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Tissue transglutaminase (TG2) plays an important role in pulmonary arterial hypertension (PAH). Previous research indicate that TG2 and protein serotonylation catalyzed by TG2 are upregulated in PAH. Serotonin transporter inhibitor fluoxetine ameliorates PAH via inhibition of protein serotonylation. It is still unknown whether PAH is inhibited through direct inhibition of TG2. Therefore, the present study aimed to investigate the effects of TG2 inhibitor cystamine on monocrotalineinduced PAH in rats. Rats were treated with monocrotaline (60 mg x [kg.sup.-1], i.p.) in combination with or without cystamine (20, 40 mg x [kg.sup.-1] x [day.sup.-1], p.o.). The results showed that compared with monocrotaline alone, combination of monocrotaline with cystamine (40 mg x [kg.sup.-1] x [day.sup.-1], p.o.) relieved right ventricle hypertrophy, inhibited pulmonary arteriolar remodeling, and down-regulated protein expression of TG2, phosphorylated protein kinase B (Akt), and extracellular regulated protein kinase (ERK) at day 21 However, except for TG2 expression, these changes were not significantly inhibited by cystamine at day 35. In addition, cystamine dose-dependently enhanced the survival rate of rats injected with monocrotaline at day 35. The findings suggest that cystamine slows but not reverses monocrotaline-induced PAH in rats, which was largely associated with the inhibition of TG2 protein expression and Akt and ERK activation. Key words: Akt, cystamine, ERK, monocrotaline, pulmonary arterial hypertension, serotonin, transglutaminase. Resume : La transglutaminase tissulaire (TG2) joue un role important dans l'hypertension pulmonaire (HP). Les recherches anterieures montrent que la TG2 et la serotonylation des proteines catalysee par la TG2 sont regulees a la hausse dans l'HP. La fluoxetine, un inhibiteur du transporteur de la serotonine, permet d'attenuer l'HP par l'intermediaire de l'inhibition de la serotonylation des proteines. Mais on ne sait toujours pas si l'HP est inhibee par l'intermediaire d'une inhibition directe de la TG2. Par consequent, les presents travaux visaient a etudier les effets de la cystamine, un inhibiteur de la TG2, sur l'HP provoquee par la monocrotaline chez le rat. Nous avons administre aux rats de la monocrotaline (60 mg x [kg.sup.-1], i.p.) seule ou en association avec de la cystamine (20, 40 mg x [kg.sup.-1] x [jour.sup.-1], p.o.). Les resultats ont montre qu'au jour 21, comparativement a la monocrotaline seule, la monocrotaline en association avec la cystamine (40 mg x [kg.sup.-1] x [jour.sup.-1], p.o.) permettait d'attenuer l'hypertrophie ventriculaire droite, d'inhiber le remodelage des arterioles pulmonaires ainsi que de reguler a la baisse l'expression proteique de la TG2, de la proteine kinase B phosphorylee (Akt) et de la proteine kinase regulee dans le compartiment extracellulaire (ERK). Cependant, a l'exception de l'expression de la TG2, ces variations n'etaient pas inhibees de facon notable par la cystamine au jour 35. De plus, la cystamine entrainait une augmentation proportionnelle a la dose du taux de survie au jour 35 chez les rats auxquels etait injectee de la monocrotaline. Les resultats laissent entendre que la cystamine entrainerait chez le rat un ralentissement de l'evolution de l'HP (mais sans son inversion), ce qui serait largement associe avec l'inhibition de l'expression proteique de la TG2 ainsi que de l'activation de l'Akt et de l'ERK. [Traduit par la Redaction] Mots-cles: Akt, cystamine, ERK, monocrotaline, hypertension pulmonaire, serotonine, transglutaminase., Introduction Pulmonary arterial hypertension (PAH) is characterized by an increase in mean pulmonary artery pressure, due to pulmonary vascular remodelling that leads to an imbalance between excessive vasoconstrictors and low [...]

Natural products have long served as both a source and inspiration for pharmaceuticals. Modifying the structure of a natural product often improves the biological activity of the compound. Metabolic engineering strategies to ferment 'unnatural' products have been enormously successful in microbial organisms. However, despite the importance of plant derived natural products, metabolic engineering strategies to yield unnatural products from complex, lengthy plant pathways have not been widely explored. Here, we show that RNA mediated suppression of tryptamine biosynthesis in Catharanthus roseus hairy root culture eliminates all production of monoterpene indole alkaloids, a class of natural products derived from two starting substrates, tryptamine and secologanin. To exploit this chemically silent background, we introduced an unnatural tryptamine analog to the production media and demonstrated that the silenced plant culture could produce a variety of novel products derived from this unnatural starting substrate. The novel alkaloids were not contaminated by the presence of the natural alkaloids normally present in C. roseus. Suppression of tryptamine biosynthesis therefore did not appear to adversely affect expression of downstream biosynthetic enzymes. Targeted suppression of substrate biosynthesis therefore appears to be a viable strategy for programming a plant alkaloid pathway to more effectively produce desirable unnatural products. Moreover, although tryptamine is widely found among plants, this silenced line demonstrates that tryptamine does not play an essential role in growth or development in C. roseus root culture. Silencing the biosynthesis of an early starting substrate enhances our ability to harness the rich diversity of plant based natural products. metabolic engineering | natural products

