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Bioassay-guided fractionation of MeOH extract from fenugreek (Trigonella foenum-graecum L.) seeds resulted in the isolation of two rat growth-hormone release stimulators in vitro, fenugreek saponin I (1) and dioscin (9), along with two new, i.e., 2 and 3, and five known analogues, i.e., 4-8. The structures of the new steroidal saponins, fenugreek saponins I, II, and III (1-3, resp.), were determined as gitogenin 3-O-beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranoside, sarsasapogenin 3-O-beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranoside, and gitogenin 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside, respectively. Fenugreek saponin I (1) and dioscin (9) caused ca. 12.5- and 17.7-fold stimulation of release, respectively, of rat growth hormone from rat pituitary cells, whereas gitogenin (5) showed moderate activity. To our knowledge, this is the first study to demonstrate that steroidal saponins stimulate rat growth-hormone release in rat pituitary cells.

To identify the active substance in the male silkworm pupae that strengthens men's vitality, the vasorelaxation activity was determined by measuring the vascular endothelial nitric oxide (eNO) produced in calf pulmonary artery endothelial (CPAE) cells treated with extracts from the pupae. Dried silkworm male pupae were extracted with ethanol and suspended in water, then partitioned with hexane, chloroform, ethylacetate, and butanol, sequentially. Among these fractions, the aqueous fraction had maximal NO production (156.87 μM/200 μl well, 10 mg/ml) and minimal cytotoxicity (IC 50 362.3 mg/ml). The vasorelaxation substances (VAS) from the aqueous fraction were isolated by a combination of gel filtration and anion-exchange chromatography on DEAE Sephadex A-25 and reverse phase-HPLC. Their chemical structures were determined on the basis of their spectroscopic parameters of EI-MS, MALDI-TOF MS, 1 H and 13 C NMR, 1 H- 1 H COSY, and GC–MS spectral data. The active substance was subsequently identified as a dimethyladenosine and dimethyladenosine-5′- l -arabinose that has phosphodiesterase (PDE) inhibition activity. This compound was shown to inhibit PDE4 activity in a dose-dependent manner. Also, it inhibited the PDE5 activity of cyclic-GMP-specific PDE5 enzyme. These results imply that dimethyladenosine may be a lead compound for the development and improvement of vasculogenic impotence drugs through phosphodiesterase inhibition and NO production in endothelial cells.

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Three new (1-3) and 17 known (4-20) iridoid and secoiridoid glycosides were isolated from a methanol extract of the rhizomes and roots of Gentiana scabra. Their chemical structures were elucidated from 1D and 2D NMR, IR absorption, and HR-ESI-MS spectra, as well as comparisons of these data with reported values. The effects of the isolated compounds on lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells were investigated. Compounds 6, 10 and 20 exhibited significant inhibitory effects on LPS-induced IL-12 p40 and IL-6 production with IC50 values of 1.62-14.29 μM. Compound 10 also showed a strong inhibitory effect on the LPS-stimulated production of TNF-α with an IC50 value of 10.45 μM.

Hericirine (I) inhibits significantly protein expression of iNOS and COX-2 and reduced NO, PEG2, TNF-α, IL-6, and IL-1β production in RAW264.7 cells exposed to LPS.

Six new (erinarols A-F, 1-6) and five known (7-11) ergostane-type sterol fatty acid esters were isolated from the methanol extract of the dried fruiting bodies of Hericium erinaceum. Their chemical structures were elucidated using chemical and physical methods as well as through comparison of NMR and mass spectral data with those reported previously. This is the first comprehensive investigation on ergostane-type sterol fatty acid esters from H. erinaceum. The isolated compounds were evaluated for their PPAR transactivational effects using a luciferase reporter system. Compounds 1 and 2 significantly activated the transcriptional activity of PPARs in a dose-dependent manner, with EC50 values of 8.2 and 6.4 μM, respectively. Moreover, compounds 1 and 2 also activated PPARα and PPARγ transcriptional activity, with stimulation from 1.3- to 3.9-fold at 20 μM concentrations.

