Impact of the lipase inhibitor orlistat on the human gut microbiota.

Orlistat, an anti-obesity agent, inhibits the metabolism and absorption of dietary fat by inactivating pancreatic lipase in the gut. The effect of orlistat on the gut microbiota of Japanese individuals with obesity is unknown. This study aimed to explore the effects of orlistat on the gut microbiota and fatty acid metabolism of Japanese individuals with obesity. Fourteen subjects with visceral fat obesity (waist circumference ≥85 cm) took orlistat orally at a dose of 60 mg, 3 times a day for 8 weeks. Body weight; waist circumference; visceral fat area; levels of short-chain fatty acids, gut microbiota, fatty acid metabolites in the feces, and gastrointestinal hormones; and adverse events were evaluated. Body weight, waist circumference, and blood leptin concentrations were significantly lower after orlistat treatment (mean ± standard deviation, 77.8 ± 9.1 kg; 91.9 ± 8.7 cm; and 4546 ± 3211 pg/mL, respectively) compared with before treatment (79.4 ± 9.0 kg; 94.4 ± 8.0 cm; and 5881 ± 3526 pg/mL, respectively). Significant increases in fecal levels of fatty acid metabolites (10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid, and 10-oxo-trans-11-octadecenoic acid) were detected. Meanwhile, no significant changes were found in abdominal computed tomography parameters, blood marker levels, or short-chain fatty acid levels in the feces. Gut microbiota analysis revealed that some study subjects had decreased abundance of Firmicutes, increased abundance of Bacteroidetes, and increased α-diversity indices (Chao1 and ACE) after 8 weeks of treatment. The levels of Lactobacillus genus and Lactobacillus gasseri were significantly higher after 8 weeks of treatment. None of the subjects discontinued treatment or experienced severe adverse events. This study suggested that orlistat might alter gut microbiota composition and affect the body through fatty acid metabolites produced by the modified gut bacteria.

Structural and Computational Analyses of the Unique Interactions of Opicapone in the Binding Pocket of Catechol O-Methyltransferase: A Crystallographic Study and Fragment Molecular Orbital Analyses.

A third-generation inhibitor of catechol O-methyltransferase (COMT), opicapone (1), has the 3-nitrocatechol scaffold as do the second-generation inhibitors such as entacapone (2) and tolcapone (3), but only 1 can sustainably inhibit COMT activity making it suitable for a once-daily regimen. These improvements should be attributed to the optimized sidechain moiety (oxidopyridyloxadiazolyl group) of 1 substituted at the 5-position of the 3-nitrocatechol ring. We analyzed the role of the sidechain moiety by solving the crystal structures of COMT/S-adenosylmethionine (SAM)/Mg/1 and COMT/S-adenosylhomocysteine (SAH)/Mg/1 complexes. Fragment molecular orbital (FMO) calculations elucidated that the dispersion interaction between the sidechains of Leu 198 and Met 201 on the ß6ß7-loop and the oxidopyridine ring of 1 were unique and important in both complexes. In contrast, the catechol binding site made a remarkable difference in the sidechain conformation of Lys 144. The ε-amino group of Lys 144 was outside of the catalytic pocket and was replaced by a water molecule in the COMT/SAH/Mg/1 complex. No nitrocatechol inhibitor has ever been reported to make a complex with COMT and SAH. Thus, the conformational change of Lys 144 found in the COMT/SAH/Mg/1 complex is the first crystallographic evidence that supports the role of Lys 144 as a catalytic base to take out a proton ion from the reaction site to the outside of the enzyme. The fact that 1 generated a complex with SAH and COMT also suggests that 1 could inhibit COMT twofold, as a typical substrate mimic competitive inhibitor and as a product-inhibition enhancer.

[Molecular Design Based on Conformational Analyses].

Today computational chemistry has become an established tool for medicinal chemists. However, softwares are becoming more sophisticated, and in order to master the tools, a wide range of fundamental competency such as thermodynamics, statistics, and physical chemistry are required in addition to chemical creativity. As a result, a software might be used as a black box. In this article, I would like to introduce what a simple computational conformation analysis can do and my experience of using it in actual wet research.

Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid-liquid phase separation of cellular biomolecules.

The development of countercurrent chromatography (CCC) technology enabled us to achieve higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol and inorganic salts (or dextrans). By eliminating the solid support matrix, all analytes can be recovered from the coiled column after the separation is completed. Recently, it has been found that droplets of biomolecules formed by liquid-liquid phase separation in cells closely relate to the transcription, regulation of signal transduction, and formation of amyloids. Meanwhile, although CCC is a separation technique based on liquid-liquid partitioning of analytes between two immiscible phases, the mechanism of separation could suggest some idea concerning the formation of biomolecule droplets in cells. This article describes the recent advances in the CCC apparatus, the coiled separation column, the choice of a suitable two-phase solvent system, and the application to separation and purification of bioactive macromolecules such as proteins and enzymes, and also discusses the possibility of CCC as a tool to reveal new mechanical roles of biomolecule droplets in the cellular environments.

Dietary Magnesium Insufficiency Induces Salt-Sensitive Hypertension in Mice Associated With Reduced Kidney Catechol-O-Methyl Transferase Activity.

[Figure: see text].

Crystal Structure of Catechol O-Methyltransferase Complexed with Nitecapone.

Catechol O-methyltransferase (COMT) is known as an important drug-target protein in the field of Parkinson's disease. All clinically approved COMT inhibitors bring a 5-substituted-3-nitrocatechol ring as a pharmacophore, and they bind to COMT with S-adenosylmethionine (SAM) and an Mg2+ ion to form a quaternary complex (COMT/SAM/Mg2+/inhibitor). However, structural information about such quaternary complexes is only available for a few inhibitors. Here, a new crystal structure of COMT complexed with nitecapone (5), SAM and Mg2+ is revealed. Comparison of the structures of these complexes indicates that conformation of the catechol binding pocket is almost constant regardless of structure of the inhibitors. The only restriction of the side chain of inhibitors (i.e., the substituent at the 5-position of 3-nitrocatechol) seems to be that it does not make steric repulsion with COMT. However, recent crystallographic and biochemical studies suggest that COMT is a flexible protein, and its conformational flexibility seems crucial for its catalytic process. Based on this information, implications of these quaternary inhibitor complexes were investigated. Met 40 in the α2α3-loop makes atomic contacts with SAM or S-adenosylhomocysteine and the 3-position of the catechol inhibitor. This interaction seems to play a critical role in the affinity of the inhibitor and to stabilize the COMT/SAM/Mg2+/nitrocatechol inhibitor complex by fixing the flexible α2α3-loop.

Transcriptomic and Proteomic Analyses Reveal the Potential Mode of Action of Chondrocyte Sheets in Hyaline Cartilage Regeneration.

Chondrocyte sheet transplantation is a novel and promising approach to treating patients who have cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration by autologous chondrocyte sheets has already been demonstrated in clinical research. In this study, the efficacy of polydactyly-derived chondrocyte sheets (PD sheets) as an allogeneic alternative to standard chondrocyte sheets was examined using an orthotopic xenogeneic transplantation model. In addition, the expression of genes and the secreted proteins in the PD sheets was analyzed using a microarray and a DNA aptamer array. The efficacy of PD sheets with respect to cartilage defects was assessed using histological scores, after which the expressions of genes and proteins exhibiting a correlation to efficacy were identified. Enrichment analysis of efficacy-correlated genes and proteins showed that they were associated with extracellular matrices, skeletal development, and angiogenesis. Eight genes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA) exhibited a positive correlation with the efficacy of PD sheets, and three genes (RARRES2, APOE, and PGF) showed a negative correlation for both transcriptomic and proteomic analyses. Among these, MIA, DKK1, and GREM1 involved in skeletal development pathways and ESM1 involved in the angiogenesis pathway exhibited a correlation between the amount of secretion and efficacy. These results suggest that these secreted factors may prove useful for predicting PD sheet efficacy and may therefore contribute to hyaline cartilage regeneration via PD sheets.

