Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Cytototoxic constituents from the bark of Chisocheton cumingianus (Meliaceae).

A new lanostane-type triterpenoid, 3ß-hydroxy-25-ethyl-lanost-9(11),24(24')-diene (1), along with 3ß-hydroxy-lanost-7-ene (2) and ß-sitosterol-3-O-acetate (3) was isolated from the stem bark of C. cumingianus. The chemical structure of the new compound was elucidated on the basis of spectroscopic data. All of the compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1-3 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 28.8 ± 0.10, 4.29 ± 0.03, and 100.18 ± 0.16 µg/ml, respectively.

Frontier studies on highly selective bio-regulators useful for environmentally benign agricultural production.

Fungal metabolites active for insects were obtained from fermentation products using okara media. The mechanisms of action of these compounds against insects were clarified using voltage clamp electrophysiology. The branching factor inducing hyphal branching in arbuscular mycorrhizal (AM) fungi was isolated from the root exudates of Lotus japonicus and identified as 5-deoxystrigol. Strigolactones were originally identified as seed germination stimulants of parasitic weeds; therefore, synthetic strigolactones were developed to exhibit the inducing activity of hyphal branching in AM fungi and diminish the stimulating activity of seed germination of parasitic weeds. Signaling molecules, acylhomoserine lactones (AHLs), in quorum sensing were identified in the fungal strain Mortierella alpina A-178, and the true producer of AHLs was clarified as symbiotic bacteria in the fungus. Since acyl-(S)-adenosylmethionine analogs may be good candidates for competitive inhibitors of AHL synthases, intermediate mimics in the biosynthesis of AHLs have been synthesized.

Removal and detoxification of iprodione in water using didodecyldimethylammonium bromide-montmorillonite organoclay and manganese dioxide.

Combination of organoclay sorption with manganese(IV) oxide (MnO2) catalyzed catechol oxidation was studied for the removal of a dicarboximide fungicide, iprodione, from water. Iprodion in water was sorbed on didodecyldimethylammonium bromide (DDAB)-modified montmorillonite (MT) organoclay and converted into the degraded product, 3,5-dichloroaniline (DCA). The degree of sorption increased by the modification with DDAB, because of the formation of a hydrophobic region for the incorporation of iprodione and negligibly interfered by coexisting MnO2. The half-life for the degradation of irodione in water at 25 °C was 7 days, whreas it reduced to 15 min in the organoclay. The activation energy, 65.4 ± 4.8 kJ mol-1, for the first-order reaction in the aqueous solution (pH 7.0) decreased to 43.9 ± 1.8 kJ mol-1 in the organoclay, indicating the catalytic activity of the organoclay that accelerates the hydrolysis reaction of iprodione. In the coexistence of appropriate amounts of MnO2 and catechol, the degraded product, DCA, reacted with oxidized products of catechol to form a water-insoluble precipitate and was successfully eliminated from water. The results obtained in the present study strongly suggest the applicability of the combined method of organoclay sorption method and MnO2-catalyzed oxidation for the diffusion control of toxic agrochemicals.

Quantitative analysis of powder samples using screen-printing techniques as sample preparation methods for LA-ICP-MS.

The combination of laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) offers a powerful tool for directly analyzing solid samples. However, LA-ICP-MS has a limitation in quantitative analyses owing to a requirement for matrix-matched standard materials. In this study, we have developed a sample preparation method that facilitates quantitative analyses by LA-ICP-MS. The sample powder is dispersed in a liquid resin and film-like samples are prepared from the resulting paste by a screen-printing technique. The sample includes the analyte spiked with internal standards and is prepared by mixing standard solutions in the sample paste. Because all reagents except for the sample powder are liquids, homogeneous samples can be easily obtained. The internal standard and concentration of the spiked analyte can be tailored for each sample, which is a requirement for accurate quantitative analyses. The amount of sample and concentration of the spiked analyte are controlled against an internal standard, enabling internal standardization without the need to have an element of known concentration in the sample. The accuracy of this method was evaluated by analyzing impurity elements in TiO2 powder; however, it is expected that other materials could also be analyzed. The versatility and flexibility of this method suggest great potential for quantitative analyses by LA-ICP-MS, for which reliable matrix-matched standard materials are required.

Interim analysis of post-marketing surveillance of ravulizumab for paroxysmal nocturnal hemoglobinuria in Japan.

Ravulizumab is a long-acting C5 inhibitor available for treating paroxysmal nocturnal hemoglobinuria (PNH). Post-marketing surveillance (PMS) was implemented following its approval in September 2019 in Japan. We report safety data obtained through to December 2021 for 218 patients and effectiveness data for 194 patients (182 switched from eculizumab and 12 complement inhibitor-naïve). Over a median follow-up of 74.4 weeks, 193 adverse events (AEs) were reported in 66/218 patients (30.3%; incidence 72.73/100 patient-years). The two most frequent AEs were anemia and pyrexia (each 3.01/100 patient-years). The incidence of serious AEs was 36.93/100 patient-years. In patients who switched from eculizumab, lactate dehydrogenase (LDH) and hemoglobin (Hb) levels were maintained over 26 weeks of ravulizumab treatment. In complement inhibitor-naïve patients, LDH decreased significantly and Hb increased significantly over 26 weeks of ravulizumab treatment. These data for Japanese patients with PNH who were naïve to complement inhibitors and patients who switched from eculizumab show that the safety and effectiveness of ravulizumab are consistent with the published clinical trial data. However, transfusion independence was less likely in patients with bone marrow failure. Further follow-up data from this PMS will help to elucidate the long-term clinical safety and effectiveness of ravulizumab for treating PNH.

