Anti-influenza activity of c60 fullerene derivatives.

The H1N1 influenza A virus, which originated in swine, caused a global pandemic in 2009, and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. Thus, the threat from influenza A remains a serious global health issue, and novel drugs that target these viruses are highly desirable. Influenza A RNA polymerase consists of the PA, PB1, and PB2 subunits, and the N-terminal domain of the PA subunit demonstrates endonuclease activity. Fullerene (C60) is a unique carbon molecule that forms a sphere. To identify potential new anti-influenza compounds, we screened 12 fullerene derivatives using an in vitro PA endonuclease inhibition assay. We identified 8 fullerene derivatives that inhibited the endonuclease activity of the PA N-terminal domain or full-length PA protein in vitro. We also performed in silico docking simulation analysis of the C60 fullerene and PA endonuclease, which suggested that fullerenes can bind to the active pocket of PA endonuclease. In a cell culture system, we found that several fullerene derivatives inhibit influenza A viral infection and the expression of influenza A nucleoprotein and nonstructural protein 1. These results indicate that fullerene derivatives are possible candidates for the development of novel anti-influenza drugs.

Anti-influenza activity of marchantins, macrocyclic bisbibenzyls contained in liverworts.

The H1N1 influenza A virus of swine-origin caused pandemics throughout the world in 2009 and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. The threat of influenza A thus remains a serious global health issue and novel drugs that target these viruses are highly desirable. Influenza A possesses an endonuclease within its RNA polymerase which comprises PA, PB1 and PB2 subunits. To identify potential new anti-influenza compounds in our current study, we screened 33 different types of phytochemicals using a PA endonuclease inhibition assay in vitro and an anti-influenza A virus assay. The marchantins are macrocyclic bisbibenzyls found in liverworts, and plagiochin A and perrottetin F are marchantin-related phytochemicals. We found from our screen that marchantin A, B, E, plagiochin A and perrottetin F inhibit influenza PA endonuclease activity in vitro. These compounds have a 3,4-dihydroxyphenethyl group in common, indicating the importance of this moiety for the inhibition of PA endonuclease. Docking simulations of marchantin E with PA endonuclease suggest a putative "fitting and chelating model" as the mechanism underlying PA endonuclease inhibition. The docking amino acids are well conserved between influenza A and B. In a cultured cell system, marchantin E was further found to inhibit the growth of both H3N2 and H1N1 influenza A viruses, and marchantin A, E and perrotein F showed inhibitory properties towards the growth of influenza B. These marchantins also decreased the viral infectivity titer, with marchantin E showing the strongest activity in this assay. We additionally identified a chemical group that is conserved among different anti-influenza chemicals including marchantins, green tea catechins and dihydroxy phenethylphenylphthalimides. Our present results indicate that marchantins are candidate anti-influenza drugs and demonstrate the utility of the PA endonuclease assay in the screening of phytochemicals for anti-influenza characteristics.

Apoptosis of human lung cancer cells by curcumin mediated through up-regulation of "growth arrest and DNA damage inducible genes 45 and 153".

The expression of "growth arrest and DNA damage inducible genes 45 and 153" is related to apoptotic induction of cells. GADD45 is an effector gene of the tumor suppressor p53, and GADD153 is associated with cellular function of cancer prevention. Curcumin, isolated from the plant Curcuma longa (LINN), has been investigated as a promising cancer preventive in food because curcumin, a phenolic and coloring compound, is widely ingested in the Indian subcontinent. However, the exact mechanisms of action of curcumin have not yet been clearly elucidated. Based on our successful results with green tea catechins as cancer preventive, we studied the relationship between the expression of GADD45 and 153 and apoptotic induction in human lung cancer cell line PC-9. In our study curcumin increased the expression of GADD45 and 153 in a p53-independent manner. Curcumin also inhibited the growth of PC-9 cells and induced G(1)/S arrest of the cell-cycle followed by strong induction of apoptosis. Treatment with GADD45 and 153 small interfering RNAs (siRNAs) inhibited the apoptotic induction in PC-9 cells by curcumin. Moreover, curcumin induced the expression of cyclin dependent kinase inhibitor genes p21 and p27, while it inhibited the expression of numerous genes, including Bcl-2, cyclin D1, CDK2, CDK4 and CDK6. All the results with PC-9 cells suggest that the up-regulation of GADD45 and 153 by curcumin is a prime mechanism in the anticancer activity of curcumin.