Effects of bovine lactoferrin on l-DOPA absorption and metabolism in mice.

Bioactive natural products, habitually ingested with milk or its derivative nutrients, have been studied for their bioavailability. In this study, we investigated the effects of the co-administration of bovine milk-derived lactoferrin (bLF) and bioactive products, with a focus on catechol-O-methyltransferase (COMT), an enzyme in the catechol metabolism. bLF showed inhibitory activity on COMT in vitro, and acidic pretreatment of bLF enhanced its inhibitory activity. Moreover, partially digested products of bLF by pepsin retained inhibitory activity. Based on these results, bLF was co-administered with levodopa (l-DOPA), which is a catechol compound and a precursor of dopamine, and the effect of bLF on l-DOPA absorption and metabolism was investigated in a mouse model. The co-administration of l-DOPA and bLF alone showed no effect on the concentration of l-DOPA in plasma. However, with the additional administration of carbidopa, the concentration of l-DOPA was significantly enhanced. Furthermore, the ratio of l-DOPA/3-O-methyldopa significantly increased. On the other hand, casein, which is a major milk protein, was not effective. In addition, COMT activity in the intestines was lowered with bLF administration. We concluded that the co-administration of bLF and carbidopa enhances the concentration of l-DOPA.

Identification of Two Phenanthrene Derivatives from Australasian Allied Species in Genus Dendrobium.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

Inhibitory Effect of Bovine Lactoferrin on Catechol-O-Methyltransferase.

Lactoferrin (LF) is a well-known multifunctional protein. In this study, we report the inhibitory potency of bovine LF (bLF) on catechol-O-methyltransferase (COMT), which catalyzes methylation of catechol substrates. We found that bLF binds to and inhibits COMT using its N-terminal region. An N-terminal peptide fragment obtained from bLF by trypsin digestion showed a higher inhibitory activity than intact bLF. A synthetic fragment of the bLF N-terminal residues 6-50, with two pairs of disulfide bonds, also showed higher inhibitory activity than intact bLF. Enzyme kinetic studies proved that bLF did not compete with S-adenosylmethionine (the methyl donor substrate) as well as methyl acceptor substrates such as dihydroxybenzoic acid, (-)-epicatechin, norepinephrine, or l-3,4-dihydroxyphenylalanine. The inhibitory potency of bLF decreased against a COMT preparation pretreated with dithiothreitol, suggesting that the oxidation status of COMT is relevant to interaction with bLF. We further confirmed that COMT activity in the cell extracts form Caco-2 and HepG2 cells was inhibited by bLF and by the synthesized fragment. Enzyme kinetic study indicated that bLF functions as a non-competitive inhibitor by binding to an allosteric surface of COMT.

Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship. Part 2.

To explore the structure-activity relationships of flavanonols on the inhibition of nitric oxide (NO) production in RAW 264.7 cells, we have prepared a series of synthetic flavanonols. In our previous study, the 2',3'-dihydroxyphenyl substructure was found to be the most potent B ring substructure among the flavanonols having 3,5,7-trihydroxychroman-4-one as the A/C ring. In this study, we examined the effect of diverse substitutions on the A ring of the 2-(2,3-dihydroxyphenyl)-3-hydroxychroman-4-one scaffold, i.e., by fixing the B ring to the 2',3'-dihydroxyphenyl substructure. Eighteen stereoisomers and 4 racemic mixtures were prepared, and their inhibitory potency on NO production in RAW 264.7 cells was tested. We observed higher inhibitory activity in the (2R,3R) stereoisomers than in the (2S,3S) stereoisomers. The presence of a hydroxy or a methoxy group at the 7-postiion enhanced the inhibitory potency, and the additional substitutions at the 6- or 8-position in the A ring increased potency and stereospecificity. A representative compound, (2R,3R)-2',3',7,8-tetrahydroxyflavanonol 5e, had an IC50 value of 17µM, whereas its (2S,3S) stereoisomer did not inhibit NO production at all at a concentration of 100µM. In this study, it was necessary to determine the absolute configuration of the stereoisomers of the synthesized flavanonols that carry methoxy substitutions in the A ring. The procedure to determine their absolute configuration by the CD excitation chirality method is also discussed.

