Yamaguchi University

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Ionic liquids (ILs) are liquids consisting entirely of ions and can be further defined as molten salts having melting points lower than 100 °C. One of the most important research areas for IL utilization is undoubtedly their energy application, especially for energy storage and conversion materials and devices, because there is a continuously increasing demand for clean and sustainable energy. In this article, various application of ILs are reviewed by focusing on their use as electrolyte materials for Li/Na ion batteries, Li-sulfur batteries, Li-oxygen batteries, and nonhumidified fuel cells and as carbon precursors for electrode catalysts of fuel cells and electrode materials for batteries and supercapacitors. Due to their characteristic properties such as nonvolatility, high thermal stability, and high ionic conductivity, ILs appear to meet the rigorous demands/criteria of these various applications. However, for further development, specific applications for which these characteristic properties become unique (i.e., not easily achieved by other materials) must be explored. Thus, through strong demands for research and consideration of ILs unique properties, we will be able to identify indispensable applications for ILs.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Cell division in many mammalian tissues is associated with specific times of day, but just how the circadian clock controls this timing has not been clear. Here, we show in the regenerating liver (of mice) that the circadian clock controls the expression of cell cycle-related genes that in turn modulate the expression of active Cyclin B1-Cdc2 kinase, a key regulator of mitosis. Among these genes, expression of wee1 was directly regulated by the molecular components of the circadian clockwork. In contrast, the circadian clockwork oscillated independently of the cell cycle in single cells. Thus, the intracellular circadian clockwork can control the cell-division cycle directly and unidirectionally in proliferating cells.

OBJECTIVE: We developed a digital image database (www.macnet.or.jp/jsrt2/cdrom_nodules.html ) of 247 chest radiographs with and without a lung nodule. The aim of this study was to investigate the characteristics of image databases for potential use in various digital image research projects. Radiologists' detection of solitary pulmonary nodules included in the database was evaluated using a receiver operating characteristic (ROC) analysis. MATERIALS AND METHODS: One hundred and fifty-four conventional chest radiographs with a lung nodule and 93 radiographs without a nodule were selected from 14 medical centers and were digitized by a laser digitizer with a 2048 x 2048 matrix size (0.175-mm pixels) and a 12-bit gray scale. Lung nodule images were classified into five groups according to the degrees of subtlety shown. The observations of 20 participating radiologists were subjected to ROC analysis for detecting solitary pulmonary nodules. Experimental results (areas under the curve, Az) obtained from observer studies were used for characterization of five groups of lung nodules with different degrees of subtlety. RESULTS: ROC analysis showed that the database included a wide range of various nodules yielding Az values from 0.574 to 0.991 for the five categories of cases for different degrees of subtlety. CONCLUSION: This database can be useful for many purposes, including research, education, quality assurance, and other demonstrations.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. METHODS: Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. RESULTS: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. CONCLUSIONS: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Individual cellular clocks in the suprachiasmatic nucleus (SCN), the circadian center, are integrated into a stable and robust pacemaker with a period length of about 24 hours. We used real-time analysis of gene expression to show synchronized rhythms of clock gene transcription across hundreds of neurons within the mammalian SCN in organotypic slice culture. Differentially phased neuronal clocks are topographically arranged across the SCN. A protein synthesis inhibitor set all cell clocks to the same initial phase and, after withdrawal, intrinsic interactions among cell clocks reestablished the stable program of gene expression across the assemblage. Na+-dependent action potentials contributed to establishing cellular synchrony and maintaining spontaneous oscillation across the SCN.

Polyoxometalates (POMs) are a subset of metal oxides with unique physical and chemical properties, which can be reliably modified through various techniques and methods to develop sophisticated materials and devices. In parallel with the large number of new crystal structures reported in the literature, the application of these POMs towards multifunctional materials has attracted considerable attention. This critical review summarizes recent progress on POM-based molecular and composite materials, and particularly highlights the emerging areas that are closely related to surface, electronic, energy, environment, life science, etc. (171 references).

