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The biological properties of elastase and Aspergillus flavus elastase inhibitor (AFLEI) from A. flavus were examined. Pathogenicity of elastase was investigated in mice immunocompromised with cyclophosphamide, cyclosporine, prednisolone and carrageenan. Compared to cyclophosphamide immunocompromised mice treated with the spores of elastase nonproducing strain, cyclophosphamide immunocompromised mice treated with the spores of elastase producing strain had a significantly shorter survival rate. Molecular mass of AFLEI was determined to be 7525.8 Da. The elastolytic activity of elastases from A. flavus, and human leukocytes were inhibited by AFLEI. The primary structure of AFLEI was determined by the Edman sequencing procedure. The search for amino acid homology with other proteins demonstrated that amino acid residues 1 to 68 of AFLEI are 100% identical to residues 20 to 87 of the hypothetical protein AFUA_3G14940 of A. fumigatus. When immunocompromised mice administered of cyclophosphamide were infected by inhalation of A. flavus then administered amphotericin B (AMPH) alone or in combination with AFLEI, survival rate tended to be higher with combination treatment than with AMPH alone. Moreover, although extensive bleeding was seen in pathology sections taken from rat lung resected 24 h after elastase was administered to the lung via the bronchus, this bleeding was inhibited by AFLEI. The X-ray analysis has revealed that the structure of this inhibitor was wedge shaped and composed of a binding loop and a scaffold protein core. As synthetic-inhibitor strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.

The anti-influenza virus drug oseltamivir has been reported to have several pharmacological actions including blocking of nicotinic acetylcholine receptor channels and activation of the dopaminergic system. These pharmacological actions highly overlap those of amantadine, another anti-influenza virus drug authorized in Japan, and ester-type local anesthetics. Moreover, oseltamivir and amantadine can clinically induce similar adverse neuropsychiatric reactions. In the present study, from the database of the Pharmaceuticals and Medical Devices Agency (PMDA), we surveyed 2576 drugs for which neuropsychiatric side effects similar to those of oseltamivir, amantadine and local anesthetics (abnormal behavior, confusion, consciousness disturbance, convulsion, delirium, delusion, hallucination, myoclonus, tremor) are listed as "clinically significant adverse reactions", and found 327 that had at least one of these adverse reactions. Other neuraminidase inhibitors (laninamivir, peramivir and zanamivir) did not elicit such adverse reactions. By discussing the pharmacological effects of drugs that elicit these adverse reactions, we propose that the similarity of adverse neuropsychiatric reactions between oseltamivir and amantadine is possibly attributable to their common pharmacological effects.

Drug resistance is a critical problem inhibiting the effective use of targeted molecular cancer therapies. Investigators have revealed a variety of resistance mechanisms, including alterations in drug targets, activation of pro-survival pathways, and the ineffective induction of cell death. The key alterations driving this resistance are likely condition-dependent, and a detailed analysis would be required to characterize these diverse alterations under a variety of conditions in order to facilitate practical precision medicine for treating individual cancer patients. We have been investigating the molecular mechanisms of anti-cancer drug resistance, and analyzed our original resistant cells against anti-mitotic kinase inhibitors. This study suggests that novel mechanisms reduce cytokinetic dysregulation caused by those inhibitors, and anti-apoptotic activities are associated with resistant phenotypes. These observations suggest that the activation of various bypass mechanisms may allow cancer cells to avoid the selective antiproliferative effect of molecularly targeted drugs, and such bypass activation mechanism would thus be a critical target for designing combination chemotherapy to overcome non-genetic drug resistance.

Influenza A and B viruses possess an enzyme "sialidase" that cleavages terminal sialic acid from glycochains. These viral sialidase proteins are highly expressed on the virus infected cells. We developed sialidase imaging probe "BTP3-Neu5Ac" that enables histochemical fluorescence staining of sialidase activity. BTP3-Neu5Ac was able to perform speedy and easy fluorescence imaging of these virus infected cells, with no needs of specific antibody and cell fixation. In addition, combination use of anti-influenza drugs (sialidase inhibitors) and BTP3-Neu5Ac resulted in selective fluorescence imaging for detection and high-efficiency isolation of drug-resistant virus. Fluorescence imaging of drug-resistant virus will be a powerful method for study of the drug-resistance mechanism, for monitoring of drug-resistant viruses. A novel tool for fluorescence imaging of viral sialidase activity is described in this review.