A new triterpenoid saponin, together with five known saponins, were isolated from the nonpolar n-hexane fraction of the leaves of Aralia elata. The structure of the new saponin, durupcoside C, was elucidated as hederagenin 3-O-beta-D-glucopyranosyl(1--3)-beta-D-glucopyranosyl(1--3)-alpha-L-arabinopyranoside on the basis of spectroscopic analysis. The known saponins were characterized as 3-O-alpha-L-rhamnopyranosyl(1--2)-alpha-L-arabinopyranosyl hederagenin 28-O-beta-D-xylopyranosyl(1--6)-beta-D-glucopyranosyl ester, hederagenin 3-O-beta-D-glucopyranosyl(1--3)-alpha-L-rhamnopyranosyl(1--2)-alpha-L-arabinopyranoside, oleanolic acid 3-O-beta-D-glucopyranosyl(1--3)-alpha-L-rhamnopyranosyl(1--2)-alpha-L-arabinopyranoside, hederagenin 3-O-alpha-L-rhamnopyranosyl(1--2)-alpha-L-arabinopyranoside (alpha-hederin), and hederagenin 3-O-beta-D-glucopyranosyl(1--3)-alpha-L-arabinopyranoside (collinsonidin). In addition, two known lipids, Arisaema glyceride 3 and ceramide mixtures were also isolated and characterized. Collinsonidin and two known lipids were isolated for the first time from this plant.

Aldose reductase, the key enzyme of the polyol pathway, is known to play important roles in diabetic complications. Therefore, inhibitors of aldose reductase would be potential agents for the prevention of diabetic complications. To evaluate the inhibitory potential of aldose reductase from Ganoderma applanatum (Polyporaceae), methanol (MeOH) and water extracts were tested for their effects on rat lens aldose reductase (RLAR). The effects of both extracts on streptozotocin (STZ)-induced diabetes in rats were also investigated. The MeOH extract exhibited a potent rat lens aldose reductase (RLAR) inhibition in vitro, and showed a significant inhibition, of not only serum glucose concentrations, but also of sorbitol accumulations in the lens, red blood cells (RBC) and sciatic nerves in STZ-induced diabetic rats. Associated with a reduction in serum glucose concentration in STZ-induced diabetic rats, this extract was found to cause a significant glucose tolerance effect. These results suggested that G. applanatum might possess constituents with antidiabetic and inhibitory effects on diabetic complications. Copyright © 2005 John Wiley & Sons, Ltd.

Four new lanostane-type triterpenes were isolated from the MeOH extract of the fruiting bodies of Ganoderma applanatum. Their structures were established as 3beta,7beta,20,23xi-tetrahydroxy-11,15-dioxolanosta-8-en-26-oic acid (1), 7beta,20,23xi-trihydroxy-3,11,15-trioxolanosta-8-en-26-oic acid (2), 7beta,23xi-dihydroxy-3,11,15-trioxolanosta-8,20E(22)-dien-26-oic acid (3), and 7beta-hydroxy-3,11,15,23-tetraoxolanosta-8,20E(22)-dien-26-oic acid methyl ester (4), respectively, by extensive spectroscopic analyses.

Five new phenolic glycosides, 2-beta-D-glucopyranosyloxy-5-hydroxyphenylacetic acid methyl ester (4), 4-hydroxy-2-[3-hydroxy-3-methylbutyl]-5-methylphenyl beta-D-glucopyranoside (5), 4-hydroxy-2-[(E)-4-hydroxy-3-methyl-2-butenyl]-5-methylphenyl beta-D-glucopyranoside (7), 4-hydroxy-2-[(2E,6Z)-8-beta-D-glucopyranosyloxy-3,7-dimethylocta-2,6-dien-1-yl]-5-methylphenyl beta-D-glucopyranoside (8), and 2,7-dimethyl-1,4-dihydronaphthalene-5,8-diol 5-O-beta-D-xylopyranosyl(1--6)-beta-D-glucopyranoside (10), were isolated from the whole plants of Pyrola japonica (Pyrolaceae), together with androsin, (-)-syringaresinol glucoside, homoarbutin, pirolatin, hyperin, monotropein and chimaphilin.

Five compounds of terpenoids and coumarins were isolated from the non-polar fraction of Artemisia apiacea by open column chromatography. Their structures were elucidated as alpha-amyrin (1), beta-amyrin (2), beta-sitosterol (3), 5,6,7-trimethoxycoumarin (4) and 6-methoxy-7,8-methylenedioxycoumarin (5) by chemical and spectroscopic analysis. This is the first report of the isolation of alpha-amyrin, beta-amyrin, 5,6,7-trimethoxycoumarin and 6-methoxy-7,8-methylenedioxycoumarin from this plant.