Structure-activity relationship of the inhibitory effects of flavonoids on nitric oxide production in RAW264.7 cells.

We isolated flavonoids from herbal specimens from the Tibetan region (Sophora yunnanensis and Rhodiola sacra) that suppress nitric oxide (NO) production in macrophages stimulated by lipopolysaccharide and interferon-γ. The isolated flavonoids carry symmetric substitutions in the B ring (R3'=R5'). We analyzed the quantitative structure-activity relationship of the inhibitory activity by comparative molecular field analysis (CoMFA) using this series of flavonoids. Use of flavonoids with symmetrical substitutions in the B ring made it simpler to align molecules because it was not necessary to consider a huge number of combinations due to the B-ring conformation. The CoMFA model, whose cross-validated q2 value was 0.705, suggested the existence of a hydroxy group at the 5-position, the choice of the A/C-ring scaffold (chromane or chromene) and electrostatic field around the B ring are important for NO inhibitory activity. Flavonoids synthesized based on the CoMFA model exhibited significant inhibitory potential against NO production, validating the predictive capability of the CoMFA model.

Identification of baseline gene expression signatures predicting therapeutic responses to three biologic agents in rheumatoid arthritis: a retrospective observational study.

BACKGROUND: According to EULAR recommendations, biologic DMARDs (bDMARDs) such as tumor necrosis factor inhibitor, tocilizumab (TCZ), and abatacept (ABT) are in parallel when prescribing to rheumatoid arthritis (RA) patients who have shown insufficient response to conventional synthetic DMARDs. However, most prediction studies of therapeutic response to bDMARDs using gene expression profiles were focused on a single bDMARD, and consideration of the results from the perspective of RA pathophysiology was insufficient. The aim of this study was to identify the specific molecular biological features predicting the therapeutic outcomes of three bDMARDs (infliximab [IFX], TCZ, and ABT) by studying blood gene expression signatures of patients before biologic treatment in a unified test platform. METHODS: RA patients who responded inadequately to methotrexate and were later commenced on any one of IFX (n = 140), TCZ (n = 38), or ABT (n = 31) as their first biologic between May 2007 and November 2011 were enrolled. Whole-blood gene expression data were obtained before biologic administration. Patients were categorized into remission (REM) and nonremission (NON-REM) groups according to CDAI at 6 months of biologic therapy. We employed Gene Set Enrichment Analysis (GSEA) to identify functional gene sets differentially expressed between these two groups for each biologic. Then, we compiled "signature scores" for these gene sets, and the prediction performances were assessed. RESULTS: GSEA showed that inflammasome genes were significantly upregulated with IFX in the NON-REM group compared with the REM group. With TCZ in the REM group, B-cell-specifically expressed genes were upregulated. RNA elongation, apoptosis-related, and NK-cell-specifically expressed genes were upregulated with ABT in the NON-REM group. Logistic regression analyses showed that "signature scores" of inflammasomes, B-cell-specifically expressed, and NK-cell-specifically expressed genes were significant, independently predictive factors for treatment outcome with IFX, TCZ, and ABT, respectively. The AUCs of ROC curves of these signature scores were 0.637, 0.796, and 0.768 for IFX, TCZ, and ABT, respectively. CONCLUSIONS: We have identified original gene expression predictive signatures uniquely underlying the therapeutic effects of IFX, TCZ, and ABT. This is, to our knowledge, the first attempt to predict therapeutic effects of three drugs concomitantly using a unified gene expression test platform.

Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship.