The human T-cell lines MT-2 and MT-4 carry the human T-cell leukemia virus type I (HTLV-I). When MT-2 and MT-4 were infected with HTLV-III, the probable etiologic agent of the acquired immune deficiency syndrome (AIDS), rapid cytopathogenic effects and cytotoxicity were observed that made it possible to titrate the biologically active virus in a plaque-forming assay. The cytopathogenic effects were preceded by the rapid induction and increase of HTLV-III antigens as revealed by immunofluorescence and immunoprecipitation. Activities of HTLV-III were neutralized by the human antibodies against the virus when immunofluorescence and plaque assays were used. Essentially the same results were obtained with the lymphadenopathy-associated virus (LAV1).

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

OBJECTIVE: This trial compared the efficacy and safety of transarterial chemoembolisation (TACE) plus sorafenib with TACE alone using a newly established TACE-specific endpoint and pre-treatment of sorafenib before initial TACE. DESIGN: Patients with unresectable hepatocellular carcinoma (HCC) were randomised to TACE plus sorafenib (n=80) or TACE alone (n=76). Patients in the combination group received sorafenib 400 mg once daily for 2-3 weeks before TACE, followed by 800 mg once daily during on-demand conventional TACE sessions until time to untreatable (unTACEable) progression (TTUP), defined as untreatable tumour progression, transient deterioration to Child-Pugh C or appearance of vascular invasion/extrahepatic spread. Co-primary endpoints were progression-free survival (PFS), which is not a conventional one but defined as TTUP, or time to any cause of death plus overall survival (OS). Multiplicity was adjusted by gatekeeping hierarchical testing. RESULTS: Median PFS was significantly longer in the TACE plus sorafenib than in the TACE alone group (25.2 vs 13.5 months; p=0.006). OS was not analysed because only 73.6% of OS events were reached. Median TTUP (26.7 vs 20.6 months; p=0.02) was also significantly longer in the TACE plus sorafenib group. OS at 1 year and 2 years in TACE plus sorafenib group and TACE alone group were 96.2% and 82.7% and 77.2% and 64.6%, respectively. There were no unexpected toxicities. CONCLUSION: TACE plus sorafenib significantly improved PFS over TACE alone in patients with unresectable HCC. Adverse events were consistent with those of previous TACE combination trials. TRIAL REGISTRATION NUMBER: NCT01217034.

Abstract The photocatalytic decomposition of phenol in oxygenated aqueous suspensions of lightly-reduced anatase TiO2, being the most satisfactory among the semiconductors investigated from the standpoint of the photocatalytic activity and stability, has been investigated at the optimum pH 3.5. The products at the initial stage of the reaction were hydroquinone, pyrocatechol, 1,2,4-benzenetriol, pyrogallol, and 2-hydroxy-1,4-benzoquinone. These intermediates underwent further photocatalytic oxidation via acids and/or aldehydes finally into CO2 and H2O. A reaction scheme involving hydroxyl radicals as real reactive species has been proposed. Although H2O2 was formed via O2\ewdot produced by electron trapping of adsorbed oxygen, its concentration remained constant at a low value during the reaction. About 0.7 mole of O2 was consumed for the consumption of one mole of phenol at the initial stage of the reaction. These results indicated that hydroxyl radicals were formed not only via holes but also via H2O2 from O2\ewdot. It was interesting from the viewpoint of wastewater treatment that phenol was completely mineralized to CO2 in the presence of TiO2 powder under solar irradiation without both aeration and mixing of the solution.

We investigated the relationship between oxidative stress and poor oocyte quality and whether the antioxidant melatonin improves oocyte quality. Follicular fluid was sampled at oocyte retrieval during in vitro fertilization and embryo transfer (IVF-ET). Intrafollicular concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in women with high rates of degenerate oocytes were significantly higher than those with low rates of degenerate oocytes. As there was a negative correlation between intrafollicular concentrations of 8-OHdG and melatonin, 18 patients undergoing IVF-ET were given melatonin (3 mg/day), vitamin E (600 mg/day) or both melatonin and vitamin E. Intrafollicular concentrations of 8-OHdG and hexanoyl-lysine adduct were significantly reduced by these antioxidant treatments. One hundred and fifteen patients who failed to become pregnant with a low fertilization rate (< or =50%) in the previous IVF-ET cycle were divided into two groups during the next IVF-ET procedure; 56 patients with melatonin treatment (3 mg/day) and 59 patients without melatonin treatment. The fertilization rate was improved by melatonin treatment compared to the previous IVF-ET cycle. However, the fertilization rate was not significantly changed without melatonin treatment. Oocytes recovered from preovulatory follicles in mice were incubated with H2O2 for 12 hr. The percentage of mature oocytes with a first polar body was significantly reduced by addition of H2O2 (300 microm). The inhibitory effect of H2O2 was significantly blocked by simultaneous addition of melatonin. In conclusion, oxidative stress causes toxic effects on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin is likely to improve oocyte quality and fertilization rates.