Real-world safety profile of eculizumab in patients with paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, or generalized myasthenia gravis: an integrated analysis of post-marketing surveillance in Japan.

Eculizumab is a C5 inhibitor approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and anti-acetylcholine receptor antibody-positive generalized myasthenia gravis (AChR + gMG) in Japan. We report integrated safety data from post-marketing surveillance in these three indications, focusing on commonly occurring adverse events (AEs) and infection-related AEs. Of 1219 patients registered, 1055 (PNH: 780; aHUS: 192; AChR + gMG: 83) had available safety data. Total eculizumab exposure was 3977.361 patient-years. AEs were reported in 74.03% of patients. AEs with an incidence of  ≥ 1.0 per 100 patient-years included hemolysis, headache, nasopharyngitis, renal impairment, anemia, pneumonia, upper respiratory tract inflammation, influenza, condition aggravated, and infection. The incidence of infection-related AEs was 21.30 per 100 patient-years, the most frequent types (≥ 1.0 per 100 patient-years) being nasopharyngitis, pneumonia, influenza, and infection. Meningococcal infections were reported in four patients (0.10 per 100 patient-years). Two patients died from meningococcal sepsis, with a mortality rate of 0.05 per 100 patient-years. This is the largest safety dataset on eculizumab in Japan derived from more than 10 years of clinical experience. No new safety signals were observed and the safety profile of eculizumab was consistent with that in previous clinical trials and international real-world safety analyses.

Production of the quorum-sensing molecules N-acylhomoserine lactones by endobacteria associated with Mortierella alpina A-178.

Gram-negative bacteria communicate with one another using N-acylhomoserine lactones (AHLs) as signaling molecules. This mechanism, known as quorum sensing (QS), is needed to develop pathogenicity, as well as symbiotic interactions with eukaryotic hosts, such as animals and plants. Increasing evidence indicates that certain bacteria, namely endobacteria, also inhabit fungal cells and establish symbiotic relationships with their hosts. However, it has not been clear whether bacterial QS acts in developing the relationships. Here we describe the isolation and identification of N-heptanoylhomoserine lactone and N-octanoylhomoserine lactone from the culture broth of the zygomycete fungus Mortierella alpina A-178. This suggested the presence of endobacteria in the fungus, as was confirmed by PCR, fluorescence in situ hybridization, and transmission electron microscopy. Two major bands obtained by PCR-denaturing gradient gel electrophoresis showed sequence identity to genes in the ß-proteobacterium Castellaniella defragrans (100 %) and the Gram-positive bacterium Cryobacterium sp. (99.8 %). The production of AHLs depended on the presence of endobacteria and was induced in response to the increase in the concentration of AHLs, suggesting that the bacterium conducts AHL-mediated QS in the fungus. This paper is the first to report the production of AHLs by endofungal bacteria and raises the possibility that QS plays roles in the development of fungus-endobacterium symbiosis.

The D3 F-box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhiza (AM) represents an ancient endosymbiosis between plant roots and Glomeromycota fungi. Strigolactones (SLs), plant-derived terpenoid lactones, activate hyphal branching of AM fungi before physical contact. Lack of SL biosynthesis results in lower colonization of AM fungi. The F-box protein, DWARF3 (D3), and the hydrolase family protein DWARF14 (D14) are crucial for SL responses in rice. Here we conducted AM fungal colonization assays with the SL-insensitive d3 and d14 mutants. The d3 mutant exhibited strong defects in AM fungal colonization, whereas the d14 mutant showed higher AM fungal colonization. As D14 has a homologous protein, D14-LIKE, we generated D14-LIKE knockdown lines by RNA interference in the wildtype and d14 background. D14 and D14-LIKE double knockdown lines exhibited similar colonization rates as those of the d14-1 mutant. D3 is crucial for establishing AM symbiosis in rice, whereas D14 and D14-LIKE are not. Our results suggest distinct roles for these SL-related components in AM symbiosis.

A new meroterpenoid, chrodrimanin C, from YO-2 of Talaromyces sp.

The new meroterpenoid, chrodrimanin C (3), together with chrodrimanins A (2) and B (1) were isolated from okara (the insoluble residue of whole soybean) that had been fermented with strain YO-2 of Talaromyces sp. Their structures were elucidated by spectroscopic methods. The partial structures of 1 essential for exhibiting insecticidal activity were investigated by using a silkworm assay. The absolute configuration of 1 was also determined.