To explore the structure-activity relationships on the inhibitory activity of flavanonols against nitric oxide (NO) production in inflammatory cells, we synthesized 19 flavanonols which shared a common 3,5,7-trihydroxychroman scaffold. A range of substitutions was included in the B ring in order to investigate the structure-activity relationship. We also succeeded in isolating stereoisomers from 16 of the flavanonols using chiral column chromatography. The inhibitory effects of these compounds on NO production were examined in RAW 264.7 cells (a murine macrophage-like cell line), which were activated by lipopolysaccharide (LPS). We only observed inhibitory activity against NO production in (2R,3R) stereoisomers, while the inhibitory activities of (2S,3S) stereoisomers were significantly weaker. We also evaluated the free radical scavenging potential of the flavanonols using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Each stereoisomer indicated the equivalent DPPH scavenging potential as expected. The radical scavenging activity was not correlated with the inhibitory activity against NO. The inhibition of NO production by flavanonols is stereospecific and cannot simply be explained by their radical scavenging activity. We propose the possible existence of a 'target' molecule for flavanonols which is involved in the production and/or regulation of NO in RAW 264.7 cells.

Molecular phylogenetics and character evolution of morphologically diverse groups, Dendrobium section Dendrobium and allies.

It is always difficult to construct coherent classification systems for plant lineages having diverse morphological characters. The genus Dendrobium, one of the largest genera in the Orchidaceae, includes ∼1100 species, and enormous morphological diversification has hindered the establishment of consistent classification systems covering all major groups of this genus. Given the particular importance of species in Dendrobium section Dendrobium and allied groups as floriculture and crude drug genetic resources, there is an urgent need to establish a stable classification system. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we analysed the macromolecular characters of the group. Phylogenetic analyses of 210 taxa of Dendrobium were conducted on DNA sequences of internal transcribed spacer (ITS) regions of 18S-26S nuclear ribosomal DNA and the maturase-coding gene (matK) located in an intron of the plastid gene trnK using maximum parsimony and Bayesian methods. The parsimony and Bayesian analyses revealed 13 distinct clades in the group comprising section Dendrobium and its allied groups. Results also showed paraphyly or polyphyly of sections Amblyanthus, Aporum, Breviflores, Calcarifera, Crumenata, Dendrobium, Densiflora, Distichophyllae, Dolichocentrum, Holochrysa, Oxyglossum and Pedilonum. On the other hand, the monophyly of section Stachyobium was well supported. It was found that many of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the result of convergence. As such, many of the sections that have been recognized up to this point were found to not be monophyletic, so recircumscription of sections is required.

Effects of the herbal medicine composition "Saiko-ka-ryukotsu-borei-To" on the function of endothelial progenitor cells in hypertensive rats.

Endothelial progenitor cells (EPCs) are known to repair vascular injuries. Recent studies suggest that Saiko-ka-ryukotsu-borei-To (SKRBT), a traditional herbal medicine that has been used to treat stress-related neuropsychiatric disorders, has protective effects on cardiovascular diseases such as hypertension and arteriosclerosis. Spontaneously hypertensive rats (SHRs) were fed diets containing lyophilized SKRBT extract for 6 weeks. Peripheral blood mononuclear cells (MNCs) were isolated and cultured to assay EPC colony formation. Oxidative stress in MNCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay and flowcytometric analyses. Treatment with SKRBT increased EPC colony numbers significantly (p<0.05) with decrease in oxidative stress and without affecting blood pressure in SHRs. Treatment with SKRBT did not reduce the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in cardiovascular organs. Serum IL-6 level was significantly reduced. SKRBT is a feasible herbal medicine that protects against cardiovascular diseases through an increase in EPC function along with anti-oxidative effects, and may affect the link between chronic inflammation and cardiovascular disease.

Genistein and daidzein, typical soy isoflavones, inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes.