A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All aptients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care.

Parasitic nematodes (roundworms) and platyhelminths (flatworms) cause debilitating chronic infections of humans and animals, decimate crop production and are a major impediment to socioeconomic development. Here we report a broad comparative study of 81 genomes of parasitic and non-parasitic worms. We have identified gene family births and hundreds of expanded gene families at key nodes in the phylogeny that are relevant to parasitism. Examples include gene families that modulate host immune responses, enable parasite migration though host tissues or allow the parasite to feed. We reveal extensive lineage-specific differences in core metabolism and protein families historically targeted for drug development. From an in silico screen, we have identified and prioritized new potential drug targets and compounds for testing. This comparative genomics resource provides a much-needed boost for the research community to understand and combat parasitic worms. Comparative study of 81 genomes of parasitic and non-parasitic worms identifies gene family births and expanded gene families at key nodes in the phylogeny that are relevant to parasitism and proteins historically targeted for drug development.

Taiji Imoto, Kazuyoshi Yagishita; A Simple Activity Measurement of Lysozyme, Agricultural and Biological Chemistry, Volume 35, Issue 7, 1 July 1971, Pages

Abstract A review of the dynamics of phase separation is presented, which focuses on the scaling assumption of the problem. Conventional linear and nonlinear theories are briefly reviewed. The problems with these conventional theories, and how the scaling idea can be used to overcome them are discussed. The growth rates of the droplets, domains, or grains are discussed in terms of the scaling assumption, and experimental tests of the scaling assumption for growth laws and scattering intensities are reviewed. To explain singular properties of the structure function and growth rate, which are observed experimentally, a strong correlation among droplets or grains on phase separations is postulated. A nonconventional form of the scattering function and a nonconventional droplet growth rate are explained qualitatively.

