scholarly journals Functional Analysis of P450 Monooxygenase SrrO in the Biosynthesis of Butenolide-Type Signaling Molecules in Streptomyces rochei

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1237
Author(s):  
Aiko Teshima ◽  
Nozomi Hadae ◽  
Naoto Tsuda ◽  
Kenji Arakawa
Keyword(s):  
Signaling Molecules ◽  
Chiral Hplc ◽  
Dependent Manner ◽  
Active Components ◽  
P450 Monooxygenase ◽  
Ligand Affinity ◽  
Chemical Structures ◽  
Shift Analysis ◽  
Monooxygenase Gene

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics lankacidin and lankamycin, and their biosynthesis is tightly controlled by butenolide-type signaling molecules SRB1 and SRB2. SRBs are synthesized by SRB synthase SrrX, and induce lankacidin and lankamycin production at 40 nM concentration. We here investigated the role of a P450 monooxygenase gene srrO (orf84), which is located adjacent to srrX (orf85), in SRB biosynthesis. An srrO mutant KA54 accumulated lankacidin and lankamycin at a normal level when compared with the parent strain. To elucidate the chemical structures of the signaling molecules accumulated in KA54 (termed as KA54-SRBs), this mutant was cultured (30 L) and the active components were purified. Two active components (KA54-SRB1 and KA54-SRB2) were detected in ESI-MS and chiral HPLC analysis. The molecular formulae for KA54-SRB1 and KA54-SRB2 are C15H26O4 and C16H28O4, whose values are one oxygen smaller and two hydrogen larger when compared with those for SRB1 and SRB2, respectively. Based on extensive NMR analysis, the signaling molecules in KA54 were determined to be 6′-deoxo-SRB1 and 6′-deoxo-SRB2. Gel shift analysis indicated that a ligand affinity of 6′-deoxo-SRB1 to the specific receptor SrrA was 100-fold less than that of SRB1. We performed bioconversion of the synthetic 6′-deoxo-SRB1 in the Streptomyces lividans recombinant carrying SrrO-expression plasmid. Substrate 6′-deoxo-SRB1 was converted through 6′-deoxo-6′-hydroxy-SRB1 to SRB1 in a time-dependent manner. Thus, these results clearly indicated that SrrO catalyzes the C-6′ oxidation at a final step in SRB biosynthesis.

Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and δ-opioid receptors

2006 ◽  
Vol 291 (1) ◽  
pp. H344-H350 ◽  
Author(s):  
Hemal H. Patel ◽  
Brian P. Head ◽  
Heidi N. Petersen ◽  
Ingrid R. Niesman ◽  
Diane Huang ◽  
...  
Keyword(s):  
Cell Death ◽  
Cardiac Myocytes ◽  
Opioid Receptors ◽  
Signaling Molecules ◽  
Blue Staining ◽  
Dependent Manner ◽  
Protective Effects ◽  
Cardiac Protection ◽  
Presence And Absence

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of δ-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-β-cyclodextrin (MβCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MβCD and MβCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 ± 3 vs. 59 ± 3% dead cells in the presence and absence of 1 μM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 ± 4 vs. 62 ± 3% dead cells, P < 0.01). MβCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MβCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage.

scholarly journals Functional Analysis of the Cytochrome P450 Monooxygenase Gene bcbot1 of Botrytis cinerea Indicates That Botrydial Is a Strain-Specific Virulence Factor

2005 ◽  
Vol 18 (6) ◽  
pp. 602-612 ◽  
Author(s):  
Verena Siewers ◽  
Muriel Viaud ◽  
Daniel Jimenez-Teja ◽  
Isidro G. Collado ◽  
Christian Schulze Gronover ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Functional Characterization ◽  
Gray Mold ◽  
Infection Process ◽  
In Planta ◽  
P450 Monooxygenase ◽  
Α Subunit ◽  
Monooxygenase Gene

The micrographic phytopathogen Botrytis cinerea causes gray mold diseases in a large number of dicotyledonous crop plants and ornamentals. Colonization of host tissue is accompanied by rapid killing of plant cells ahead of the growing hyphen, probably caused by secretion of nonspecific phytotoxins, e.g., the sesquiterpene botrydial. Although all pathogenic strains tested so far had been shown to secrete botrydial and although the toxin causes comparable necrotic lesions as infection by the fungus, the role of botrydial in the infection process has not been elucidated so far. Here, we describe the functional characterization of bcbot1, encoding a P450 monooxygenase and provide evidence that it is involved in the botrydial pathway, i.e., it represents the first botrydial biosynthetic gene identified. We show that bcbot1 is expressed in planta and that expression in vitro and in planta is controlled by an α-subunit of a heterotrimeric GTP-binding protein, BCG1. Deletion of bcbot1 in three standard strains of B. cinerea shows that the effect on virulence (on several host plants) is strain-dependent; only deletion in one of the strains (T4) led to reduced virulence.