SCOPE: Previous reports suggest that soy isoflavones have multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about whether soy isoflavones can inhibit the downregulation of adiponectin and their molecular mechanisms. In the present study, we demonstrate that genistein (Gen) or daidzein (Dai) can significantly inhibit the downregulation of adiponectin expression via unique and different molecular mechanisms. METHODS AND RESULTS: Pretreatment with Gen or Dai significantly inhibited the tumor necrosis factor-alpha (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Gen inhibited the TNF-α-induced c-Jun-NH(2) -terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on JNK1 with Gen or Dai clearly supported our experimental results. However, Dai did not significantly inhibit JNK signaling. Dai did, however, inhibit the TNF-α-induced downregulation of forkhead box-containg protein O1, which is also involved in adiponectin expression. CONCLUSION: These data demonstrate that: (i) both Gen and Dai significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes; (ii) Gen is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin; and (iii) Dai can inhibit the downregulation of adiponectin by restoring the TNF-α-mediated reduction of forkhead box-containg protein O1 protein expression.

Analysis of EMG signals of patients with essential tremor focusing on the change of tremor frequency.

Essential tremor is a disorder that causes involuntary oscillations in patients while engaging in actions or while maintaining a posture. ET patients have serious difficulties in performing activities of daily living such as eating food, drinking water, and writing. We have thus been developing an EMG-controlled exoskeletal robot to suppress tremors. The EMG signal of ET patients involves a mix of voluntary movement and tremor signals. To control the exoskeletal robot accurately, tremor signals must be removed from the patient's EMG signal. To date, we have been developing a filter to remove tremor signals from the patient's EMG. The design of this filter was based on the hypothesis that the rectified tremor signals are able to be approximated by a powered sine wave. This filter was found to have a large effect on removing tremor signals. However, tremor signals are generated both while performing voluntary movement and while maintaining a posture, and the filter was attenuating both signals. To control this robot accurately, the signal generated while performing voluntary movement is expected not to be attenuated. To accomplish this, we try to use a parameter that reflects a state of the patient's movement, performing a voluntary movement or maintaining a posture, as a switch to activate the powered sine filter. This paper provides an analysis of the favorable parameters. We focus on two parameters: the peak-to-peak interval of the rectified EMG signal, and the interval of the flat and low amplitude area of the rectified EMG signal. Through evaluation, it is affirmed that both parameters change with the state of the patient's movement. However, the latter parameter is superior to the former in terms of variability, which indicates that the interval of the flat and low amplitude area of the rectified EMG signal is a more favorable parameter to promote control of the exoskeletal robot. As a future work, we will mount the parameter to the algorithm and evaluate the robotic system.

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy.

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

Copy number loss of (src homology 2 domain containing)-transforming protein 2 (SHC2) gene: discordant loss in monozygotic twins and frequent loss in patients with multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a sporadic disease. Its pathogenesis may involve multiple genetic and nongenetic factors, but its etiology remains largely unknown. We hypothesized that the genome of a patient with MSA would demonstrate copy number variations (CNVs) in the genes or genomic regions of interest. To identify genomic alterations increasing the risk for MSA, we examined a pair of monozygotic (MZ) twins discordant for the MSA phenotype and 32 patients with MSA. RESULTS: By whole-genome CNV analysis using a combination of CNV beadchip and comparative genomic hybridization (CGH)-based CNV microarrays followed by region-targeting, high-density, custom-made oligonucleotide tiling microarray analysis, we identified disease-specific copy number loss of the (Src homology 2 domain containing)-transforming protein 2 (SHC2) gene in the distal 350-kb subtelomeric region of 19p13.3 in the affected MZ twin and 10 of the 31 patients with MSA but not in 2 independent control populations (p = 1.04 × 10-8, odds ratio = 89.8, Pearson's chi-square test). CONCLUSIONS: Copy number loss of SHC2 strongly indicates a causal link to MSA. CNV analysis of phenotypically discordant MZ twins is a powerful tool for identifying disease-predisposing loci. Our results would enable the identification of novel diagnostic measure, therapeutic targets and better understanding of the etiology of MSA.