Increases in metabolic rate and core temperature are common responses to severe injury. We have investigated the hypothesis that these responses are due to increases in substrate cycling. A substrate cycle exists when opposing, nonequilibrium reactions catalyzed by different enzymes are operating simultaneously. At least one of the reactions must involve the hydrolysis of ATP. Thus, a substrate cycle both liberates heat and increases energy expenditure, yet there is not net conversion of substrate to product. In studies in volunteers (n = 18) and in patients with severe burns who were in a hypermetabolic state (n = 18), we used stable-isotope tracers to quantify substrate cycling in the pathways of glycolysis and gluconeogenesis and a cycle involving the simultaneous breakdown and synthesis of stored triglyceride (triglyceride-fatty acid cycle). The total rates of triglyceride-fatty acid and glycolytic-gluconeogenic cycling were elevated in the patients by 450 and 250 percent, respectively (P less than 0.01). An infusion of propranolol in the patients greatly reduced triglyceride-fatty acid cycling but did not affect gluconeogenic-glycolytic cycling. We conclude that increased substrate cycling contributes to the increased thermogenesis and energy expenditure following severe burns and that the increased triglyceride-fatty acid cycling is due to beta-adrenergic stimulation.

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.

No abstract

Multiple pathogenic mechanisms by whichHelicobacter pylori infection induces gastric cancer have been established in the last two decades. In particular, aberrant DNA methylation is induced in multiple driver genes, which inactivates them. Methylation profiles in gastric cancer are associated with specific subtypes, such as microsatellite instability. Recent comprehensive and integrated analyses showed that many cancer-related pathways are more frequently altered by aberrant DNA methylation than by mutations. Aberrant DNA methylation can even be present in noncancerous gastric mucosae, producing an ''epigenetic field for cancerization.'' Mechanistically, H. pylori-induced chronic inflammation, but not H. pylori itself, plays a direct role in the induction of aberrant DNA methylation. The expression of three inflammation-related genes, Il1b, Nos2, and Tnf, is highly associated with the induction of aberrant DNA methylation. Importantly, the degree of accumulated aberrant DNA methylation is strongly correlated with gastric cancer risk. A recent multicenter prospective cohort study demonstrated the utility of epigenetic cancer risk diagnosis for metachronous gastric cancer. Suppression of aberrant DNA methylation by a demethylating agent was shown to inhibit gastric cancer development in an animal model. Induction of aberrant DNA methylation is the major pathway by which H. pylori infection induces gastric cancer, and this can be utilized for translational opportunities.

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. ). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo-and deoxyribonucleoside 5-monophosphates and ribonucleoside 3-monophosphates with highest affinity to 3-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P 20 -25 ). YfbR was strictly specific to deoxyribonucleoside 5-monophosphates, whereas YjjG showed narrow specificity to 5-dTMP, 5-dUMP, and 5-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di-and triphosphates: YfbR was equally sensitive to both di-and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.DNA and RNA synthesis requires a continuous and balanced supply of the four deoxyribonucleotides and four ribonucleotides. A network of biosynthetic and catabolic enzymes regulates the size of each pool with the enzymes ribonucleotide reductase, nucleoside kinases, and nucleotidases playing the main roles (1). By opposing the phosphorylation of nucleosides by kinases, intracellular 5Ј-nucleotidases participate in substrate cycles that regulate the cellular levels of ribo-and deoxyribonucleoside monophosphates and thereby all ribo-and deoxyribonucleotide pools (1, 2).Nucleoside monophosphate phosphohydrolases or nucleotidases (EC 3.1.3.5 and EC 3.1.3.6) are phosphatases that specifically dephosphorylate nucleoside monophosphates to nucleosides and inorganic phosphate. Seven mammalian 5Ј-nucleotidases have been identified through cloning and biochemical characterization. These enzymes differ in tissue specificity, subcellular localization, primary structure, and substrate specificity (2). All act on nucleoside monophosphates producing free nucleosides and P i . Each natural nucleotide can be the substrate for several nucleotidases, because they have overlapping specificities. The ubiquitous ecto-5Ј-nucleotidase, eN, 1 is anchored to the surface of the plasma membrane, and AMP is considered to be its major physiological substrate (3). Five other nucleotidases, including dNT-1, occur in the cytosol and one (dNT-2) occurs in mitochondria. dNT-1 and dNT-2 show a preference for the dephosphorylation of dUMP and dTMP (4). The "high K m -nucleotidase" cN-II prefers GMP and IMP (5), the cytosolic nucleotidases cN-IA and cN-IB have a preference for dephosphorylation of AMP (6, 7), and PN-1 (or cN-III) is most active with CMP and UMP (8). For bovine cN-II...