Drugs for the treatment of osteoporosis improve bone strength by inhibiting bone resorption or stimulating bone formation. New drugs already using some action mechanism such as bisphosphonate or parathyroid hormone-related peptide analog are being developed in Japan. In addition, new drugs with new action mechanisms such as cathepsin K inhibitor or anti-sclerostin antibody are also being developed and it has been reported that they have good potential to increase bone mineral density.

Over the three decades, advances in the fields of cancer genetics and immunology have elucidated mechanisms that cause the growth of cutaneous malignancies. This has contributed to the rational design of molecular target therapies. In this review, the molecular pathways and cancer immunology critical to the development of cutaneous malignancies are discussed. Included is a review of the following novel pharmacologic agents; BRAF inhibitors, MEK inhibitors, anti-CTLA-4 antibody and anti-PD-1 antibodies for melanoma, cetuximab and gefitinib for squamous cell carcinoma, vismodegib for basal cell carcinoma and nevoid basal cell carcinoma syndrome, imiquimod for extramammary Paget's disease, imatinib for dermatofibrosarcoma protuberans and vorinostat and mogamulizumab for cutaneous T-cell lymphoma.

Poly(ADP-ribose) polymerases(PARP) synthesize the ADP-ribose polymers onto proteins and play a role in DNA repair. PARP inhibitors block the repair of single-strand breaks, which in turn gives rise to double-strand breaks during DNA replication. Thus, PARP inhibitors elicit synthetic lethality in cancer with BRCA1/2 loss-of-function mutations that hamper homologous recombination repair of double-strand breaks. Olaparib, the first-in-class PARP inhibitor, was approved for treatment of BRCA-mutated ovarian cancer in Europe and the United States in 2014. Other PARP inhibitors under clinical trials include rucaparib, niraparib, veliparib, and the "PARP-trapping" BMN-673. BRCA1/2 sequencing is an FDA-approved companion diagnostics, which predicts the cancer vulnerability to PARP inhibition. Together, synthetic lethal PARP inhibition is a novel promising strategy for cancer intervention even in cases without prominent driver oncogenes.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine kinase belonging to the mitogen-activated protein kinase (MAPK) kinase kinase family. ASK1 is activated in response to various stresses, such as reactive oxygen species (ROS), tumor necrosis factor (TNF) alpha, lipopolysaccharide (LPS) and endoplasmic reticulum (ER) stress, and plays pivotal roles in a wide variety of cellular responses, including cell death, differentiation and inflammation. Recent studies have shown that ASK1-MAPK signaling pathway has key roles in human diseases induced by the dysfunction of cellular stress responses. In this review, we provide an overview of the current understanding of ASK1 as a potential therapeutic target for human diseases.

This review describes the synthesis and evaluation of novel nucleic acid derivatives performed by our research group to date. We developed a new method for the synthesis of 2-alkoxyadenosine analogs via nonaqueous diazotization-dediazoniation reactions. By applying these reactions, we effectively synthesized four types of carbocyclic oxetanocin analogs (2-alkoxy-C.OXT-A). The angiogenic activities of these compounds were evaluated using human umbilical vein endothelial cells. This resulted in increased activities of the analogs, especially of 2-methoxy-C.OXT-A and 2-isopropoxy-C.OXT-A, at a concentration of 100 μM; they showed angiogenic potency similar to or greater than that of vascular endothelial growth factor. We also synthesized and evaluated a novel series of uracil derivatives carrying a 3,5-dimethylbenzyl group at the N(3)-position and acting as non-nucleoside HIV-1 reverse transcriptase inhibitors. Some of these compounds showed good-to-moderate inhibitory activity, with EC₅₀ values in the submicromolar range. Among them, the analog 6-amino-1-(4-picolyl)-uracil showed significant HIV-1 reverse transcriptase inhibition, with an EC₅₀ value of 0.03 μM and a high selectivity index of 2863.