New role of (-)-epicatechin in enhancing the induction of growth inhibition and apoptosis in human lung cancer cells by curcumin.

Curcumin, a phenolic compound isolated from the plant Curcuma longa (Linn), is ingested every day in the Indian subcontinent and is well reported to possess cancer-preventive activity. To achieve effective cancer prevention with curcumin, we need to find a new method to enhance the effects of curcumin in the diet. Based on our evidence that (-)-epicatechin (EC), an inert catechin, enhances the cancer-preventive activity of green tea catechins, we studied the enhancing effects of EC on inductions of growth inhibition and apoptosis in human lung cancer cell lines PC-9 and A549 with curcumin. The combination of curcumin with EC significantly increased the inhibition of cell growth compared with curcumin or EC alone. The combination similarly increased both apoptosis and expression of GADD153 and GADD45 genes, associated with their enhanced protein production. Knockdown of GADD153 or GADD45 by small interfering RNA abrogated the apoptosis induction and growth inhibition induced by the combination, indicating the crucial role of their upregulation. Treatments of PC-9 cells with c-Jun-NH(2)-kinase inhibitor SP600125, with p38 mitogen-activated protein kinase inhibitor SB202190 and with PD98059 (extracellular signal-regulated kinase 1/2 inhibitor) all increased the upregulation of GADD153 and GADD45 genes by the combination. Because EC was previously shown to enhance the incorporation of EGCG into PC-9 cells, we think that EC has similar effects on curcumin. This report is the first report on the enhancing effects of EC on curcumin, and the data suggest that EC plays a significant role in the enhancement of the cancer-preventive activity of curcumin in the diet.

Green tea catechins inhibit the endonuclease activity of influenza A virus RNA polymerase.

The influenza A RNA polymerase possesses endonuclease activity to digest the host mRNA. Thus this endonuclease domain can be a target of anti-influenza A virus drug. Here we report that green tea catechins inhibit this viral endonuclease activity and that their galloyl group is important for their function. Docking simulations revealed that catechins with galloyl group fit well into the active pocket of the endonuclease domain to enable stable binding. Our results provide useful data that make it possible to refine and optimize catechin-based drug design more readily for stability.

Structural basis of the influenza A virus RNA polymerase PB2 RNA-binding domain containing the pathogenicity-determinant lysine 627 residue.

Because the influenza A virus has an RNA genome, its RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNA primers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 A tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique "phi"-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this phi loop and found that lysine 627 is located in the phi loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNA binding of viral RNA polymerases.

Identification of a vesicular aspartate transporter.

Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes and is exocytosed and stimulates neighboring cells by binding to specific cell receptors. Although evidence increasingly supports the occurrence of aspartergic neurotransmission, this process is still debated because the mechanism for the vesicular storage of aspartate is unknown. Here, we show that sialin, a lysosomal H(+)/sialic acid cotransporter, is present in hippocampal synaptic vesicles and pineal SLMVs. RNA interference of sialin expression decreased exocytosis of aspartate and glutamate in pinealocytes. Proteoliposomes containing purified sialin actively accumulated aspartate and glutamate to a similar extent when inside positive membrane potential is imposed as the driving force. Sialin carrying a mutation found in people suffering from Salla disease (R39C) was completely devoid of aspartate and glutamate transport activity, although it retained appreciable H(+)/sialic acid cotransport activity. These results strongly suggest that sialin possesses dual physiological functions and acts as a vesicular aspartate/glutamate transporter. It is possible that people with Salla disease lose aspartergic (and also the associated glutamatergic) neurotransmission, and this could provide an explanation for why Salla disease causes severe neurological defects.

Identification of a vesicular nucleotide transporter.

ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.

Secretion of L-glutamate from osteoclasts through transcytosis.

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

Vesicular inhibitory amino acid transporter is expressed in gamma-aminobutyric acid (GABA)-containing astrocytes in rat pineal glands.

