Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity

The reactive industrial chemicals acrylamide (AA) and N-methylolacrylamide (MAA) are neurotoxic and carcinogenic in animals, MAA showing a lower potency than AA. The causative agent in AA-induced carcinogenesis is assumed to be the epoxy metabolite, glycidamide (GA), which in contrast to AA gives rise to stable adducts to DNA. The causative agent in MAA induced carcinogenesis is so far not studied. The two AAs were studied in mice and rats using analysis of hemoglobin (Hb) adducts as a measure of in vivo doses and the in vivo micronucleus (MN) assay as an end-point for chromosome damage. Male CBA mice were treated by intraperitoneal (i.p.) injection of three different doses and male Sprague-Dawley rats with one dose of each AA. Identical adducts were monitored from the two AAs [N-(2-carbamoylethyl)valine] and the respective epoxide metabolites [N-(2-carbamoyl-2-hydroxyethyl)valine]. Per unit of administered amount, AA gives rise to higher (three to six times) Hb adduct levels than MAA in mice and rats. Mice exhibit, compared with rats, higher in vivo doses of the epoxy metabolites, indicating that AAs were more efficiently metabolized in the mice. In mouse the two AAs induced dose-dependent increases in both Hb adduct level and MN frequency in peripheral erythrocytes. Per unit of administered dose MAA showed only half the potency for inducing micronuclei compared with AA, although the MN frequency per unit of in vivo dose of measured epoxy metabolite was three times higher for MAA than for AA. No increase in MN frequency was observed in rat bone marrow erythrocytes, after treatment with either AA. This is compatible with a lower sensitivity of the rat than of the mouse to the carcinogenic action of these compounds.     

Cryptic speciation in Fusarium subglutinans

Fusarium isolates that form part of the Gibberella fujikuroi species complex have been classified using either a morphological, biological, or phylogenetic species concept. Problems with the taxonomy of Fusarium species in this complex are mostly experienced when the morphological and biological species concepts are applied. The most consistent identifications are obtained with the phylogenetic species concept. Results from recent studies have presented an example of discordance between the biological and phylogenetic species concepts, where a group of F. subglutinans sensu stricto isolates, i.e., isolates belonging to mating population E of the G. fujikuroi complex, could be sub-divided into more than one phylogenetic lineage. The aim of this study was to determine whether this sub-division represented species divergence or intraspecific diversity in F. subglutinans. For this purpose, we included 29 F. subglutinans isolates belonging to the E-mating population that were collected from either maize or teosinte, from a wide geographic range. DNA sequence data for six nuclear regions in each of these isolates were obtained and used in phylogenetic concordance analyses. These analyses revealed the presence of two major groups representing cryptic species in F. subglutinans. These cryptic species were further sub-divided into a number of smaller groups that appear to be reproductively isolated in nature. This suggests not only that the existing F. subglutinans populations are in the process of divergence, but also that each of the resulting lineages are undergoing separation into distinct taxa. These divergences did not appear to be linked to geographic origin, host, or phenotypic characters such as morphology.     

The Mitochondrial Genome of Acropora tenuis (Cnidaria; Scleractinia) Contains a Large Group I Intron and a Candidate Control Region

The complete nucleotide sequence of the mitochondrial genome of the coral Acropora tenuis has been determined. The 18,338 bp A. tenuis mitochondrial genome contains the standard metazoan complement of 13 protein-coding and two rRNA genes, but only the same two tRNA genes (trnM and trnW) as are present in the mtDNA of the sea anemone, Metridium senile. The A. tenuis nad5 gene is interrupted by a large group I intron which contains ten protein-coding genes and rns; M. senile has an intron at the same position but this contains only two protein-coding genes. Despite the large distance (about 11.5 kb) between the 5?-exon and 3?-exon boundaries, the A. tenuis nad5 gene is functional, as we were able to RT-PCR across the predicted intron splice site using total RNA from A. tenuis. As in M. senile, all of the genes in the A. tenuis mt genome have the same orientation, but their organization is completely different in these two zoantharians: The only common gene boundaries are those at each end of the group I intron and between trnM and rnl. Finally, we provide evidence that the rns-cox3 intergenic region in A. tenuis may correspond to the mitochondrial control region of higher animals. This region contains repetitive elements, and has the potential to form secondary structures of the type characteristic of vertebrate D-loops. Comparisons between a wide range of Acropora species showed that a long hairpin predicted in rns-cox3 is phylogenetically conserved, and allowed the tentative identification of conserved sequence blocks.     

