Control of Glycolytic Flux by AMP-Activated Protein Kinase in Tumor Cells Adapted to Low pH

Tumor cells grow in nutrient- and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. This adaptation process commonly results in a tumor phenotype that displays a high rate of aerobic glycolysis and aggressive tumor characteristics. The glucose regulatory molecule, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), is a bifunctional enzyme that is central to glycolytic flux and is downstream of the metabolic stress sensor AMP-activated protein kinase (AMPK), which has been suggested to modulate glycolysis and possibly activate isoforms of PFKFB, specifically PFKFB3 expressed in tumor cells. Our results demonstrated that long-term low pH exposure induced AMPK activation, which resulted in the up-regulation of PFKFB3 and an increase in its serine residue phosphorylation. Pharmacologic activation of AMPK resulted in an increase in PFKFB3 as well as an increase in glucose consumption, whereas in contrast, inhibition of AMPK resulted in the down-regulation of PFKFB3 and decreased glycolysis. PFKFB3 overexpression in DB-1 tumor cells induced a high rate of glycolysis and inhibited oxygen consumption, confirming its role in controlling glycolytic flux. These results show that low pH is a physiological stress that can promote a glycolytic phenotype commonly associated with tumorigenesis. The implications are that the tumor microenviroment contributes to tumor growth and treatment resistance.     

Response of murine bone marrow granulocyte-macrophage colony-forming units to hyperthermia in situ

A detailed understanding of how bone marrow stem cell progenitors are affected by heat is prerequisite to predicting how whole-body or regional hyperthermia protocols may affect bone marrow function. This investigation reports the reproductive integrity of murine tibial bone marrow granulocyte-macrophage colony-forming units (CFU-GM) after in situ hyperthermia. Heat was applied by water bath immersion of the leg of male BALB/c mice anesthetized with 90 mg/kg pentobarbital given subcutaneously. Tibial and rectal temperatures were monitored in representative animals by microthermocouples (tip diameter approximately 100 microns). By approximately 3 min after immersion of the limb, marrow temperature was within 0.3 degree C of water bath temperature (O'Hara et al., Int. J. Hyperthermia 5, 589-601, 1989) and was within 0.1 degree C by 5 min after immersion. The CFU-GM were cultured in "lung-conditioned" McCoy's 5A medium supplemented with 15% fetal calf serum and 0.3% Bacto agar. In situ heating of tibial marrow to exposure temperatures of 42, 42.5, 43, 44, and 45 degrees C gave D0's (+/- 95% CI) of 91 +/- 44, 44 +/- 27, 27 +/- 2.2, 16 +/- 6, and 7 +/- 4 min, respectively. Heating to 41.5 degrees C for up to 180 min did not result in cytotoxicity. Development of thermotolerance after approximately 100 min of heating was apparent by the presence of a "resistant tail" of the 42 degrees C survival curve. A plot of D0 vs water bath temperature was bimodal with an inflection point at approximately 42.5 degrees C. The inactivation enthalpy for temperatures above 42.5 degrees C was 586 kJ/mol (140 kcal/mol) and for temperatures below 42.5 degrees C was estimated to be 1205 kJ/mol (288 kcal/mol). These results show that CFU-GM can be heated predictably in situ, can be inactivated with thermal exposures as low as 42 degrees C, and are capable of developing thermotolerance. These findings underscore the necessity to understand stem cell inactivation by hyperthermia in situ prior to widespread implementation of clinical hyperthermia protocols where bone marrow may be included in the treatment field.     

Novel Protein-Protein Contacts Facilitate mRNA 3′-Processing Signal Recognition by Rna15 and Hrp1

Precise 3′-end processing of mRNA is essential for correct gene expression, yet in yeast, 3′-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3′-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3′-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.     

Fine structure of polysaccharide microcrystals

Summary Negative staining showed the presence of microcrystals in various polysaccharides. Cellulose microcrystals from Valoniopsis, Vaucheria, and an unidentified tunicate had widths of 20, 27, and 30 Å, respectively. Mannan microcrystals from Acetabularia were 10x25 Å and were oriented in linear arrays with their long axis perpendicular to the array axis. dichotomosiphon and Caulerpa xylans had respective microcrystal widths of 22 and 24 Å. All microcrystals appeared as component part of microfibrils.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5'-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5'-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5'-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.     

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.     

Immediate Increase in Food Intake Following Exercise Messages

Communications to stimulate weight loss include exercise‐promotion messages that often produce unsatisfactory results due to compensatory behavioral and metabolic mechanisms triggered by physical activity. This research investigated potential automatic facilitation of eating immediately after exercise messages in the absence of actual exercise. Two controlled experiments demonstrated greater than control food intake following exposure to print messages typical of exercise campaigns as well as subliminal presentation of action words associated with exercise (e.g., “active”). These inadvertent effects may explain the limited efficacy of exercise‐promotion programs for weight loss, particularly when systematic dietary guidelines are absent.     

A common polygenic basis for quinine and PROP avoidance in mice

Inbred strains of mice (Mus musculus) differ greatly in ability to taste various bitter compounds. For some compounds, the differences result from allelic variation at a single locus. However, segregation patterns incompatible with monogenic inheritance have been found for quinine avoidance. The Soa bitter sensitivity locus exerts some influence on this phenotype, but an unknown number of other loci also contribute. Relative avoidance patterns for quinine sulfate in panels of naive inbred strains resembled avoidance patterns for 6-n-propyl-2- thiouracil (PROP), suggesting a common genetic basis. In particular, C57BL/6J mice strongly avoided both 0.1 mM quinine sulfate and 1 mM PROP in two-bottle preference tests, whereas C3H/HeJ mice were indifferent to both. Therefore, 12 BXH/Ty recombinant inbred strains, derived from these strains, were tested with both solutions to begin identification of the unknown bitter loci. Naive mice were tested for four consecutive days with each compound (order counterbalanced). Some BXH/Ty strain means resembled those of the parent strains, but others were intermediate. This indicated recombination among loci affecting avoidance, and therefore polygenic inheritance. The strain means were highly correlated across compounds (r = 0.98), suggesting that the same polygenes controlled both phenotypes. The BXH/Ty means for both compounds were then compared with the strain genotypes at 212 chromosome position markers distributed throughout the genome. Eight markers on five chromosomes (3, 6, 7, 8 and 9) yielded significant correlations. Six of the markers were correlated with both phenotypes, again suggesting common polygenic inheritance. The marker with the highest correlation was Prp, tightly linked to Soa on chromosome 6. The correlated marker regions likely contain quantitative trait loci affecting bitter avoidance. The phenotypic similarity of PROP to quinine, rather than to phenylthiourea, apparently stemming from a common polygenic basis, indicates a difference between mice and humans in gustatory organization related to bitters.