Aconitum jaluense Komar. (Ranunculaceae) is one of the Aconitum plants growing in Korean peninsula. An investigation of the alkaloidal constituents of this species led to the isolation of seven C19-norditerpenoid and a C20-diterpenoid alkaloid. Three of them have been identified as neoline, mesaconitine, and hypaconitine, which were isolated from this plant collected from Mt. Bultasan in the north part. The other five alkaloids were determined as lipomesaconitine, lipohypaconitine, 15alpha-hydroxyneoline, hokbusine A, and napelline, which have not been found in this plant. Structures of those alkaloids were determined on the basis of their spectral data. It is of interest to note that a comparison of the present work and the previous report showed some differences in the alkaloidal contents.

A phthalide, levistolide A (1), and six coumarins, demethylsuberosin (2), fraxetin (3), (-)-marmesinin (4), 3'( S)- O-β-D-glucopyranosyl-3’,4'-dihydroxanthyletin (5), 3'( R)- O-β-D-glucopyranosyl-3’,4'-dihydroxanthyletin (6), and isopraeroside IV (7) were isolated from the methanolic extract of the roots of Angelica tenuissima Nakai. Their chemical structures were confirmed by comparing spectroscopic and reported data. All seven compounds were isolated for the first time from this plant source. The anti-allergic activities of compounds 1-7 were examined using human mast cells, and compounds 1-3 at 10 μM potently suppressed IL-6 expression and inhibited histamine release from human mast cells by more than 30%.

Erinarols G-J and 10 known ergostane-type sterols were isolated from a methanol extract of the dried fruiting bodies of Hericium erinaceum. Their chemical structures were elucidated using extensive spectroscopic analyses including 1D and 2D NMR experiments and HR-ESI-MS analysis, as well as through comparison with previously reported data. Anti-inflammatory effects of the isolated compounds were evaluated in terms of inhibition of tumor necrosis factor α (TNF-α) and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells. The results showed that erinarols H and J, as well as 2 of the ergostane-type sterols exhibited inhibitory activity against TNF-α secretion, with inhibition values ranging from 33.7% to 43.3% at 10 μM. Erinarols J and three ergostane-type sterols exhibited significant inhibitory effects against NO production, with inhibition values ranging from 38.4% to 71.5% at 10 μM.

The piscidin-family, one of antimicrobial peptides (AMPs) mainly distributed in fish, is crucial effectors of fish innate immune response. Piscidin-family typically has broad-spectrum antimicrobial activity and the ability to modulate the immune response. In this study, we identified moronecidin (Rbmoro) included in piscidin-family from rock bream and investigated its gene expression using quantitative real-time PCR and biological activity (including antimicrobial and cytotoxic activity). The coding region of Rbmoro was 204 bp encoding 67 amino acid residues. Tertiary structure prediction of Rbmoro showed an amphipathic α-helical structure. Rbmoro gene was widely expressed in different tissues of healthy fish. Additionally, Rbmoro gene expression was induced in all tested tissues after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. We synthesized mature peptide of Rbmoro based on amino acid sequence of its AMP 12 domain, and the synthetic peptide appeared broad-spectrum antimicrobial activity to various bacteria. However, the synthetic peptide has weak haemolytic activity against fish erythrocytes. These results suggest that Rbmoro might play an important role in innate immune response of rock bream.

Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we analysed the recombinant protein produced from rock bream thioredoxin 1 cDNA (rRbTRx1). To verify the immune-stimulatory effect of this recombinant protein, changes in the expression level of several genes were investigated using the cDNA microarray chips in rock bream peripheral blood leukocytes stimulated with rRbTRx1. Furthermore, the immune responses of rock bream to Streptococcus iniae FKC (formalin-killed cell) vaccination alone or in combination with recombinant proteins were analysed after an experimental challenge with living S. iniae. Microarray analysis showed that 237 unique genes were upregulated more than two-fold after rRbTRx1 stimulation. Serum agglutination titres were relatively low; however, the FKC vaccine still conferred protection against S. iniae. Moreover, the adverse effects showed no statistically significant difference between fish injected with a concentration and non-injected fish. After experimental challenge to the rock bream by injection with living bacteria (S. iniae), the relative percent survival in the vaccinated groups with FKC + rRbTRx1 was significantly higher than that of the vaccinated groups with FKC alone, which were 85.9% and 68.2%, respectively. This indicated that the recombinant protein as an adjuvant showed synergism with the injected vaccine in rock bream.

