Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.     

Spatial pattern of allozyme variation in a contact zone of Pinus Ponderosa and P. arizonica (Pinaceae)

The spatial distribution of genotypes for nine polymorphic allozyme loci was examined in a contact zone between Pinus ponderosa var. scopulorum and another tree regarded as either a separate species, Pinus arizonica, or variety, Pinus ponderosa var. arizonica, in southern Arizona. Previous work had identified a steep elevational cline for a key taxonomic trait, number of leaf-needles per fascicle, on the south slope of Mt. Lemmon. The present results indicate that the taxa are not fully interbreeding in this contact zone, because allozyme genotypes are considerably more spatially structured than expected for the dispersal characteristics of pines. The amount of spatial differentiation is also much less than that observed for needle number. It appears that this is due to the lack of differentiation for allozyme gene frequencies for the two types of trees, which is further evidenced by analysis of samples from two other populations away from the contact zone. It is likely that if the two taxa were isolated in the past, it was not for long enough nor complete enough to allow mutation-drift to create substantial differentiation between them. Another possible explanation is that introgression after recontact is so advanced that any differences have been erased throughout the Santa Catalina mountain range.     

Under how wide a set of conditions will nonrandom mating occur in Raphanus sativus (Brassicaceae)?

Studies of the weedy annual Raphanus sativus have demonstrated that nonrandom mating, a prerequisite for sexual selection, can occur in greenhouse plants. To determine whether this nonrandom mating pattern can occur under a wide range of conditions, including conditions that might occur in the field, we considered variation in both maternal condition and pollen load size. Maternal condition was varied by altering the watering regime. Pollen load size was varied from approximately 26 to 343 pollen grains per stigma. At the smallest pollen load size, patterns of seed paternity were altered in two of the three pollen donor pairs; seed paternity became more equal among donors. For one of three pollen donor pairs, seed paternity was more divergent among donors on stressed maternal plants. Finally, for one pollen donor pair, rank order of pollen donor performance changed from the medium to the small pollen loads on stressed vs. control maternal plants. Thus, some field conditions may alter patterns of nonrandom mating in wild radish.     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

Chlorophyll b to chlorophyll a energy transfer kinetics in the CP29 antenna complex: a comparative femtosecond absorption study between native and reconstituted proteins

The energy transfer processes between Chls b and Chls a have been studied in the minor antenna complex CP29 by femtosecond transient absorption spectroscopy. Two samples were analyzed: the native CP29, purified from higher plants, and the recombinant one, reconstituted in vitro with the full pigment complement. The measurements indicate that the transfer kinetics in the two samples are virtually identical, confirming that the reconstituted CP29 has the same spectroscopic properties as the native one. In particular, three lifetimes (150 fs, 1.2 ps, and 5-6 ps) were identified for Chl b-652 nm to Chl a energy transfer and at least one for Chl b-640 nm (600-800 fs). Considering that the complexes bind two Chls b per polypeptide, the observation of more than two lifetimes for the Chl b to Chl a energy transfer, in both samples, clearly indicates the presence of the so-called mixed Chl binding sites--sites which are not selective for Chl a or Chl b, but can accommodate either species. The kinetic components and spectra are assigned to specific Chl binding sites in the complex, which provides further information on the structural organization.     

Structure-based design of novel nonpeptide inhibitors of the Src SH2 domain:phosphotyrosine mimetics exploiting multifunctional group replacement chemistry

A series of novel nonpeptide inhibitors of the pp60(c-Src) (Src) SH2 domain is described that exploit multifunctional group replacement of the phenylphosphate moiety of phosphotyrosine (pTyr). Relative to an x-ray structure of citrate complexed to the pTyr binding site of the Src SH2 domain, these nonpeptide ligands illustrate the systematic replacement of the phosphate group by multiple nonhydrolyzable, mono- or dianionic functionalities. Specifically, several phenylalanine (Phe) analogs incorporating key 4' and 3' substituents were synthesized and incorporated into a bicyclic benzamide template previously reported (W. C. Shakespeare et al., Proceedings of the National Academy of Science USA, 2000, Vol. 97, pp. 9373-9378). These pTyr mimetics included 4',3'-diphosphono-Phe (Dpp), 4',3'-dicarboxymethyloxy-Phe (Dcp), and 4'-phosphono-3'-carboxymethyloxy-Phe (Cpp). Noteworthy were nonpeptide inhibitors 8-11 that were 5- to 10-fold more potent than the cognate tetrapeptide ligand Ac-pTyr-Glu-Glu-Ile-NH(2) in binding to the Src SH2 domain.     

