Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles

The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points. A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed. By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle. We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA. In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA. The DNA is more accessible in regions near the particle boundary. We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity. Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine. Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle. The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.     

Synthesis of a more stable osmium ammine electron-dense DNA stain

Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.     

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.     

Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina

Summary When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish. We have carried out a morphologic and immunologic study of the emergence of this population in vitro. Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein. SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture. Not all of the cells that attach flatten. As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them. The flattened cells are positive for Vimentin by IF within 10 min of attachment. The percent of fluorescent cells found on the substrate is constant during the time in culture. This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate. When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate. Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized. Thus, the same cells that contained Vimentin in the retina now express it as flat cells. this supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Müller cells. We have also observed the emergence of a population of cells with short (0.5μm) microvilli that appear within 8 h of culture. They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters. This research was supported in part by grant EY-04892 and RR-0715 from the National Institutes of Health, Bethesda, MD, and a grant from the W.W. Smith Foundations     

Normal human colon cells suppress malignancy when fused with colon cancer cells

Summary Normal human colon mucosa cells and cells obtained from histologically normal tissues near that cancer were fused with human colon cancer cells. Resultant hybrid populations of normal and malignant cell fusions behaved as nonmalignant cells in culture, were unable to grow in soft agar, did not express tumor-associated antigens, and were nontumorigenic in nude mice. Autofusion of the cancer cell population led to a phenotype intermediate between normal and malignant cells. That is, the cultures had a much lower plating efficiency in soft agar, and the tumors had a longer latency and slower growth rate in nude mice. This is the first cell culture system to demonstrate that normal epithelial cells can suppress malignancy of their autologous cancer cells, and is a prelude to more extensive studies of genetic events involved in malignant conversion of human colonic epithelium. This study was supported by The University of Texas Health Science Center at San Antonio Center for Human Cell Biotechnology and a graduate student stipend (T. J.) from the Department of Cellular and Structural Biology.     

Social and reproductive behavior ofChaetodontoplus mesoleucus (Pomacanthidae) at Bantayan island,Philippines, with notes on pomacanthid relationships

Chaetodontoplus mesoleucus occurs at Bantayan Island in a habitat of small patches of mixed scleractinian and alcyonacean corals of low diversity and simple structure. Male-female pairs were predominant, and the sex-ratio showed only a slight skew towards females. However, the presence of single male, two-female social groups demonstrates that the species is polygamous. Small size of social groups is attributed to a preference for a habitat lacking structural complexity. The species did not occur on complex coral reefs. Social and spawning behavior are nearly identical to that of most pomacanthids for which data are available, and although sex-change was not demonstrated, size-related dominance hierarchies and close phylogenetic relationships to sex-changing pomacanthids suggest protogynous hermaphroditism in this species. Behaviorally,C. mesoleucus appears quite similar to a large group of species proposed herein to represent a generalized pomacanthid behavioral type. Divergences from this generalized type by members ofGenicanthus, eastern PacificHolacanthus, and western AtlanticPomacanthus are discussed. Evidence is given to suggest the phylogenetic derivation of the subgenusCentropyge (genusCentropyge) from an ancestor of the subgenusXiphipops type. Color dimorphism and “rendezvous sites” are briefly discussed.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium.

Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs. Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs. An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus. One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs. The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal. The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa. Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.     

Orthopoxvirus fusion inhibitor glycoprotein SPI-3 (open reading frame K2L) contains motifs characteristic of serine proteinase inhibitors that are not required for control of cell fusion.

The cowpox virus (CPV) SPI-3 gene (open reading frame K2L in vaccinia virus) is one of three orthopoxvirus genes whose products are members of the serpin (serine proteinase inhibitor) superfamily. The CPV SPI-3 gene, when overexpressed by using the vaccinia virus/T7 expression system, synthesized two proteins of 50 and 48 kDa. Treatment with the N glycosylation inhibitor tunicamycin converted the two SPI-3 proteins to a single 40-kDa protein, close to the size of 42 kDa predicted from the DNA sequence, suggesting that the SPI-3 protein, unlike the other two orthopoxvirus serpins, is a glycoprotein. Immunoblotting with an anti-SPI-3 antibody showed that the SPI-3 protein is synthesized early in infection prior to DNA replication. SPI-3 inhibits cell-cell fusion during infections with both CPV and vaccinia virus. A transfection assay was devised to test engineered mutants of SPI-3 for the ability to inhibit fusion. Two mutants with C-terminal deletions of 156 and 70 amino acids were completely inactive in fusion inhibition. Site-directed mutations were constructed near the C terminus of SPI-3, in or near the predicted reactive-site loop which is conserved in inhibitory serpins. Substitutions within the loop at the P1 to P1' positions and P5 to P5' positions, inclusive, did not result in any loss of activity, nor did changes at the P17 to P10 residues in the stalk of the reactive loop. Therefore, SPI-3 does not appear to control cell fusion by acting as a serine proteinase inhibitor.