Recent studies have established the existence of substrate cycles in humans, but factors regulating the rate of cycling have not been identified. We have therefore investigated the acute response of glucose/glucose-6P-glucose (glucose) and triglyceride/fatty acid (TG/FA) substrate cycling to the infusion of epinephrine (0.03 gg/kg min) and glucagon. The response to a high dose glucagon infusion (2 gg/kg -min) was tested, as well as the response to a low dose infusion (5 ng/kg. min), with and without the simultaneous infusion of somatostatin (0.1 gg/kg min) and insulin (0.1 mU/kg min).Additionally, the response to chronic prednisone (50 mg/d) was evaluated, both alone and during glucagon (low dose) and epinephrine infusion. Finally, the response to hyperglycemia, with insulin and glucagon held constant by somatostatin infusion and constant replacement of glucagon and insulin at basal rates, was investigated. Glucose cycling was calculated as the difference between the rate of appearance (R.) of glucose as determined using 24k-and 6,642-glucose as tracers. TG/FA cycling was calculated by first determining the R. glycerol with d5-glycerol and the R. FFA with 11-'3Cqpalmitate, then subtracting R. FFA from three times R. glycerol.The results indicate that glucagon stimulates glucose cycling, and this stimulatory effect is augmented when the insulin response to glucagon infusion is blocked. Glucagon had minimal effect on TG/FA cycling. In contrast, epinephrine stimulated TG/FA cycling, but affected glucose cycling minimally. Prednisone had no direct effect on either glucose or TG/FA cycling, but blunted the stimulatory effect of glucagon on glucose cycling. Hyperglycemia, per se, had no direct effect on glucose or TG/FA cycling. Calculations revealed that stimulation of TG/FA cycling theoretically amplified the sensitivity of control of fatty acid flux, but no such amplification was evident as a result of the stimulation of glucose cycling by glucagon.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nM, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nM compared with 108 nM). Kinetic studies of eIF4F and eIFiso4F with VPg show ϳ2.6-fold faster association for eIFiso4F⅐VPg as compared with eIF4F⅐VPg. The dissociation rate was ϳ2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F⅐VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.Most eukaryotic mRNAs contain a cap structure m 7 GpppX 2 (where X is any nucleotide) at the 5Ј-end that is required for efficient initiation of translation. The cap serves as the binding site for initiation factors eIF4F or eIFiso4F, an isozyme form of eIF4F present in higher plants, either of which is involved in the assembly of the initiation complex. Alternative translation mechanisms have been shown to take place under a unique set of circumstances. One such example is that of viral infection. Viruses employ different strategies for preferential translation of their mRNAs often using internal ribosome entry sites (IRES).Recently attention has focused on the possible role in translation initiation of the viral protein linked to the genome (VPg) of potyvirus (1, 2). It has been suggested that VPg may serve as an analog of the m 7 G cap of the mRNAs and plays a role in mRNA translation because it interacts with the cap-binding proteins eIF4E and eIFiso4E (3, 4), subunits of eIF4F and eIFiso4F. Potyviruses have a messenger-polarity single-strand RNA genome, a poly(A) tail, and VPg covalently attached to the 5Ј terminus (1, 5), which upon infection of a suitable host is cleaved from the genome RNA by an unknown cellular enzyme. The 5Ј-pUpU-processed RNA serves as a template for viral protein synthesis as well as early rounds of RNA synthesis (6). Potyviruses have been shown to be capable of cap-independent translation (7) making the role of VPg in translation uncertain. Studies support a biological role for the VPg linked to the viral RNA in virions. The covalent tyrosine residue-mediated linkage between ...