Gamma-aminobutyric acid (GABA) is an inhibitory amino acid and acts as an intercellular transmitter in the central nervous system and peripheral tissues. In pineal glands, GABA is supposed to be a paracrine-like modulator of secretion of melatonin, although its mode of action, especially the sites of GABA signal appearance, is unknown. Vesicular inhibitory amino acid transporter (VIAAT) is a potential marker for the GABAergic phenotype. Here we presented evidence that VIAAT is expressed in GFAP-expressing astrocytes and a subpopulation of OX42-expressing microglia, but not in pinealocytes in cultured cells of rat pineal glands. The VIAAT-expressing cells also exhibit GABA immunoreactivity. Essentially the same results were obtained for pineal glands. These results suggest that GABA is stored and secreted from astrocytes and a subpopulation of microglia in pineal glands.

A novel variant of ionotropic glutamate receptor regulates somatostatin secretion from delta-cells of islets of Langerhans.

Many metabolic factors affect the secretion of insulin from beta-cells and glucagon from alpha-cells of the islets of Langerhans to regulate blood glucose. Somatostatin from delta-cells, considered a local inhibitor of islet function, reduces insulin and glucagon secretion by activating somatostatin receptors in islet cells. Somatostatin secretion from delta-cells is increased by high glucose via glucose metabolism in a similar way to insulin secretion from beta-cells. However, it is unknown how low glucose triggers somatostatin secretion. Because L-glutamate is cosecreted with glucagon from alpha-cells under low-glucose conditions and acts as a primary intercellular messenger, we hypothesized that glutamate signaling triggers the secretion of somatostatin. In this study, we showed that delta-cells express GluR4c-flip, a newly identified splicing variant of GluR4, an (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptor of rat. After treatment with L-glutamate, AMPA, or kainate, secretion of somatostatin from isolated islets was significantly stimulated under low-glucose conditions. The glutamate-dependent somatostatin secretion was Ca(2+) dependent and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Somatostatin in turn inhibited the secretion of L-glutamate and glucagon from alpha-cells. These results indicate that L-glutamate triggers somatostatin secretion from delta-cells by way of the GluR4c-flip receptor under low-glucose conditions. The released somatostatin may complete the feedback inhibition of alpha-cells. Thus, alpha- and delta-cells may communicate with each other through L-glutamate and somatostatin signaling.

Metabotropic glutamate receptor type 4 is involved in autoinhibitory cascade for glucagon secretion by alpha-cells of islet of Langerhans.

In islets of Langerhans, L-glutamate is stored in glucagon-containing secretory granules of alpha-cells and cosecreted with glucagon under low-glucose conditions. The L-glutamate triggers secretion of gamma-aminobutyric acid (GABA) from beta-cells, which in turn inhibits glucagon secretion from alpha-cells through the GABAA receptor. In the present study, we tested the working hypothesis that L-glutamate functions as an autocrine/paracrine modulator and inhibits glucagon secretion through a glutamate receptor(s) on alpha-cells. The addition of L-glutamate at 1 mmol/l; (R,S)-phosphonophenylglycine (PPG) and (S)-3,4-dicarboxyphenylglycine (DCPG), specific agonists for class III metabotropic glutamate receptor (mGluR), at 100 micromol/l; and (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I) at 50 micromol/l inhibited the low-glucose-evoked glucagon secretion by 87, 81, 73, and 87%, respectively. This inhibition was dose dependent and was blocked by (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG), a specific antagonist of class III mGluR. Agonists of other glutamate receptors, including kainate and quisqualate, had little effectiveness. RT-PCR and immunological analyses indicated that mGluR4, a class III mGluR, was expressed and localized with alpha- and F cells, whereas no evidence for expression of other mGluRs, including mGluR8, was obtained. L-Glutamate, PPG, and ACPT-I decreased the cAMP content in isolated islets, which was blocked by CPPG. Dibutylyl-cAMP, a nonhydrolyzable cAMP analog, caused the recovery of secretion of glucagon. Pertussis toxin, which uncouples adenylate cyclase and inhibitory G-protein, caused the recovery of both the cAMP content and secretion of glucagon. These results indicate that alpha- and F cells express functional mGluR4, and its stimulation inhibits secretion of glucagon through an inhibitory cAMP cascade. Thus, L-glutamate may directly interact with alpha-cells and inhibit glucagon secretion.