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5.2E: Osmosis - Biology

5.2E: Osmosis - Biology



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LEARNING OBJECTIVES

  • Describe the process of osmosis and explain how concentration gradient affects osmosis

Osmosis and Semipermeable Membranes

Osmosis is the movement of water through a semipermeable membrane according to the concentration gradient of water across the membrane, which is inversely proportional to the concentration of solutes. Semipermeable membranes, also termed selectively permeable membranes or partially permeable membranes, allow certain molecules or ions to pass through by diffusion.

While diffusion transports materials across membranes and within cells, osmosis transports only water across a membrane. The semipermeable membrane limits the diffusion of solutes in the water. Not surprisingly, the aquaporin proteins that facilitate water movement play a large role in osmosis, most prominently in red blood cells and the membranes of kidney tubules.

Mechanism of Osmosis

Osmosis is a special case of diffusion. Water, like other substances, moves from an area of high concentration to one of low concentration. An obvious question is what makes water move at all? Imagine a beaker with a semipermeable membrane separating the two sides or halves. On both sides of the membrane the water level is the same, but there are different concentrations of a dissolved substance, or solute, that cannot cross the membrane (otherwise the concentrations on each side would be balanced by the solute crossing the membrane). If the volume of the solution on both sides of the membrane is the same but the concentrations of solute are different, then there are different amounts of water, the solvent, on either side of the membrane. If there is more solute in one area, then there is less water; if there is less solute in one area, then there must be more water.

To illustrate this, imagine two full glasses of water. One has a single teaspoon of sugar in it, whereas the second one contains one-quarter cup of sugar. If the total volume of the solutions in both cups is the same, which cup contains more water? Because the large amount of sugar in the second cup takes up much more space than the teaspoon of sugar in the first cup, the first cup has more water in it.

Returning to the beaker example, recall that it has a mixture of solutes on either side of the membrane. A principle of diffusion is that the molecules move around and will spread evenly throughout the medium if they can. However, only the material capable of passing through the membrane will diffuse through it. In this example, the solute cannot diffuse through the membrane, but the water can. Water has a concentration gradient in this system. Thus, water will diffuse down its concentration gradient, crossing the membrane to the side where it is less concentrated. This diffusion of water through the membrane—osmosis—will continue until the concentration gradient of water goes to zero or until the hydrostatic pressure of the water balances the osmotic pressure. In the beaker example, this means that the level of fluid in the side with a higher solute concentration will go up.

Key Points

  • Osmosis occurs according to the concentration gradient of water across the membrane, which is inversely proportional to the concentration of solutes.
  • Osmosis occurs until the concentration gradient of water goes to zero or until the hydrostatic pressure of the water balances the osmotic pressure.
  • Osmosis occurs when there is a concentration gradient of a solute within a solution, but the membrane does not allow diffusion of the solute.

Key Terms

  • solute: Any substance that is dissolved in a liquid solvent to create a solution
  • osmosis: The net movement of solvent molecules from a region of high solvent potential to a region of lower solvent potential through a partially permeable membrane
  • semipermeable membrane: A type of biological membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally by specialized facilitated diffusion

Abstract

Breeding and rearing some of the clownfishes most commonly used in the aquarium trade actually represent an economical and ecological tool for broadening development. Culture of clownfish species in low-saline water is still in its infancy. Salinity of the culture environment is one of the more relevant parameters affecting fish physiology, modifying food intake and growth performance in many fish species. The objective of this study was to breed skunk clownfish (Amphiprion akallopisos) in aquarium condition, document the embryonic development, determine the upper and lower lethal salinities of juveniles, tolerance of five different salinities (20, 25, 30, 35, and 40 ppt) and their effect on the survival rate of larvae. Higher (53–55 ppt) and lower (3–6 ppt) salinities produced loss of appetite and movement, finally leading to mortality in juveniles. In a ninety six hour experiment, larvae showed 100% survival at the salinities of 30 (control) and 35 ppt and 88% survival in 40 ppt salinity and 76% survivals in 20 and 25 ppt. In conclusion juveniles of A. akallopisos exhibit satisfactory rates of survival and no signs of stress in high (up to 53 ppt) and low saline (up to 6 ppt) waters. These results demonstrate that using such salinities, which can reduce the incidence of diseases and mortality, does not produce significant physiological alterations in this species. In addition, descriptive studies on embryonic development and mass scale larval rearing were also carried out during the present study.


5.2E: Osmosis - Biology

Apr. 2017 The 5th Asian Graduate Student Symposium on Membrane Engineering, The Best Student Oral Presentation Award at AGSM5, Characterization of separation function of Amphotericin B for biomimetic water purification membrane

Toru Takai, Daisuke Saeki, Kazuo Kumagai, Hideto Matsuyama

Published Papers

Asuka Inada, Kazuo Kumagai, Hideto Matsuyama

Elsevier BV, Dec. 2020, Separation and Purification Technology, 252, 117462 - 117462

Asuka Inada, Tomoki Takahashi, Kazuo Kumagai, Hideto Matsuyama

© 2019 American Chemical Society. To develop a forward osmosis (FO) process, selection of draw solutes (DSs) is a critical factor in determining water permeability of the process. In this search for novel high-performance DSs, various morpholine derivatives were investigated for their thermoresponsive potential. 4-Butylmorpholine (BuMP) showed a preferable minimum lower critical solution temperature for the FO process (31.7 °C). The dilute phase of BuMP after phase separation at 70 °C showed a low concentration (3.3 wt %) and low osmotic pressure (3.16 bar). In the FO flux test, the water permeability and reverse solute flux of BuMP (55.0 wt %, 28 bar) against water were Jw 2.09 L m-2 h-1 and Js 14.0 g m-2 h-1, respectively. Using 0.6 M NaCl (model seawater) as feed solution, BuMP (94.6 wt %) could extract water from this model seawater (Jw 0.56 L m-2 h-1). These results indicate a high potential for MP derivatives as DSs and provide new guidance for their development for FO desalination.

03 Jun. 2019, Industrial and Engineering Chemistry Research, 58 (27), 12253 - 12260, English

Asuka Inada, Kenichiro Yumiya, Tomoki Takahashi, Kazuo Kumagai, Yoko Hashizume, Hideto Matsuyama

© 2018 Elsevier B.V. Recently, forward osmosis (FO) is attracting research attention once again. In a FO process, it is important to develop a draw solution (DS) with a high osmotic pressure and low solute leakage. In this study, thermoresponsive star-shaped oligomers with a glycerol backbone were developed as new draw solutes for FO. A series of glycerol-oligo(ethylene oxide)-block-oligo(butylene oxide) (GEB) oligomers were systematically designed and synthesized. The average degrees of polymerization of ethylene oxide (EO m) and butylene oxide (BO n) units of GEmBn were varied to control the hydrophilic/hydrophobic balance of the molecule. Aqueous solutions of GEBs were evaluated in terms of their osmotic pressures, phase diagrams, and viscosities. Most of them showed a lower critical solution (LCST)-type phase separation at temperatures below 60 °C. The osmotic pressure of 68 wt% GE7B3 (concentration of the dense phase after phase separation at 60 °C) was 74 bar, about 2.6 times higher than that of seawater. Moreover, the leakage of GE7B3 was much lower than that of conventional draw solutes. The osmotic pressure of the dilute phase of a GE7B3 solution at 60 °C was less than 2 bar, implying reduced energy consumption during post-processing by low-pressure reverse osmosis to collect pure water.

15 Mar. 2019, Journal of Membrane Science, 574, 147 - 153, English

Riyo Maruki Imamura, Kazuo Kumagai, Hirofumi Nakano, Takayoshi Okabe, Tetsuo Nagano, Hirotatsu Kojima

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

SAGE PUBLICATIONS INC, Mar. 2019, SLAS DISCOVERY, 24 (3), 284 - 294, English

Masaaki Ito, Shin-ichiro Egashira, Kazuki Yoshida, Tomoko Mineno, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Michio Ui, Isao Matsuoka

Aims: The P2Y(6) nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y(6) receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y(6) receptor.Main methods: High-throughput screening (HTS) was used to study the effects of various compounds on human P2Y(6) receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2+ concentration ([Ca2+]i) levels using an FDSS7000 real-time fluorescence detector. IL-8 concentration was measured by enzyme-linked immunosorbent assay.Key findings: Among structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y(6) receptor were identified. Further studies using a dose-response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y(6) receptor inhibitor TIM-38, which inhibited UDP-induced [Ca2+] elevation in a dose-dependent manner. TIM-38 had an IC60 value of 43 ptM and inhibited P2Y(6) without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM-38 inhibited UDP-induced interleukin-8 release in a dose-dependent manner without affecting releases caused by other stimulus such as interleukin-1 beta or tumour necrosis factor-alpha. Analyses of TIM-38 derivatives revealed that the nitro moiety is vital to P2Y(6) receptor inhibition.Significance: TIM-38 acts as a novel structural antagonist of P2Y(6) receptor and may be a good lead compound for developing a P2Y(6) receptor-targeted anti-inflammatory drug. (C) 2017 Published by Elsevier Inc.