We are studying the medicinal synthetic chemistry of biomolecular component mimics such as carbohydrates, nucleosides, amino acids, and peptides. In this review, the synthesis and biological activities of iminosugars as carbohydrate mimics are discussed. Glycosidases and glycosyltransferases are involved in a wide range of anabolic and catabolic process, including digestion, the lysosomal catabolism of glycoconjugates, glycoprotein biosynthesis. Hence, modifying or blocking these processes in vivo using inhibitors is a topic of great interest from the therapeutic point of view. Iminosugars are sugars in which the endocyclic oxygen is replaced by a basic nitrogen atom. They are regarded as transition state mimics in certain types of enzyme reactions. This makes the field of iminosugars as carbohydrate mimics an exciting area of research. We synthesized all of the stereoisomers of polyhydroxypiperidines such as fagomine, 1-deoxynojirimycine, and isofagomine. In addition, their both enantiomers, as substrates for a variety of glycosidases were evaluated. Secondly, the asymmetric synthesis of α-1-C-alkyl-arabinoiminofuranoses was achieved by asymmetric allylic alkylation, RCM, and Negishi cross coupling as key reactions. Surprisingly, the L-forms showed a quite potent inhibitory activity toward rat intestinal maltase, while the activities of the D-forms were much weaker. Some of the prepared L-forms showed potent inhibitory activities towards intestinal maltase, with IC₅₀ values comparable to those of commercial drugs such as acarbose, voglibose, and miglitol, which are used in the treatment of type 2 diabetes. Among them, the inhibitory activity towards intestinal sucrase of α-1-C-L-butylarabinoiminofuranose was quite strong towards intestinal sucrase compared to the above commercial drugs.

This review discusses the importance of quantum chemical interactions in biomolecules for medicinal science and their relevance to the author's β-secretase (BACE1) inhibitor drug discovery research. Although molecular mechanics/dynamics (MM/MD) methods are available in many in silico design tools used for drug discovery, they cannot accurately evaluate quantum effects between biomolecules and drugs. The key roles of biomolecular quantum chemical interactions in drug discovery are discussed using the arginine side chain as an example. Arginine is recognized as a charged amino acid in commonly used drug design software, unlike other amino acids with π-electron orbitals, such as phenylalanine, tyrosine, and tryptophan. Quantum chemical interactions via the arginine side chain are crucial for molecular recognition, and are found in many X-ray crystal structures, such as protein-protein, protein homodimer, RNA aptamer-protein, and enzyme-inhibitor complexes. This review describes the essential role of quantum chemical interactions via the arginine side chain in the mechanism of BACE1 inhibition, and proposes an "electron donor/acceptor bioisostere" concept for medicinal science based on quantum chemical interactions. Several potent BACE1 inhibitors, as well as the first peptides with BACE1 inhibiting activity were designed and synthesized based on studies of quantum chemical interactions via arginine side chain and the "electron donor bioisostere" concept.

Co-culture of the fungal pathogen Cryptococcus neoformans with Staphylococcus aureus results in the death of the fungus, caused by the adherence to the latter. The present study found that the molecules responsible for this adherence were capsular glucuronoxylomannan (GXM) (present on C. neoformans) and a glycolytic enzyme triosephosphate isomerase (TPI) (present on S. aureus). The mannan backbone of GXM and purified TPI interacted in vitro. GXM-bound TPI molecules were identified by immunoelectron microscopy. The death of C. neoformans was accompanied by decreased actin turnover, increased accumulation of reactive oxygen species, and DNA fragmentation. This process may also be influenced by the Rho/Rho-associated coiled-coil-forming kinase (ROCK) pathway and enhanced expression of voltage-dependent ion-selective channels. Taken together, these results suggest that Rho-ROCK signaling may play a role via the mitochondrial pathway in the induction of C. neoformans apoptosis-like cell death after its adherence to S. aureus adherence.

