Rat liver microsomal progesterone metabolism: evidence for differential troleandomycin and pregnenolone 16 alpha-carbonitrile inductive effects in the cytochrome P-450 III family

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.     

Nanosecond fluorescence of tryptophans in cytochrome P-450scc (CYP11A1): effect of substrate binding.

Fluorescence of eight tryptophan residues in cytochrome P-450scc with bound endogenous cholesterol could be fitted with a two component model: a single exponential and a "top-hat" distribution of lifetimes as the second component. The short-lived component (tau 1 about 700 ps) does not change significantly upon binding of substrate (22R-hydroxycholesterol). The parameters of the long-lived component (central lifetime tau m about 3.4 ns) change upon binding of carbon monoxide and substrate. 22R-hydroxycholesterol binding broadens the distribution of the long-lived component; that is the heterogeneity of the Trp environment is increased when this substrate displaces the endogenous cholesterol.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Age and sex-dependent effects of landscape cover and trapping on the spatial genetic structure of the stone marten (<Emphasis Type="Italic">Martes foina</Emphasis>)

Maintenance of genetic variation is of critical importance for wild populations since low levels limit the species’ ability to respond to different threats (diseases, predators, environmental changes) in both the long and the short term. Human activities could impact the genetic variation of wild species in multiple ways, including via fragmentation and harvesting. We used an individual-based landscape genetics approach to describe the impact of landscape elements and trapping pressure on the spatial genetic structure of a large sample (n = 370) of the stone marten (Martes foina) in central-eastern France (Bresse). An analysis of isolation-by-resistance using a causal modeling approach showed an influence of landscape cover and/or trapping pressure on gene flow according to age and sex class. Overall, the connectivity in the study area is provided mainly by vegetation cover, while roads and open areas partially impede it. Unexpectedly for this “urban adapter” species, buildings could reduce gene flow. We also emphasized the sex-dependent effect of trapping on gene flow. Genetic differentiation in males was influenced by trapping pressure and landscape structure while only the latter influenced genetic differentiation in females. A stronger isolation by distance in males than in females suggested that at the scale of the study area, males are more exposed to trapping pressure, which reduces effective dispersal. Overall, the combination of both landscape and trapping costs might create an ‘ecological trap’ that could disrupt gene flow, leading to a north–south division in the study area.     

Engineering high α‐amylase levels in wheat grain lowers Falling Number but improves baking properties

Late maturity α‐amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α‐amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α‐amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS‐affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α‐amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100‐fold higher than the wild‐type resulted in low FN similar to those seen in LMA‐ or PHS‐affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small‐scale 10‐g baking tests. In these small‐scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard‐related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α‐amylase on end product quality.     

Cellular localisation of a water-soluble fullerene derivative

Fullerenes are a new class of compounds with potential uses in biology and medicine and many insights were made in the knowledge of their interaction with various biological systems. However, their interaction with organised living systems as well as the site of their potential action remains unclear. In this work, we have demonstrated that a fullerene derivative could cross the external cellular membrane and it localises preferentially to the mitochondria. We propose that our finding supports the potential use of fullerenes as drug delivery agents as their structure mimics that of clathrin known to mediate endocytosis.     

Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength

High-molecular-weight glutenin subunits (HMW-GS) are important determinants of wheat dough quality as they confer visco-elastic properties to the dough required for mixing and baking performance. With this important role, the HMW-GS alleles are key markers in breeding programs. In this work, we present the use of a PCR marker initially designed to discriminate Glu1 Bx7 and Glu1 Bx17 HMW-GS. It was discovered that this marker also differentiated two alleles, originally both scored as Glu1 Bx7, present in the wheat lines CD87 and Katepwa respectively, by a size polymorphism of 18 bp. The marker was scored across a segregating doubled-haploid (DH) population (CD87 × Katepwa) containing 156 individual lines and grown at two sites. Within this population, the marker differentiated lines showing the over-expression of the Glu1 Bx7 subunit (indicated by the larger PCR fragment), derived from the CD87 parent, relative to lines showing the normal expression of the Glu1 Bx7 subunit, derived from the Katepwa parent. DNA sequence analysis showed that the observed size polymorphism was due to an 18 bp insertion/deletion event at the C-terminal end of the central repetitive domain of the Glu1 Bx 7 coding sequence, which resulted in an extra copy of the hexapeptide sequence QPGQGQ in the deduced amino-acid sequence of Bx7 from CD87. When the DH population was analysed using this novel Bx7 PCR marker, SDS PAGE and RP HPLC, there was perfect correlation between the Bx7 PCR marker results and the expression level of Bx7. This differentiation of the population was confirmed by both SDS-PAGE and RP-HPLC. The functional significance of this marker was assessed by measuring key dough properties of the 156 DH lines. A strong association was shown between lines with an over expression of Bx7 and high dough strength. Furthermore, the data demonstrated that there was an additional impact of Glu-D1 alleles on dough properties, with lines containing both over-expressed Bx7 and Glu-D1 5+10 having the highest levels of dough strength. However, there was no statistically significant epistatic interaction between Glu-B1 and Glu-D1 loci.Communicated by J.W. Snape     

The interaction of TOGp with microtubules and tubulin

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.     

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.     

Where to sleep in a rural landscape? A comparative study of resting sites pattern in two syntopic Martes species

The niche‐complementarity hypothesis predicts that two sympatric species must differ in their requirements for one of the three main ecological dimensions (i.e. habitat use, diet, and activity time) to coexist. European pine marten Martes martes and stone marten M. foina are syntopic medium‐sized mustelids with very similar morphology and ecology for which resting sites are a key resource. To better understand how these species coexist, we investigated whether key features of their resting site pattern (number of resting sites, area over which they are distributed, main habitat type used for resting) differed. We used diurnal telemetry to identify resident individuals (e.g. spatially stable individuals over time) and to locate them during resting periods in a fragmented forested area in France. Stone marten used fewer resting sites distributed over a smaller surface area than pine marten. Most stone marten resting sites were located in open habitat (83%) in the proximity of human habitations, whereas pine martens rested almost exclusively in forest (98%). Sex, age, and season explained some variability in both the number of resting sites and the probability of resting within forested habitat for stone marten but not pine marten. The area covered by resting sites was larger in males than in females, but age modulated this difference in an opposite way for the two species. Such a pattern was expected given the intra‐sexual territoriality and the reproductive phenology of these species. Overall, stone marten showed higher inter‐individual variability in resting site pattern than pine marten. The particular pattern observed in subadult male stone martens during summer (increase in resting site surface area and in the probability to rest in forest) may reflect an attempt to settle in forests, and we discuss these implications in the context of interspecific competition.