Inhibition of endosomal/lysosomal degradation increases the infectivity of human immunodeficiency virus

Productive entry of human immunodeficiency virus type 1 (HIV-1) into a host cell is believed to proceed via fusion of the viral envelope with the host cell's plasma membrane. Interestingly, the majority of HIV-1 particles that bind to the cell surface are taken up by the host cell via endocytosis; however, this mode of internalization generally does not result in infection. Presumably, virus particles remain trapped in the endocytic pathway and are eventually degraded. Here, we demonstrate that treatment of cells with various pharmacological agents known to elevate the pH of endosomes and lysosomes allows HIV-1 to efficiently enter and infect the host cell. Pretreatment of cells with bafilomycin A1 results in up to a 50-fold increase in the infectivity of HIV-1(SF2). Similarly, pretreatment of target cells with amantadine, concanamycin A, concanamycin B, chloroquine, and ammonium chloride resulted in increases in HIV-1 infectivity ranging between 2- and 15-fold. Analysis of receptor and coreceptor expression, HIV-long terminal repeat (LTR) transactivation, and transduction with amphotropic-pseudotyped murine leukemia virus (MLV)-based vectors suggests that the increase in infectivity is not artifactual. The increased infectivity under these conditions appears to be due to the ability of HIV-1 and MLV particles to enter via the endocytic pathway when spared from degradation in the late endosomes and lysosomes. These results could have significant implications for the administration of current and future lysosmotropic agents to patients with HIV disease.     

Mechanistic studies of a retaining alpha-galactosyltransferase from Neisseria meningitidis

Lipopolysaccharyl alpha-galactosyltransferase from Neisseria meningitidis catalyzes the transfer of a galactosyl moiety from the activated donor UDP-Gal to glycoconjugates to yield an elongated saccharide product with net retention of anomeric configuration relative to the donor substrate. Through kinetic analyses in which the concentrations of both substrates are independently varied and through inhibition studies with dead-end analogues of both substrates and with the oligosaccharide product, we have demonstrated that this enzyme follows an ordered bi-bi kinetic mechanism. Various aspects of the chemical mechanism including the possible formation of a covalent glycosyl-enzyme intermediate were also probed using an assortment of strategies. While the results of these investigations were unable to clearly delineate the chemical mechanism of this enzyme, they provide important insights into the catalytic machinery surrounding the events involved in catalysis.     

Soy isoflavones' osteoprotective role in postmenopausal women: mechanism of action

Ovarian hormone deficiency is a major risk factor for osteoporosis. Current therapies emphasize the use of antiresorptive agents, such as estrogen, calcitonin, and bisphosphonates. These therapies are associated with certain risks and side effects making compliance a major obstacle. Recent findings suggest that a class of synthetic and naturally occurring compounds, selective estrogen receptor modulators, e.g. raloxifene and soy isoflavones can offer attractive alternatives. Evidence for bone-sparing effects of isoflavones relies mainly on animal findings supported by a limited number of human studies. These observations suggest that isoflavones exert their effects on bone by stimulating bone formation and at the same time suppressing bone resorption. However, the precise osteoprotective mechanism of isoflavones remains uncertain and awaiting further clarification. From a clinical point of view, larger and longer duration studies are warranted to enable us to draw clear conclusions in regards to the role of isoflavones on bone.     

Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry

Comparative analysis has long been utilized in biological research to interpret protein interactions in both drug na?ve versus drug challenged and normal versus diseased tissues. The technology of proteomics today allows researchers to provide insight into old and still open questions related to biological mechanisms while offering the opportunity to discover novel details in cellular lifecycles. Perhaps the most powerful way to execute these differential displays is in the combination of two-dimensional (2-D) gel electrophoresis and mass spectrometry. While these two techniques together are well suited for abundant and soluble proteins found in cells, rare proteins and integral membrane proteins are still problematic. Recently, a series of novel zwitterionic detergents has been reported in the literature that shows a substantial improvement in solubilizing integral membrane proteins. We show that the amidosulfobetaine, 4-octylbenzol amidosulfobetaine, is better than 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) at solubilizing both an ion channel and a G-protein coupled receptor (GPCR), while another amidosulfobetaine, myristic amidosulfobetaine (ASB-14), was better than CHAPS at solubilizing a GPCR. Neither membrane protein was visible after staining with colloidal Coomassie blue, silver nor Sypro Ruby. However, a comparison against a duplicate immunoblot allowed for the localization and identification of the ion channel from a 2-D gel by liquid chromatography-tandem mass spectrometry.     