Janus kinase 2 (JAK2) is an essential non-receptor type tyrosine kinase for various cytokine signals. In 2005, a somatic JAK2 mutation (V617F) was found in the majority of myeloproliferative neoplasm (MPN) patients. It has been shown that the V617F mutation caused the constitutive activation of JAK2, exhibiting the cytokine-independent survival and proliferation of Ba/F3 cells. In addition, tumorigenesis was induced after a transplantation of Ba/F3 cells expressing JAK2 V617F mutant in nude mice, suggesting that JAK2 V617F mutant behaves as a potent oncogene product. We found that JAK2 V617F mutant causes aberrant activation of a transcription factor c-Myc, which is critical for the JAK2 V617F mutant-caused oncogenic activities. In the screening of genes which expression was induced by JAK2 V617F mutant, we detected the significant induction of target genes of c-Myc such as Aurora kinase A (Aurka) and ornithine decarboxylase (ODC). Interestingly, JAK2 V617F mutant enhanced resistance to cisplatin (CDDP)-induced DNA damage and ectopic expression of Aurka in Ba/F3 cells exhibited similar resistance to CDDP. Conversely, knockdown and inhibition of Aurka in cells expressing JAK2 V617F mutant abolished the resistance to CDDP, suggesting that Aurka is most likely critical for resistance to DNA damage in cells transformed by JAK2 V617F mutant. In addition, we found that ODC inhibitor, DL-α-difluoromethylornithine (DFMO) prevented the proliferation of the JAK2 V617F mutant-induced transformed cells. Taking these observations together, c-Myc plays an essential role in JAK2 V617F mutant-induced hematopoietic disorder and would be a good target for the treatment of MPN.

P-glycoprotein (P-gp), one of the important drug-efflux pumps, is known to affect pharmacokinetics and pharmacodynamics of P-gp substrate drugs. We have previously reported that intestinal P-gp expression levels are transiently decreased in streptozotocin (STZ)-induced type 1 diabetic mouse model. Herein, we examined the analgesic effects of orally administered morphine and its pharmacokinetic properties under diabetic conditions, specifically focusing on the involvement of intestinal P-gp in a type 1 diabetic mouse model. Type 1 diabetes was induced in male ddY mice by an i.p. injection of STZ (230 mg/kg). We assessed the oral morphine analgesia using the tail-flick test. Serum and brain morphine content were determined on a HPLC-ECD system. Intestinal P-gp expression levels were significantly decreased on day 9 after STZ administration. On the other hands, oral morphine analgesia, and serum and brain morphine content were significantly increased on day 9 after STZ administration. The decrease in the intestinal P-gp expression levels were suppressed by aminoguanidine, a specific iNOS inhibitor. Interestingly, the increase in the analgesic effect of morphine, as well as serum and brain morphine content, was suppressed by aminoguanidine. Conversely, there was no change in the analgesic effect obtained with subcutaneous morphine in STZ-treated mice. In conclusions, our findings suggest that the oral morphine analgesia is dependent on intestinal P-gp expression, and that may be one of the problems against obtaining stable pharmacological effects of morphine in diabetic patients.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle disease, characterized by progressive skeletal muscle atrophy and weakness. DMD is caused by mutations in the dystrophin gene, which encodes for the cytoskeletal protein dystrophin. DMD is one of the most common types of muscular dystrophies, affecting approximately 1 in 3,500 boys. There is no complete cure for this disease. Clinical trials for gene transfer therapy as a treatment for DMD have been performed but mainly in animal models. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) was found to be induced in grouped necrotic muscle fibers of DMD patients and animal models, mdx mice, and DMD dogs. We found an orally active H-PGDS inhibitor (HQL-79) and determined the 3D structure of the inhibitor-human H-PGDS complex by X-ray crystallography. Oral administration of HQL-79 markedly suppressed prostaglandin D 2 (PGD 2 ) production, reduced necrotic muscle volume, and improved muscle strength in mdx dystrophic mice. Based on the high-resolution 3D structures of the inhibitor-H-PGDS complex, we designed alternative H-PGDS inhibitors, which were 100- to 3000-times more potent than HQL-79, as assessed by in vitro and in vivo analyses. We used these novel inhibitors for the treatment of DMD dogs and confirmed that oral administration of these inhibitors prevented skeletal muscle atrophy and weakness by decreasing PGD 2 production. These results indicate that PGD 2 , synthesized by H-PGDS, is involved in the expansion of muscle necrosis in DMD. Thus, inhibition of H-PGDS by using inhibitors is a novel therapy for DMD.