PERGAMON-ELSEVIER SCIENCE LTD, Jul. 2017, LIFE SCIENCES, 180, 137 - 142, English

Seiji Ishii, Kenji Fukui, Satoshi Yokoshima, Kazuo Kumagai, Youko Beniyama, Tetsuya Kodama, Tohru Fukuyama, Takayoshi Okabe, Tetsuo Nagano, Hirotatsu Kojima, Takato Yano

The main components of the quorum-sensing system are expected to be favorable targets for drug development to combat various chronic infectious diseases. ComA of Streptococcus is an ATP-binding cassette transporter containing a peptidase domain (PEP), which is essential for the quorum-sensing signal production. Using high-throughput screening, we found a potent small molecule that suppressed the S. mutans quorum-sensing pathway through inhibition of PEP activity. The compound effectively attenuated the biofilm formation and competence development of S. mutans without inhibiting cell growth. The kinetic and structural studies with this molecule and a related compound unexpectedly revealed an allosteric site of PEP. This relatively hydrophobic site is thought to undergo large structural changes during the catalytic process. These compounds inhibit PEP activity by binding to and suppressing the structural changes of this site. These results showed that PEP is a good target for inhibitors of the Streptococcus quorum-sensing system.

NATURE PUBLISHING GROUP, Jun. 2017, SCIENTIFIC REPORTS, 7, 4029, English

Ke Liu, Naoko Kunii, Megumi Sakuma, Atsumi Yamaki, Satoru Mizuno, Mayu Sato, Hiromichi Sakai, Sayaka Kado, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Yasuhito Shirai, Fumio Sakane

Diacylglycerol kinase (DGK) consists of 10 isozymes. The alpha-isozyme enhances the proliferation of cancer cells. However, DGK alpha facilitates the nonresponsive state of immunity known as T-cell anergy therefore, DGK alpha enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGK alpha activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl) amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGK alpha with an IC50 value of 0.6 mu M. CU-3 targeted the catalytic region, but not the regulatory region, of DGK alpha. CU-3 competitively reduced the affinity of DGK alpha for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGK alpha and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Mar. 2016, JOURNAL OF LIPID RESEARCH, 57 (3), 368 - 379, English

Ke Liu, Naoko Kunii, Megumi Sakuma, Atsumi Yamaki, Satoru Mizuno, Mayu Sato, Hiromichi Sakai, Sayaka Kado, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Yasuhito Shirai, Fumio Sakane

Diacylglycerol kinase (DGK) consists of 10 isozymes. The alpha-isozyme enhances the proliferation of cancer cells. However, DGK alpha facilitates the nonresponsive state of immunity known as T-cell anergy therefore, DGK alpha enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGK alpha activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl) amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGK alpha with an IC50 value of 0.6 mu M. CU-3 targeted the catalytic region, but not the regulatory region, of DGK alpha. CU-3 competitively reduced the affinity of DGK alpha for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGK alpha and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Mar. 2016, JOURNAL OF LIPID RESEARCH, 57 (3), 368 - 379, English

2015, コンバーテック, 43 (12), 10 - 13, Japanese

Masayuki Isa, Daisuke Asanuma, Shigeyuki Namiki, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Kenzo Hirose

Overexpression of growth factor receptors in cancers, e.g., human epidermal growth factor receptor 2 (HER2) in ovarian and breast cancers, is associated with aggressiveness. A possible strategy to treat cancers that overexpress those receptors is blockade of receptor signaling by inducing receptor internalization and degradation. In this study, we developed a cell-based high-throughput screening (HTS) system to identify small molecules that induce HER2 internalization by employing our recently developed acidic-pH-activatable probe in combination with protein labeling technology. Our HTS system enabled facile and reliable quantification of HER2 internalization with a Z' factor of 0.66 and a signal-to-noise ratio of 44.6. As proof of concept, we used the system to screen a similar to 155,000 small-molecule library and identified three hits that induced HER2 internalization and degradation via at least two distinct mechanisms. This HTS platform should be adaptable to other disease-related receptors in addition to HER2.

AMER CHEMICAL SOC, Oct. 2014, ACS CHEMICAL BIOLOGY, 9 (10), 2237 - 2241, English

Megumi Tarui, Hideo Shindou, Kazuo Kumagai, Ryo Morimoto, Takeshi Harayama, Tomomi Hashidate, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Takahide Nagase, Takao Shimizu

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jul. 2014, JOURNAL OF LIPID RESEARCH, 55 (7), 1386 - 1396, English

Megumi Tarui, Hideo Shindou, Kazuo Kumagai, Ryo Morimoto, Takeshi Harayama, Tomomi Hashidate, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Takahide Nagase, Takao Shimizu

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jul. 2014, JOURNAL OF LIPID RESEARCH, 55 (7), 1386 - 1396, English

Kenta Mosallanejad, Yusuke Sekine, Seiko Ishikura-Kinoshita, Kazuo Kumagai, Tetsuo Nagano, Atsushi Matsuzawa, Kohsuke Takeda, Isao Naguro, Hidenori Ichijo

During infection with an RNA virus, the DExD/H-box RNA helicases RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) activate the interferon regulatory factor 3 (IRF3), nuclear factor kappa B (NF-kappa B), c-Jun amino-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways through an unknown mechanism involving the adaptor protein MAVS (mitochondrial antiviral signaling). We used a Drosophila misexpression screen to identify DEAH-box polypeptide 15 (DHX15) as an activator of the p38 MAPK pathway. Human DHX15 contributed to the activation of the NF-kappa B, JNK, and p38 MAPK pathways, but not the IRF3 pathway, in response to the synthetic double-stranded RNA analog poly(I:C) (polyinosinic-polycytidylic acid), and DHX15 was required for optimal cytokine production in response to poly(I:C) and infection with RNA virus. DHX15 physically interacted with MAVS and mediated the MAVS-dependent activation of the NF-kappa B and MAPK pathways. Furthermore, DHX15 was required for poly(I:C)-and RNA virus-dependent, MAVS-mediated apoptosis. Thus, our findings indicate that, in RIG-I-like receptor signaling, DHX15 specifically stimulates the NF-kappa B and MAPK pathways downstream of MAVS and contributes to MAVS-mediated cytokine production and apoptosis.

AMER ASSOC ADVANCEMENT SCIENCE, Apr. 2014, SCIENCE SIGNALING, 7 (323), ra40, English

Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z'-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators. (C) 2013 Elsevier Inc. All rights reserved.

ACADEMIC PRESS INC ELSEVIER SCIENCE, Feb. 2014, ANALYTICAL BIOCHEMISTRY, 447, 146 - 155, English

Nobuo Hosotani, Kazuo Kumagai, Shigeyuki Honda, Akira Ito, Takuro Shimatani, Ikutaro Saji

A new peptaibol compound, SPF-5506-A(4), was isolated from the fermentation broth of Trichoderma sp. SPF-5506. The chemical structure of the 14-residue peptide was determined by MS, NMR and amino acid sequence analyses. The absolute configuration of amino acid residues in the acid hydrolysate was determined by Marfey's method. The structure of SPF-5506-A4 was established as Ac-Aib-L-Asn-L-Ile-Aib-L-Pro-L-Ser-L-IleAib-L-Pro-L-LeU-L-Leu-Aib-L-Pro-L-leucinol. The compound inhibited amyloid P-peptide formation in primary guinea pig cerebral cortex neuron cell culture dose-dependently with an IC50 of 0.1 mu g/ml. Cytotoxicity was not observed at concentrations of < 3 mu g/ml.

