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Diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF), has a high affinity for heparin. We studied the effect of heparin-like molecules on the binding of diphtheria toxin (DT) to DTR/proHB-EGF. Mutant Chinese hamster ovary (CHO) cells deficient in heparan sulfate (HS) proteoglycans were about 15 times less sensitive to DT than wild type CHO-K1 cells. When free heparan sulfate or heparin was added to the culture medium, DT sensitivity of the mutant cells was fully restored. Studies of binding of 125I-labeled DT to HS-deficient CHO cells transfected with human DTR/proHB-EGF cDNA indicated that the increased sensitivity to DT after addition of heparin is due to increased binding of DT to cells. Vero cells display a relatively large amount of heparan sulfate residues compared to CHO-K1 cells or L cells. Enhancement of DT binding by the addition of heparin was also observed with CHO-K1 cells and L cells that had been transfected with human DTR/proHB-EGF cDNA, but the degree of enhancement was less than that observed with the HS-deficient CHO cells. Addition of heparin did not affect DT binding or DT sensitivity of Vero cells. Heparin-dependent binding was observed when intact Vero cells were treated with heparitinase or when the cell membrane was solubilized with a neutral detergent. Scatchard plot analysis for the binding of DT to a recombinant HB-EGF in vitro and to L cells expressing human DTR/proHB-EGF revealed that heparin increases the affinity of DTR/proHB-EGF for DT but does not change the number of binding sites. Although DRAP27/CD9 is known to enhance DT binding to DTR/proHB-EGF, the results indicate that heparin and DRAP27/CD9 increase DT binding by independent mechanisms. Thus, heparin-like molecules, probably in the form of heparan sulfate proteoglycan on the cell surface, are a third factor required for maximal DT binding activity of cells.

Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma.

D-amino acid oxidase (DAO) is a flavoenzyme that exists in the kidney, liver and brain of mammals. This enzyme catalyzes the oxidation of D-amino acids to the corresponding α-keto acid, hydrogen peroxide and ammonia. Recently D-serine, one of the substrates of DAO, has been found in the mammalian brain, and shown to be a co-agonist of the N-methyl-D-aspartate (NMDA) receptor in glutamate neurotransmission. In this study, we investigated the metabolism of extracellular D-serine and the effects of D-serine metabolites to study the pathophysiological role of DAO. Treatment with a high dose of D-serine induced the cell death in dose-dependent manner in DAO-expressing cells. Moreover, overexpression of DAO in astroglial cells induced the enhanced cytotoxicity. The treatment with 1 mM beta-hydroxypyruvate (HPA), uniquely produced from the D-serine metabolism by DAO activity, also induced cell death, comprising apoptosis, in the astroglial cell, but not in the other cells derived from liver and kidney. Taken together, we consider that high dose of extracellular D-serine induced cell death by the production of not only hydrogen peroxide but also HPA as a result of DAO catalytic activity in astroglial cell. Furthermore, this cytotoxicity of HPA is observed uniquely in astroglial cells expressing DAO.

Chiral synthesis of (−)-nupharamine and (+)-3-epinupharamine, based on elaboration of the intramolecular asymmetric Diels-Alder reaction of N-acylnitroso compounds and the introduction of the furyl group in a fully stereocontrolled manner, is described.

A general protocol for the enantiogenic total syntheses of a series of the 5-substituted 8-methylindolizidine class of alkaloids from the arrow poison frog, i.e., (-)-indolizidines 205A (1), 207A (2), 209B (3), and 235B (4), is described, in which a key step is the asymmetric intramolecular Diels-Alder reaction of the chiral N-acylnitroso compound 8 leading to the bicyclic oxazinolactam 7 which was utilized as a versatile common chiral intermediate for the preparation of these alkaloids

d-Amino acid oxidase (DAO) is a peroxisomal flavoenzyme that catalyzes oxidative deamination of a wide range of d-amino acids. Among the possible substrates of DAO in vivo, d-serine is proposed to be a neuromodulator of the N-methyl-d-aspartate (NMDA) type glutamate receptor. The gene for DAO was reported to be associated with schizophrenia. Since DAO is expected to be one of the key enzymes in the regulation of NMDA neurotransmission, the modulation of the enzyme activity is expected to be therapeutical for neuronal disorders. In search of the pathophysiological role of DAO, we analyzed the distribution of DAO mRNA and protein in the rat and human brain. In rat, the distribution of DAO mRNA was newly detected in choroid plexus (CP) epithelial cells in addition to glial cells of pons, medulla oblongata, and especially Bergmann glia of cerebellum. Moreover, to investigate how DAO expression level is altered in schizophrenia, we performed immunohistochemistry in the human brain. In agreement with the results in the rat brain, the immunoreactivity for DAO was detected in glial cells of rhombencephalon and in CP. Furthermore, higher level of DAO expression was observed in schizophrenic CP epithelial cells than that in non-schizophrenic cases. These results suggest that an increase in DAO expression in parts of the brain is involved in aberrant d-amino acid metabolism. In particular, gene expression of DAO in CP suggests that DAO may regulate d-amino acid concentration by modulating the cerebrospinal fluid and may be regarded as a potential therapeutic target for schizophrenia.

Chiral synthesis of (−)-nupharamine and (+)-3-epinupharamine, based on elaboration of the intramolecular asymmetric Diels-Alder reaction of N-acylnitroso compounds and the introduction of the furyl group in a fully stereocontrolled manner, is described.