The Journal of Antibiotics (2014) 67, 727–729; doi:10.1038/ja.2014.57; published online 7 May 2014Fruiting bodies of Hericium erinaceum (Hericiaceae) are known as atraditional edible mushroom. The main constituents of H. erinaceum,which include polysaccharides, aromatic compounds and fatty acids,have been investigated previously, and their bioactive effects are wellknown.

Seven compounds, including three neolignans 1–3, a norlignan 4, and three diterpenoids 5–7, were isolated from the feces of Trogopterus xanthipes. Structures of these compounds were identified by 1D and 2D NMR as well as MS. The absolute configurations of compounds 1, 2, and 4 were determined by comparing CD spectra and optical rotations. Among the isolated compounds, 1–3 were novel and subsequently named trogopterins A, B, and C, respectively. Likewise, compound 4 was isolated from nature for the first time. Cytotoxic activities of compounds 1–4 were evaluated. Compounds 1–3 exhibited moderate cytotoxic activities against HL-60 cells with IC50 values of 34.77–45.68 μM.

Mammalian sperm cells must swim long distances to fertilize the egg. The powerful burst of hyperactivated motility, which is necessary for successful fertilization, requires calcium influx via CatSper, sperm flagellar Ca2+ channel. We characterized ultrastructures of CatSper channels and Ca2+ signaling molecules in single flagella. We find that the CatSper channel, together with other signaling molecules, organizes four linear Ca2+ domains along the flagella. This unique structure focuses tyrosine phosphorylation in time and space during capacitation. We looked for motility-correlative molecular phenotypes in capacitation-induced heterogeneous sperm populations. Hyperactivated motility correlates with the intact CatSper domains, the presence of P-Tyr, and the efficient sperm migration in female reproductive tract. Our results offer a new view of spatiotemporal organization of signal transduction in flagella that augments the mechanistic insights of Ca2+ in motility regulation for mammalian spermatozoa.

A molecular lens of the nonresonant dipole force formed by focusing a nanosecond IR laser pulse has been applied to benzene and CS2 molecular beams. Using the velocity map imaging technique for molecular ray tracing, characteristic molecular lens parameters including the focal length (f ), minimum beam width (W), and distance to the minimum beam width position (D) were determined. The laser intensity dependence of the observed lens parameters was in good agreement with theoretical predictions. W was independent of the laser peak intensity (I0), whereas f and D varied linearly with 1/I0. The differences in lens parameters between the molecular species were well correlated with the polarizability per mass values of the molecules. A high chromatographic resolution of Rs=0.84 was achieved between the images of benzene molecular beams undeflected and deflected by the lens. The possibilities for a new type of chromatography are discussed.

The 26S pro-teasome is composed of 20S core and 19S regulator. Since pro-teasomes interact primarily with endogenous proteins, inhi-bition of the proteolytic action of the proteasome may block the signaling action of the transcription factor NF- κB and, thus, inhibit the completion of the cell cycle and hence the mitotic proliferation of cancerous cells, leading to cell death by apopto-sis and inhibition of angiogenesis and metastasis.

Preclinical evaluation has shown that cancer cells are more sensitive to the proapoptotic effects of proteasome inhibition than normal cells. Therefore, several groups of proteasome inhibitors such as peptide aldehydes, peptide boronates, nonpeptide inhibitors, peptide vinyl sulfones, and epoxyketones, have been developed and are now widely used as research tools to study the role of the UPP in various cellular processes.Currently, only two proteasome inhibitors, bortezomib and NPI-0052, have been reported in clinical trials.

(MYC-1590) wassubjected to chemical investigation. An organic extract fromcultures of this isolate showed potent antifungal activity indisk diffusion assays. Fractionation of the crude EtOAcextract of solid-substrate fermentation culture of MYC-1590by silica gel column chromatography, followed by repeatedreverse phase HPLC, afforded two new metabolites; 6,8-dimethoxy-4,5-dimethyl-3-methyleneisochroman-1-one (

Summary Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel ( CatSpers ) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility. PaperFlick

We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting. Paclitaxel (Taxol®) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11–27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