New chrodrimanin congeners, chrodrimanins D-H, from YO-2 of Talaromyces sp.

Four new meroterpenoids, named chrodrimanins D-G (4-7), and one known compound, renamed chrodrimanin H (8), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with the YO-2 strain of Talaromyces sp. Their structures were elucidated by spectroscopic methods. Chrodrimanins D (4), E (5), and F (6) showed insecticidal activity against silkworms with respective LD(50) values of 20, 10, and 50 µg/g of diet.

Staphylococcus aureus requires cardiolipin for survival under conditions of high salinity.

BACKGROUND: The ability of staphylococci to grow in a wide range of salt concentrations is well documented. In this study, we aimed to clarify the role of cardiolipin (CL) in the adaptation of Staphylococcus aureus to high salinity. RESULTS: Using an improved extraction method, the analysis of phospholipid composition suggested that CL levels increased slightly toward stationary phase, but that this was not induced by high salinity. Deletion of the two CL synthase genes, SA1155 (cls1) and SA1891 (cls2), abolished CL synthesis. The cls2 gene encoded the dominant CL synthase. In a cls2 deletion mutant, Cls1 functioned under stress conditions, including high salinity. Using these mutants, CL was shown to be unnecessary for growth in either basal or high-salt conditions, but it was critical for prolonged survival in high-salt conditions and for generation of the L-form. CONCLUSIONS: CL is not essential for S. aureus growth under conditions of high salinity, but is necessary for survival under prolonged high-salt stress and for the generation of L-form variants.

Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi form mutualistic, symbiotic associations with the roots of more than 80% of land plants. The fungi are incapable of completing their life cycle in the absence of a host root. Their spores can germinate and grow in the absence of a host, but their hyphal growth is very limited. Little is known about the molecular mechanisms that govern signalling and recognition between AM fungi and their host plants. In one of the first stages of host recognition, the hyphae of AM fungi show extensive branching in the vicinity of host roots before formation of the appressorium, the structure used to penetrate the plant root. Host roots are known to release signalling molecules that trigger hyphal branching, but these branching factors have not been isolated. Here we have isolated a branching factor from the root exudates of Lotus japonicus and used spectroscopic analysis and chemical synthesis to identify it as a strigolactone, 5-deoxy-strigol. Strigolactones are a group of sesquiterpene lactones, previously isolated as seed-germination stimulants for the parasitic weeds Striga and Orobanche. The natural strigolactones 5-deoxy-strigol, sorgolactone and strigol, and a synthetic analogue, GR24, induced extensive hyphal branching in germinating spores of the AM fungus Gigaspora margarita at very low concentrations.

[A case of advanced colon cancer successfully treated with cetuximab diagnosed as EGFR-positive by liver biopsy].

Cetuximab, a monoclonal antibody used to target the epidermal growth factor receptor(EGFR), was approved for refractory and metastatic colon cancer expressing EGFR, and the EGFR expression must be confirmed. But the EGFR expression may become false negative by immunohistochemistry. When we used a past operation specimen for a search in particular, a correct evaluation may be difficult for prolonged formalin fixation. We report a case successfully treated by cetuximab, who was diagnosed as EGFR-negative by the past operation specimen, but as EGFR-positive by the liver biopsy specimen. A 51- year old woman with multiple organ metastases, who had experienced failure with prior oxaliplatin, irinotecan(CPT-11), 5- FU and bevacizumab regimens, was administered cetuximab plus CPT-11 because it was EGFR-positive by liver biopsy, and the tumor was obviously reduced. It is useful to obtain another specimen such as by liver biopsy, when the EGFR expression is negative by a past operation specimen.

Structural requirements of strigolactones for hyphal branching in AM fungi.

Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C-D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C-D part.

Facile preparation of deuterium-labeled N-acylhomoserine lactones as internal standards for isotope dilution mass spectrometry.

N-Acylhomoserine lactones (AHLs) are widely conserved signal molecules that mediate quorum sensing in Gram-negative bacteria. In this study, deuterium-labeled AHLs were prepared for use as internal standards for isotope dilution mass spectrometry. Their utility in the sensitive and precise quantification of AHLs in culture supernatants of bacteria by GC/MS was demonstrated.

Inhibitory activity against urease of quercetin glycosides isolated from Allium cepa and Psidium guajava.

Methanolic extracts of edible plants and seaweeds were tested for their inhibitory activity against Jack bean urease. Quercetin-4'-O-beta-D-glucopyranoside was isolated from Allium cepa as a urease inhibitor with an IC(50) value of 190 microM-. Quercetin and two quercetin glycosides, avicularin and guaijaverin, were isolated from Psidium guajava as urease inhibitors with respective IC(50) values of 80 microM-, 140 microM-, and 120 microM-.

Determination of absolute structures of cyclic peptides, PF1171A and PF1171C, from unidentified ascomycete OK-128.

Two cyclic peptides, PF1171A (1) and PF1171C (2), were isolated from okara that had been fermented with unidentified ascomycete OK-128. Their absolute configurations were determined by Marfey's method. These peptides showed paralytic activity against silkworms.