A study of 2,4,6-trinitrotoluene inhibition of benzo[a]pyrene uptake and activation in a microbial mutagenicity assay.

A number of in vitro and in vivo studies have determined that binary and complex mixtures may interact to produce a toxicity that could not be predicted based on the individual chemicals. The present study was conducted with a binary mixture of model compounds to investigate possible interactions affecting their mutagenicity. The compounds included Benzo[a]pyrene (BAP), a polycyclic aromatic hydrocarbon that is an indirect-acting mutagen of great environmental concern, and 2,4,6-Trinitrotoluene (TNT), a nitro-aromatic compound that is a direct-acting mutagen frequently found as a soil contaminant at munitions sites. This study indicated that a binary mixture of BAP and TNT failed to induce the positive mutagenic response in Salmonella typhimurium strain TA98 characteristic of either compound alone. Spectrofluorometric analysis of BAP, and kinetic analyses of 3HBAP uptake in the presence or absence of TNT using TA98 cells that were treated or untreated with activated rat liver microsomes were performed. In cells preloaded with BAP, cellular BAP fluorescence was rapidly suppressed in the presence of TNT. Mass spectroscopy of BAP and TNT mixtures revealed a number of products, believed to be the result of complexation and nitration, that may account for the antagonistic action of TNT on BAP-induced mutagenicity in TA98 cells. Further, kinetic studies indicated that TNT inhibited the incorporation of BAP into cells.

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43.

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mM 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mM 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1-3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mM 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mM octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3-5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 microM monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mM 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.

Cellular effects of misscheduled brlA, abaA, and wetA expression in Aspergillus nidulans.

Asexual sporulation in the fungus Aspergillus nidulans is controlled, in part, by a central regulatory pathway composed of the brlA, abaA, and wetA genes. The coding region of each of these genes was fused, in frame, to the threonine-inducible alcohol dehydrogenase promotor then stably incorporated into the A. nidulans genome at the argB locus. Misscheduled expression of each of these developmental genes interfered with normal growth and sporulation. Expression of brlA or abaA terminated vegetative growth and led to anomalous deposition of wall material at the hyphal apex and septa. brlA alone induced the formation of phialide-like structures and the production of conidia.

Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation.

The Aspergillus nidulans CAN41 transcription unit is activated by the brlA regulatory gene early during development of the asexual reproductive apparatus, the conidiophore. Disruption of CAN41 results in a novel mutant phenotype in which conidiophore cells and spores lack an external wall layer, the rodlet layer, making them less hydrophobic than in the wild type and leading to inefficient spore dispersal. The rodletless mutation defines a new locus on chromosome III, rodA. rodA encodes a small, moderately hydrophobic polypeptide containing 8 cysteines arranged in a pattern similar to that observed in three hydrophobic cell wall proteins from the Holobasidiomycete Schizophyllum commune. We propose that the Aspergillus and Schizophyllum 8-cysteine polypeptides define a class of secreted, hydrophobic, fungal cell wall proteins that are important in the formation and function of aerial structures such as conidiophores and mushrooms.

abaA controls phialide differentiation in Aspergillus nidulans.

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. Loss of function mutations in the abacus A (abaA) regulatory locus result in formation of aberrant conidiophores that fail to produce conidia. Wild-type conidiophores form two tiers of sterigmata. The first tier, metulae, divide to produce the second tier, phialides. Phialides are sporogenous cells that produce conidia through a specialized apical budding process. We have examined conidiophore development in an abaA- strain at the ultrastructural level. The results showed that in the mutant metulae produce supernumerary tiers of cells with metula-like, rather than phialide-like, properties. Temperature shift experiments with an abaA14ts strain demonstrated that abaA+ function induced phialide formation by the aberrant abacus cells and was continuously required for maintenance of phialide function. In the absence of abaA+ activity, metulae simply proliferated and later developmental steps never occurred. We conclude that abaA+ directs the differentiation of phialides and is continuously required for maintenance of their function.

Conidium differentiation in Aspergillus nidulans wild-type and wet-white (wetA) mutant strains.

Conidium (asexual spore) differentiation in wild-type and the wet-white (wetA) mutant of Aspergillus nidulans was compared in intact chains of successively older conidia. Carbohydrate cytochemistry helped define three stages (Stages I, II, and III) of wild-type conidium maturation on the basis of changes in the ultrastructure and composition of the conidium wall. Conidia of the wetA6 mutant strain formed normally but failed to mature during Stages II and III. Specifically, the inner wall layer of wetA6 conidia did not condense during Stage II and two wall layers that stained for carbohydrates did not form during the transition to Stage III. Concomitantly, wetA6 conidia formed large cytoplasmic vacuoles and underwent lysis. The wetA gene appears to have a conidium-specific function for the modification of the conidium wall during Stages II and III. These modifications of the conidium wall are essential for the stability of mature, dormant conidia.