Oncofetal H19 RNA promotes tumor metastasis

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-β and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-β treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.     

Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.     

Response of root respiration and root exudation to alterations in root C supply and demand in wheat

Rising atmospheric CO₂ concentrations have highlighted the importance of being able to understand and predict C fluxes in plant-soil systems. We investigated the responses of the two fluxes contributing to below-ground efflux of plant root-dependent CO₂, root respiration and rhizomicrobial respiration of root exudates. Wheat (Triticum aestivum L., var. Consort) plants were grown in hydroponics at 20°C, pulse-labelled with ¹⁴CO₂ and subjected to two regimes of temperature and light (12 h photoperiod or darkness at either 15°C or 25°C), to alter plant C supply and demand. Root respiration was increased by temperature with a Q ₁₀ of 1.6. Root exudation was, in itself, unaltered by temperature, however, it was reduced when C supply to the roots was reduced and demand for C for respiration was increased by elevated temperature. The rate of exudation responded much more rapidly to the restriction of C input than did respiration and was approximately four times more sensitive to the decline in C supply than respiration. Although temporal responses of exudation and respiration were treatment dependent, at the end of the experimental period (2 days) the relative proportion of C lost by the two processes was conserved despite differences in the magnitude of total root C loss. Approximately 77% of total C and 67% of ¹⁴C lost from roots was accounted for by root respiration. The ratio of exudate specific activity to CO₂ specific activity converged to a common value for all treatments of 2, suggesting that exudates and respired CO₂were not composed of C of the same age. The results suggest that the contributions of root and rhizomicrobial respiration to root-dependent below-ground respiration are conserved and highlight the dangers in estimating short-term respiration and exudation only from measurements of labelled C. The differences in responses over time and in the age of C lost may ultimately prove useful in improving estimates of root and rhizomicrobial respiration.     

Vascular Cholinesterases and Choline Uptake in Isolated Rat Forebrain Microvessels: A Possible Link

The two parameters of the active [methyl-3H]choline uptake into isolated rat forebrain microvessels, Km and Vmax, were determined for 1-, 3-, 10-, and 24-month-old Charles River male rats and compared with the activities of the enzymes choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) in these microvessels over the same time course. The value of Km remained constant over the entire period, but that of Vmax increased from 8.5 +/- 1.0 to 80.6 +/- 16.4 nmol g-1 (mean +/- SEM) over the first 3 months of life. Over the same period, the increase in ChAT activity, from an initial value of 7.1 +/- 1.6 to 10.2 +/- 0.3 nmol g-1 min-1, was not proportional to that of choline uptake. Levels of BuChE activity (0.9-1.3 mumol g-1 min-1) were almost unchanged throughout the entire 24-month period, but those of AChE showed a steady and significant increase from 1 to 24 months, remaining relatively high at senescence (4.7 mumol g-1 min-1), when choline uptake had decreased to one-third of its optimal value. Selective inhibition of AChE with 1,5-bis(4-allyldimethylammonium-phenyl)pentan-3-one dibromide (0.5 microM) in unruptured capillaries from 3-month-old rats resulted in a decrease in Vmax of choline uptake from approximately 81 to 59 nmol g-1 min-1 or with 9-amino-1,2,3,4-tetrahydroacridine (10 microM) in capillaries from 2-month-old rats from approximately 30 to 15 nmol g-1 min-1. Selective inhibition of BuChE with tetraisopropyl pyrophosphoramide (100 microM) resulted in an increase in Vmax from approximately 81 to 96 nmol g-1 min-1. It is possible that the two vascular enzyme systems are coupled to a hypothetical endothelial choline transporter, but with an action opposite to each other.     