Optical analysis of ink and other contaminants in process waters

TAPPI Journal ◽  
2012 ◽  
Vol 11 (8) ◽  
pp. 51-58
Author(s):  
ANTTI HAAPALA ◽  
MIKA KÖRKKÖ ◽  
ELISA KOIVURANTA ◽  
JOUKO NIINIMÄKI
Keyword(s):  
Optically Active ◽  
Process Water ◽  
Paper Machine ◽  
Optical Analysis ◽  
Direct Process ◽  
Active Components ◽  
Water Contaminants ◽  
Dependent Measurement ◽  
Measurement Methodology

Analysis methods developed specifically to determine the presence of ink and other optically active components in paper machine white waters or other process effluents are not available. It is generally more interest¬ing to quantify the effect of circulation water contaminants on end products. This study compares optical techniques to quantify the dirt in process water by two methods for test media preparation and measurement: direct process water filtration on a membrane foil and low-grammage sheet formation. The results show that ink content values obtained from various analyses cannot be directly compared because of fundamental issues involving test media preparation and the varied methodologies used to formulate the results, which may be based on different sets of assumptions. The use of brightness, luminosity, and reflectance and the role of scattering measurements as a part of ink content analysis are discussed, along with fine materials retention and measurement media selection. The study concludes with practical tips for case-dependent measurement methodology selection.

Amelioration of Carbon Tetrachloride-Induced Liver Injury by Citrus Leaf Extract

2019 ◽  
Vol 17 (4) ◽  
pp. 426-431
Author(s):  
Jin Xuezhu ◽  
Li Jitong ◽  
Nie Leigang ◽  
Xue Junlai
Keyword(s):  
Carbon Tetrachloride ◽  
Leaf Extract ◽  
Hepatic Injury ◽  
The Body ◽  
Dependent Manner ◽  
Weight Changes ◽  
Citrus Leaf ◽  
Dose Dependent ◽  
And Oxidative Stress

The main purpose of this study is to investigate the role of citrus leaf extract in carbon tetrachloride-induced hepatic injury and its potential molecular mechanism. Carbon tetrachloride was used to construct hepatic injury animal model. To this end, rats were randomly divided into 4 groups: control, carbon tetrachloride-treated, and two carbon tetrachloride + citrus leaf extract-treated groups. The results show that citrus leaf extract treatment significantly reversed the effects of carbon tetrachloride on the body weight changes and liver index. Besides, treatment with citrus leaf extract also reduced the levels of serum liver enzymes and oxidative stress in a dose-dependent manner. H&E staining and western blotting suggested that citrus leaf extract could repair liver histological damage by regulating AMPK and Nrf-2.

The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

1999 ◽  
Vol 73 (6) ◽  
pp. 4575-4581 ◽  
Author(s):  
Masahiko Makino ◽  
Satoshi Shimokubo ◽  
Shin-Ichi Wakamatsu ◽  
Shuji Izumo ◽  
Masanori Baba
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Virus Type ◽  
Spastic Paraparesis ◽  
Tropical Spastic Paraparesis ◽  
Normal Donor ◽  
Dependent Manner ◽  
T Lymphotropic Virus

ABSTRACT The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4+ T cells and CD8+ T cells in a viral dose-dependent manner. However, the proliferation level of CD4+ T cells was five- to sixfold higher than that of CD8+ T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4+ T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4+ and CD8+ T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP.

scholarly journals Role of MDA5 in regulating CXCL10 expression induced by TLR3 signaling in human rheumatoid fibroblast-like synoviocytes

2021 ◽  
Author(s):  
Tatsuro Saruga ◽  
Tadaatsu Imaizumi ◽  
Shogo Kawaguchi ◽  
Kazuhiko Seya ◽  
Tomoh Matsumiya ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Neutralizing Antibody ◽  
Poly I:C ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Inflammatory Reactions ◽  
Polyinosinic:Polycytidylic Acid ◽  
Tlr3 Signaling ◽  
Fibroblast Like Synoviocytes

