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.     

The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.     

Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques

The response of cytosolic calcium [Ca2+]i to angiotensin II (AII) and potassium (K+) in individual rat glomerulosa cells was determined using the calcium-sensitive fluorescent dye, fura-2 and digital imaging. Control (4 mM K+) cytosolic calcium levels were generally in the 80-120 nM range and increased monotonically as [K+] was increased from 4 to 12 mM. There was no delay in the onset of the response. In most cells the [Ca2+]i decreased from its peak after 3-4 min, even in the presence of superfusate containing elevated K+. The time course of the change in [Ca2+]i in response to AII stimulation, on the other hand, was more variable. It was most often characterized by an early decrease followed by a large delayed increase. The response also was observed to decline during sustained AII stimulation. The majority of the cells showed some response to one or the other secretagogue with a sizeable minority (25%) having an increase in [Ca2+]i in excess of 200%. While the majority showed a response, the cell to cell variation was substantial. Finally, the pattern of cytosolic calcium increase sometimes showed a marked dependence on the secretagogue used, with different regions of the same cell being more strongly affected by one agent or the other. A few cells (10%) responded to AII only at one pole, establishing a large concentration gradient of calcium across the cell. Because of differences in time course, pattern, and degree of responsiveness, it is likely that the mechanisms underlying the Ca2+ elevation with K+ and AII are different.     

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.