CLONING,EXPRESSION, AND CHARACTERIZATION OF A HISTONE‐LIKE PROTEIN FROM THE MARINE DINOFLAGELLATE LINGULODINIUM POLYEDRUM (DINOPHYCEAE)1

Although nucleosomes and histones are lacking in dinoflagellate nuclei, small basic histone‐like proteins have been reported, but their function(s) is unknown. In this study we cloned and sequenced a gene for a histone‐like protein from the dinoflagellate Lingulodinium polyedrum (Stein) Dodge (HLp) (formerly Gonyaulax polyedra Stein) and investigated its post‐translational modification and DNA‐binding activities. HLp appears to be acetylated in L. polyedrum, and we identified several L. polyedrum proteins that possess histone acetyltransferase activity and may be responsible for this modification. HLp binds weakly to L. polyedrum DNA but to certain specific sequences with higher affinity, consistent with its having a regulatory function.     

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.     

Nonidentical alkylation sites in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

These studies establish the specificity of 3,3,3-trifluorobromoacetone for reaction with the active site cysteines of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and suggest the potential use of trifluoroacetonyl groups as 19F nuclear magnetic resonance probes for study of symmetry relations between the four protomers of the enzyme. The alkylation of the holoenzyme follows biphasic kinetics and indicates either preexistent or induced nonequivalence among the sites; these effects are not predisposed by a low coenzyme/enzyme ratio. Two additional alkylation sites not at the active centers are created by acylation with beta-(2-furyl)acryloyl phosphate: it is concluded that pseudosubstrates cause an intramolecular rearrangement which exposes two sulfhydryl functions besides those of the active site (Cys-149).     

Carbon-13 nuclear magnetic resonance studies of the interaction of specifically labeled (90%-13C) oxytocin and arginine vasopressin with neurophysins.

Oxytocin and arginine vasopressin have been synthesized, via solid phase techniques, enriched to 90% ¹³C in the 2-carbon of their C-terminal glycinamide residues. In the presence of an approximately equimolar amount of bovine neurophysin II, the ¹³C nuclear magnetic resonance signal due to the enriched carbon in oxytocin shows a broadening which is highly dependent on both the temperature and the concentration of the neurophysin-oxytocin complex. The linewidth varies from 3 hz, observed at a protein concentration of 11mg/ml and a temperature of 37°, to 120 hz for a protein concentration of 65 mg/ml at 6°C. Similar results were obtained with arginine vasopressin. The results indicate that under conditions of low protein concentration and high temperature, the glycinamide residues of oxytocin and arginine vasopressin bound to neurophysin possess significant internal motion, while lowering the temperature and/or raising the protein-hormone concentration reduces this internal motion, probably concommitant with association of the protein-hormone complex into higher molecular weight aggregates.     

19F NMR studies of the binding of 5-fluoro-2′-deoxyuridylate to thymidylate synthase

Summary In the presence of thymidylate synthase, the ¹⁹F signal of 5-fluoro-2-deoxyuridylate is shifted upfield 0.6 ppm or 4.5 ppm depending on the enzyme preparation used. The bands at these positions represent different species of binary complex. When either binary complex is reacted with methylenetetrahydrofolate a ternary complex is formed with a ¹⁹F signal shifted 12.5 ppm upfield and broadened to 120 Hz.Substitution of the hydrogen atoms of the methylene group of methylenetetrahydrofolate with deuterium atoms results in line-narrowing of the spectrum of the ternary complex from 120 to 80 Hz indicating the close proximity of the methylene group to the fluorine atom in the ternary complex. A model compound, 5-fluoro-6-hydroxy-5-methyl-5, 6-dihydrouracil, gives a chemical shift in the same direction and of similar magnitude to that seen with the ternary complex.     

Info-gap robust-satisficing model of foraging behavior: do foragers optimize or satisfice?

In this note we compare two mathematical models of foraging that reflect two competing theories of animal behavior: optimizing and robust satisficing. The optimal-foraging model is based on the marginal value theorem (MVT). The robust-satisficing model developed here is an application of info-gap decision theory. The info-gap robust-satisficing model relates to the same circumstances described by the MVT. We show how these two alternatives translate into specific predictions that at some points are quite disparate. We test these alternative predictions against available data collected in numerous field studies with a large number of species from diverse taxonomic groups. We show that a large majority of studies appear to support the robust-satisficing model and reject the optimal-foraging model.