The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Summary The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible). This work was submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Environmental Science, Institute of Environmental Sciences.     

Evidence that Northern Pioneering Pines with Tuberculate Mycorrhizae are Unaffected by Varying Soil Nitrogen Levels

Tuberculate mycorrhizae on Pinus contorta (lodgepole pine) have previously been shown to reduce acetylene, but an outstanding question has been to what degree these structures could meet the nitrogen requirements of the tree. We compared the growth, tissue nitrogen contents, and stable nitrogen isotope ratios of P. contorta growing in gravel pits to the same species growing on adjacent intact soil. Trees growing in severely nitrogen deficient gravel pits had virtually identical growth rates and tissue nitrogen contents to those growing on intact soil that had nitrogen levels typical for the area. δ¹⁵N values for trees in the gravel pits were substantially lower than δ¹⁵N values for trees on intact soil, and isotope ratios in vegetation were lower than the isotope ratios of the soil. The form of soil nitrogen in the gravel pits was almost exclusively nitrate, while ammonium predominated in the intact soil. Discrimination against ¹⁵N during plant uptake of soil nitrate in the highly N-deficient soil should be weak or nonexistent. Therefore, the low δ¹⁵N in the gravel pit trees suggests that trees growing in gravel pits were using another nitrogen source in addition to the soil. Precipitation-borne nitrogen in the study area is extremely low. In conjunction with our other work, these findings strongly suggests that P. contorta and its microbial symbionts or associates fix nitrogen in sufficient amounts to sustain vigorous tree growth on the most nitrogen-deficient soils.     

Stabilization,solubilization, partial purification and some properties of cytochrome P-450 present in CaCl2-precipitated human placental microsomes

Two forms of a carbon monoxide-binding cytochrome were found to exist in CaCl₂-precipitated microsomal subfractions of human placental homogenates at term. These exhibited absorption maxima at wavelengths of 450 nm and 421–432 nm. Conversion of P-450 to the degradation product could be prevented by resuspension of the microsomes in buffered solutions containing 20% glycerol and 10^?4M dithiothreitol. Solubilization of such resuspended microsomes with sodium cholate could be effected with minimal degradation. The solubilized pigment then could be partially purified by differential fractionation with ammonium sulfate. The partially purified cytochromes did not appear to bind desmethylimpramine but did bind aniline and nicotinamide to yield atypical type II difference spectra with maxima at 435 nm and minima at 416 nm. A type I difference spectrum could be elicited with androstenedione and a spectral dissociation constant (K_S) of 4.7×10^?8M was obtained. Androstenedione also appeared to effectively prevent the binding of carbon monoxide to the cytochrome.     

Gene dosage studies with pleiotropic mutants of Serratia marcescens superactive in the synthesis of marcescin A and certain other exocellular proteins

Summary Pleiotropic mutants of Serratia marcescens have been isolated. They synthesize greater quantities of the bacteriocin marcescin A and exocellular lipase and exhibit higher rates of spontaneous induction of prophage than does the wild-type strain. These mutants were found to contain more marcescin A plasmid DNA than the parent strain and, furthermore, this increase in plasmid DNA was observed to be proportional to the increase in synthesis of marcescin A. From these results it is proposed that the mutation functions via a gene-dosage effect (at least in the case of bacteriocin synthesis) and causes an elevated synthesis of bacteriocin plasmid DNA.A preliminary report of this work was presented to the 1972 Summer Meeting of the Gesellschaft für Physiologische Chemie held in Bochum, Germany (Timmis and Winkler, 1972).     

Independence between modification of genetic position effects and formation of lampbrush loops by the Y chromosome of Drosophila hydei

Summary The phenotype of the variegation position effect white-mottled-2 in Drosophila hydei is modified by supernumerary Y chromosomes and by fractions thereof. Different translocated Y fragments have varying degrees of effectiveness in suppressing the mutant phenotype in the mottled eyes. In fragments derived from similar regions of the Y chromosome the suppressive ability is related to their cytological lengths. In contrast, fragments derived from distinctive regions of the Y chromosome differ markedly in their effectiveness, and these differences are not necessarily correlated with the cytological length. In particular, fragments of the distal region of Y^L are more effective in enhancing the wild phenotype than are proximal fragments of similar size.The mutation white-mottled-2 is accompanied by a complex rearrangement of the X chromosome. This inhibits crossing over between large regions of the X chromosome in structural heterozygotes; it causes also a delay of development and a considerable reduction of viability in homozygous females and hemizygous males. XO males are inviable. The inviability of these males is partially covered by Y fragments. With respect to viability, the fragments show similar regional differences in effectiveness as in the modification of the mottled phenotype.There is also a parental effect on the modulation of the white-mottled-2 phenotype.There is no correlation between the activity of Y chromosomal factors on spermiogenesis and the activity of Y factors on the modification of the variegation position effect. Suppression of Y chromosomal sites which normally unfold lampbrush loops during the spermatocyte stage and whose activity has previously been shown to be indispensible for normal differentiation of the male germ line cells does not result in any visible alterations of the effectiveness on the mottling. So there is obviously independence between these two different genetic activities of Y chromosomal factors.     

ULTRASTRUCTURAL ASPECTS OF TWO SPECIES OF GUTTULINOPSIS

Two acrasid cellular slime molds. Guttulinopsis vulgaris and G. nivea, are compared at the ultrastructural level. The amoebae of the two species are indistinguishable except for the presence of intranuclear fibers in G. vulgaris. Both species share some unusual features, including: plate-like cristae in the mitochondria, production of microbody-like organelles in the perinuclear space, spores with thin bilaminar walls, and stalks containing microfilaments bound in striated bundles. These and other observations are discussed with regard to the development of the sorocarps and the relationship of the genus to other members of the Acrasida.