Xenopus LSm proteins bind U8 snoRNA via an internal evolutionarily conserved octamer sequence

U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.     

A paradigm for therapy-induced microenvironmental changes in solid tumors leading to drug resistance

Intrinsic alterations in the tumor microenvironment are known to contribute to various forms of drug resistance. For example, tumor hypoxia, due to abnormal or sluggish blood flow within areas of solid tumors, can result in both microenvironment-mediated radiation and chemotherapeutic drug resistance. In contrast, acquired resistance to chemotherapy is generally considered to be the result of the gradual selection of mutant subpopulations having genetic mutations and biochemical alterations responsible for the resistant phenotype. Here we present a paradigm for therapyinduced microenvironment-mediated acquired drug resistance. It is based on the results showing that tumor cells appear to be heterogeneous in their relative dependence on adjacent tumor-associated vasculature for survival. Some tumor cells are highly vessel dependent, whereas some are significantly less so, and thus can survive in more hypoxic regions of tumors, distal from such tumor vessels. Hence, it is possible that variant tumor cells that are less vessel dependent may therefore be selected for over time by successful antiangiogenic drug therapies. This results in loss of response or attenuated responses to the therapy. Preliminary evidence is summarized in support of this hypothesis, using paired human colon cancer (HCT116) cell lines that contain two copies of either the wild-type or the disrupted p53 tumor suppressor gene. The mutant cells were found to be less responsive to antiangiogenic therapy, compared to the wild-type cells, and could be progressively selected for in mixed cell populations. Because p53 inactivation can lead to resistance to hypoxia-mediated apoptosis, the results suggest that a protracted and successful antiangiogenic therapy may create more hypoxic tumor microenvironments, thereby creating the necessary conditions to accelerate the selection of mutant tumor cells that are more adept in surviving and growing in such environments. As such, consideration might be given to the combined use of bioreductive hypoxic cell cytotoxic drugs and angiogenesis inhibitors to prolong the efficacy of antiangiogenic therapeutics.     

Role of conserved transmembrane cationic amino acids in the prostaglandin transporter PGT

The prostaglandin transporter "PGT" interacts electrostatically with its anionic substrate, based on inhibition by the disulfonic stilbenes [Chan, B. S. (1998) J. Biol. Chem. 273, 6689-6697], inhibition by the thiol-reactive anion sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570], and the requirement for a negatively charged 1-position carboxyl on the substrate [Itoh, S. (1996) Mol. Pharm. 50, 736-742]. Here we found that modification of positively charged residues on wild-type PGT by arginine- and lysine-specific reagents significantly inhibited transport. We previously found that the binding site of PGT is formed, at least in part, by its membrane-spanning segments [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570]. Three charged residues within predicted transmembrane spans (E78, R560, and K613) are conserved in PGT and in related transporters. Substitution of the anionic residue E78 (E78D and E78C) produced an essentially functional transporter, whereas substitution of the cationic residues with neutral residues (R560N and K613Q) resulted in poorly functional transporters. Immunoblotting revealed similar expression levels of wild-type and mutant transporters, and immunostaining indicated correct targeting. Conservative charge substitutions (R560K, K613R, and K613H) resulted in generally functional transporters. In contrast, R560N was nonfunctional, whereas the substrate affinity of K613G decreased greater than 50-fold. Conservative substitutions retaining the charge at position 613 (K613R and K613H) restored the substrate affinity, suggesting a direct role of K613 in substrate binding. Double-neutral mutants E78G/R560C and E78G/K613C were inactive, indicating that these residues are not simply charge-paired. Our results suggest that an arginine at position 560 is critical for maximal substrate translocation, and that a positively charged side chain at position 613 contributes to electrostatic binding of the anionic substrate.