Stereoselective Formation and Metabolism of 4-Hydroxy-Retinoic Acid Enantiomers by Cytochrome P450 Enzymes

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.     

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-β (TGF-β) through the intrinsic pathway and to tumor necrosis factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.     

An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the "gatekeeper" residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3(M385G) can also specifically utilize N(6)-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.     

The chemokine receptor CCR2 is not required for successful initiation of labor in mice

Chemokine-driven neutrophil and monocyte recruitment into the uterus and cervix has been proposed to initiate labor. Chemokines that bind CXCR2 direct neutrophil migration and are induced during labor in humans. The chemokine CCL2, induced in the uterus by endocrine and mechanical signals, has been proposed to drive CCR2-dependent monocyte homing to the uterus to contribute to the initiation of labor. However, no direct evidence indicates that chemokines or their receptors play indispensable roles in labor-associated inflammation, and the impact of leukocyte infiltration on labor is unclear. Here, we have quantified expression of the principal monocyte- and neutrophil-attracting chemokines in the uteri of term pregnant (Day 18) and laboring wild-type mice. None of the neutrophil attractants we assayed were up-regulated with labor. Strikingly, however, Ccl2 was markedly increased, and this was concomitant with increased expression of Ccr2, the myeloid marker Itgam (also known as Cd11b), the monocyte/macrophage marker Emr1 (also known as F4/80). Moreover, in CCR2-deficient mice, this labor-associated increase in Itgam and Emr1 was not seen, consistent with the monocyte-trafficking defects that exist in these animals. Nonetheless, laboring CCR2-deficient and wild-type uteri showed similarly enhanced expression of the myometrial activation markers Gja1 and Oxtr (commonly known as connexin 43 and oxytocin receptor, respectively), and CCR2-deficient mice had gestation lengths, litter sizes, and fetal and placental weights no different from those of their wild-type counterparts. Thus, whereas labor is associated with an inflammatory response in gestational tissues, CCR2-dependent leukocyte recruitment into the mouse uterus is dispensable for the initiation of successful labor.     

The population genetics of Quechuas, the largest native South American group: autosomal sequences, SNPs, and microsatellites evidence high level of diversity

Elucidating the pattern of genetic diversity for non-European populations is necessary to make the benefits of human genetics research available to individuals from these groups. In the era of large human genomic initiatives, Native American populations have been neglected, in particular, the Quechua, the largest South Amerindian group settled along the Andes. We characterized the genetic diversity of a Quechua population in a global setting, using autosomal noncoding sequences (nine unlinked loci for a total of 16 kb), 351 unlinked SNPs and 678 microsatellites and tested predictions of the model of the evolution of Native Americans proposed by (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496). European admixture is <5% and African ancestry is barely detectable in the studied population. The largest genetic distances were between African versus Quechua or Melanesian populations, which is concordant with the African origin of modern humans and the fact that South America was the last part of the world to be peopled. The diversity in the Quechua population is comparable with that of Eurasian populations, and the allele frequency spectrum based on resequencing data does not reflect a reduction in the proportion of rare alleles. Thus, the Quechua population is a large reservoir of common and rare genetic variants of South Amerindians. These results are consistent with and complement our evolutionary model of South Amerindians (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496), proposed based on Y-chromosome data, which predicts high genomic diversity due to the high level of gene flow between Andean populations and their long-term effective population size.     

Flow preferences of individual blacknose dace (Rhinichthys atratulus); influence of swimming ability and environmental history

Fish from lotic environments generally have a variety of flow velocities available to them in their immediate environment. Other than prey availability or predator presence, little is known about what factors determine where in this mosaic of flows an individual fish will choose to locate. Since individuals of a species can have substantially different swimming abilities, and interspecific differences in flow velocity selection have been related to differential swimming abilities, one possibility is that an animal??s physical condition constrains the flow environments it chooses to occupy. Additionally, since the flow in an animal??s environment can contribute to swimming ability, there could also be environmental control over flow selection behavior. This study examined whether flow velocity selection by individual blacknose dace (Rhinichthys atratulus) is a repeatable trait in the laboratory, and whether it is a function of either the animal??s swimming ability or the magnitude of flow in their home stream reach. Blacknose dace from two populations, collected from each of two separate reaches with substantially different flows from within their home streams, exhibited significantly repeatable flow velocity selection over the course of 1?day in the laboratory. The flow velocity selected by the fish varied significantly among individual dace. Some of this variance was accounted for by fish from the slower stream reaches choosing significantly faster flows than did those from faster reaches. There were no significant differences in flow selection behavior between populations. There was also no relationship between sprinting ability and the flow velocity selected by a fish.     

Uranium accumulation by aquatic plants from uranium-contaminated water in Central Portugal

Several species of plants have developed a tolerance to metal that enables them to survive in metal contaminated and polluted sites. Some of these aquatic plants have been reported to accumulate significant amounts of specific trace elements and are, therefore, useful for phytofiltration. This work focuses the potential of aquatic plants for the phytofiltration of uranium (U) from contaminated water. We observed that Callitriche stagnalis, Lemna minor, and Fontinalis antipyretica, which grow in the uraniferous geochemical province of Central Portugal, have been able to accumulate significant amounts of U. The highest concentration of U was found in Callitriche stagnalis (1948.41 mg/kg DW), Fontinalis antipyretica (234.79 mg/kg DW), and Lemna minor (52.98 mg/kg DW). These results indicate their potential for the phytofiltration of U through constructed treatment wetlands or by introducing these plants into natural water bodies in the uraniferous province of Central Portugal.     

Stressed but stable: canopy loss decreased species synchrony and metabolic variability in an intertidal hard-bottom community

The temporal stability of aggregate community properties depends on the dynamics of the component species. Since species growth can compensate for the decline of other species, synchronous species dynamics can maintain stability (i.e. invariability) in aggregate properties such as community abundance and metabolism. In field experiments we tested the separate and interactive effects of two stressors associated with storminess--loss of a canopy-forming species and mechanical disturbances--on species synchrony and community respiration of intertidal hard-bottom communities on Helgoland Island, NE Atlantic. Treatments consisted of regular removal of the canopy-forming seaweed Fucus serratus and a mechanical disturbance applied once at the onset of the experiment in March 2006. The level of synchrony in species abundances was assessed from estimates of species percentage cover every three months until September 2007. Experiments at two sites consistently showed that canopy loss significantly reduced species synchrony. Mechanical disturbance had neither separate nor interactive effects on species synchrony. Accordingly, in situ measurements of CO(2)-fluxes showed that canopy loss, but not mechanical disturbances, significantly reduced net primary productivity and temporal variation in community respiration during emersion periods. Our results support the idea that compensatory dynamics may stabilise aggregate properties. They further suggest that the ecological consequences of the loss of a single structurally important species may be stronger than those derived from smaller-scale mechanical disturbances in natural ecosystems.