Cathepsins are lysosomal cysteine proteases belonging to the papain family, whose members play important roles in normal metabolism for the maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin H (RbCTSH) cDNAs were identified by expressed sequence tag analysis of a lipopolysaccharide-stimulated rock bream liver cDNA library. The full-length RbCTSH cDNA (1326 bp) contained an open reading frame of 978 bp encoding 325 amino acids. The presence of an ERFNIN-like motif was predicted in the propeptide region of RbCTSH. Furthermore, multiple alignments showed that the EPQNCSAT region was well conserved among other cathepsin H sequences. Phylogenetic analysis revealed that RbCTSH is most closely related to Nile tilapia cathepsin H. RbCTSH was expressed significantly in the intestine, spleen, head kidney and stomach. RbCTSH mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. All examined tissues of fish infected with Edwardsiella tarda, Streptococcus iniae and red sea bream iridovirus (RSIV) showed significant increases in RbCTSH expression compared to the control. In the kidney and spleen, RbCTSH mRNA expression was upregulated markedly following infection with bacterial pathogens. These findings indicate that RbCTSH plays an important role in the innate immune response of rock bream. Furthermore, these results provide important information for the identification of other cathepsin H genes in various fish species.

The resolution of fluorescence microscopy had traditionally been limited to ~200–300 nm due to the diffraction of light. Recently, this resolution limit has been broken using mainly two classes of methods, one of which utilizes photoswitching of fluorophores to temporally separate the spatially overlapping images of individual molecules such that the positions of these molecules can be precisely determined. A sub-diffraction-limit image can then be reconstructed from these molecular coordinates. With relatively simple optical instrumentation and sample preparation, this class of methods has improved the spatial resolution of far-field optical microscopy by more than an order of magnitude, achieving resolutions down to sub-10 nm range for biological specimens. Three-dimensional, multicolor, and live-cell super-resolution imaging has been demonstrated. In this chapter, we provide an overview of this class of optical nanoscopy, primarily in terms of the imaging principle, the spatial/temporal resolution, the different imaging schemes, the photoswitchable probes, the applications, and future directions.

Ejaculated mammalian spermatozoa gain the capacity to fertilize the egg within the female reproductive tract. Called capacitation, these changes to sperm include alterations in motility, membrane composition, and widespread protein tyrosine phosphorylation. Hyperactivated motility, the amplified, asymmetric movement of the tail, requires the sperm tail-specific Ca2+ channel, Cationic channel of sperm (CatSper). Calcium-mediated intraflagellar signaling is poorly understood, in part because flagellar diameters are less than 1 μm, preventing the resolution of specific molecular structures by conventional fluorescence microscopy. Here, we employed three-dimensional stochastic optical reconstruction microscopy (STORM) to determine how CatSper channel complex and other Ca2+ signaling molecules are organized. We find that the CatSper channel forms four linear nanodomains with calcium signaling molecules near the plasma membrane along the flagella. Strikingly, lack of the CatSper channel results in delocalization of Ca2+ signaling molecules. The spatial organization of protein components in CatSper signaling provides insights into Ca2+ signal transduction in regulation of sperm motility.

Fungal endophytes have attracted attention due to their functional diversity. Secondary metabolites produced by Pestalotiopsis sydowiana from a halophyte, Phragmites communis Trinus, were investigated. Eleven compounds, including four penicillide derivatives (1–4) and seven α-pyrone analogues (5–10) were isolated from cultures of P. sydowiana. The compounds were identified based on spectroscopic data. The inhibitory activities against the 20S proteasome were evaluated. Compounds 1–3, 5, and 9–10 showed modest proteasome inhibition activities, while compound 8 showed strong activity with an IC50 of 1.2 ± 0.3 μM. This is the first study on the secondary metabolites produced by P. sydowiana and their proteasome inhibitory activities. The endophytic fungus P. sydowiana might be a good resource for proteasome inhibitors.

