David R. Evans

Planetary radio astronomy observations from Voyager 2 near Saturn

Planetary radio astronomy measurements obtained by Voyager 2 near Saturn have added further evidence that Saturnian kilometric radiation is emitted by a strong dayside source at auroral latitudes in the northern hemisphere and by a weaker source at complementary latitudes in the southern hemisphere. These emissions are variable because of Saturns rotation and, on longer time scales, probably because of influences of the solar wind and Dione. The electrostatic discharge bursts first discovered by Voyager 1 and attributed to emissions from the B ring were again observed with the same broadband spectral properties and an episodic recurrence period of about 10 hours, but their occurrence frequency was only about 30 percent of that detected by Voyager 1. While crossing the ring plane at a distance of 2.88 Saturn radii, the spacecraft detected an intense noise event extending to above 1 megahertz and lasting about 150 seconds. The event is interpreted to be a consequence of the impact, vaporization, and ionization of charged, micrometer-size G ring particles distributed over a vertical thickness of about 1500 kilometers.

Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway.

Pyrimidine nucleotides play a critical role in cellular metabolism serving as activated precursors of RNA and DNA, CDPdiacylglycerol phosphoglyceride for the assembly of cell membranes and UDP-sugars for protein glycosylation and glycogen synthesis (1–3). In addition, uridine nucleotides act via extracellular receptors to regulate a variety of physiological processes (4). There are two routes to the synthesis of pyrimidines; nucleotides can be recycled by the salvage pathways or synthesized de novo from small metabolites (Fig. 1). Most cells have several specialized passive and active transporters (5, 6) that allow the reutilization of preformed pyrimidine nucleosides and bases. The relative contribution of the de novo and salvage pathways depends on cell type and developmental stage. In general, the activity of the de novo pathway is low in resting or fully differentiated cells where the need for pyrimidines is largely satisfied by the salvage pathways (7). In contrast, de novo pyrimidine biosynthesis (Fig. 1) is indispensable in proliferating cells in order to meet the increased demand for nucleic acid precursors and other cellular components. Consequently, the activity of the de novo pathway is subject to elaborate growth state-dependent control mechanisms. Pyrimidine biosynthesis is invariably up-regulated in tumors and neoplastic cells (8), and the pathway has been linked to the etiology or treatment of several other disorders including AIDS (9), diabetes (10), and various autoimmune diseases (11) such as rheumatoid arthritis. This review focuses on the structure and regulation of the pyrimidine biosynthetic complexes and the interplay of the diverse control mechanisms operative in mammalian cells.

Regulation of carbamoyl phosphate synthetase by MAP kinase.

The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo , CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.

Voyager 2 radio observations of Uranus

Within distances to Uranus of about 6 x 106 kilometers (inbound) and 35 x 106 kilometers (outbound), the planetary radio astronomy experiment aboard Voyager 2 detected a wide variety of radio emissions. The emission was modulated in a period of 17.24 � 0.01 hours, which is identified as the rotation period of Uranus magnetic field. Of the two poles where the axis of the off-center magnetic dipole (measured by the magnetometer experiment aboard Voyager 2) meets the planetary surface, the one closer to dipole center is now located on the nightside of the planet. The radio emission generally had maximum power and bandwidth when this pole was tipped toward the spacecraft. When the spacecraft entered the nightside hemisphere, which contains the stronger surface magnetic pole, the bandwidth increased dramatically and thereafter remained large. Dynamically evolving radio events of various kinds embedded in these emissions suggest a Uranian magnetosphere rich in magnetohydrodynamic phenomena.

Voyager Planetary Radio Astronomy at Neptune

Detection of very intense short radio bursts from Neptune was possible as early as 30 days before closest approach and at least 22 days after closest approach. The bursts lay at frequencies in the range 100 to 1300 kilohertz, were narrowband and strongly polarized, and presumably originated in southern polar regions ofthe planet. Episodes of smooth emissions in the frequency range from 20 to 865 kilohertz were detected during an interval of at least 10 days around closest approach. The bursts and the smooth emissions can be described in terms of rotation in a period of 16.11 � 0.05 hours. The bursts came at regular intervals throughout the encounter, including episodes both before and after closest approach. The smooth emissions showed a half-cycle phase shift between the five episodes before and after closest approach. This experiment detected the foreshock of Neptunes magnetosphere and the impacts of dust at the times of ring-plane crossings and also near the time of closest approach. Finally, there is no evidence for Neptunian electrostatic discharges.

FUNCTIONAL EXPRESSION OF HUMAN PP2AC IN YEAST PERMITS THE IDENTIFICATION OF NOVEL C-TERMINAL AND DOMINANT-NEGATIVE MUTANT FORMS

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac ofSaccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.

Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.

Dihydroorotase from the hyperthermophile Aquifiex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.

In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187–190) are consistent with a partial channeling of the intermediate at 37u2009°C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-l-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

Eukaryotic initiation factor 4E degradation during brain ischemia.

Abstract: Suppression of protein synthesis in the brain following an ischemic insult has been thought to occur because of inhibition of translation initiation. All eukaryotic mRNAs, with the exception of heat‐shock transcripts, require the activity of eukaryotic initiation factor (eIF) 4E for formation of the translation initiation complex, and eIF‐4E availability is rate‐limiting. The response of brain eIF‐4E concentration and phosphorylation following decapitation ischemia was studied in rat brain homogenates after electrophoresis and western blotting with antibodies against eIF‐4E and phosphoserine, respectively. There was no change in level of eIF‐4E after 5 min of ischemia (p = 0.82 vs. time 0), but it had decreased 32 (p = 0.01) and 57% (p = 0.006) after 10 and 20 min of ischemia, respectively. There was no loss of serine phosphorylation on eIF‐4E beyond signal loss observed due to degradation of the protein itself (p = 0.31). In vitro exposure of eIF‐4E to activated μ‐calpain resulted in a 50% loss in 10 min of eIF‐4E on western blots. If active eIF‐4E is required for translation of its own mRNA, degradation of this protein during ischemia, possibly by activated μ‐calpain, could be a direct mechanism of irreversible neuronal injury, and the rate of proteolysis of eIF‐4E could place an upper time limit on the maximal duration of global brain ischemia compatible with neurologic recovery.