BackgroundChronic treatment with selective serotonin (5-HT) reuptake inhibitors (SSRIs) facilitates adult neurogenesis and reverses the state of maturation in mature granule cells (GCs) in the dentate gyrus (DG) of the hippocampus. Recent studies have suggested that the 5-HT4 receptor is involved in both effects. However, it is largely unknown how the 5-HT4 receptor mediates neurogenic effects in the DG and, how the neurogenic and dematuration effects of SSRIs interact with each other.ResultsWe addressed these issues using 5-HT4 receptor knockout (5-HT4R KO) mice. Expression of the 5-HT4 receptor was detected in mature GCs but not in neuronal progenitors of the DG. We found that chronic treatment with the SSRI fluoxetine significantly increased cell proliferation and the number of doublecortin-positive cells in the DG of wild-type mice, but not in 5-HT4R KO mice. We then examined the correlation between the increased neurogenesis and the dematuration of GCs. As reported previously, reduced expression of calbindin in the DG, as an index of dematuration, by chronic fluoxetine treatment was observed in wild-type mice but not in 5-HT4R KO mice. The proliferative effect of fluoxetine was inversely correlated with the expression level of calbindin in the DG. The expression of neurogenic factors in the DG, such as brain derived neurotrophic factor (Bdnf), was also associated with the progression of dematuration. These results indicate that the neurogenic effects of fluoxetine in the DG are closely associated with the progression of dematuration of GCs. In contrast, the DG in which neurogenesis was impaired by irradiation still showed significant reduction of calbindin expression by chronic fluoxetine treatment, suggesting that dematuration of GCs by fluoxetine does not require adult neurogenesis in the DG.ConclusionsWe demonstrated that the 5-HT4 receptor plays an important role in fluoxetine-induced adult neurogenesis in the DG in addition to GC dematuration, and that these phenomena are closely associated. Our results suggest that 5-HT4 receptor-mediated phenotypic changes, including dematuration in mature GCs, underlie the neurogenic effect of SSRIs in the DG, providing new insight into the cellular mechanisms of the neurogenic actions of SSRIs in the hippocampus.

A series of R and S enantiomers of 2-aminopurine methylenecyclopropane analogues of nucleosides was synthesized. Two diastereoisomeric lipophilic phosphate prodrugs derived from R and S enantiomers of 2,6-diaminopurine analogue were also prepared. Enantioselectivity (diastereoselectivity in case of prodrugs) of in vitro antiviral effects was investigated with human and murine cytomegalovirus (HCMV and MCMV, respectively), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV). Strong differences in enantioselectivity were found between the R and S enantiomers of adenine analogue and enantiomeric 2-aminopurine analogues. Thus, the enantiomers of adenine analogue were equipotent against HCMV but not MCMV, where the S enantiomer is strongly preferred. The same S preference was found throughout the 2-aminopurine series for both HCMV and MCMV. In contrast, R-synadenol in HIV-1 assays was the best agent, whereas the S enantiomers of moderately effective 2-amino-6-cyclopropylamino and 2-amino-6-methoxypurine analogues were preferred. Little enantiomeric preference was found for R and S enantiomers of synadenol and the corresponding enantiomers of 2,6-diaminopurine analogue against HBV. A mixed pattern of enantioselectivity was observed for EBV depending on the type of host cells and assay. Against VZV, the R and S enantiomers of adenine analogue were equipotent or almost equipotent, but throughout the series of 2-aminopurine analogues a distinct preference for the S enantiomers was found. The stereoselectivity pattern of both diastereoisomeric prodrugs mostly followed enantioselectivity of the parent analogues. The varying enantioselectivities in the series of purine methylenecyclopropane analogues are probably a consequence of differences in the mechanisms of action in different virus/host cell systems.

Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a, 95-96% ee) is described. Racemic synadenol (1a + 2a) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a) and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound gave acetate 6. Reaction with N, N-dimethylchloromethyleneammonium chloride led to 6-chloropurine derivative 7. Ammonolysis furnished (S)-(+)-synadenol (2a). Absolute configuration of 1a was established by two methods: (i) synthesis from (R)-methylenecyclopropanecarboxylic acid (8) and (ii) X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. Racemic methylenecyclopropanecarboxylic acid (10) was resolved by a modification of the described procedure. The R-enantiomer 8 was converted to ethyl ester 13 which was brominated to give vicinal dibromides 14. Reduction with diisobutylaluminum hydride then furnished alcohol 15 which was acetylated to the corresponding acetate 16. Alkylation-elimination procedure of adenine with 16 yielded acetates 17 and 18. Deprotection with ammonia afforded a mixture of Z- and E-isomers 1a and 19 of the R-configuration. Comparison with products 1a and 2a by chiral HPLC established the R-configuration of (-)-synadenol (1a). These results were confirmed by X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. The latter forms a pseudosymmetric dimer with adenine-adenine base pairing in the lattice with the nucleobase in an anti-like conformation. Enantiomers 1a and 2a exhibit varied enantioselectivity toward different viruses. Both enantiomers are equipotent against human cytomegalovirus (HCMV) and varicella zoster virus (VZV). The S-enantiomer 2a is somewhat more effective than R-enantiomer 1a in herpes simplex virus 1 and 2 (HSV-1 and HSV-2) assays. By contrast, enantioselectivity of antiviral effect is reversed in Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1) assays where the R-enantiomer 1a is preferred. In these assays, the S-enantiomer 2a is less effective (EBV) or devoid of activity (HIV-1).