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues. In mammals, these cytokines can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in the sequence, and include the CXC(α), CC(β), C(γ), and CX3C(δ) classes. We identified CXC chemokine cDNA, designated RbCXC, isolated using expressed sequence tag analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCXC cDNA (742 bp) contained an open reading frame of 342 bp encoding 114 amino acids. Results from phylogenetic analysis showed that RbCXC was strictly separated into a distinct clade compared to other known CXC chemokine subgroups. RbCXC was significantly expressed in the trunk kidney, liver, spleen, gill, peripheral blood leukocytes (PBLs), and head kidney. Rock bream PBLs were stimulated with several mitogens, including LPS and polyinosinic-polycytidylic acid (poly I:C), which significantly induced the expression of RbCXC mRNA. RbCXC mRNA expression was examined in several tissues under conditions of bacterial and viral challenge. Experimental challenges revealed that all examined tissues from fish infected with Edwardsiella tarda and red sea bream iridovirus showed significant increases in RbCXC expression compared to the control. In the case of Streptococcus iniae infection, RbCXC mRNA expression was markedly upregulated in the kidney, spleen, and liver. In addition, a maltose binding protein fusion recombinant RbCXC (∼53 kDa) was produced in an Escherichia coli expression system and purified. Subsequently, the addition of purified recombinant RbCXC (rRbCXC) to kidney leukocytes was examined to investigate the impact of proliferative and chemotactic activity. The rRbCXC induced significant kidney leukocyte proliferation and attraction at concentrations ranging from 10 to 300 μg/mL, suggesting that it can be utilised as an immune stimulant and/or molecular adjuvant to enhance the immunological effects of vaccines.

The mechanism underlying glaucoma remains controversial, but apoptosis caused by increased levels of reactive oxygen species (ROS) is thought to play a role in its pathogenesis. We investigated the effects of compounds isolated from Psoralea corylifolia on oxidative stress-induced cell death in vitro and in vivo. Transformed retinal ganglion cells (RGC-5) were treated with l-buthione-(S,R)-sulfoximine (BSO) and glutamate in the presence or with pre-treatment with compound 6, bakuchiol isolated from P. corylifolia. We observed reduced cell death in cells pre-treated with bakuchiol. Moreover, bakuchiol inhibited the oxidative stress-induced decrease of mitochondrial membrane potential (MMP, ΔΨm). Furthermore, while intracellular Ca(2+) was high in RGC-5 cells after exposure to oxidative stress, bakuchiol reduced these levels. In an in vivo study, in which rat retinal damage was induced by intravitreal injection of N-methyl-d-aspartate (NMDA), bakuchiol markedly reduced translocation of AIF and release of cytochrome c, and inhibited up-regulation of cleaved caspase-3, cleaved caspase-9, and cleaved PARP. The survival rate of retinal ganglion cells (RGCs) 7days after optic nerve crush (ONC) in mice was significantly decreased; however, bakuchiol attenuated the loss of RGCs. Moreover, bakuchiol attenuated ONC-induced up-regulation of apoptotic proteins, including cleaved PARP, cleaved caspase-3, and cleaved caspase-9. Bakuchiol also significantly inhibited translocation of mitochondrial AIF into the nuclear fraction and release of mitochondrial cytochrome c into the cytosol. These results demonstrate that bakuchiol isolated from P. corylifolia has protective effects against oxidative stress-induced retinal damage, and may be considered as an agent for treating or preventing retinal degeneration.

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.

We identified the CC chemokine cDNA designated as RbCC1 (CC chemokine 1 in rock bream, Oplegnathus fasciatus), which was isolated using expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCC1 cDNA (850 bp) contained an open reading frame (ORF) of 366 bp encoding 122 amino acids. Results from our phylogenetic analysis demonstrated that the RbCC1 was closest relationship to the orange-spotted grouper and Mi-iyu croaker CC chemokines located within the fish CC chemokine group. RbCC1 was significantly expressed in the intestine, spleen, liver, and PBLs (peripheral blood leukocytes). Rock bream PBLs were stimulated with several mitogens, LPS and Con A/PMA which significantly induced the expression of RbCC1 mRNA in the PBLs. The RbCC1 mRNA expression in several tissues under conditions of bacterial and viral challenge was examined. The experimental challenge revealed that the kidney and spleen of fish infected with Streptococcus iniae showed the most significant increases in RbCC1 expression compared to the control. In the case of RSIV infection, the RbCC1 mRNA expression was markedly up-regulated in the liver. In this study, recombinant RbCC1 (approximately 53 kDa) was produced using an Escherichia coli expression system followed by purification. Subsequently, the addition of purified rRbCC1 was examined to investigate the impact on the proliferative and chemotactic activity on kidney leukocytes from rock bream. The results demonstrated that the rRbCC1 induces significant biological activity on kidney leukocyte proliferation and attraction at concentrations in the range of 10–300 μg/mL and suggests that rRbCC1 could be utilized as an immune-stimulant and/or molecular adjuvant to enhance the immune effects of vaccines.

