoring of left ventricular function allows a more reliable assessment of therapy efficacy.