AbstractC-X-C motif chemokine 10 (CXCL10) is an inflammatory chemokine and a key molecule in the pathogenesis of rheumatoid arthritis (RA). Melanoma differentiation-associated gene 5 (MDA5) is an RNA helicase that plays a role in innate immune and inflammatory reactions. The details of the regulatory mechanisms of CXCL10 production and the precise role of MDA5 in RA synovitis have not been fully elucidated. The aim of this study was to examine the role of MDA5 in regulating CXCL10 expression in cultured human rheumatoid fibroblast-like synoviocytes (RFLS). RFLS was stimulated with Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA mimetic. Expression of interferon beta (IFN-β), MDA5, and CXCL10 was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay. A neutralizing antibody of IFN-β and siRNA-mediated MDA5 knockdown were used to determine the role of these molecules in regulating CXCL10 expression downstream of TLR3 signaling in RFLS. Poly I:C induced IFN-β, MDA5, and CXCL10 expression in a concentration- and time-dependent manner. IFN-β neutralizing antibody suppressed the expression of MDA5 and CXCL10, and knockdown of MDA5 decreased a part of CXCL10 expression (p < 0.001). The TLR3/IFN-β/CXCL10 axis may play a crucial role in the inflammatory responses in RA synovium, and MDA5 may be partially involved in this axis.

scholarly journals Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dasol Kim ◽  
Hui-Yun Hwang ◽  
Eun Sun Ji ◽  
Jin Young Kim ◽  
Jong Shin Yoo ◽  
...  
Keyword(s):  
Metabolic Regulation ◽  
Elongation Factor ◽  
Dependent Manner ◽  
Diet Induced Obesity ◽  
Metabolic Dysregulation ◽  
Autophagy Induction ◽  
Transcription Factor Eb ◽  
Mitochondrial Elongation

AbstractDisorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

scholarly journals Active Components from Cassia abbreviata Prevent HIV-1 Entry by Distinct Mechanisms of Action

2021 ◽  
Vol 22 (9) ◽  
pp. 5052
Author(s):  
Yue Zheng ◽  
Xian-Wen Yang ◽  
Dominique Schols ◽  
Mattia Mori ◽  
Bruno Botta ◽  
...  
Keyword(s):  
Crude Extract ◽  
Female Genital Tract ◽  
Binding Activity ◽  
Sub Saharan Africa ◽  
Active Components ◽  
Chemical Structures ◽  
3D Space ◽  
In Silico Study ◽  
Sub Saharan ◽  
Hiv 1

Cassia abbreviata is widely used in Sub-Saharan Africa for treating many diseases, including HIV-1 infection. We have recently described the chemical structures of 28 compounds isolated from an alcoholic crude extract of barks and roots ofC. abbreviata, and showed that six bioactive compounds inhibit HIV-1 infection. In the present study, we demonstrate that the six compounds block HIV-1 entry into cells: oleanolic acid, palmitic acid, taxifolin, piceatannol, guibourtinidol-(4α®8)-epiafzelechin, and a novel compound named as cassiabrevone. We report, for the first time, that guibourtinidol-(4α®8)-epiafzelechin and cassiabrevone inhibit HIV-1 entry (IC50 of 42.47 µM and 30.96 µM, respectively), as well as that piceatannol interacts with cellular membranes. Piceatannol inhibits HIV-1 infection in a dual-chamber assay mimicking the female genital tract, as well as HSV infection, emphasizing its potential as a microbicide. Structure-activity relationships (SAR) showed that pharmacophoric groups of piceatannol are strictly required to inhibit HIV-1 entry. By a ligand-based in silico study, we speculated that piceatannol and norartocarpetin may have a very similar mechanism of action and efficacy because of the highly comparable pharmacophoric and 3D space, while guibourtinidol-(4α®8)-epiafzelechin and cassiabrevone may display a different mechanism. We finally show that cassiabrevone plays a major role of the crude extract of CA by blocking the binding activity of HIV-1 gp120 and CD4.

scholarly journals GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1802
Author(s):  
Nayoung Kim ◽  
Mi Yeon Kim ◽  
Woo Seon Choi ◽  
Eunbi Yi ◽  
Hyo Jung Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Negative Regulator ◽  
Target Cells ◽  
Dependent Manner ◽  
Cell Functions ◽  
Cell Based Therapy ◽  
Activating Receptors ◽  
Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

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