Six triterpenoids, two sterols, and two phenolic constituents were isolated from the methanol extract of sclerotia of Inonotus obliquus (Hymenochaetaceae). Their chemical structures were identified as lanosterol (1), 3β-hydroxylanosta-8,24-dien-21-al (3), inotodiol (5), trametenolic acid (6), lanosta-8,25-dien-3,22,24-triol (7), β-sitosterol (2), ergosterolperoxide (4), betulin (8), 2,5-dihydroxy-benzaldehyde (9), and 3,4-dihydroxybenzalacetone (10) on the basis of spectroscopic methods and comparison with the literature. Among these compounds, 9 was isolated for the first time from this mushroom. To the best of our knowledge, the present study marks the first chemical investigation of I. obliquus from Alaska. Among the isolated compounds, compounds 1, 3, 4, 5, 6, 8, 9, and 10 were evaluated for in vitro cytotoxic activity against A 549 (human alveolar basal epithelial carcinoma cells), L1210 (mouse lymphocytic leukemia cells), COLO 205 (colorectal adenocarcinoma cells), MCF-7 (breast cancer cells), and HL-60 (human leukemia) cancer cell lines. 3β-Hydroxylanosta-8,24-dien-21-al (3) and tramentenolic acid (6) exhibited modest cytotoxic effects against L1210 cells with IC50s of 62.5 and 34.4 μM, and betulin (8) showed weak cytotoxic effects against A549 and HL-60 with IC50s of 81.2 and 87.5 μM, respectively. In addition, 3,4-dihydroxybenzalacetone (10) exhibited the strongest cytotoxic activities against A549 and HL-60 cells with IC50 values of 23.6 and 21.7 μM, respectively.

Phomalevones A-C (1-3), three new com-pounds with bis-dihydroxanthone and bis-benzophenone systems, were isolated from cultures of a Hawaiian isolate of Phoma sp. (MYC-1734 = NRRL 39060; Cucurbitariaceae). The structures of 1-3 were determined by analysis of NMR and MS data. The absolute configurations of the sp(3) stereocenters in the monomeric unit of 1 were assigned by application of Mosher's method, and overall absolute configurations were proposed on the basis of ECD data using both computational methods and comparisons with literature data for model compounds. All three compounds showed antibacterial activity, and compounds 2 and 3 also exhibited antifungal effects.

A new neo-clerodane diterpenoid, barbatellarine F (1), was isolated from the CHCl3-soluble fraction of the aerial part of Scutellaria barbata. The chemical structure of the new compound was elucidated by detailed analysis of NMR and MS data. Compound 1 was a C-13 epimer of the previously reported compound barbatellarine E, which was confirmed by NOESY data as well as optical rotation.

The new sesquiterpenoid curcuzederone (I) is isolated from the rhizome of curcuma zedoaria along with a known flavonoid.

This letter reports a straightforward means of collecting two-dimensional electronic (2D-E) spectra using optical tools common to many research groups involved in ultrafast spectroscopy and quantum control. In our method a femtosecond pulse shaper is used to generate a pair of phase stable collinear laser pulses which are then incident on a gas or liquid sample. The pulse pair is followed by an ultrashort probe pulse that is spectrally resolved. The delay between the collinear pulses is incremented using phase and amplitude shaping and a 2D-E spectrum is generated following Fourier transformation. The partially collinear beam geometry results in perfectly phased absorptive spectra without phase twist. Our approach is much simpler to implement than standard non-collinear beam geometries, which are challenging to phase stabilize and require complicated calibrations. Using pulse shaping, many new experiments are now also possible in both 2D-E spectroscopy and coherent control.

Science often advances only as quickly as technology allows. This trend is especially evident in the field of molecular spectroscopy, where the science of studying molecular structures, dynamics, and chemical reactions has progressed in step with the technology of manipulating electromagnetic fields. For example, nuclear magnetic resonance (NMR) spectroscopy, which is now used to determine the structures of proteins, was made possible by radio-frequency technology developed in the 1950s. Likewise, laser-guided control of excited-state chemistry became possible in the 1990s with technology for temporally shaping visible and

A new method for collecting 2D-IR spectra that utilizes both a pump-probe beam geometry and a mid-IR pulse shaper is used to gain a fuller understanding of fiber formation in the human islet amyloid polypeptide (hIAPP). We extract structural kinetics in order to better understand aggregation in hIAPP, the protein component of the amyloid fibers found to inhibit insulin production in type II diabetes patients.

