Isolation of Fluorescent Benzoxazines with Neuroprotective Activity from the Olive Weevil Pimelocerus perforatus.

The weevil Pimelocerus perforatus poses a serious pest problem for olive cultivation in Japan. Two new racemic fluorescent benzoxazines, designated as pimeforazine A ((±)-1) and pimeforazine B ((±)-2), were successfully isolated from P. perforatus. Their structures, including the absolute configurations of their resolved enantiomers, were determined using spectroscopic methods, single-crystal X-ray diffraction, and electronic circular dichroism calculations. The neuroprotective activity of the isolated compounds was evaluated against hydrogen peroxide-induced cellular damage in SH-SY5Y human neuroblastoma cells. Compounds (±)-1 and (±)-2 exhibited neuroprotective effects.

CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation.

CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.

Janohigenins: Long-chain anacardic acid derivatives with neuroprotective activity from Ophiopogon japonicus seeds.

Eight hitherto undescribed long-chain anacardic acid derivatives, janohigenins, were isolated from the endosperm of Ophiopogon japonicus seed, and their structures were elucidated employing spectroscopic and chemical methods. The neuroprotective activity of the isolated compounds was evaluated against rotenone-induced cellular damage in SH-SY5Y human neuroblastoma cells. Janohigenins exhibited noticeable neuroprotection at 1 µM.

Secondary metabolites from cultures of the ant pathogenic fungus Ophiocordyceps irangiensis BCC 2728.

Five new compounds, iranginins A-E (1-5), together with sixteen known compounds were isolated from the insect pathogenic fungus Ophiocordyceps irangiensis BCC 2728. The structures and the absolute configurations of the new compounds were established by spectroscopic analyses, the application of modified Mosher's method (for 2), ECD calculation (for 5), and X-ray crystallographic analysis (for 4). LL-Z1640-5 and mucorisocoumarin C were active against Mycobacterium tuberculosis (MIC 41.7 and 85.0 µM, respectively), while peyroisocoumarin D exhibited cytotoxic activity (IC50 65.6 µM).

Asaroidoxazines from the Roots of Asarum asaroides Induce Apoptosis in Human Neuroblastoma Cells.

Plants in the family Aristolochiaceae contain phenanthrene skeleton-containing chemical constituents that exhibit nephrotoxic, carcinogenic, mutagenic, anti-inflammatory, and cytotoxic effects. Two new phenanthrene-containing 1,2-oxazin-6-ones, designated as asaroidoxazine A (1) and asaroidoxazine B (2), and a known aristolactam, 5-methoxyaristololactam I (3), were isolated from the roots of Asarum asaroides. The structures of compounds 1 and 2 were determined using spectroscopic methods and X-ray crystallography. Treatment of SH-SY5Y human neuroblastoma cells with 1 µM of asaroidoxazine A (1) induced nuclear condensation as well as caspase-3/7 activation, indicating that this compound is a strong apoptosis inducer in neuronal cells. This is the first report of apoptosis induction by phenanthrene-containing oxazines.

Natural dolapyrrolidone: Isolation and absolute stereochemistry of a substructure of bioactive peptides.

During the course of our chemical analysis of the hydrophilic fractions from marine cyanobacterium Moorena producens, we have isolated natural dolapyrrolidone (Dpy, 1), a natural pyrrolidone derived from phenylalanine, for the first time as a single compound. Compound 1, with an (S)-l absolute stereochemistry, was previously identified as a substructure that is common among several bioactive natural peptides. Surprisingly, the absolute stereochemistry of the isolated natural 1, determined through total synthesis, was (R)-d. This result was unambiguously determined by HPLC analysis using a chiral stationary column by comparing the retention times of the natural 1 and authentic samples of synthetic enantiomers. To verify the unexpected result, the absolute stereochemistry of the natural 1 was confirmed by X-ray crystallographic analysis of Pt-complex derivative using the synthetic enantiomer.

Lasianosides A-E: New Iridoid Glucosides from the Leaves of Lasianthus verticillatus (Lour.) Merr. and Their Antioxidant Activity.

The genus Lasianthus (Rubiaceae) consists of approximately 180 species, of which the greatest species diversity is found in tropical Asia. Some of the Lasianthus species have been used in folk medicine to treat tinnitus, arthritis, fever, and bleeding. Lasianthus verticillatus (Lour.) Merr. (Syn. Lasianthus trichophlebus auct. non Hemsl.) is a shrub, branchlets terete about 1.5-3 m in height. This paper studies the chemical composition of the leaves of L. verticillatus for the first time, which resulted in the isolation of five undescribed iridoid glucosides, lasianosides A-E (1-5), together with three known compounds (6-8). The undescribed structures of isolated compounds (1-5) were characterized by physical and spectroscopic data analyses, including one-dimensional (1D) and two-dimensional (2D) NMR, IR, UV, and high-resolution electrospray ionization mass spectra (HR-ESI-MS). Furthermore, the electronic circular dichroism data determined the absolute configurations of the new compounds. The free radical scavenging properties of isolated compounds was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and their cytotoxicity was assessed toward human lung cancer cell line A549 by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the isolated compounds, 3 and 4 displayed potent radical scavenging activities with IC50 values of 30.2 ± 1.8 and 32.0 ± 1.2 µM, which were comparable to that of Trolox (29.2 ± 0.39 µM), respectively, while 5 possessed moderate activity with an IC50 value of 46.4 ± 2.3 µM. None of the isolated compounds exerted cytotoxicity against human cell line A549. As a result, lasianosides C, D, and E have the potential to be non-toxic safe antioxidant agents.

Altercrasins A⁻E, Decalin Derivatives, from a Sea-Urchin-Derived Alternaria sp.: Isolation and Structural Analysis Including Stereochemistry.