Small GTPase Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca2+-sensitizing effects in extensively Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor (Gong, M. C., Iizuka, K., Nixon, G., Browne, J. P., Hall, A., Eccleston, J. F., Sugai, M., Kobayashi, S., Somlyo, A. V., and Somlyo, A. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1340–1345). Here we demonstrate the contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca2+. These effects of constitutively active Rho-kinase were wortmannin (a potent myosin light chain kinase inhibitor)-insensitive. Immunoblot analysis revealed that the amount of native Rho-kinase was markedly lower in Triton X-100-permeabilized tissue than in intact tissue. Our results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independently of the Ca2+-calmodulin-dependent myosin light chain kinase pathway. Small GTPase Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca2+-sensitizing effects in extensively Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor (Gong, M. C., Iizuka, K., Nixon, G., Browne, J. P., Hall, A., Eccleston, J. F., Sugai, M., Kobayashi, S., Somlyo, A. V., and Somlyo, A. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1340–1345). Here we demonstrate the contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca2+. These effects of constitutively active Rho-kinase were wortmannin (a potent myosin light chain kinase inhibitor)-insensitive. Immunoblot analysis revealed that the amount of native Rho-kinase was markedly lower in Triton X-100-permeabilized tissue than in intact tissue. Our results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independently of the Ca2+-calmodulin-dependent myosin light chain kinase pathway. Smooth muscle contraction is primarily regulated by the levels of phosphorylation of myosin light chain (MLC), 1The abbreviations used are: MLC, myosin light chain; GTPγS, guanosine 5′-[γ-thio]triphosphate; Rho-kinase, Rho-associated serine/threonine kinase; CaM, calmodulin; CAT, the catalytic subunit of recombinant Rho-kinase; WM, wortmannin; DTT, dithiothreitol. which has heretofore been considered to be governed by a Ca2+-calmodulin (CaM)-dependent MLC kinase pathway (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 2Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593-603Crossref PubMed Google Scholar, 3Hartshorne D.J. Johnson D.R. Physiology of the Gastrointestinal Tract. 1. Raven Press, New York1987: 423-482Google Scholar, 4Sellers J.R. Adelstein R.S. Boyer P. Krebs E.G. The Enzyme. 18. Academic Press, San Diego, CA1987: 381-418Google Scholar). However, as the use of Ca2+ indicator revealed that the force/Ca2+ratio is variable, the Ca2+-CaM-dependent MLC kinase pathway cannot solely account for the mechanisms of agonist- or GTPγS-induced increases in the force/Ca2+ ratio, so-called Ca2+ sensitization (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 5Bradley A.B. Morgan K.G. J. Physiol. ( London ). 1987; 385: 437-448Crossref PubMed Scopus (133) Google Scholar, 6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 7Himpens B. Kitazawa T. Somlyo A.P. Pflügers Arch. Gen. Physiol. 1990; 417: 21-28Crossref Scopus (153) Google Scholar, 8Kitazawa T. Kobayashi S. Horiuchi K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 9Kubota Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar). Thus, an additional mechanism that can regulate Ca2+ sensitization of smooth muscle has been proposed. Using membrane permeabilization of smooth muscle, the possibility that monomeric Ras family G-proteins, such as Rho, contribute to Ca2+ sensitization of smooth muscle was demonstrated (10Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1991; 267: 8719-8722Google Scholar, 11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar, 12Otto B. Steusloff A. Just I. Aktories K. Pfitzer G. J. Physiol. ( London ). 1996; 496: 317-329Crossref PubMed Scopus (106) Google Scholar). Direct activation of G-proteins by the application of GTPγS (8Kitazawa T. Kobayashi S. Horiuchi K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 9Kubota Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar), agonists (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 5Bradley A.B. Morgan K.G. J. Physiol. ( London ). 1987; 385: 437-448Crossref PubMed Scopus (133) Google Scholar, 6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 7Himpens B. Kitazawa T. Somlyo A.P. Pflügers Arch. Gen. Physiol. 1990; 417: 21-28Crossref Scopus (153) Google Scholar, 8Kitazawa T. Kobayashi S. Horiuchi K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar), and GTP-activated Rho (10Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1991; 267: 8719-8722Google Scholar, 11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar, 12Otto B. Steusloff A. Just I. Aktories K. Pfitzer G. J. Physiol. ( London ). 