Influence of lipophilicity on the interactions of hydroxy stilbenes with cytochrome P450 3A4

Resveratrol, a polyphenol found in red wine, was recently suggested to act as an irreversible, mechanism-based inactivator of cytochrome P450 3A4 (CYP3A4). We found a significant inhibition of human CYP3A4-dependent transformation of cyclosporine by resveratrol, with IC50 = 4.5 microM. We studied the kinetics parameters of CYP3A4 transformation of resveratrol and structurally related, naturally occurring stilbenes. Resveratrol, piceid, resveratroloside, 5,4'-dihydroxy-3-O-methoxystilbene, and 5,3-dihydroxy-4'-O-methoxystilbene were all shown to inhibit hydroxylation of testosterone by CYP3A4. Both methoxy-stilbenes had lower IC50 values, ranging from 0.43 to 0.47 microM, suggesting that lipophilicity rather than number or positions of free hydroxyls (3,5 or 5,4') determines the CYP3A4 inhibition capacity of polyphenols. In line with these findings, both glucosyl-stilbenes were found to be weak inhibitors of CYP3A4. The affinity of the enzyme towards methoxy-stilbenes, expressed as apparent Km, was indeed higher than those for the parent resveratrol and its glucosides, in CYP3A4 reaction mixtures. Vmax values were similar, except for piceid. These results support the role of lipophilicity in the interaction of polyphenols with CYP3A4. It is suggested that selective structural modifications of substrates add significantly to knowledge acquired through molecular modifications of the enzyme.     

Co-operation despite disagreement: from politics to healthcare

Political interaction among citizens who hold opposing moral views commonly requires reaching beyond toleration, toward actual co-operation with policies one opposes. On the more personal level, however, regarding (e.g.) interactions between healthcare providers and patients, several authors emphasise the importance of preserving integrity. But those who oppose any 'complicity in evil' often wrongly conflate instances in which the other's position is (and should be) totally rejected with instances of legitimate, although deep, disagreement. Starting with a striking example from the context of a particular tradition, I argue generally that in the latter sort of disagreements, talk of 'complicity' should be largely replaced with a more co-operative moral stance, grounded in a pluralistic framework. Co-operation Despite Disagreement (CDD) should be sought either for institutional reasons – akin to the political – or for relational reasons. CDD involves sharing another's perspective and sometimes calls for adopting another's moral judgements in preference to one's own. I seek to identify some of the conditions and circumstances that would justify such a shift, particularly in scenarios involving assistance, such as physician-assisted suicide (PAS) or the role of an anaesthesiologist in abortion. This discussion is meant to provide examples of the kind of second-order reasons appropriate for determining the terms for CDD – in distinction from first-order considerations (e.g., the much-contested 'active/passive' distinction) which are likely to be the subject of the initial disagreement and hence cannot serve to resolve it.     

Effect of Manganese on Lignin Degradation by Pleurotus ostreatus during Solid-State Fermentation

Lignin degradation by Pleurotus ostreatus was studied under solid-state fermentation (SSF) in chemically defined medium containing various levels of Mn. Degradation of [¹⁴C]lignin prepared from cotton branches to soluble products, as well as its mineralization to ¹⁴CO₂, was enhanced by the addition of Mn. The effect of malonate on lignin mineralization was most marked during the first 10 days of SSF, in a treatment amended with 73 μM Mn. A high concentration of Mn (4.5 mM) caused inhibition of both fungal growth and mineralization rates during the first 2 weeks of incubation. Addition of malonate reversed this effect because of chelation of Mn. Mn was found to precipitate in all treatments, with or without the addition of malonate. α-Keto-γ-methiolbutyric acid cleavage to ethylene, an indication of ^. OH production, was observed as early as 3 days of incubation in all treatments.     

Multiplexing schemes for generic SNP genotyping assays.

Association studies in populations relate genomic variation among individuals with medical condition. Key to these studies is the development of efficient and affordable genotyping techniques. Generic genotyping assays are independent of the target SNPs and offer great flexibility in the genotyping process. Efficient use of such assays calls for identifying sets of SNPs that can be interrogated in parallel under constraints imposed by the genotyping technology. In this paper, we study problems arising in the design of genotyping experiments using generic assays. Our problem formulation deals with two main factors that affect the genotyping cost: the number of assays used and the number of PCR reactions required for sample preparation. We prove that the resulting computational problems are hard, but provide approximate and heuristic solutions to these problems. Our algorithmic approach is based on recasting the multiplexing problems as partitioning and packing problems on a bipartite graph. We tested our algorithmic approaches on an extensive collection of synthetic data and on data that was simulated using real SNP sequences. Our results show that the algorithms achieve near-optimal designs in many cases and demonstrate the applicability of generic assays to SNP genotyping.     