A novel central nervous system (CNS) selective neurokinin-1 (NK(1)) receptor antagonist, (2S,3S)-3-[(1R)-6-methoxy-1-methyl-1-trifluoromethylisochroman-7-yl]-methylamino-2-phenylpiperidine 'CJ-17,493' (compound (+)-1), was synthesized stereoselectively using a kinetic resolution by lipase-PS as a key step. Compound (+)-1 displayed high and selective affinity (K(i)=0.2 nM) for the human NK(1) receptor in IM-9 cells, potent activity in the [Sar(9), Met(O(2))(11)]SP-induced gerbil tapping model (ED(50)=0.04 mg/kg, s.c.) and in the ferret cisplatin (10mg/kg, i.p.)-induced anti-emetic activity model (vomiting: ED(90)=0.07 mg/kg, s.c.), all levels of activity comparable with those of CP-122,721. In addition, compound (+)-1 exhibited linear pharmacokinetics rather than the super dose-proportionality of CP-122,721 and this result provides a potential solution for the clinical issue observed with CP-122,721.

1,1-Dialkyl-2-perfluoroalkyl compounds, which are potential metabolically stable bioisosteres of the tert-alkyl moiety, have been synthesized from the corresponding tertiary alcohols using titanium (IV) chloride-dimethylzinc or trimethylaluminium as the source of the methyl group. The synthetic methods proved to be versatile for synthesizing 1,1-dimethyl-2,2,2-trifluoroethyl compounds and analogs, including compounds containing aromatic and heterocyclic rings.

1,1-Dialkyl-2-perfluoroalkyl compounds, which are potential metabolically stable bioisosteres of the tert-alkyl moiety, have been synthesized from the corresponding tertiary alcohols using titanium (IV) chloride-dimethylzinc or trimethylaluminium as the source of the methyl group. The synthetic methods proved to be versatile for synthesizing 1,1-dimethyl-2,2,2-trifluoroethyl compounds and analogs, including compounds containing aromatic and heterocyclic rings.

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that has a high affinity for heparin and heparan sulfate. While interactions with heparin are thought to modulate the biological activity of HB-EGF, the precise role of the heparin-binding domain has remained unclear. We analyzed the activity of wild-type HB-EGF and a mutant form lacking the heparin-binding domain (DeltaHB) in the presence or absence of heparin. The activity of the EGF-like domain of HB-EGF was determined by measuring binding to diphtheria toxin (DT) as well as the growth factor activity in EGF receptor-expressing cells. The binding affinity of DeltaHB for DT was much higher than that of wild-type HB-EGF in the absence of heparin. The binding affinity of HB-EGF for DT was increased by addition of exogenous heparin and reached the level close to the affinity of DeltaHB, whereas that of DeltaHB was not affected. Moreover, the growth factor activity of DeltaHB was much higher than that of wild-type HB-EGF in the absence of heparin but was not affected by addition of exogenous heparin, whereas HB-EGF had increased growth factor activity with added heparin. These results indicate that the heparin-binding domain suppresses the activity of the EGF-like domain of HB-EGF and that association of heparin with HB-EGF via this domain removes the suppressive effect. Thus, we conclude that the heparin-binding domain serves as a negative regulator of this growth factor.

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.

EC cells are characterized by their potent capacity to differentiate into several cell types, such as mesoderm-like cells, endoderm-like cells, or ectoderm-like cells. By subtracting the mRNAs expressed by one EC cell clone, F9 cells, with the mRNAs expressed by another EC cell clone, P19 cells, we identified six novel genes that are expressed selectively by F9 cells. One of these genes (Nucling) encodes a polypeptide of 1411 amino acids containing an ankyrin repeat, aspartyl protease motif, a leucine zipper motif, and two t-SNARE coiled-coil domains. Northern blot analyses revealed the Nucling mRNA to be detected predominantly in heart, liver, kidney and testis, but not in brain or spleen. Immunostaining analyses revealed a unique feature of Nucling that the transiently expressed protein forms aggregates exclusively around nuclear membranes. Moreover, the expression level of the Nucling gene transcript increases progressively during the early developmental stages in mice, and specifically at cardiomuscular differentiation in vitro and in vivo. These results suggest that Nucling may play some role in the gene regulation of cell differentiation during embryonal development.

CD9 associates with a diphtheria toxin receptor (DTR) that is identical to the membrane-anchored form of heparin-binding EGF-like growth factor. We determined the region of CD9 important for upregulation activity. Human and monkey CD9 upregulates DT binding activity of DTR, while mouse CD9 has no upregulation activity. Transfection of chimeric constructs comprising monkey and mouse CD9s showed that the human sequence between Ala156 and Asp183 is essential for the upregulation activity. Studies of mutants, replacing a single amino acid within the region between Ala156 and Asp183 of monkey CD9 with the corresponding amino acid residue in mouse CD9, revealed that substitution of Gly158 is critical for the reduction of the upregulation activity and secondly for the substitution of Val159 and Thr175. These three amino acid residues were deduced to be located on the head domain of the second extracellular loop, suggesting that interactions of CD9 with DTR or DT at the domain containing these three amino acids were important for the upregulation of DT binding.

Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.

The total synthesis of (–)-indolizidines 205A and 235B, alkaloids from the arrow poison-frog, via a common chiral oxazino-lactam, prepared by an asymmetric intramolecular Diels–Alder reaction of an N-acylnitroso intermediate, is described.