Bone homeostasis is maintained by delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. Bisphosphonates are widely used nowadays by suppressing bone resorption to treat patients with osteoporosis, which results from high bone turnover, causing excessive bone resorption phase. While bisphosphonates increase bone mineral density and improve back pain due to spinal compression fracture, they may have some problems such as osteonecrosis of jaw and excessive suppression of bone turnover. Cathepsin K inhibitor, which has a new mechanism in addition to function of suppressing bone resorption, is recently focused. Cathepsin K is a protease which specifically expresses in osteoclasts and plays an important role in resolution of bone collagen. Cathepsin K inhibitor has a potential of inhibiting bone resorption without suppressing bone formation and could be an attractive therapeutic target of osteoporosis.

Furanocoumarins (FCs) such as bergamottin (BG) and 6',7'-dihydroxybergamottin (DHBG) contained in grapefruits are known to be cytochrome P450 3A4 (CYP3A4) inhibitors. These are contained in larger quantity in peel than in pulp, and therefore, processed peel products possibly have strong CYP3A4 inhibitory activity. The CYP3A4 inhibitory potency of these processed peel products, however, remains to be elucidated. The FC content and CYP3A inhibitory activities of various processed fruit peel products were investigated. CYP3A inhibitory activities of crystallized grapefruit peel, grapefruit marmalade, lemon peel and bitter orange slice were close to that of 100% grapefruit juice, while the activities of yuzu slice, pomelo (buntan) marmalade and crystallized iyokan peel were very weak, 1/8-1/20 of 100% grapefruit juice. The maximum BG content was 5.6 µg/g in lemon peel. The maximum DHBG content was 7.2 µg/g in crystallized grapefruit peel, about 1/30 that of raw peel. Grapefruit marmalade and crystallized grapefruit peel contained similar amounts of FCs to 100% grapefruit juice, but FCs were not detected in pomelo (buntan) marmalade or crystallized iyokan peel. Good correlation (r=0.78) was observed between the FC contents of these peel products and those CYP3A inhibitory activities. Preparation of homemade grapefruit marmalade and crystallized peel revealed that considerably lower DHBG content in these products and lower CYP3A inhibitory activity than anticipated were attributable to outflow of DHBG to broth during boiling of the raw peel.

Laninamivir is a novel neuraminidase inhibitor of influenza viruses and it has been reported that its prodrug, CS-8958 shows a long-lasting characteristics. Using viruses isolated in Nagasaki of pandemic (H1N1) 2009 influenza virus which cause pandemic in 2009, it was shown that laninamivir has a strong inhibitory activities against their neuraminidases and virus replication in cultured cells, and strong binding stability to the virus NA. Furthermore, a single intranasal administration of CS-8958 showed a superior reduction of virus load in lungs in mouse infection model. These suggest that CS-8958 will work as a long-acting neuraminidase inhibitor to an infection with pandemic (H1N1) 2009 influenza viruses as well.

Amantadine resistance has become increasingly common among recent seasonal influenza viruses. In addition, almost all pandemic (H1N1) 2009 viruses are resistant to this drug. Between 2007 and early 2009, the prevalence of oseltamivir-resistant seasonal H1N1 variants that possess the His274Tyr neuraminidase mutation had dramatically increased, raising concerns regarding the utility of oseltamivir. Fortunately, most of the pandemic (H1N1) 2009 viruses were sensitive to neuraminidase inhibitors. In this paper, drug resistance among recent influenza virus isolates is comprehensively reviewed, especially from the standpoint of the molecular basis of drug resistance.

Against pandemic influenza A (H1N1) 2009, anti-influenza drugs were useful to save the disease aggravation and reduce the mortality. In addition to the existing anti-influenza drugs, novel drugs, including neuraminidase inhibitors (peramivir and laninamivir) and RNA polymerase inhibitors (favipiravir), also have clinical potentials to suppress pandemic influenza A (H1N1) 2009 virus as well as seasonal influenza virus.

