Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Spatial clumping of food increases its monopolization and defense by convict cichlids, Cichlasoma nigrofasciatum

The hypothesis that resource monopolization and defense increaseas the spatial clumping of resources increases was tested usinggroups of three convict cichlids competing for 120 Daphnia magnaprey. Spatial clumping was manipulated by varying the distance(3, 20, or 40 cm) between three tubes through which the preyappeared. As predicted, monopolization of prey (percentage eatenby the dominant fish) and frequency of aggression (chases perminute) by dominant fish increased significantly as the distancebetween the tubes decreased. However, there was no evidenceof individual flexibility in the aggressiveness (percentageof conspecifics chased) of dominant fish across treatments.Differences among dominant fish in aggressiveness were positivelycorrelated with their ability to monopolize prey, but the strengthof the correlation decreased as the distance between the tubesincreased. Aggression appears to be a more effective mechanismof interference competition when resources are clumped thanwhen resources are dispersed.     

Amino acid sequence of photosystem I subunit IV from the cyanobacterium Synechocystis PCC 6803

We describe here the complete amino acid sequence of photosystem I subunit IV from Synechocystis 6803. The molecular mass of 8.0 kDa is lower than in higher plants and Chlamydomonas, due to the lack of a characteristic, proline-rich, N-terminal sequence. The remaining sequence exhibits a good conservation, with a hydrophilic and strongly basic N-tenninal head followed by two hydrophobic domains. There is no possibility of classical membrane-spanning alpha helices. This component is likely to be one of the most stroma accessible subunits of photosystem I.     

From the beginning to the present state of molecular microbial pathogenesis—A tribute to Pascale Cossart

The universe of Molecular Microbial Pathogenesis is filled with many female and male stars. But there are two particularly bright shining supernovae-like stars: the late Stanley Falkow and the very lively and creative Pascale Cossart. These two outstanding luminaries, surrounded by numerous planets, do not only belong to different scientific generations but their splendor also comes from very different scientific concepts. Stanley Falkow, often referred to as the ‘Father of Molecular Microbial Pathogenesis’, made many groundbreaking contributions to this field by addressing almost all important bacterial pathogens. Pascale Cossart, who could be called in analogy the ‘Queen of Modern Molecular Microbial Pathogenesis’ by combining the Microbiology and Cell Biology, concentrates in her similarly impressive scientific work essentially on a single bacterial species which she studied and still studies in great depth: the facultative intracellular bacterial pathogen Listeria monocytogenes—and the vast majority of her most prominent publications deals with this pathogen in almost all facets. It is certainly not an exaggeration to say that she together with her co-workers and collaborators developed this model bacterium into a paradigm among the intracellular bacterial pathogens.     

Essential oil variation and antioxidant capacity of Mentha pulegium populations and their relation to ecological factors

Mentha pulegium L. is an aromatic herb belonging to the Lamiaceae family, a wild plant which is distributed in different areas of Iran. In this research, we evaluated the variability of essential oil content and compositions of 12 M. pulegium populations. Essential oils were analyzed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) methods. The essential oils content varied from 0.22 to 1.63% w/w within different populations. Twenty-nine compounds were identified which represent 83.4–98.7% of the total essential oil. The most significant essential oil compounds among the studied population were identified using the principal components analysis (PCA-biplot). According to the PCA-biplot, the major compounds were pulegone (2.5–51.7%), menthone (0.2–25.3%), limonene (0.0–35.4%), 1,8-cineol (0.0–33.4%), piperitenone oxide (0.2–55.2%), and trans-piperitone epoxide (0.0–28.5%). Besides, hierarchical cluster analysis indicated that the studied populations were classified into two main clusters based on the essential oil components. The canonical correspondence analysis (CCA) indicated that some environmental factors could influence the phytochemical constituents as well as the antioxidant activity. The temperature and altitude were effective environmental factors with regards to 1–8 cineol, limonene and menthone content, while average rainfall was the most effective factor with respect to trans-piperitone epoxide, piperitenone oxide, and pulegone content. Our results consequently showed that environmental factors had a significant effect on the essential oil content and its antioxidant activity in M. pulegium populations.     

Cytokeratin immunoreactivity in lobular intraepithelial neoplasia.

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34betaE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34betaE12. We conclude that the reactivity seen in the cells of LIN with 34betaE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.     

Relationship of Aerial Broad Band Reflectance to Meloidogyne incognita Density in Cotton

Aerial images were obtained on 22 July 1999 and 4 August 2000 from five cotton sites infested with Meloidogyne incognita. Images contained three broad bands representing the green (500-600 nm), red (600-700 nm), and near-infrared (700-900 nm) spectrum. Soil samples were collected and assayed for nematodes in the fall at these sites. Sampling locations were identified from images, by locating the coordinates of a wide range of light intensity (measured as a digital number) for each single band, and combinations of bands. There was no single band or band combination in which reflectance consistently predicted M. incognita density. In all 10 site-year combinations, the minimum number of samples necessary to estimate M. incognita density within 25% of the population mean was greater when sampling by reflectance-based classes (3 to 4 per site) than sampling based on the entire site as one unit. Two sites were sampled at multiple times during the growing season. At these sites, there was no single time during the growing season optimal to take images for nematode sampling. Aerial infrared photography conducted during the growing season could not be used to accurately determine fall population densities of M. incognita.     

The phylogeny of Patrinieae sensu Graebner (Valerianaceae) revisited: additional evidence from ndhF sequence data

 DNA sequences of both 5′ and 3′ regions of the plastid ndhF gene were generated in order to study the position of Patrinia and Nardostachys, to check the potential paraphyletic nature of Patrinieae, and to evaluate the possible link between the tribe and Linnaeaceae. Parsimony analysis showed very strong support for Patrinia as sister to all members of Valerianaceae (including Nardostachys) and indicated the paraphyletic nature of the tribe Patrinieae. Additionally, trees were constructed from available rbcL data separately and supplemented with ndhF sequences. Topologies of these combined cladograms are in agreement with the ndhF phylogeny, suggesting that the traditionally circumscribed Patrinieae can no longer be recognized but must be considered as part of a basal grade in Valerianaceae. Parsimony analysis based on a morphological data set supported a monophyletic Patrinieae; combination with the molecular data showed a paraphyletic Patrinieae. Furthermore, the possible link between Patrinieae and Linnaeaceae is evaluated. Received July 12, 2001 Accepted February 25, 2002     

Field Hypothesis on the Self-regulation of Gene Expression

The mechanism of the self-regulation of gene expression in living cells is generally explained by considering complicated networks of key-lock relationships, and in fact there is a large body of evidence on a hugenumber of key-lock relationships. However, in the present article we stress that with the network hypothesis alone it is impossible to fully explain the mechanism of self-regulation in life. Recently, it has been established that individual giant DNA molecules, larger than several tens of kilo base pairs, undergo a large discrete transition in their higher-order structure. It has become clear that nonspecific weak interactions with various chemicals, suchas polyamines, small salts, ATP and RNA, cause on/off switching in the higher-order structure of DNA. Thus, the field parameters of the cellular environment should play important roles in the mechanism of self-regulation, in addition to networks of key and locks. This conformational transition induced by field parameters may be related to rigid on/off regulation, whereas key-lock relationships may be involved in a more flexible control of gene expression.