As the number of patients treated with acyclovir (ACV) has increased, increasing numbers of ACV-resistant (ACV r ) herpes simplex virus (HSV) and varicella-zoster virus strains have been isolated, mainly from immunocompromised patients (8). The selectivity of ACV as an antiherpesvirus drug is based on its specific interaction with virus-encoded enzymes, thymidine kinase (TK) and DNA polymerase (DNA Pol). Therefore, the mechanisms responsible for ACV resistance are mutations in the TK and/or DNA Pol polypeptides (1). A previous largescale clinical study on ACV r HSV strains isolated from patients infected with human immunodeficiency virus indicated that 96% of ACV r HSV mutants were low producers of, or deficient in, TK activity (TK Ϫ ), with 4% being TK mutants with an altered substrate specificity. No DNA Pol mutants were isolated (12).It is generally believed that ACV r strains arise from naturally occurring mutations during DNA replication and are selected by ACV both in vitro (cell culture experiments) and in vivo (patients) (2). This hypothesis was formed from the following observations: (i) without exposure to ACV, approximately 0.3 to 20 ACV r mutants occur in 10 4 PFU of clinical HSV-1 isolates (11,19,26,28) and of a laboratory strain grown from one plaque (2); (ii) ACV r isolates under ACV selective pressure represent only a small proportion of the viable virions in the original virus population (19); and (iii) no ACV mutagenic activity has been detected in previous studies (3, 30).However, there is still a possibility that ACV does influence the development of ACV r strains during ACV treatment. To address the question of whether or not ACV induces mutation, we chose penciclovir (PCV), which is similar to ACV both in structure and in its need for phosphorylation by virus TK for its anti-HSV action, as a control drug and isolated a series of ACV r and PCV-resistant (PCV r ) strains emerging during serial passages of HSV-1 in the presence of ACV or PCV. Sequencing of the TK and DNA Pol genes showed differential mutation patterns in the ACV r and PCV r isolates, suggesting that one, if not both, of the drugs had a differential effect on the generation of drug-resistant mutants. MATERIALS AND METHODS Cells and viruses.A human osteosarcoma TK-deficient cell line, 143B/ TK Ϫ neo R , was kindly supplied by Riken Cell Bank, Tsukuba, Japan. Human embryo lung (HEL) fibroblasts, Vero, and 143B/TK Ϫ neo R cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with 10% calf serum. The VR-3 strain of HSV-1 described previously (6) was obtained from the American Type Culture Collection, Rockville, Md. This virus strain was plaque purified three times, grown in HEL cells for less than three passages, and stored in small portions at Ϫ80°C.Isolation of drug-resistant viruses. ACV r and PCV r HSV-1 strains were isolated by serial passage of the reference VR-3 strain in the presence of increasing concentrations of ACV or PCV, as follows. Twenty PFU of the VR-3 strain were inoculated onto HEL cell mon...

Twenty 2-thiopyrimidine nucleoside analogues were synthesized and examined for inhibitory activity against herpes simplex virus (HSV) type 1 and 2, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and thymidine kinase-deficient HSV (HSV-TK-) replication in vitro. 2-thiouracil (thymine) arabinoside, 2'-deoxy-2-thiouridine (or 2-thiothymidine) and their 5-halogenated derivatives showed anti-HSV activity in both RPM18226 (human B-lymphoblastoid cells) and MRC-5 (human embryo lung cells). 2'-deoxy-5-halogenated-2-thiocytidines were also inhibitory against HSV, whereas 2-thiocytosine arabinoside and its derivatives were not inhibitory against HSV replication, except 5-bromo and 5-iodo congeners (TN-31, TN-32). Substitution of the halogen atom at the 5-position of the pyrimidine rings to an atom with a higher molecular weight increased anti-HSV and VZV activities, except for the anti-HSV activity of 2-thiouracil arabinosides. 2'-deoxy-5-methyl-, and 2'-deoxy-5-iodo-2-thiouridines (TN-17, TN-44) showed the most potent anti-HSV activity, and 2'-deoxy-5-chloro- and 2'-deoxy-5-bromo-2-thiocytidines were potent inhibitors of VZV replication. However, none of the compounds inhibited HCMV and HSV-TK- replication. TN-31 and TN-32 were shown to inhibited HCMV and HSV-TK- as well as HSV and VZV replication. The cytotoxicity of the 2-thio-pyrimidine nucleoside analogues was less than that of the 2-oxy-congeners of the compounds (5-iodo-2'-deoxyuridine, 5-iodo-2'-deoxycytidine, thymine arabinoside and cytosine arabinoside). The selectivity index of 2'-deoxy-5-iodo-2-thiouridine (TN-44) was higher than that of 5-iodo-deoxyuridine. TN-17 and TN-44 were not cytotoxic to resting or stimulated human peripheral blood mononuclear cells at 400 microM, although TN-32 was cytotoxic at a concentration of 20 microM.

Gene expression of human immunodeficiency virus (HIV) depends on a host cellular transcription factors including nuclear factor-gB (NF-~B

A series of 1,3-dioxolanyluracil analogues was prepared from the dioxolane intermediates 2, and their anti-Epstein Barr virus (anti-EBV) activities were determined. The potency of L-dioxolane uracil nucleosides against EBV replication is dependent on the substituents at the 5-position in the following decreasing order: I > Br > Cl > CH3 > CF3 > F. The most active and selective analogue was the iodo derivative (L-I-OddU) with an EC50 value of 0.03 microM and an EC90 value of 0.16 microM. There was no cytotoxicity or depletion of mitochondrial DNA in cells after exposure to L-I-OddU at 50 microM. The action against EBV replication in H1 cells is time-dependent, and EBV DNA in cells treated with L-I-OddU could rebound to pretreatment levels once the drug was removed. In view of the potent antiviral activity plus favorable toxicity profiles, L-I-OddU may be potentially useful for the treatment of EBV-related infectious diseases as well as for delaying the onset or decreasing the incidence of EBV-associated cancers.

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