1996; 496: 317-329Crossref PubMed Scopus (106) Google Scholar) could exert Ca2+-sensitizing effects on saponin- or β-escin-permeabilized smooth muscle. However, the activated Rho failed to induce Ca2+ sensitization of extensively Triton X-100-permeabilized smooth muscle (11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar). Considering that extensive Triton X-100-permeabilization allows higher molecular weight compounds to diffuse from the cytosol of smooth muscle of the rabbit portal vein (13Iizuka K. Ikebe M. Somlyo A.V. Somlyo A.P. Cell Calcium. 1994; 16: 431-445Crossref PubMed Scopus (49) Google Scholar), important diffusible factor(s) for the Ca2+ sensitization of smooth muscle might be lost during extensive permeabilization by Triton X-100, an event that would result in no response to activated Rho. We have recently reported that Rho-kinase, which is activated by GTP-bound active form of Rho (14Leung T. Manser E. Tan E. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar, 16Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (792) Google Scholar), phosphorylates not only MLC, thereby activating myosin ATPase (17Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar), but also myosin phosphatase, thus inactivating it in vitro (18Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J.H. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2436) Google Scholar). These findings in a cell-free system, plus the previous reports of G-protein-mediating Ca2+ sensitization as described above, suggest that Rho-kinase may induce contraction and concomitant MLC phosphorylation of the smooth muscle. We examined the effects of the constitutively active form of Rho-kinase on smooth muscle extensively permeabilized by Triton X-100 and attempted to determine if Rho-kinase would be the factor lost during extensive Triton X-100 permeabilization. The catalytic subunit of recombinant Rho-kinase (CAT; molecular mass is about 80 kDa) was expressed as a glutathione S-transferase fusion protein and purified using a baculovirus system and a glutathione-Sepharose column (17Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar). The kinase activity of the elute was determined by phosphorylation assay using S6 peptide as a substrate (15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar) in buffer containing 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, and 100 μm ATP (0.5–20 Gbq/mmol). CaM was purified from bovine brain by previously described method (19Walsh M.P. Valentine K.A. Ngai P.K. Carruthers C.A. Hollenberg M.D. Biochem. J. 1984; 224: 117-127Crossref PubMed Scopus (107) Google Scholar). Other materials and chemicals were obtained from commercial sources. Small strips of male rabbit (2–2.5 kg) portal veins were manually dissected (50–100 μm wide and 0.5–1 mm long), connected to an isometric force transducer (UL-2GR, Minebea, Japan), and mounted in a well (200 μl) on a bubble plate (6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar). After recording contractions evoked by 118 mm K+, the strips were incubated in relaxing solution, followed by 0.5% Triton X-100 for 20 min at 25 °C. The solutions have been described in detail elsewhere (6Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar). CaM (0.5 μm) was added to all reactive solutions for experiments with chemical permeabilization. 0.5% Triton X-100-permeabilized and intact rabbit portal veins were homogenized in extraction buffer containing 50 mm Tris-HCl, pH 7.2, 400 mm NaCl, 2 mm EGTA, 1 mm EDTA, 1 mm DTT, 0.1 μm p-amidinophenylmethanesulfonyl fluoride hydrochloride, 10 μg/ml leupeptin, and 1 mm benzamidine. Each extract was centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207240) Google Scholar) followed by immunoblotting (21Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 471-510Google Scholar). Anti-Rho-kinase rabbit polyclonal antibodies were generated against the glutathioneS-transferase fusion-catalytic domain of Rho-kinase (15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar), and anti-MLC rabbit polyclonal antibodies were provided by Dr. J. T. Stull (University of Texas Southwestern Medical Center, Dallas, TX). Immunostained proteins were visualized by Supersignal (Pierce). Densities of bands of immunostained proteins were quantitated using a scanning densitometer, Densitograph (Atto, Tokyo, Japan). After treatment with 5.1 μg/ml (0.06 μm) CAT and/or 10 μm wortmannin, the fringe-like strips of the rabbit portal veins permeabilized by Triton X-100 were quickly placed in a frozen slurry of acetone containing 10% trichloroacetic acid and 10 mm DTT to terminate the contractile responses. After depletion of trichloroacetic acid, the strips were homogenized in urea sample buffer containing 20 mm Tris base, 22 mmglycine, pH 8.6, 8 m urea, 10 mm DTT, 10% sucrose, and 0.1% bromphenol blue. The extracts were subjected to glycerol-urea polyacrylamide gel electrophoresis following immunoblotting using anti-MLC antibodies as documented (22Persechini A.K. Kamm K.E. Stull J.T. J. Biol. Chem. 1986; 261: 6293-6299Abstract Full Text PDF PubMed Google Scholar). Immunostained proteins were visualized colorimetrically with 4-chloro-1-naphthol and subjected to densitometrical quantitation. Introduction of CAT, which is not only the constitutively active form of Rho-kinase but also the highly homologous domain among rat (14Leung T. Manser E. Tan E. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar), bovine (15Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar), human (16Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (792) Google Scholar), and mouse Rho-associated kinases (23Nakagawa O. Fujisawa K. Ishizaki T. Saito Y. Nakao K. Narumiya S. FEBS Lett. 1996; 392: 189-193Crossref PubMed Scopus (651) Google Scholar), into the cytosol of the extensively Triton X-100-permeabilized rabbit portal vein smooth muscle provoked a contraction both at a constant cytosolic Ca2+ (pCa 6.5; Fig.1 a) and at a nominally zero cytosolic Ca2+ buffered with 10 mm EGTA (pCa ≪ 8.0; Fig. 1 c). CAT exerted contraction, whereas the vehicle had no effect on the force (Fig. 1, a andc). These contractions were completely reversed by wash out of CAT, contrary to those induced by 10 μm microcystin-LR (24Somlyo A.P. Kitazawa T. Himpens B. Matthijs G. Horiuti K. Kobayashi S. Goldman Y.E. Somlyo A.V. Adv. Protein Phosphatases. 1989; 5: 181-195Google Scholar, 25Gong M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar) (Fig. 1 b). In the absence of cytosolic Ca2+ at pCa ≪ 8.0, the CAT-induced contraction was also reversible (data not shown). In neither intact nor α-toxin-permeabilized strips of the portal vein did CAT exert the contractile effects (data not shown). These observations are interpreted to mean that constitutively active CAT could be introduced into the cytosol of the smooth muscle only by extensive membrane permeabilization to induce a reversible contraction. MLC phosphorylation mediated by Ca2+-CaM-dependent MLC kinase pathway plays a primary role in smooth muscle contraction through myosin-actin-interaction and the consequent activation of myosin ATPase (2Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593-603Crossref PubMed Google Scholar, 3Hartshorne D.J. Johnson D.R. Physiology of the Gastrointestinal Tract. 1. Raven Press, New York1987: 423-482Google Scholar, 4Sellers J.R. Adelstein R.S. Boyer P. Krebs E.G. The Enzyme. 18. Academic Press, San Diego, CA1987: 381-418Google Scholar). To investigate involvement of Ca2+-CaM-dependent MLC kinase pathway in the CAT-induced contraction, we examined the effects of wortmannin (WM), a potent MLC kinase inhibitor (26Nakanishi S. Kakita S. Takahashi I. Kawahara K. Tsukuda E. Sano T. Yamada K. Yoshida M. Kase H. Matsuda Y. Hashimoto Y. Nonomura Y. J. Biol. Chem. 1992; 267: 2157-2163Abstract Full Text PDF PubMed Google Scholar) on force development induced by cumulative application of CAT (Fig. 2). In the presence of cytosolic Ca2+ at pCa 6.5, in which Ca2+-CaM-dependent MLC kinase should be active, 10 μm WM shifted the dose-response curve down and to the right. In the absence of cytosolic Ca2+ at pCa ≪ 8.0, in which MLC kinase would be hardly activated, 10 μm WM did not affect CAT-induced force development. In the absence of CAT, treatment of 10 μm WM completely inhibited the cytosolic Ca2+-provoked contraction atpCa 6.5, in the Triton X-100-permeabilized fibers. Considering our finding that WM did not affect the activity of CAT up to 100 μm in vitro (data not shown), this WM-sensitive component of CAT-induced contraction at pCa 6.5 seemed to be due to inhibition of the Ca2+-provoked contraction through the Ca2+-CaM-dependent MLC kinase pathway but not related to the CAT-mediated pathway. All these observations suggest that the CAT-induced contraction of smooth muscle of rabbit portal vein permeabilized by Triton X-100 is modulated independently by the Ca2+-CaM-dependent MLC kinase pathway. To clarify whether CAT induces contraction with a concomitant increase in levels of MLC phosphorylation, we examined the effects of CAT on MLC phosphorylation, using immunoblotting with anti-MLC polyclonal antibody (Fig. 3). As shown in lanes 1–3 of Fig.3 a, at pCa ≪ 8.0, monophosphorylation of MLC was detected only in the presence of CAT and was insensitive to 10 μm WM (42.77 ± 9.22% of the total amount of immunostained MLC (n = 4) in the absence of WM, 35.95 ± 3.39% (n = 4; p > 0.05) in the presence of WM, respectively). At pCa 6.5, shown inlanes 4–6 of Fig. 3 a, CAT potentiated the level of monophosphorylation of MLC (60.33 ± 1.42%, n= 4), which was partially inhibited by 10 μm WM (26.05 ± 5.18%, n = 4, p < 0.01). Based on the statistical analysis and the results in Fig. 2, this WM-sensitive component of CAT-induced MLC phosphorylation atpCa 6.5 also seemed to be due to inhibition of Ca2+-CaM-dependent MLC kinase activity. These results are consistent with that of counterparts of the effects of CAT on the contractile responses (Fig. 3 b). It was concluded that CAT potentiates