Benzene induces gene-duplicating but not gene-inactivating mutations at the glycophorin A locus in exposed humans.

Occupational exposure to benzene is known to cause leukemia, but the mechanism remains unclear. Unlike most other carcinogens, benzene and its metabolites are weakly or nonmutagenic in most simple gene mutation assays. Benzene and its metabolites do, however, produce chromosomal damage in a variety of systems. Here, we have used the glycophorin A (GPA) gene loss mutation assay to evaluate the nature of DNA damage produced by benzene in 24 workers heavily exposed to benzene and 23 matched control individuals in Shanghai, China. The GPA assay identifies stem cell or precursor erythroid cell mutations expressed in peripheral erythrocytes of MN-heterozygous subjects, distinguishing the NN and N phi mutant variants. A significant increase in the NN GPA variant cell frequency (Vf) was found in benzene-exposed workers as compared with unexposed control individuals (mean +/- SEM, 13.9 +/- 1.7 per million cells vs. 7.4 +/- 1.1 per million cells in control individuals; P = 0.0002). In contrast, no significant difference existed between the two groups for the N phi Vf (9.1 +/- 0.9 vs. 8.8 +/- 1.8 per million cells; P = 0.21). Further, lifetime cumulative occupational exposure to benzene was associated with the NN Vf (P = 0.005) but not with the N phi Vf (P = 0.31), suggesting that NN mutations occur in longer-lived bone marrow stem cells. NN variants result from loss of the GPA M allele and duplication of the N allele, presumably through recombination mechanisms, whereas NO variants arise from gene inactivation, presumably due to point mutations and deletions. Thus, these results suggest that benzene produces gene-duplicating mutations but does not produce gene-inactivating mutations at the GPA locus in bone marrow cells of humans exposed to high benzene levels. This finding is consistent with data on the genetic toxicology of benzene and its metabolites and adds further weight to the hypothesis that chromosome damage and mitotic recombination are important in benzene-induced leukemia.

Glycine-induced long-term potentiation is associated with structural and functional modifications of α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors

Global long-term potentiation (LTP) was induced in organotypic hippocampal slice cultures by a brief application of 10 mM glycine. Glycine-induced LTP was occluded by previous theta burst stimulation-induced potentiation, indicating that both phenomena share similar cellular processes. Glycine-induced LTP was associated with increased [3H]α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) binding in membrane fractions as well as increased amount of a selective spectrin breakdown product generated by calpain-mediated spectrin proteolysis. Antibodies against the C-terminal (C-Ab) and N-terminal (N-Ab) domains of GluR1 subunits were used to evaluate structural changes in AMPA receptor properties resulting from glycine-induced LTP. No quantitative or qualitative changes were observed in Western blots from membrane fractions prepared from glycine-treated slices with C-Ab. In contrast, Western blots stained with N-Ab revealed the formation of a 98-kDa species of GluR1 subunits as well as an increased amount of immunoreactivity after glycine-induced LTP. The amount of spectrin breakdown product was positively correlated with the amount of the 98-kDa species of GluR1 after glycine treatment. Functional modifications of AMPA receptors were evaluated by determining changes in the effect of pressure-applied AMPA on synaptic responses before and after glycine-induced LTP. Glycine treatment produced a significant increase in AMPA receptor function after potentiation that correlated with the degree of potentiation. The results indicate that LTP induction produces calpain activation, truncation of the C-Ab domain of GluR1 subunits of AMPA receptors, and increased AMPA receptor function. They also suggest that insertion of new receptors takes place after LTP induction.

Establishment of an animal–bacterial association: Recruiting symbiotic vibrios from the environment

While most animal–bacterial symbioses are reestablished each successive generation, the mechanisms by which the host and its potential microbial partners ensure tissue colonization remain largely undescribed. We used the model association between the squid Euprymna scolopes and Vibrio fischeri to examine this process. This light organ symbiosis is initiated when V. fischeri cells present in the surrounding seawater enter pores on the surface of the nascent organ and colonize deep epithelia-lined crypts. We discovered that when newly hatched squid were experimentally exposed to natural seawater, the animals responded by secreting a viscous material from the pores of the organ. Animals maintained in filtered seawater produced no secretions unless Gram-negative bacteria, either living or dead, were reintroduced. The viscous material bound only lectins that are specific for either N-acetylneuraminic acid or N-acetylgalactosamine, suggesting that it was composed of a mucus-containing matrix. Complex ciliated fields on the surface of the organ produced water currents that focused the matrix into a mass that was tethered to, and suspended above, the light organ pores. When V. fischeri cells were introduced into the seawater surrounding the squid, the bacteria were drawn into its fluid-filled body cavity during ventilation and were captured in the matrix. After residing as an aggregate for several hours, the symbionts migrated into the pores and colonized the crypt epithelia. This mode of infection may be an example of a widespread strategy by which aquatic hosts increase the likelihood of successful colonization by rarely encountered symbionts.

Genetic separation of tumor growth and hemorrhagic phenotypes in an estrogen-induced tumor.

Chronic administration of estrogen to the Fischer 344 (F344) rat induces growth of large, hemorrhagic pituitary tumors. Ten weeks of diethylstilbestrol (DES) treatment caused female F344 rat pituitaries to grow to an average of 109.2 +/- 6.3 mg (mean +/- SE) versus 11.3 +/- 1.4 mg for untreated rats, and to become highly hemorrhagic. The same DES treatment produced no significant growth (8.9 +/- 0.5 mg for treated females versus 8.7 +/- 1.1 for untreated females) or morphological changes in Brown Norway (BN) rat pituitaries. An F1 hybrid of F344 and BN exhibited significant pituitary growth after 10 weeks of DES treatment with an average mass of 26.3 +/- 0.7 mg compared with 8.6 +/- 0.9 mg for untreated rats. Surprisingly, the F1 hybrid tumors were not hemorrhagic and had hemoglobin content and outward appearance identical to that of BN. Expression of both growth and morphological changes is due to multiple genes. However, while DES-induced pituitary growth exhibited quantitative, additive inheritance, the hemorrhagic phenotype exhibited recessive, epistatic inheritance. Only 5 of the 160 F2 pituitaries exhibited the hemorrhagic phenotype; 36 of the 160 F2 pituitaries were in the F344 range of mass, but 31 of these were not hemorrhagic, indicating that the hemorrhagic phenotype is not merely a consequence of extensive growth. The hemorrhagic F2 pituitaries were all among the most massive, indicating that some of the genes regulate both phenotypes.

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells.

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.