JAPAN ANTIBIOTICS RESEARCH ASSOC, Mar. 2007, JOURNAL OF ANTIBIOTICS, 60 (3), 184 - 190, English

Shinjiro Kaneko, Akio Iwanami, Masaya Nakamura, Akiyoshi Kishino, Kaoru Kikuchi, Shinsuke Shibata, Hirotaka J. Okano, Takeshi Ikegami, Ayako Moriya, Osamu Konishi, Chikao Nakayama, Kazuo Kumagai, Toru Kimura, Yasufumi Sato, Yoshio Goshima, Masahiko Taniguchi, Mamoru Ito, Zhigang He, Yoshiaki Toyama, Hideyuki Okano

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

NATURE PUBLISHING GROUP, Dec. 2006, NATURE MEDICINE, 12 (12), 1380 - 1389, English

T Shimatani, N Hosotani, M Ohnishi, K Kurnagai, Saji, I

Two new human chymase inhibitors, SPF-32629A and B, were isolated from the cultured broth of Penicillium sp. SPF-32629. These structures were determined by spectroscopic methods and identified as new pyridone compounds. SPF-32629B was the carboxylated derivative of SPF-32629A. SPF-32629A and B specifically inhibited human chymase among four serine proteases tested with the IC50 of 0.25 and 0.42 mu g/ml, respectively.

JAPAN ANTIBIOTICS RESEARCH ASSOC, Jan. 2006, JOURNAL OF ANTIBIOTICS, 59 (1), 29 - 34, English

N Hosotani, K Kumagai, H Nakagawa, T Shimatani, Saji, I

Seven new antimycin antibiotics, named antimycins A(10), A(11), A(12), A(13), A(14), A(15) and A(16), were isolated from the fermentation broth of strains of Streptomyces spp. SPA-10191 and SPA-8893, along with known antimycins A(1), A(2), A(3) and A(4). The structures of the new antimycins were determined by spectral analyses, including 2D NMR techniques. These compounds exhibited antifungal activity against Candida utilis.

JAPAN ANTIBIOTICS RESEARCH ASSOC, Jul. 2005, JOURNAL OF ANTIBIOTICS, 58 (7), 460 - 467, English

N Hosotani, K Kumagai, H Nakagawa, T Shimatani, Saji, I

Two new 24-membered macrolides, SPA-6952A and B, were isolated from the fermentation broth of Streptomyces sp. SPA-6952. The structures of the new macrolides were determined by spectral analyses, including 2D NMR techniques. These compounds exhibited cytotoxic activity against human promyelocytic leukemia HL-60 cells.

JAPAN ANTIBIOTICS RESEARCH ASSOC, Jun. 2005, JOURNAL OF ANTIBIOTICS, 58 (6), 409 - 411, English

K Kikuchi, A Kishino, O Konishi, K Kumagai, N Hosotani, Saji, I, C Nakayama, T Kimura

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 muM (IC50=0.16 muM). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Oct. 2003, JOURNAL OF BIOLOGICAL CHEMISTRY, 278 (44), 42985 - 42991, English

S Kaneko, A Iwanami, M Nakamura, S Miyao, A Kishino, K Kikuchi, T Kimura, K Kumagai, Y Toyama, H Okano

MARY ANN LIEBERT INC PUBL, Oct. 2003, JOURNAL OF NEUROTRAUMA, 20 (10), 1133 - 1133, English

K Kumagai, N Hosotani, K Kikuchi, T Kimura, Saji, I

A new semaphorin inhibitor xanthofulvin was isolated from the cultured broth of a fungus Penicillium sp. SPF-3059 along with a known compound vinaxanthone by solvent extraction and bioassay-guided fractionation. The tautomeric structure of xanthofulvin was determined by spectroscopic analyses. The two compounds exhibited significant semaphorin inhibitory activity with IC50 values of 0.09 and 0.1 mug/ml, respectively, in semaphorin3A-induced growth cone collapse assay using cultured chick dorsal root ganglia neurons.

JAPAN ANTIBIOT RES ASSN, Jul. 2003, JOURNAL OF ANTIBIOTICS, 56 (7), 610 - 616, English

J Nagamine, R Nagata, H Seki, N Nomura-Akimaru, Y Ueki, K Kumagai, M Taiji, H Noguchi

SM-130686, an oxindole derivative, is a novel orally active GI I secretagogue (GI IS) which is structurally distinct from previously reported GHSs such as MK-677, NN703 and hexarelin. SM-130686 stimulates GH release from cultured rat pituitary cells in a dose-dependent manner. Half-maximum stimulation was observed at a concentration of 6.3 +/- 3.4 nM. SM-130686-induced GH release was inhibited by a GHS antagonist, but not by a GH-releasing hormone antagonist. SM-130686 dose-dependently inhibited the binding of radiolabeled ligand, S-35-MK-677, to human GHS receptor 1a (IC50 = 1.2 nM). This indicates that SM-130686 stimulates GH release through the GHS receptor. The effect of a single oral administration of SM-130686 on GH release in pentobarbital-anesthetized rats was studied. After treatment with 10 mg/kg SM-130686, plasma GH concentrations measured by radioimmunoassay significantly increased, reaching a peak at 20-45 min, and remained above baseline during the experimental period (60 min). The anabolic effect of repetitive SM-130686 adininistration was studied in rats. Rats received 10 mg/kg SM-130686 orally twice a day and were weighed every day for 9 days. At day 9 there was a significant increase in both the body weight and the fat free mass (19.5 +/- 2.1 and 18-1 +/- 7.5 g respectively). Serum IGF-I concentration was also significantly elevated 6 h after the last dose of SM-130686. An. endogenous GHS ligand for the GHS receptor has recently been identified from stomach extract and designated as ghrelin. The GH-releasing activity in vitro relative to ghrelin (100%) was about 52% for SM-130686. It is likely that SM-130686 is a partial agonist for the GHS receptor. In summary, we describe here an orally active GHS, SM-130686, which acts through the GHS receptor. Repetitive administration of SM-130686 to rats, similar to repetitive administration of GH, significantly increased the fat free mass by an amount almost equal to the gain in body weight.

SOC ENDOCRINOLOGY, Dec. 2001, JOURNAL OF ENDOCRINOLOGY, 171 (3), 481 - 489, English

T Tokunaga, WE Hume, T Umezome, K Okazaki, Y Ueki, K Kumagai, S Hourai, J Nagamine, H Seki, M Taiji, H Noguchi, R Nagata

A series of substituted oxindole derivatives was synthesized and evaluated for growth hormone (GH) releasing activity using cultured rat pituitary cells. (+)-6-Carbamoyl-3-(2-chlorophenyl)(2-diethylaminoethyl)-4-trifluoromethyloxindole (SM-130686, 37S) was found to have potent activity (EC50 = 3.0 nM), while the other enantiomer 37R had reduced activity. The absolute configuration of 37S was confirmed by X-ray crystallographic analysis. Compound 37S showed a good pharmacokinetic profile in rats with 28% oral bioavailability at 10 mg/kg and excellent in vivo activity as evidenced by a significant weight gain after 4 days of oral administration at 10 mg/kg twice a day. Compound 37S displaced the binding of S-35-MK-677 to human GHS-R with an IC50 value of 1.2 +/- 0.2 nM.

AMER CHEMICAL SOC, Dec. 2001, JOURNAL OF MEDICINAL CHEMISTRY, 44 (26), 4641 - 4649, English

R Nagata, T Tokunaga, WE Hume, T Umezome, K Okazaki, Y Ueki, K Kumagai, J Nagamine, H Seki, M Taiji, H Noguchi

AMER CHEMICAL SOC, Aug. 2001, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 222, U670 - U670, English

R Nagata, T Tokunaga, EW Hume, K Okazaki, Y Ueki, K Kumagai, J Nagamine, H Seki, M Taiji, H Noguchi

AMER CHEMICAL SOC, Aug. 2000, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 220, U591 - U591, English

K Kumagai, K Shono, H Nakayama, Y Ohno, Saji, I

A potent sigma (sigma) receptor ligand was isolated from the culture broth of Streptomyces longispororuber #525. The active compound was identified to be (2R-trans)-2-butyl-5-heptylpyrrolidine by spectroscopic and chemical studies. The compound exhibited high affinity and selectivity for sigma receptors. The IC50 values toward sigma(1), sigma(2) and dopamine D-2 receptors were 2.0, 22.7 and more than 40,000 nM, respectively. Its (2S-trans)- and (+/-)-cis-isomers, both synthesized, were also found to be high affinity sigma ligands.

JAPAN ANTIBIOT RES ASSN, May 2000, JOURNAL OF ANTIBIOTICS, 53 (5), 467 - 473, English

S MASUTOMO, A INOUE, K KUMAGAI, R MURAI, S MITSUDA

Pseudomonas sp, B21C9, a new nitrile-degrading microorganism, was isolated from a soil sample. By the hydrolysis of (RS)-isopropyl-4'-chlorophenylacetonitrile using cells of B21C9, (S)-2-isopropyl-4'-chlorophenylacetic acid having excellent optically purity could be obtained, It appeared that the microbial hydrolysis proceeded via stepwise reactions by a nitrile hydratase having poor (S)-selectivity and an amidase having strict (S)-selectivity.