In order to find out the seeds of antitumor agents, we focused on potential bioactive materials from marine-derived microorganisms. Marine products include a number of compounds with unique structures, some of which may exhibit unusual bioactivities. As a part of this study, we studied metabolites of a strain of Alternaria sp. OUPS-117D-1 originally derived from the sea urchin Anthocidaris crassispina, and isolated five new decalin derivatives, altercrasins A-E (1-5). The absolute stereostructure of altercrasins A (1) had been decided by chemical transformation and the modified Mosher's method. In this study, four decalin derivatives, altercrasins B-E (2-5) were purified by silica gel chromatography, and reversed phase high-performance liquid chromatography (RP HPLC), and their structures were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic analyses. The absolute configuration of them were deduced by the comparison with 1 in the NMR chemical shifts, NOESY correlations, and electronic circular dichroism (ECD) spectral analyses. As a result, we found out that compound pairs of 1/2 and 4/5 were respective stereoisomers. In addition, their cytotoxic activities using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines showed that 4 and 5 exhibit potent cytotoxicity, in especially, the activity of 4 was equal to that of 5-fluorouracil.

Spontaneous emulsification and self-propulsion of oil droplets induced by the synthesis of amino acid-based surfactants.

It is well known that oil droplets in or on water exhibit spontaneous movement induced by surfactants, and this self-propulsion is regarded as an important factor in droplet-based models for a living cell. We report here an oil-droplet system spontaneously producing amino acid-based surfactants, which are then utilized for the droplets' self-propulsion. Thus this system is an active system capable of producing the fuel for the propulsion by itself, which can be used as a conceptual model for cell metabolism.

Cyclohelminthol X, a Hexa-Substituted Spirocyclopropane from Helminthosporium velutinum yone96: Structural Elucidation, Electronic Circular Dichroism Analysis, and Biological Properties.

Helminthosporium velutinum yone96 produces cyclohelminthol X (1), a unique hexa-substituted spirocyclopropane. Although its molecular formula and NMR spectral data resemble those of AD0157, being isolated from marine fungus Paraconiothyrium sp. HL-78-gCHSP3-B005, our detailed analyses disclosed a totally different structure. Chemical shift calculations and electronic circular dichroism spectral calculations were quite helpful to establish the structure, when those were performed based on density functional theory. The carbon framework of cyclohelminthols I-IV is found at the C1-C8 propenylcyclopentene substructure of 1. Thus, 1 is assumed to be biosynthesized by cyclopropanation between an oxidized form of cyclohelminthol IV and a succinic anhydride derivative 4. Cytotoxicity for two cancer cell lines and proteasome inhibition efficiency are measured.

Effects of sex steroid hormones and their metabolites on neuronal injury caused by oxygen-glucose deprivation/reoxygenation in organotypic hippocampal slice cultures.

In this study, protective actions of the sex steroid hormones, progesterone, testosterone, and 17ß-estradiol, against oxygen-glucose deprivation (OGD)/reoxygenation-induced neuronal cell death were examined using rat organotypic hippocampal slice cultures. Progesterone, testosterone, and 17ß-estradiol significantly attenuated neuronal cell death elicited by OGD/reoxygenation. While the neuroprotection conferred by progesterone was not affected by SU-10603, an inhibitor of cytochrome P45017α, finasteride, a 5α-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone, partially reversed the neuroprotection induced by progesterone. The progesterone metabolite, allopregnanolone attenuated neuronal injury induced by OGD/reoxygenation. Pretreatment with letrozole, a cytochrome P450 aromatase inhibitor or 4-hydroxyphenyl-1-naphthol, a 17ß-hydroxysteroid dehydrogenase 2 inhibitor showed no effect on testosterone-mediated neuroprotection, while finasteride completely abolished the protective action of testosterone. Treatment with 5α-dihydrotestosterone significantly suppressed neuronal injury. Pretreatment with mifepristone, a progesterone receptor antagonist and hydroxyflutamid, an androgen receptor antagonist significantly diminished the neuroprotective effects of progesterone and testosterone, respectively. ICI182,780, an estrogen receptor antagonist, showed no effect on neuroprotection mediated by 17ß-estradiol. Pretreatment with actinomycin D or cycloheximide clearly abolished the neuroprotective effects of progesterone and testosterone, while actinomycin D and cycloheximide did not show any effect on neuroprotection mediated by 17ß-estradiol. Taken together, progesterone protects neurons via progesterone receptor-dependent genomic pathway, and allopregnanolone is involved in progesterone-mediated neuroprotection. Testosterone and its metabolite 5α-dihydrotestosterone protect neurons via the genomic pathway of the androgen receptor. Metabolism of sex steroid hormones in the brain might complicate their protective actions in the brain.

Chiroptical molecular propellers based on hexakis(phenylethynyl)benzene through the complexation-induced intramolecular transmission of local point chirality.

We designed hexakis(phenylethynyl)benzene derivatives with a tertiary amide group on each blade to achieve a helically biased propeller arrangement through the complexation-induced intramolecular transmission of point chirality. A hydrogen-bonding ditopic guest was captured at two amide groups, and thus could pair two neighboring blades to form a supramolecular cyclic structure, in which an auxiliary chiral group associated with a blade acted as a chiral handle to control the helical bias, while the chiral auxiliary did not exert any helical influence on the dynamic helicity in the absence of a guest due to the high flexibility of each blade.

A sensitive method based on fluorescence-detected circular dichroism for protein local structure analysis.

We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca(2+) and/or interacting with binding peptides. Because FDCD appears to reflect the protein's local structure around the fluorophore, it may provide a useful means for "pinpoint analysis" of protein structures.