Recombinant perciform GnRH-R activates different signaling pathways in fish and mammalian heterologous cell lines

Perciforms have three forms of gonadotropin-releasing hormone (GnRH) in their brain. All three GnRHs are potent secretogogues for luteinizing hormone (LH) from the pituitary. The pivotal role of GnRH-R-GnRH interactions in reproductive homeostasis is well established; however, there is a paucity of information on how a GnRH-R responds to the three endogenous GnRH forms in a perciform species. In this study, a recombinant pituitary GnRH-R from striped bass (stb) was expressed in a mammalian cell line (COS-7) and a fish cell line (CHSE-214). Activation of the signaling pathways was monitored by reporter gene (luciferase) based assays, which were specific for cAMP-PKA or Ca 2+/calmodulin kinase (activated via c-fos promoter) signaling pathways. The stbGnRH-R expressed in two different cell lines triggered different downstream signaling in response to the treatments with chicken (c) GnRH II. Interestingly, when endogenous GnRHs were used in combinations, the luciferase activity was significantly attenuated in transfected CHSE-214 cells.     

An antagonist to GnRH in the control of reproduction in teleost fish: dopaminergic inhibition. Ancestral origin and differential conservation within vertebrates?

In mammals, the neurohormonal control of the pituitary gonadotropes is provided by the gonadoliberin GnRH. Several studies on teleost fish indicate that a single positive control by GnRH is not a general rule among vertebrates. Peter and colleagues presented the first evidence of an inhibitory neurohormonal factor, "GRIF" (gonadotropin-release inhibiting factor). They induced a preovulatory LH surge by injuring particular brain areas in the goldfish. Subsequent in vivo and in vitro studies identified dopamine as GRIF, and neuroanatomical investigations have demonstrated that dopaminergic neurones in the anterior preoptic area projecting to the pituitary represent the anatomical substrate for GRIF activity. An inhibitory role of dopamine on the control of LH and ovulation/spermiation has been evidenced in many adult teleosts, including its implications for aquaculture. However, dopamine does not play an inhibitory role in all adult teleosts. As regards the early stages of gametogenesis and especially the control of puberty, a role for dopamine has been suggested or rejected depending on species. The European eel has a unique life cycle with a long prepubertal stage, which has made it a useful model to demonstrate the key-role of dopamine in the control of puberty. Data from tetrapods suggest that the role of dopamine as a GRIF is not restricted to the teleosts, but that it may have an ancient evolutionary origin, and has been differentially conserved throughout vertebrate evolution.     

Use of GnRHa-delivery systems for the control of reproduction in fish

The most commonly observed reproductivedysfunctions in cultured fish are theunpredictability of final oocyte maturation(FOM) in females, and the diminished volume andquality of sperm in males. Gonadotropin-releasing hormone agonists (GnRHa)have been used extensively in order tostimulate the release of pituitary luteinizinghormone (LH) required to induce FOM, ovulationand spermiation. Because multiple hormonaltreatments are often necessary for a successfulresponse, fish must be monitored and handledextensively, which is labor intensive,stressful to the fish and can often result inbroodstock mortalities. To ameliorate thisproblem, sustained-release delivery systems forGnRHa have been developed during the last twodecades and have been increasingly applied incontrolling reproduction of a variety ofcultured fish. Solid implants of cholesterolor poly[ethylene-vinyl acetate], andbiodegradable microspheres ofpoly[lactide-glycolide] or poly[fatty aciddimer-sebasic acid] release GnRHa for a periodof time (from a few days to many weeks.) GnRHa-delivery systems do not causedesensitization of the pituitary gonadotrophsin fish, and by stimulating a sustainedelevation of plasma LH they induce the naturalprogression of plasma steroid increasesassociated with FOM and spermiation. Thismethod has been used with very encouragingresults in females of more than 40 culturedspecies and has been effective in inducing FOM,ovulation or spawning in fish with synchronous,group-synchronous and asynchronous ovariandevelopment. In males, GnRHa-delivery systemshave been tested in more than 20 species,producing significant increases in miltproduction for up to 5 weeks. Future researchshould focus on the optimization of thistechnology in terms of (a) using the mostpotent GnRHa, (b) identifying the mostappropriate GnRHa release kinetics according tothe reproductive biology of different species,and (c) determining minimum effective doses. Developments in these areas will greatlyenhance the effectiveness and efficiency ofGnRHa-delivery systems, while at the same timereducing their cost thus making them moreaffordable to the aquaculture industry.