the contractile response by increasing the extent of monophosphorylation of MLC. To determine if native Rho-kinase is one of the cofactors diffusible during permeabilization by Triton X-100, we examined the amounts of native Rho-kinase in intact and permeabilized fibers by immunoblot analysis using rabbit polyclonal antibodies against Rho-kinase. To standardize the densitometrical value, the ratio of densitometrical quantification of immunostaining of Rho-kinase to that of MLC was calculated in both intact and permeabilized fibers. As shown in Fig.4, the amounts of native Rho-kinase in the Triton X-100-permeabilized rabbit portal vein were markedly lower than those in intact tissue (0.06 ± 0.01 (n = 4) for permeabilized sample, 0.95 ± 0.02 (n = 4) for intact sample, respectively), whereas the amounts of the possible cytoskeletal proteins, such as MLC and myosin heavy chain in permeabilized fibers were similar to the counterparts of intact fibers. These results confirm that extensive permeabilization by Triton X-100 allows for the loss of cytosolic proteins, including Rho-kinase, whereas cytoskeletal proteins such as myosin are stable. Based on all of these findings taken together plus evidence that the direct activation of G-proteins did not exert contractile effects on the extensively Triton X-100-permeabilized smooth muscle (11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar), we consider that Rho-kinase may be a valid candidate for the key molecule in G-protein-mediating smooth muscle contraction and may be the molecule lost during extensive permeabilization by Triton X-100. We demonstrate here what seems to be the evidence that Rho-kinase is a direct on the contractile of smooth muscle, independently of the Ca2+-CaM-dependent MLC kinase pathway. for MLC kinase Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar, K. Ikebe M. Somlyo A.V. Somlyo A.P. Cell Calcium. 1994; 16: 431-445Crossref PubMed Scopus (49) Google Scholar), we no that the of kinases to the cytosol of permeabilized smooth muscle directly contractile responses with findings with the inhibition of myosin may be the mechanism of the G-protein-mediating Ca2+ sensitization of smooth muscle contraction (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1731) Google Scholar, 9Kubota Y. Nomura M. Kamm K.E. Mumby M.C. Stull J.T. Am. J. Physiol. 1992; 262: C405-C410Crossref PubMed Google Scholar, A.P. Kitazawa T. Himpens B. Matthijs G. Horiuti K. Kobayashi S. Goldman Y.E. Somlyo A.V. Adv. Protein Phosphatases. 1989; 5: 181-195Google Scholar, 25Gong M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar, T. Masuo M. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar), the CAT-induced contraction of smooth muscle permeabilized by Triton X-100 may be also mediated by the inhibition of myosin is by our previous finding that Rho-kinase inhibited the activity of myosin through of subunit in vitro (18Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J.H. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2436) Google Scholar). However, at cytosolic zero contraction of the permeabilized smooth muscle was by an MLC kinase inhibitor M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar), whereas the CAT-induced contraction was insensitive to it of the MLC kinase inhibitor to and myosin contractions the that myosin inhibition cannot account for the CAT-induced contraction at the cytosolic zero Ca2+. this together with our that Rho-kinase directly the phosphorylation of MLC and myosin in vitro (17Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar), we suggest that the of CAT-induced contraction of Triton X-100-permeabilized rabbit portal vein might be a concomitant monophosphorylation of MLC directly induced by CAT independently of a Ca2+-CaM-dependent MLC kinase pathway. We that Rho-kinase is considered a valid key molecule in G-protein-mediating Ca2+ sensitization of smooth muscle contraction. We Dr. J. T. Stull for the D. J. and M. P. for and M. for on the

In response to herbivory, plants emit specific blends of herbivore-induced plant volatiles (HIPVs). HIPVs mediate sizable arrays of interactions between plants and arthropods, microorganisms, undamaged neighboring plants or undamaged sites within the plant in various ecosystems. HIPV profiles vary according to the plant and herbivore species, and the developmental stages and conditions of the live plants and herbivores. To understand the regulatory mechanisms underling HIPV biosynthesis, the following issues are reviewed here: (i) herbivore-induced formation of plant volatile terpenoids and green leaf volatiles; (ii) initial activation of plant responses by feeding herbivores; and (iii) the downstream network of the signal transduction. To understand the ecological significance of HIPVs, we also review case studies of insect-plant and inter-/intraplant interactions mediated by HIPVs that have been documented in the field and laboratory in recent years.