TAYLOR & FRANCIS LTD, Apr. 1995, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 59 (4), 720 - 722, English

K KUMAGAI, K TOKUNAGA, M TSUTSUMI, K IKUTA

We recently showed that S1, a sequence-scrambled form of CD4 CDR3-related synthetic peptide, has more potent inhibitory activities on HIV-1 replication and HIV-1-induced syncytium formation than the original form. In this study, a series of derivatives of S1 were synthesized and their anti-HIV-1 activities were evaluated. A D-isomer was as potent as S1, and a homodimer was 10- to 18-fold more potent than SI. The increased antiviral activity of the dimer peptide was related to alpha-helix formation, as detected by circular dichroism.

ELSEVIER SCIENCE BV, Sep. 1993, FEBS LETTERS, 330 (2), 117 - 121, English

K KUMAGAI, A FUKUI, S TANAKA, M IKEMOTO, K MORIGUCHI, S NABESHIMA

A new macrolide antibiotic, PC-766B, was isolated from the cells of Nocardia brasiliensis SC-4710 by acetone extraction, and purified by gel filtration, silica gel chromatography, HPLC and TLC. The structure of PC-766B was determined by NMR spectral analysis to be a new class of the hygrolidin family antibiotics. PC-766B had a 16-membered macrocyclic lactone ring, a 6-membered hemiketal ring and a 2-deoXy-D-rhamnose moiety. DL-alpha-Tocopherol, known as an antioxidant agent, significantly improved the stability of PC-766B and prevented the decomposition of PC-766B during the storage of the antibiotic.

JAPAN ANTIBIOT RES ASSN, Jul. 1993, JOURNAL OF ANTIBIOTICS, 46 (7), 1139 - 1144, English

K KUMAGAI, K TAYA, A FUKUI, M FUKASAWA, M FUKUI, S NABESHIMA

An actinomycete strain SC-4710, a new soil isolate, was found to produce a new macrolide antibiotic, PC-766B. Chemotaxonomic analysis of the producing organism revealed that the cells of SC-4710 had type IV cell wall, type A whole cell sugar pattern, type PII phospholipids, menaquinone MK-8(H-4), cellular fatty acids comprising straight-chain saturated, unsaturated and tuberculostearic acids, and mycolic acids. The strain was identified as Nocardia brasiliensis (Lindenberg) Pinoy. The antibiotic, PC-766B, was active against Gram-positive bacteria, and some fungi and yeasts, but inactive against Gram-negative bacteria. It also showed antitumor activity against murine tumor cells in vitro and in vivo, and a weak inhibitory activity against Na+, K+-ATPase in vitro.

JAPAN ANTIBIOT RES ASSN, Jun. 1993, JOURNAL OF ANTIBIOTICS, 46 (6), 972 - 978, English

K OHKI, K KUMAGAI, S MITSUDA, T TAKANO, T KIMURA, K IKUTA

We have previously identified CD4 peptides that exhibited blocking activity on human immunodeficiency virus type 1 (HIV-1) infection, i.e. CD4(68-130) and CD4(66-92) which include the region corresponding to the third complementarity-determining region of IgG. Here we describe a unique peptide derived from CD4(66-92), altered in amino acid sequence but not in composition, which was found to have increased anti-HIV-1 activity. The acidic amino acid residues in this scrambled peptide, S1, localized at the N-terminus, while in the native peptide they clustered at the C-terminus. On the other hand, a second scrambled peptide, S2, in which the acidic amino acid residues were fully dispersed, did not show any anti-HIV-1 activity. However, we could not identify any correlation between CD4(66-92) and S1 peptides by their hydrophobic or circular dichroism spectrum analyses. The results provide insight into the mechanisms of HIV-1 gp120 and CD4 interaction and may be useful as a new approach to AIDS therapy.

BUTTERWORTH-HEINEMANN LTD, Apr. 1993, VACCINE, 11 (6), 682 - 686, English

K KUMAGAI, M KATO, S NABESHIMA, S TSUYUMU

New antibiotics, T1801 A, B, C, and D, were isolated from the culture broth of Pseudomonas sp. SC-1801. Their structures were found by spectroscopic analyses to be tri- and tetra(methylthio) derivatives of hydroquinone (T1801 A and C) or p-benzoquinone (T1801 B and D). They are new quinone and hydroquinone antibiotics and are active against Gram-positive bacteria, some fungi, and yeasts.

TAYLOR & FRANCIS LTD, Sep. 1992, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 56 (9), 1439 - 1442, English

K. Kumagai, S. Nabeshima, S. Kato, M. Watanabe, K. Ikuta

We have previously shown that liposomes containing fragment A of diphtheria toxin, which were prepared by the detergent-dialysis method with egg phosphatidylcholine, phosphatidylserine and cholesterol, possess a selective killing activity against human immunodeficiency virus (HIV)-1-infected cells, but not against uninfected cells (Ikuta et al., 1987). Since the liposomes were found to be unstable in human plasma in vitro, we prepared improved liposomes by the extrusion method with dimyristoylphosphatidylcholine instead of egg phosphatidylcholine. These liposomes were found to be very stable in human plasma, and also possessed the selective killing activity against HIV-1 -infected cells. In addition, it was found that the fragment A in the liposomes was not necessary for the selective cell killing activity. The cell killing activity and selectivity of HIV-1 -infected cells of the liposomes were remarkably affected by cholesterol content and the acyl chain length of the saturated fatty acid of phosphatidylcholines. These data suggest that membranes of liposomes can interact specifically with HIV-1 -infected cells, but not with uninfected cells, resulting in the inhibition of cell proliferation.

1991, Antiviral Chemistry and Chemotherapy, 2 (3), 143 - 148, English

K. Kumagai, K. Nakada, K. Kikuchi, H. Mori

Urinary excretion of renal tubular cell enzymes, N-acetyl-β-D-glucosaminidase (NAG) and γ-glutamyl transpeptidase (γ-GTP) was evaluated to elucidate the renal cell damage before, during and after the induced hypotension with trimetaphan (TMP), nitroglycerin (TNG) and prostaglandin E1 (PGE1) under halothane-nitrous oxide-oxygen anesthesia in patients undergoing neurosurgery. Significant increases in excretion of NAG and γ-GTP were observed in TNG group. Urinary excretion of these two enzymes in TMP group tended to increase, but its increases were not statistically significant. In PGE1 treated group, there was no tendency for urinary NAG excretion, while γ-GTP excretion tended to decrease compared with that observed before the induced hypotension. Among these three drugs, TNG exerted the most significant effect on urinary excretion of both enzymes and TMP ranked next. Effect of PGE1 on urinary enzyme excretion was weakest. According to the general opinions, degree in stray of renal tubular cell enzyme into urine is thought to parallel with the injury of renal tubular cells. Therefore, the finding obtained in this study suggests that the induced hypotension with PGE1 has a less harmful effect on renal tubular cells.


1 INTRODUCTION

Lignocellulosic biomass is an abundant, renewable resource and is considered a promising option to contribute to the biomass demand of a growing bioeconomy (Kumar, 2014 Lewandowski, 2016 ). Over the last decade, the potential to produce transportation fuels from lignocellulosic feedstocks has been accentuated (van der Weijde et al., 2013 ). This trend is supported by the advancement of conversion technologies and coincides with ambiguity in sustainability considerations of sugar- or starch-based biofuels (McKechnie, Pourbafrani, Saville, & MacLean, 2015 ). Controversies related to these first generation feedstocks include concerns about food competition, land use change or intensive agricultural inputs and have directed the research focus towards lignocellulose-based solutions (Falano, Jeswani, & Azapagic, 2014 ). At present, first commercialization ventures of lignocellulosic ethanol production in Europe can be observed (Clariant International Ltd, 2017a, 2017b ) and thus potential impacts have to be analysed and considered.