Effects of acid-induced esophagitis on esophageal smooth muscle

Acid-induced esophagitis is associated with sustained longitudinal smooth muscle (LSM) contraction and consequent esophageal shortening. In addition, LSM strips from opossums with esophagitis are hyper-responsive, while the circular smooth muscle (CSM) contractility is impaired. To determine the origin of these changes, studies were performed on esophageal smooth muscle cells isolated from opossum esophagi perfused intraluminally on 3 consecutive days with either saline (control; n = 8) or HCl (n = 9). CSM and LSM cells, obtained by enzymatic digestion, were exposed to various concentrations of carbachol (CCh) and fixed. CCh induced concentration-dependent contraction of both LSM and CSM cells. CCh-induced LSM cell contraction was not different between control and esophagitis animals; however, there was marked attenuation in the CCh-induced contraction of CSM cells from esophagitis animals. Morphological studies revealed significant hypertrophy of the CSM cells. These findings suggest that impaired CSM contractility can be attributed at least in part to alterations to the CSM cell itself. In contrast, hyper-contractility demonstrated in LSM strips is likely related to factors in the surrounding tissue.     

Genetic structure of age classes in Camellia japonica (Theaceae)

Camellia japonica L. (Theaceae), an insect- and bird-pollinated, broad-leaved evergreen tree, is widely distributed in Japan and the southern Korean peninsula. The species has a relatively even age distribution within populations, which may influence the spatial genetic structure of different age classes relative to species with typical L-shaped age distributions. To determine whether the internal spatial genetic structure found in seedlings and young individuals carries over into adults, we used allozyme loci, F-statistics, spatial autocorrelation statistics (Moran's I), and coancestry measures to examine changes in genetic structure among seven age classes in a population (60-m x 100-m area) in southern Korea. In seedlings, weak but significant positive values of Moran's I-statistics and coancestry measures were found for distances less than 14 m, which is consistent with a mechanism of limited seed dispersal combined with overlapping seed shadows. This spatial structure, however, dissipates in older age classes, and in adults genetic variation has an essentially random spatial distribution. Morisita's index of dispersion of individuals in each age class showed that seedlings and juveniles are more highly clustered than are older individuals. These results suggest that self-thinning changes the spatial relationships of individuals, and thus genotypes. A multilocus estimate of FST (0.008) shows a small but statistically significant difference in allele frequencies among age classes. In summary, intrapopulation genetic structure within and among age classes of C. japonica was significant but weak. Despite presumably limited seed dispersal, weak spatial genetic structure in juveniles suggests overlapping seed shadows followed by self-thinning during recruitment. The present study also demonstrates that studies of spatial genetic structure focusing on limited numbers of generations may not be sufficient to reveal the entire picture of genetic structure in populations with overlapping generations.     

Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

The Escherichia coli twin-arginine translocase: conserved residues of TatA and TatB family components involved in protein transport

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. In this report we have studied the functions of conserved residues within the structurally related TatA and TatB proteins. Our results demonstrate that there are two regions within each protein of high sequence conservation that are critical for efficient Tat translocase function. The first region is the interdomain hinge between the transmembrane and the amphipathic alpha-helices of TatA and TatB proteins. The second region is within the amphipathic helices of TatA and TatB. In particular an invariant phenylalanine residue within TatA proteins is essential for activity, whereas a string of glutamic acid residues on the same face of the amphipathic helix of TatB is important for function.