Visceral inflammation, including that arising from bladder inflammation, reduces the threshold to sensation of innocuous or noxious stimuli applied to peripheral structures (referred hyperalgesia). Cystitis may induce transient or persistent plastic changes mediated by neurotrophins, particularly nerve growth factor (NGF), which contribute to increased nociceptive input. In this study, acute or subacute cystitis was induced in female rats by one or three (at 72-h intervals) 400-[micro]1 intravesical instillations of 1 mM acrolein. Sensitivity of the hindpaws to mechanical and thermal stimuli was determined before and 4, 24, 48, 72, and 96 h after treatment. Other groups of rats were treated with intravesical or intrathecal k252a [a nonspecific antagonist of tyrosine kinase (trk) receptors, including trkA, the high-affinity receptor for NGF] before the first or third acrolein instillation. Some rats were intraperitoneally injected with specific NGF-neutralizing antiserum or normal serum before acrolein instillation. Acute and subacute cystitis induced mechanical, but not thermal, referred hyperalgesia that was attenuated by intravesical pretreatment with k252a. Systemic treatment with NGF-neutralizing antiserum before instillation of acrolein suppressed subsequent mechanical referred hyperalgesia. Expression of NGF was increased within the bladder by acute or subacute cystitis and in L6/S dorsal root ganglia by subacute cystitis. These results suggest that the bladder-derived NGF acting via trk receptors at least partially mediates peripheral sensitization to mechanical stimuli associated with acute and subacute acrolein-induced cystitis. k252a; peripheral nociception; visceral inflammation; hyperalgesia

Staurosporine isolated from Streptomyces sp. TP-A0274 is a member of the family of indolocarbazole alkaloids that exhibit strong antitumor activity. A key step in staurosporine biosynthesis is the formation of the indolocarbazole core by intramolecular C-C bond formation and oxidative decarboxylation of chromopyrrolic acid (CPA) catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we report x-ray crystal structures of CPA-bound and -free forms of StaP. Upon substrate binding, StaP adopts a more ordered conformation, and conformational rearrangements of residues in the active site are also observed. Hydrogen-bonding interactions of two carboxyl groups and T-shaped [pi][pi] interactions with indole rings hold the substrate in the substrate-binding cavity with a conformation perpendicular to the heme plane. Based on the crystal structure of StaP-CPA complex, we propose that C-C bond formation occurs through an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I [Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738-740]. The subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry. heme | staurosporine | rebeccamycin | secondary metabolism

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2006.06.004 Byline: Anne E. King (1), Michael A. Ackley (2), Carol E. Cass (3)(4), James D. Young (5), Stephen A. Baldwin (1) Abstract: Hydrophilic purine and pyrimidine nucleosides rely on specialized carrier proteins for their membrane translocation. The recent identification of two gene families encoding equilibrative and concentrative nucleoside transporters in mammals and other organisms has provided the essential breakthrough to a more complete understanding of the biological significance of nucleoside transport. Although nucleoside salvage is a primary function of these proteins, recent data indicate functions beyond metabolic recycling. In brain and spinal cord, for example, nucleoside transporters have the potential to regulate synaptic levels of neuroactive purines such as adenosine and, thereby, indirectly modulate physiological processes through G-protein-coupled purine P1 receptors. As described in this review, recent research indicates novel putative functions for CNS nucleoside transporters in sleep, arousal, drug and alcohol addiction, nociception and analgesia. The therapeutic use of nucleoside analogue drugs and nucleoside transporter inhibitors in viral, neoplastic, cardiovascular and infectious disease is also described. Author Affiliation: (1) Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK (2) Pfizer Global R&D, Sandwich, Kent CT13 9NJ, UK (3) Membrane Protein Research Group, Department of Oncology, University of Alberta, Alberta T6G 2H7, Canada (4) Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada (5) Membrane Protein Research Group, Department of Physiology, University of Alberta, Alberta T6G 2H7, Canada

ABSTRACT Phyllanthus niruri has been found to exhibit marked inhibitory effect on hepatitis B virus evident by its exhaustive utility in cases of chronic jaundice. However, till date, research has [...]

Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome.

Plants produce a variety of secondary metabolites, some of which are used as pharmaceuticals or are health promoting as food components. Recent genetic studies on the flavonoid biosynthetic pathway show that transcription factors are efficient new molecular tools for plant metabolic engineering to increase the production of valuable compounds. The use of specific transcription factors would avoid the time-consuming step of acquiring knowledge about all enzymatic steps of a poorly characterized biosynthetic pathway. Although genetic approaches are difficult for most plant species, promoter studies of single-pathway genes and T-DNA activation tagging are feasible alternative approaches for isolating transcription factors, as illustrated for terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Nearly 500 alkaloids have been detected in skin extracts from frogs of the family Dendrobatidae. All seem to have been sequestered unchanged into skin glands from alkaloid-containing arthropods. Ants, beetles, and millipedes seem to be the source of decahydroquinolines, certain izidines, coccinellines, and spiropyrrolizidine oximes. But the dietary source for a major group of frog-skin alkaloids, namely the pumiliotoxins (PTXs), alloPTXs, and homoPTXs, remained a mystery. In hopes of revealing an arthropod source for the PTX group, small arthropods were collected from eight different sites on a Panamanian island, where the dendrobatid frog (Dendrobates pumilio) was known to contain high levels of two PTXs. The mixed arthropod collections from several sites, each representing up to 20 arthropod taxa, contained PTX 307A and/or alloPTX 323B. In addition, the mixed arthropod collections from several sites contained a 5,8-disubstituted indolizidine (205A or 235B), representing another class of alkaloids previously unknown from an arthropod. An ant alkaloid, decahydroquinoline 195A, was detected in the mixed arthropod collections from several sites. Thus, 'combinatorial bioprospecting' demonstrates that further collection and analysis of individual taxa of leaf-litter arthropods should reveal the taxa from which PTXs, alloPTXs, and 5,8-disubstituted indolizidines are derived.

Smoothened (Smo), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Although the cellular mechanisms that regulate Smo function remain unclear, the direct inhibition of Smo by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved. Here we demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle in a manner that antagonizes cyclopamine action. In addition, we have identified four small molecules that directly inhibit Smo activity but are structurally distinct from cyclopamine. Functional and biochemical studies of these compounds provide evidence for the small molecule modulation of Smo through multiple mechanisms and yield insights into the physiological regulation of Smo activity. The mechanistic differences between the Smo antagonists may be useful in the therapeutic manipulation of Hh signaling.

Research has been conducted on the anhydrovinblastine. The preparation of this compound from the parent alkaloid leurosine has been carried out and discussed.

Research has been conducted on daphmanidin A, an alkaloid with the fused-hexacyclic skeleton. The isolation of this alkaloid from Daphniphyllum teijsmanii leaves has been carried out and the use of the spectroscopic data in investigating the alkaloid's structures is described.

Research has been conducted on the marine natural alkaloid haliclamine A. The synthesis of this alkaloid has been carried out via the use of thiophene chemistry and Zincke procedure in constructing primary amines of the bis-pyridinium macrocycle and the details are reported.

Research has been conducted on the quaternary indole alkaloids isolated from Aspidosperma subincanum Mart barks. The study of these alkaloids' structure and stereochemistry carried out via chemical means and spectrum analysis is presented.

Research has been conducted on the manzamine alkaloids' core ABC ring system. The synthesis of this system has been carried out starting from arecoline and the results indicate that this synthesis involves the Claisen rearrangement and a stereoselective azomethine ylide dipolar cycloaddition reaction.

Research has been conducted on the cis- and trans-disubstituted octahydroquinolinone ring systems with stereogenic centers. The stereodivergent synthesis of these systems has been carried out via the stereodivergent and stereoselective construction of trisubstituted piperidine ring systems using Michael addition reaction to enaminoesters.

Research has been conducted on the amaryllidaceae alkaloid buflavine and its regioisomer. The synthesis of these compounds which involves enecarbamate formation, hydrogenation and sequential Meyers' biaryl coupling is described.

Research has been conducted on the pumiliotoxin alkaloids. The preparation of these alkaloids has been carried out via the use of palladium(0)-catalyzed cross-coupling reactions between vinyl iodides and homoallylic organozincs and the results are reported.

Research has been conducted on the sebastianines A and B, alkaloids from the ascidian Cystodytes dellechiajei. The structure of these alkaloids has been investigated via the use of spectrum analysis and the results indicate that existence of the ring system for both compounds.