In the production of lignocellulosic ethanol via biochemical pathways, cellulose and hemicellulose serve as sugar source for the fermentation reactions. For an efficient conversion, several process steps are required: Pretreatment, (enzymatic) hydrolysis, fermentation and product stream purification. Pretreatment is required to overcome the lignocellulose recalcitrance and to increase accessibility of the polysaccharides to the degrading enzymes (Torres, van der Weijde, Dolstra, Visser, & Trindade, 2013 ). Various methods exist, deploying (combinations of) physical, physicochemical, chemical and biological approaches (Cao, Sun, Liu, Yin, & Wu, 2012 da Costa Sousa, Chundawat, Balan, & Dale, 2009 Murnen et al., 2007 Prasad, Sotenko, Blenkinsopp, & Coles, 2016 ). These processes and techniques differ in efficiency and material/energy demands and have various advantages and drawbacks with respect to production of inhibitory by-products and further process steps (Achinas & Euverink, 2016 Sims, Taylor, Jack, & Mabee, 2008 ). For instance, the efficiencies of subsequent hydrolysis processes strongly depend on the previous treatment. During hydrolysis, cellulose and (partially) hemicellulose are hydrolysed to obtain soluble monomeric sugar molecules for the further conversion to ethanol. Today, these processes are most commonly performed by means of enzymatic reactions (Achinas & Euverink, 2016 ). However, enzymatic activity can be negatively affected by highly recalcitrant lignocellulosic structures, as well as adsorption to and inhibition by lignin (Agbor, Cicek, Sparling, Berlin, & Levin, 2011 Cao et al., 2012 Klein-Marcuschamer, Oleskowicz-Popiel, Simmons, & Blanch, 2010 ). Accordingly, an efficient pretreatment process alters the lignocellulosic structure and decreases the adherence of lignin to cellulose and enzymes, in order to facilitate optimal sugar release (Klein-Marcuschamer et al., 2010 Padmanabhan et al., 2016 ). Free sugar monomers can then be metabolized and degraded to ethanol by means of microorganisms such as yeasts (e.g., Saccharomyces cerevisiae) or bacteria (e.g., Zymomonas mobilis). As only sugars are utilized for ethanol production, the third lignocellulosic main component, lignin, can be used as a by-product. For instance, it can be combusted for heat and power generation, along with other residues of the refinery process (Humbird et al., 2011 ).

Lignocellulosic feedstock may be supplied from diverse sources, including agricultural residues and dedicated biomass crops. For a sustainable provision of sufficient biomass on an industrial level, several prerequisites have to be considered. Most importantly, feedstocks are required to provide high yields within low-input management schemes (only small amounts of mineral nutrients and pesticides applied van der Weijde et al., 2013 ). Among others, miscanthus is considered a crop that potentially fulfils these requirements. It is a perennial rhizomatous C4 grass originating from East Asia (Cadoux, Riche, Yates, & Machet, 2012 ).

Plants can grow up to four metres and yield high amounts of biomass in a range of conditions (Brosse, Dufour, Meng, Sun, & Ragauskas, 2012 Lewandowski et al., 2016 van der Weijde et al., 2013 ). Due to an efficient nutrient relocation mechanism and an extensive root system, miscanthus is characterized by efficient resource use and the potential to sequester atmospheric carbon (Clifton-Brown, Breuer, & Jones, 2007 Lewandowski & Schmidt, 2006 ).

The biochemical conversion of lignocellulosic biomass to ethanol for fuel provision has been studied intensively from an environmental perspective. A wide range of feedstocks and conversion processes have been analysed by means of life cycle assessments (LCA). LCA is a method for assessing the environmental impacts of products throughout their life cycle including the raw material acquisition, production, transportation as well as end use and disposal. Morales, Quintero, Conejeros, and Aroca ( 2015 ) reviewed 60 LCA studies of lignocellulosic ethanol production from diverse feedstocks. They demonstrated that most studies focus on global warming potential and revealed a clear reduction potential when lignocellulosic instead of fossil feedstocks are used.

This was shown for agricultural residues, such as corn stover or wheat straw, as well as for dedicated energy crops. For instance, switchgrass, a perennial grass comparable to miscanthus, offers a reduction potential of 53%–93% of greenhouse gas (GHG) emissions in comparison to petrol. Furthermore, various studies observed lower impacts on ozone layer depletion and heavy metal emission-related toxicities. However, for other impact categories, such as acidification, eutrophication and ecotoxicity trends are less clear and depend strongly on feedstock types. Despite the fact, that a wide variety of feedstocks have been analysed, only a few have focused on the environmental performance of miscanthus ethanol. Some exceptions, considering the biochemical conversion, include studies from the USA and Europe (Cronin et al., 2016 Dwivedi et al., 2015 Falano et al., 2014 Meyer, Wagner, & Lewandowski, 2017 Scown et al., 2012 ). Although it is deemed a promising option for a sustainable provision of biomass, the cultivation of miscanthus on marginal lands for biofuel production has only been considered in a single study. This study focused on the Mediterranean region and did not consider the conversion pathway in detail (Schmidt, Fernando, Monti, & Rettenmaier, 2015 ), although the environmental performance of lignocellulosic ethanol is strongly influenced by the design of the conversion process. A large variety of options exist, mainly differing in the pretreatment step and influencing conversion conditions, yields, energy requirement, material demand and by-product generation (Falano et al., 2014 McKechnie et al., 2015 ).

Hence, this study aims to analyse the environmental performance of ethanol production from miscanthus at a marginal site and to contrast the results with the performance at an average-yield site. To do so, an environmental LCA of lignocellulosic ethanol from miscanthus is performed. The cradle-to-grave analysis follows the ISO norms 14040 and 14044 (ISO, 2006a, 2006b ). It enables the identification of main drivers for different environmental impacts and the evaluation of variation due to site-specific factors (incl. biomass composition and by-product generation). In addition, the study analyses the GHG emission reduction potential in comparison with petrol. In order to consider differences in the conversion process, the analysed systems extend to the refinery and three relevant pretreatment techniques were compared.

The combination of two European miscanthus cultivation scenarios—a marginal site in the UK and an average site in Germany—and three refinery models provided six production pathways for the analysis. With this approach, the study contributes to the identification of favourable pathways for the production of lignocellulosic ethanol and gives insights into the potential role of marginal areas for the sustainable production of biofuels.


5.2E: Osmosis - Biology

2017年04月 The 5th Asian Graduate Student Symposium on Membrane Engineering, The Best Student Oral Presentation Award at AGSM5, Characterization of separation function of Amphotericin B for biomimetic water purification membrane

Toru Takai, Daisuke Saeki, Kazuo Kumagai, Hideto Matsuyama

Asuka Inada, Kazuo Kumagai, Hideto Matsuyama

Elsevier BV, 2020年12月, Separation and Purification Technology, 252, 117462 - 117462

Asuka Inada, Tomoki Takahashi, Kazuo Kumagai, Hideto Matsuyama

© 2019 American Chemical Society. To develop a forward osmosis (FO) process, selection of draw solutes (DSs) is a critical factor in determining water permeability of the process. In this search for novel high-performance DSs, various morpholine derivatives were investigated for their thermoresponsive potential. 4-Butylmorpholine (BuMP) showed a preferable minimum lower critical solution temperature for the FO process (31.7 °C). The dilute phase of BuMP after phase separation at 70 °C showed a low concentration (3.3 wt %) and low osmotic pressure (3.16 bar). In the FO flux test, the water permeability and reverse solute flux of BuMP (55.0 wt %, 28 bar) against water were Jw 2.09 L m-2 h-1 and Js 14.0 g m-2 h-1, respectively. Using 0.6 M NaCl (model seawater) as feed solution, BuMP (94.6 wt %) could extract water from this model seawater (Jw 0.56 L m-2 h-1). These results indicate a high potential for MP derivatives as DSs and provide new guidance for their development for FO desalination.

2019年06月03日, Industrial and Engineering Chemistry Research, 58 (27), 12253 - 12260, 英語

Asuka Inada, Kenichiro Yumiya, Tomoki Takahashi, Kazuo Kumagai, Yoko Hashizume, Hideto Matsuyama

© 2018 Elsevier B.V. Recently, forward osmosis (FO) is attracting research attention once again. In a FO process, it is important to develop a draw solution (DS) with a high osmotic pressure and low solute leakage. In this study, thermoresponsive star-shaped oligomers with a glycerol backbone were developed as new draw solutes for FO. A series of glycerol-oligo(ethylene oxide)-block-oligo(butylene oxide) (GEB) oligomers were systematically designed and synthesized. The average degrees of polymerization of ethylene oxide (EO m) and butylene oxide (BO n) units of GEmBn were varied to control the hydrophilic/hydrophobic balance of the molecule. Aqueous solutions of GEBs were evaluated in terms of their osmotic pressures, phase diagrams, and viscosities. Most of them showed a lower critical solution (LCST)-type phase separation at temperatures below 60 °C. The osmotic pressure of 68 wt% GE7B3 (concentration of the dense phase after phase separation at 60 °C) was 74 bar, about 2.6 times higher than that of seawater. Moreover, the leakage of GE7B3 was much lower than that of conventional draw solutes. The osmotic pressure of the dilute phase of a GE7B3 solution at 60 °C was less than 2 bar, implying reduced energy consumption during post-processing by low-pressure reverse osmosis to collect pure water.