More than 60 percents of breast cancers occur in post menopausal women and most of their initial stages are hormone dependent. For the treatment of estrogen dependent breast tumors, mainly two treatment tools are available, one is selective estrogen receptor modulator (SERM), and the other is aromatase inhibitor (AI). Although these drugs are clinically valuable, the existence of resistant tumors againt these two treatments is one of the most serious matters. The latest studies clarify that to inhibit the local formation of estrogen is more important than to decrease the estrogen dose in plasma. Steroid sulfatase (STS) is mainly expressed in local breast carcinoma tissues and is one of the most promising targets to inhibit the local formation of estrogens.

Several lines of evidence indicate that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer's disease. Inhibition or modulation of gamma-secretase, that is a responsible enzyme for the Abeta production, is one of the plausible therapeutics for Alzheimer's disease. However, the gamma-secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain of several membrane protein including APP and Notch receptor. Thus, development of drugs that regulate the production of Abeta without affecting the Notch signaling is now demanding. Extensive drug screening and development allow that some secretase inhibitors and modulators have advanced into late-phase clinical trials, whereas the molecular mechanisms of Notch-sparing effect by these compounds effect still remain unknown. Identification of the molecular targets and mechanisms of these compounds using chemical biological approaches is currently underway. This review focuses on the recent development of inhibitors/modulators and provides a direction for the effective treatment of AD through inhibition/modulation of the gamma-secretase activity.

Hepatitis C virus (HCV) develops persistent infection in most infected patients, and eventually cause chronic hepatitis, liver cirrhosis and then hepatocellular carcinoma. The combination therapy of PEG-IFN and ribavirin improves the efficacy in many patients, while it does not lead to sufficient achievements in genotype1b patients. To invent new anti-HCV reagent, we focused on host factors which HCV take advantage of in its life-cycle. We identified serine palmitoyltransferase inhibitor as anti-HCV reagent through high-through put screenig using HCV replicon cells. Moreover, we evaluate the anti-HCV effect of SPT-inhibitor in vivo with humanized chimeric mice. SPT-inhibitor led to rapid decline in serum HCV-RNA of about 1-2log within 8 day, futhermore the combination therapy of SPT-inhibitor and PEG-IFN achieved about 3log reduction in serum HCV-RNA. At last, we investigated the mechanism of anti-HCV effect of SPT-inhibitor. It has been reported that sphingolipids and cholesterol compose the lipid raft, in which the replication of HCV occur. We investigated the influence of SPT-inhibitor to lipid rafts by analysing the detergent resistant membrane (DRM). The analysis proved that SPT inhibitor got HCV RNA dependent RNA polymerase (NS5B) to move to detergent soluble fraction from DRM, and Biacore analysis indicated the binding of sphingomyelin to NS5B. These results suggested SPT inhibitor got NS5B to release from replication complex.

Ethanol is a deleterious agent that causes various kinds of neuronal damage to both the developing and adult brain. Recent research on alcoholism implicates impaired function of neural stem cell (NSC) in the pathogenesis of ethanol-induced brain dysfunction. We previously reported that the differentiation of NSCs into neurons was significantly influenced by ethanol. We also found that neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/ REST) binding activity potentiated by ethanol underlies the mechanism of ethanol inhibition of neuronal differentiation. Epigenetics refers to post-translational modifications of DNA and nuclear proteins that produce lasting alterations in patterns of gene expression. Epigenetic mechanism plays a critical role of neuronal plasticity and there is clear evidence that dysfunction of epigenetic mechanism also contributes to neurological and psychiatric illness. We will review epigenetic regulation in pathogenesis of psychiatric illness including alcoholism. We also demonstrated that trichostatin A, histone deacetylase inhibitor, reduced the ethanol-induced suppression of neuronal differentiation of NSCs. We suggest that ethanol alters the function of neural differentiation through the mechanism of potentiation of NRSF/REST binding and histone modifications.

Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

Axons of central neurons can regenerate in a peripheral nervous system milieu, by macrophage activation or by inhibition of Rho pathways. Among retinal ganglion cells (RGCs) with axons regenerating into a transplanted peripheral nerve segment, Alpha/Y cells have the highest ability to regenerate axons and their axons extend fastest. OFF-center RGCs regenerate axons less effectively and are more vulnerable to axotomy than ON-center ones. RGC axons extended beyond the crushed site when oxidized galectine-1, a macrophage activator, was vitreously injected. Rho inhibitors were added to cultured retinae to learn their effect on axonal regeneration. Neurite extension from the retinae was promoted with 10-300 microM Y-27632 or 1-20 microM Y-39983 whereas glial processes extended in 100-300 microM Y-27632, 1-20 microM Y-39983 or 10-100 microM Fasudil. The injection of 100 microM Y-27632 or 10 microM Y-39983 into the vitreous and crush site resulted in robust regeneration of RGC axons while 10 microM Y-276932 caused fewer regenerated fibers and Fasudil caused no fibers. Thus neurite extension may be crucial for axonal regeneration and glial process extension may be beneficial for the regeneration.

Injury to the peripheral nerves often induces produces spontaneous pain, hyperalgesia (increased responsiveness to noxious stimuli), and allodynia (painful responses to normally innocuous stimuli). In contrast to inflammatory pain, the currently available therapeutics for neuropathic pain are either relatively ineffective or accompanied by considerable side effects. Numerous animal models of chronic pain following nerve injury have been introduced. All these neuropathic pain models are generated by partial nerve injury, where a few primary afferents are axotomized, while the others are spared. Among these models, the L5 spinal nerve ligation (SNL) model is unique because in this model, the L4 dorsal root ganglion (DRG) neurons are clearly separated from the axotomized L5 DRG neurons. Previous studies have focused considerable attention on the directly damaged primary afferents and their influence on the activity of the dorsal horn neurons. However, increasing evidence suggests that DRG neurons with intact axons also exhibit alterad excitability and gene expression, and these changes might play functional roles in the pathomechanisms of neuropathic pain. For example, L5 SNL increases the expression of substance P, calcitonin gene-related peptide, brain-derived neurotrophic factor, and the transient receptor potential ion channels TRPV1 and TRPA1 in the uninjured L4 DRG neurons. Furthermore, compelling evidence suggests that the glial cells in the spinal cord may also play a role in the pathogenesis of neuropathic pain. Recent studies have shown that peripheral nerve injury results in the activation of mitogen-activated protein kinases (MAPK) in spinal glial cells and that MAPK inhibitors diminish nerve injury-induced pain hypersensitivity. This review mainly focuses on the DRG neurons and spinal glial cells and will review the roles of MAPK in the nociceptive pathways for neuropathic pain.

Inhibitors of xanthine oxidoreductase decrease production of uric acid, thus they act as hypouricemic drugs. Allopurinol, a prototypical xanthine oxidoreductase inhibitor, has been widely prescribed for treatment of gout and hyperuricemia. However, severe side effects of allopurinol may occur in patients with renal insufficiency. Recently, novel nonpurine selective inhibitors of xanthine oxidoreductase have been developed as potential alternatives to allopurinol. They have different inhibition mechanisms, utilizing the enzyme structure and the reaction mechanism. Such variation of the inhibition mechanism affects/in vivo/hypouricemic effects of the inhibitors.

The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, suppress cell proliferation and induce apoptosis in various cancer cell lines. However, the effects of statins in head and neck carcinoma have not been reported. In this study, we investigated the mechanism by which fluvastatin induces apoptosis in HSC-3 cells. An increase in caspase-3 activity was observed. The apoptosis induced by fluvastatin was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP) to the cell culture. When we examined the survival signals at the time of apoptotic induction, we also found that fluvastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Moreover, we also found that U0126, a MEK1/2 inhibitor, induces apoptosis in HSC-3 cells. These results suggested that fluvastatin induces apoptosis by inhibiting GGPP biosynthesis and consequently decreasing the level of phosphorylated ERK1/2. The results of this study also indicate that fluvastatin may be used as an anticancer agent for tongue carcinoma.