Cyanobacteria, blue-green algae, are a rich source of bioactive secondary metabolites with many potential applications. The ubiquitin-proteasome proteolytic system plays an important role in selective protein degradation and regulates cellular events including apoptosis. Cancer cells are more sensitive to the proapoptotic effects of proteasome inhibition than normal cells. Thus, proteasome inhibitors can be potential anticancer agents. Cyanobacteria have been shown to be a rich source of highly effective inhibitors of proteases. A proteasome inhibitor was screened from an extract of the culture of Scytonema hofmanni on the basis of its inhibitory activity, which led to the isolation of nostodione A with an IC(50) value of 50 microM. Its structure was determined by spectroscopic methods such as 1H-NMR and ESI-MS spectral analyses.

The capability of 2D IR spectroscopy for elucidating time-evolving structures is enhanced by a programmable mid-IR pulse shaper that greatly improves the ease, speed and accuracy of data collection. We demonstrate our technique on a model metal carbonyl compound and then apply it to study the structural disorder of amyloid fibers composed of aggregated human islet amyloid polypeptide (hIAPP).

A new norditerpene alkaloid named 8-O-methylhypaconine (1) was isolated along with twelve known alkaloids from the underground parts of an unknown species of Aconitum plant culti vated in Korea. Among the known alkaloids, two dianthramide glucosides, N-(2'-beta-glucopyra nosyl-5'-hydroxysalicyl)-5-hydroxyanthranilic acid methyl ester (2) and N-(2'-beta-glucopyranosyl-5'-hydroxysalicyl)-5-hydroxy-6-methoxyanthranilic acid methyl ester (3), were isolated from Aconitum plants for the first time. The structures were established on the basis of chemical and spectroscopic methods.

The capability of 2D IR spectroscopy to elucidate time-evolving structures is enhanced by a programmable mid-IR pulse shaper that greatly improves the ease, speed, and accuracy of data collection. Traditional ways of collecting 2D IR spectra are difficult to implement, cause distorted peak shapes, and result in poor time resolution and/or phase problems. We report on several methods for collecting 2D IR spectra by using a computer-controlled germanium acoustooptic modulator that overcomes the above problems. The accuracy and resolution of each method is evaluated by using model metal carbonyl compounds that have well defined lineshapes. Furthermore, phase cycling can now be employed to largely alleviate background scatter from heterogeneous samples. With these methods in hand, we apply 2D IR spectroscopy to study the structural diversity in amyloid fibers of aggregated human islet amyloid polypeptide (hIAPP), which is involved with type 2 diabetes. The 2D IR spectra reveal that the β-sheet fibers have a large structural distribution, as evidenced by an inhomogeneously broadened β-sheet peak and strong coupling to random coil conformations. Structural diversity is an important characteristic of hIAPP because it may be that partly folded peptides cause the disease. This experiment on hIAPP is an example of how computer generation of 2D IR pulse sequences is a key step toward automating 2D IR spectroscopy, so that new pulse sequences can be implemented quickly and a diverse range of systems can be studied more easily.

We report selective population of the excited vibrational levels of the ${T}_{1u}$ CO-stretching mode in $\mathrm{W}(\mathrm{CO}{)}_{6}$ using phase-tailored, femtosecond mid-IR ($5.2\text{ }\text{ }\ensuremath{\mu}\mathrm{m}$, $1923\text{ }\text{ }{\mathrm{cm}}^{\ensuremath{-}1}$) pulses. An evolutionary algorithm was used to optimize specific vibrational populations. Stimulated emission peaks, indicative of population inversion, could be induced. Systematic truncation of each optimized pulse allowed for increased understanding of the excitation mechanism. The pulses and techniques developed herein will have broad applications in controlling ground state chemistry and enhancing vibrational spectroscopies.

A germanium acousto-optic modulator (Ge AOM) is utilized in high resolution phase and amplitude shaping of IR light between 2 and 18 µm. Shaped waveforms centered at 4.9 µm are demonstrated in both the time and frequency domains. The phase stability of the shaped pulse is measured by cross-correlation with an unshaped mid-IR pulse, indicating that such shaped pulses can be readily incorporated into phase sensitive experiments. This Ge AOM will enable new experiments in 2D IR spectroscopy and coherent control on the ground electronic state.