2019年03月15日, Journal of Membrane Science, 574, 147 - 153, 英語

Riyo Maruki Imamura, Kazuo Kumagai, Hirofumi Nakano, Takayoshi Okabe, Tetsuo Nagano, Hirotatsu Kojima

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

SAGE PUBLICATIONS INC, 2019年03月, SLAS DISCOVERY, 24 (3), 284 - 294, 英語

Masaaki Ito, Shin-ichiro Egashira, Kazuki Yoshida, Tomoko Mineno, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Michio Ui, Isao Matsuoka

Aims: The P2Y(6) nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y(6) receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y(6) receptor.Main methods: High-throughput screening (HTS) was used to study the effects of various compounds on human P2Y(6) receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2+ concentration ([Ca2+]i) levels using an FDSS7000 real-time fluorescence detector. IL-8 concentration was measured by enzyme-linked immunosorbent assay.Key findings: Among structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y(6) receptor were identified. Further studies using a dose-response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y(6) receptor inhibitor TIM-38, which inhibited UDP-induced [Ca2+] elevation in a dose-dependent manner. TIM-38 had an IC60 value of 43 ptM and inhibited P2Y(6) without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM-38 inhibited UDP-induced interleukin-8 release in a dose-dependent manner without affecting releases caused by other stimulus such as interleukin-1 beta or tumour necrosis factor-alpha. Analyses of TIM-38 derivatives revealed that the nitro moiety is vital to P2Y(6) receptor inhibition.Significance: TIM-38 acts as a novel structural antagonist of P2Y(6) receptor and may be a good lead compound for developing a P2Y(6) receptor-targeted anti-inflammatory drug. (C) 2017 Published by Elsevier Inc.

PERGAMON-ELSEVIER SCIENCE LTD, 2017年07月, LIFE SCIENCES, 180, 137 - 142, 英語

Seiji Ishii, Kenji Fukui, Satoshi Yokoshima, Kazuo Kumagai, Youko Beniyama, Tetsuya Kodama, Tohru Fukuyama, Takayoshi Okabe, Tetsuo Nagano, Hirotatsu Kojima, Takato Yano

The main components of the quorum-sensing system are expected to be favorable targets for drug development to combat various chronic infectious diseases. ComA of Streptococcus is an ATP-binding cassette transporter containing a peptidase domain (PEP), which is essential for the quorum-sensing signal production. Using high-throughput screening, we found a potent small molecule that suppressed the S. mutans quorum-sensing pathway through inhibition of PEP activity. The compound effectively attenuated the biofilm formation and competence development of S. mutans without inhibiting cell growth. The kinetic and structural studies with this molecule and a related compound unexpectedly revealed an allosteric site of PEP. This relatively hydrophobic site is thought to undergo large structural changes during the catalytic process. These compounds inhibit PEP activity by binding to and suppressing the structural changes of this site. These results showed that PEP is a good target for inhibitors of the Streptococcus quorum-sensing system.

NATURE PUBLISHING GROUP, 2017年06月, SCIENTIFIC REPORTS, 7, 4029, 英語

Ke Liu, Naoko Kunii, Megumi Sakuma, Atsumi Yamaki, Satoru Mizuno, Mayu Sato, Hiromichi Sakai, Sayaka Kado, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Yasuhito Shirai, Fumio Sakane

Diacylglycerol kinase (DGK) consists of 10 isozymes. The alpha-isozyme enhances the proliferation of cancer cells. However, DGK alpha facilitates the nonresponsive state of immunity known as T-cell anergy therefore, DGK alpha enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGK alpha activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl) amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGK alpha with an IC50 value of 0.6 mu M. CU-3 targeted the catalytic region, but not the regulatory region, of DGK alpha. CU-3 competitively reduced the affinity of DGK alpha for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGK alpha and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2016年03月, JOURNAL OF LIPID RESEARCH, 57 (3), 368 - 379, 英語

LIU Ke, KUNII Naoko, SAKUMA Megumi, YAMAKI Atsumi, MIZUNO Satoru, SATO Mayu, SAKAI Hiromichi, KADO Sayaka, KUMAGAI Kazuo, KOJIMA Hirotatsu, OKABE Takayoshi, NAGANO Tetsuo, SHIRAI Yasuhito, SAKANE Fumio

Diacylglycerol kinase (DGK) consists of 10 isozymes. The alpha-isozyme enhances the proliferation of cancer cells. However, DGK alpha facilitates the nonresponsive state of immunity known as T-cell anergy therefore, DGK alpha enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGK alpha activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl) amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGK alpha with an IC50 value of 0.6 mu M. CU-3 targeted the catalytic region, but not the regulatory region, of DGK alpha. CU-3 competitively reduced the affinity of DGK alpha for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGK alpha and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2016年03月, Journal of Lipid Research, 57 (3), 368 - 379, 英語

2015年, コンバーテック, 43 (12), 10 - 13, 日本語

Masayuki Isa, Daisuke Asanuma, Shigeyuki Namiki, Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Kenzo Hirose

Overexpression of growth factor receptors in cancers, e.g., human epidermal growth factor receptor 2 (HER2) in ovarian and breast cancers, is associated with aggressiveness. A possible strategy to treat cancers that overexpress those receptors is blockade of receptor signaling by inducing receptor internalization and degradation. In this study, we developed a cell-based high-throughput screening (HTS) system to identify small molecules that induce HER2 internalization by employing our recently developed acidic-pH-activatable probe in combination with protein labeling technology. Our HTS system enabled facile and reliable quantification of HER2 internalization with a Z' factor of 0.66 and a signal-to-noise ratio of 44.6. As proof of concept, we used the system to screen a similar to 155,000 small-molecule library and identified three hits that induced HER2 internalization and degradation via at least two distinct mechanisms. This HTS platform should be adaptable to other disease-related receptors in addition to HER2.

AMER CHEMICAL SOC, 2014年10月, ACS CHEMICAL BIOLOGY, 9 (10), 2237 - 2241, 英語

Megumi Tarui, Hideo Shindou, Kazuo Kumagai, Ryo Morimoto, Takeshi Harayama, Tomomi Hashidate, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Takahide Nagase, Takao Shimizu

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2014年07月, JOURNAL OF LIPID RESEARCH, 55 (7), 1386 - 1396, 英語

Megumi Tarui, Hideo Shindou, Kazuo Kumagai, Ryo Morimoto, Takeshi Harayama, Tomomi Hashidate, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano, Takahide Nagase, Takao Shimizu

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2014年07月, JOURNAL OF LIPID RESEARCH, 55 (7), 1386 - 1396, 英語

Kenta MOSALLANEJAD, Yusuke SEKINE, Seiko Ishikura-KINOSHITA, Kazuo KUMAGAI, Tetsuo NAGANO, Atsushi MATSUZAWA, Kohsuke TAKEDA, Isao NAGURO, Hidenori ICHIJO

During infection with an RNA virus, the DExD/H-box RNA helicases RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) activate the interferon regulatory factor 3 (IRF3), nuclear factor kappa B (NF-kappa B), c-Jun amino-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways through an unknown mechanism involving the adaptor protein MAVS (mitochondrial antiviral signaling). We used a Drosophila misexpression screen to identify DEAH-box polypeptide 15 (DHX15) as an activator of the p38 MAPK pathway. Human DHX15 contributed to the activation of the NF-kappa B, JNK, and p38 MAPK pathways, but not the IRF3 pathway, in response to the synthetic double-stranded RNA analog poly(I:C) (polyinosinic-polycytidylic acid), and DHX15 was required for optimal cytokine production in response to poly(I:C) and infection with RNA virus. DHX15 physically interacted with MAVS and mediated the MAVS-dependent activation of the NF-kappa B and MAPK pathways. Furthermore, DHX15 was required for poly(I:C)-and RNA virus-dependent, MAVS-mediated apoptosis. Thus, our findings indicate that, in RIG-I-like receptor signaling, DHX15 specifically stimulates the NF-kappa B and MAPK pathways downstream of MAVS and contributes to MAVS-mediated cytokine production and apoptosis.

AMER ASSOC ADVANCEMENT SCIENCE, 2014年04月, Science Signaling, 7 (323), ra40, 英語

Kazuo Kumagai, Hirotatsu Kojima, Takayoshi Okabe, Tetsuo Nagano

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z'-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators. (C) 2013 Elsevier Inc. All rights reserved.

ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年02月, ANALYTICAL BIOCHEMISTRY, 447, 146 - 155, 英語

Nobuo Hosotani, Kazuo Kumagai, Shigeyuki Honda, Akira Ito, Takuro Shimatani, Ikutaro Saji

A new peptaibol compound, SPF-5506-A(4), was isolated from the fermentation broth of Trichoderma sp. SPF-5506. The chemical structure of the 14-residue peptide was determined by MS, NMR and amino acid sequence analyses. The absolute configuration of amino acid residues in the acid hydrolysate was determined by Marfey's method. The structure of SPF-5506-A4 was established as Ac-Aib-L-Asn-L-Ile-Aib-L-Pro-L-Ser-L-IleAib-L-Pro-L-LeU-L-Leu-Aib-L-Pro-L-leucinol. The compound inhibited amyloid P-peptide formation in primary guinea pig cerebral cortex neuron cell culture dose-dependently with an IC50 of 0.1 mu g/ml. Cytotoxicity was not observed at concentrations of < 3 mu g/ml.

JAPAN ANTIBIOTICS RESEARCH ASSOC, 2007年03月, JOURNAL OF ANTIBIOTICS, 60 (3), 184 - 190, 英語

Shinjiro Kaneko, Akio Iwanami, Masaya Nakamura, Akiyoshi Kishino, Kaoru Kikuchi, Shinsuke Shibata, Hirotaka J. Okano, Takeshi Ikegami, Ayako Moriya, Osamu Konishi, Chikao Nakayama, Kazuo Kumagai, Toru Kimura, Yasufumi Sato, Yoshio Goshima, Masahiko Taniguchi, Mamoru Ito, Zhigang He, Yoshiaki Toyama, Hideyuki Okano

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

NATURE PUBLISHING GROUP, 2006年12月, NATURE MEDICINE, 12 (12), 1380 - 1389, 英語

T Shimatani, N Hosotani, M Ohnishi, K Kurnagai, Saji, I

Two new human chymase inhibitors, SPF-32629A and B, were isolated from the cultured broth of Penicillium sp. SPF-32629. These structures were determined by spectroscopic methods and identified as new pyridone compounds. SPF-32629B was the carboxylated derivative of SPF-32629A. SPF-32629A and B specifically inhibited human chymase among four serine proteases tested with the IC50 of 0.25 and 0.42 mu g/ml, respectively.

JAPAN ANTIBIOTICS RESEARCH ASSOC, 2006年01月, JOURNAL OF ANTIBIOTICS, 59 (1), 29 - 34, 英語

N Hosotani, K Kumagai, H Nakagawa, T Shimatani, Saji, I

Seven new antimycin antibiotics, named antimycins A(10), A(11), A(12), A(13), A(14), A(15) and A(16), were isolated from the fermentation broth of strains of Streptomyces spp. SPA-10191 and SPA-8893, along with known antimycins A(1), A(2), A(3) and A(4). The structures of the new antimycins were determined by spectral analyses, including 2D NMR techniques. These compounds exhibited antifungal activity against Candida utilis.

JAPAN ANTIBIOTICS RESEARCH ASSOC, 2005年07月, JOURNAL OF ANTIBIOTICS, 58 (7), 460 - 467, 英語

N Hosotani, K Kumagai, H Nakagawa, T Shimatani, Saji, I

Two new 24-membered macrolides, SPA-6952A and B, were isolated from the fermentation broth of Streptomyces sp. SPA-6952. The structures of the new macrolides were determined by spectral analyses, including 2D NMR techniques. These compounds exhibited cytotoxic activity against human promyelocytic leukemia HL-60 cells.

JAPAN ANTIBIOTICS RESEARCH ASSOC, 2005年06月, JOURNAL OF ANTIBIOTICS, 58 (6), 409 - 411, 英語

K Kikuchi, A Kishino, O Konishi, K Kumagai, N Hosotani, Saji, I, C Nakayama, T Kimura

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 muM (IC50=0.16 muM). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2003年10月, JOURNAL OF BIOLOGICAL CHEMISTRY, 278 (44), 42985 - 42991, 英語

S Kaneko, A Iwanami, M Nakamura, S Miyao, A Kishino, K Kikuchi, T Kimura, K Kumagai, Y Toyama, H Okano

MARY ANN LIEBERT INC PUBL, 2003年10月, JOURNAL OF NEUROTRAUMA, 20 (10), 1133 - 1133, 英語

K Kumagai, N Hosotani, K Kikuchi, T Kimura, Saji, I

A new semaphorin inhibitor xanthofulvin was isolated from the cultured broth of a fungus Penicillium sp. SPF-3059 along with a known compound vinaxanthone by solvent extraction and bioassay-guided fractionation. The tautomeric structure of xanthofulvin was determined by spectroscopic analyses. The two compounds exhibited significant semaphorin inhibitory activity with IC50 values of 0.09 and 0.1 mug/ml, respectively, in semaphorin3A-induced growth cone collapse assay using cultured chick dorsal root ganglia neurons.

JAPAN ANTIBIOT RES ASSN, 2003年07月, JOURNAL OF ANTIBIOTICS, 56 (7), 610 - 616, 英語

J Nagamine, R Nagata, H Seki, N Nomura-Akimaru, Y Ueki, K Kumagai, M Taiji, H Noguchi

SM-130686, an oxindole derivative, is a novel orally active GI I secretagogue (GI IS) which is structurally distinct from previously reported GHSs such as MK-677, NN703 and hexarelin. SM-130686 stimulates GH release from cultured rat pituitary cells in a dose-dependent manner. Half-maximum stimulation was observed at a concentration of 6.3 +/- 3.4 nM. SM-130686-induced GH release was inhibited by a GHS antagonist, but not by a GH-releasing hormone antagonist. SM-130686 dose-dependently inhibited the binding of radiolabeled ligand, S-35-MK-677, to human GHS receptor 1a (IC50 = 1.2 nM). This indicates that SM-130686 stimulates GH release through the GHS receptor. The effect of a single oral administration of SM-130686 on GH release in pentobarbital-anesthetized rats was studied. After treatment with 10 mg/kg SM-130686, plasma GH concentrations measured by radioimmunoassay significantly increased, reaching a peak at 20-45 min, and remained above baseline during the experimental period (60 min). The anabolic effect of repetitive SM-130686 adininistration was studied in rats. Rats received 10 mg/kg SM-130686 orally twice a day and were weighed every day for 9 days. At day 9 there was a significant increase in both the body weight and the fat free mass (19.5 +/- 2.1 and 18-1 +/- 7.5 g respectively). Serum IGF-I concentration was also significantly elevated 6 h after the last dose of SM-130686. An. endogenous GHS ligand for the GHS receptor has recently been identified from stomach extract and designated as ghrelin. The GH-releasing activity in vitro relative to ghrelin (100%) was about 52% for SM-130686. It is likely that SM-130686 is a partial agonist for the GHS receptor. In summary, we describe here an orally active GHS, SM-130686, which acts through the GHS receptor. Repetitive administration of SM-130686 to rats, similar to repetitive administration of GH, significantly increased the fat free mass by an amount almost equal to the gain in body weight.

SOC ENDOCRINOLOGY, 2001年12月, JOURNAL OF ENDOCRINOLOGY, 171 (3), 481 - 489, 英語

T Tokunaga, WE Hume, T Umezome, K Okazaki, Y Ueki, K Kumagai, S Hourai, J Nagamine, H Seki, M Taiji, H Noguchi, R Nagata

A series of substituted oxindole derivatives was synthesized and evaluated for growth hormone (GH) releasing activity using cultured rat pituitary cells. (+)-6-Carbamoyl-3-(2-chlorophenyl)(2-diethylaminoethyl)-4-trifluoromethyloxindole (SM-130686, 37S) was found to have potent activity (EC50 = 3.0 nM), while the other enantiomer 37R had reduced activity. The absolute configuration of 37S was confirmed by X-ray crystallographic analysis. Compound 37S showed a good pharmacokinetic profile in rats with 28% oral bioavailability at 10 mg/kg and excellent in vivo activity as evidenced by a significant weight gain after 4 days of oral administration at 10 mg/kg twice a day. Compound 37S displaced the binding of S-35-MK-677 to human GHS-R with an IC50 value of 1.2 +/- 0.2 nM.

AMER CHEMICAL SOC, 2001年12月, JOURNAL OF MEDICINAL CHEMISTRY, 44 (26), 4641 - 4649, 英語

R Nagata, T Tokunaga, WE Hume, T Umezome, K Okazaki, Y Ueki, K Kumagai, J Nagamine, H Seki, M Taiji, H Noguchi

AMER CHEMICAL SOC, 2001年08月, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 222, U670 - U670, 英語

R Nagata, T Tokunaga, EW Hume, K Okazaki, Y Ueki, K Kumagai, J Nagamine, H Seki, M Taiji, H Noguchi

AMER CHEMICAL SOC, 2000年08月, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 220, U591 - U591, 英語

K Kumagai, K Shono, H Nakayama, Y Ohno, Saji, I

A potent sigma (sigma) receptor ligand was isolated from the culture broth of Streptomyces longispororuber #525. The active compound was identified to be (2R-trans)-2-butyl-5-heptylpyrrolidine by spectroscopic and chemical studies. The compound exhibited high affinity and selectivity for sigma receptors. The IC50 values toward sigma(1), sigma(2) and dopamine D-2 receptors were 2.0, 22.7 and more than 40,000 nM, respectively. Its (2S-trans)- and (+/-)-cis-isomers, both synthesized, were also found to be high affinity sigma ligands.

JAPAN ANTIBIOT RES ASSN, 2000年05月, JOURNAL OF ANTIBIOTICS, 53 (5), 467 - 473, 英語

S MASUTOMO, A INOUE, K KUMAGAI, R MURAI, S MITSUDA

Pseudomonas sp, B21C9, a new nitrile-degrading microorganism, was isolated from a soil sample. By the hydrolysis of (RS)-isopropyl-4'-chlorophenylacetonitrile using cells of B21C9, (S)-2-isopropyl-4'-chlorophenylacetic acid having excellent optically purity could be obtained, It appeared that the microbial hydrolysis proceeded via stepwise reactions by a nitrile hydratase having poor (S)-selectivity and an amidase having strict (S)-selectivity.

TAYLOR & FRANCIS LTD, 1995年04月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 59 (4), 720 - 722, 英語

K KUMAGAI, K TOKUNAGA, M TSUTSUMI, K IKUTA

We recently showed that S1, a sequence-scrambled form of CD4 CDR3-related synthetic peptide, has more potent inhibitory activities on HIV-1 replication and HIV-1-induced syncytium formation than the original form. In this study, a series of derivatives of S1 were synthesized and their anti-HIV-1 activities were evaluated. A D-isomer was as potent as S1, and a homodimer was 10- to 18-fold more potent than SI. The increased antiviral activity of the dimer peptide was related to alpha-helix formation, as detected by circular dichroism.

ELSEVIER SCIENCE BV, 1993年09月, FEBS LETTERS, 330 (2), 117 - 121, 英語

K KUMAGAI, A FUKUI, S TANAKA, M IKEMOTO, K MORIGUCHI, S NABESHIMA

A new macrolide antibiotic, PC-766B, was isolated from the cells of Nocardia brasiliensis SC-4710 by acetone extraction, and purified by gel filtration, silica gel chromatography, HPLC and TLC. The structure of PC-766B was determined by NMR spectral analysis to be a new class of the hygrolidin family antibiotics. PC-766B had a 16-membered macrocyclic lactone ring, a 6-membered hemiketal ring and a 2-deoXy-D-rhamnose moiety. DL-alpha-Tocopherol, known as an antioxidant agent, significantly improved the stability of PC-766B and prevented the decomposition of PC-766B during the storage of the antibiotic.

JAPAN ANTIBIOT RES ASSN, 1993年07月, JOURNAL OF ANTIBIOTICS, 46 (7), 1139 - 1144, 英語

K KUMAGAI, K TAYA, A FUKUI, M FUKASAWA, M FUKUI, S NABESHIMA

An actinomycete strain SC-4710, a new soil isolate, was found to produce a new macrolide antibiotic, PC-766B. Chemotaxonomic analysis of the producing organism revealed that the cells of SC-4710 had type IV cell wall, type A whole cell sugar pattern, type PII phospholipids, menaquinone MK-8(H-4), cellular fatty acids comprising straight-chain saturated, unsaturated and tuberculostearic acids, and mycolic acids. The strain was identified as Nocardia brasiliensis (Lindenberg) Pinoy. The antibiotic, PC-766B, was active against Gram-positive bacteria, and some fungi and yeasts, but inactive against Gram-negative bacteria. It also showed antitumor activity against murine tumor cells in vitro and in vivo, and a weak inhibitory activity against Na+, K+-ATPase in vitro.

JAPAN ANTIBIOT RES ASSN, 1993年06月, JOURNAL OF ANTIBIOTICS, 46 (6), 972 - 978, 英語

K OHKI, K KUMAGAI, S MITSUDA, T TAKANO, T KIMURA, K IKUTA

We have previously identified CD4 peptides that exhibited blocking activity on human immunodeficiency virus type 1 (HIV-1) infection, i.e. CD4(68-130) and CD4(66-92) which include the region corresponding to the third complementarity-determining region of IgG. Here we describe a unique peptide derived from CD4(66-92), altered in amino acid sequence but not in composition, which was found to have increased anti-HIV-1 activity. The acidic amino acid residues in this scrambled peptide, S1, localized at the N-terminus, while in the native peptide they clustered at the C-terminus. On the other hand, a second scrambled peptide, S2, in which the acidic amino acid residues were fully dispersed, did not show any anti-HIV-1 activity. However, we could not identify any correlation between CD4(66-92) and S1 peptides by their hydrophobic or circular dichroism spectrum analyses. The results provide insight into the mechanisms of HIV-1 gp120 and CD4 interaction and may be useful as a new approach to AIDS therapy.

BUTTERWORTH-HEINEMANN LTD, 1993年04月, VACCINE, 11 (6), 682 - 686, 英語

K KUMAGAI, M KATO, S NABESHIMA, S TSUYUMU

New antibiotics, T1801 A, B, C, and D, were isolated from the culture broth of Pseudomonas sp. SC-1801. Their structures were found by spectroscopic analyses to be tri- and tetra(methylthio) derivatives of hydroquinone (T1801 A and C) or p-benzoquinone (T1801 B and D). They are new quinone and hydroquinone antibiotics and are active against Gram-positive bacteria, some fungi, and yeasts.

TAYLOR & FRANCIS LTD, 1992年09月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 56 (9), 1439 - 1442, 英語

K. Kumagai, S. Nabeshima, S. Kato, M. Watanabe, K. Ikuta

We have previously shown that liposomes containing fragment A of diphtheria toxin, which were prepared by the detergent-dialysis method with egg phosphatidylcholine, phosphatidylserine and cholesterol, possess a selective killing activity against human immunodeficiency virus (HIV)-1-infected cells, but not against uninfected cells (Ikuta et al., 1987). Since the liposomes were found to be unstable in human plasma in vitro, we prepared improved liposomes by the extrusion method with dimyristoylphosphatidylcholine instead of egg phosphatidylcholine. These liposomes were found to be very stable in human plasma, and also possessed the selective killing activity against HIV-1 -infected cells. In addition, it was found that the fragment A in the liposomes was not necessary for the selective cell killing activity. The cell killing activity and selectivity of HIV-1 -infected cells of the liposomes were remarkably affected by cholesterol content and the acyl chain length of the saturated fatty acid of phosphatidylcholines. These data suggest that membranes of liposomes can interact specifically with HIV-1 -infected cells, but not with uninfected cells, resulting in the inhibition of cell proliferation.

1991年, Antiviral Chemistry and Chemotherapy, 2 (3), 143 - 148, 英語

K. Kumagai, K. Nakada, K. Kikuchi, H. Mori

Urinary excretion of renal tubular cell enzymes, N-acetyl-β-D-glucosaminidase (NAG) and γ-glutamyl transpeptidase (γ-GTP) was evaluated to elucidate the renal cell damage before, during and after the induced hypotension with trimetaphan (TMP), nitroglycerin (TNG) and prostaglandin E1 (PGE1) under halothane-nitrous oxide-oxygen anesthesia in patients undergoing neurosurgery. Significant increases in excretion of NAG and γ-GTP were observed in TNG group. Urinary excretion of these two enzymes in TMP group tended to increase, but its increases were not statistically significant. In PGE1 treated group, there was no tendency for urinary NAG excretion, while γ-GTP excretion tended to decrease compared with that observed before the induced hypotension. Among these three drugs, TNG exerted the most significant effect on urinary excretion of both enzymes and TMP ranked next. Effect of PGE1 on urinary enzyme excretion was weakest. According to the general opinions, degree in stray of renal tubular cell enzyme into urine is thought to parallel with the injury of renal tubular cells. Therefore, the finding obtained in this study suggests that the induced hypotension with PGE1 has a less harmful effect on renal tubular cells.