Inhibition of fructose-1,6-bisphosphatase by a new class of allosteric effectors

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.     

Additive effects of beta-alanine and sodium bicarbonate on upper-body intermittent performance

We examined the isolated and combined effects of beta-alanine (BA) and sodium bicarbonate (SB) on high-intensity intermittent upper-body performance in judo and jiu-jitsu competitors. 37 athletes were assigned to one of four groups: (1) placebo (PL)+PL; (2) BA+PL; (3) PL+SB or (4) BA+SB. BA or dextrose (placebo) (6.4 g day^?1) was ingested for 4 weeks and 500 mg kg^?1 BM of SB or calcium carbonate (placebo) was ingested for 7 days during the 4th week. Before and after 4 weeks of supplementation, the athletes completed four 30-s upper-body Wingate tests, separated by 3 min. Blood lactate was determined at rest, immediately after and 5 min after the 4th exercise bout, with perceived exertion reported immediately after the 4th bout. BA and SB alone increased the total work done in +7 and 8 %, respectively. The co-ingestion resulted in an additive effect (+14 %, p < 0.05 vs. BA and SB alone). BA alone significantly improved mean power in the 2nd and 3rd bouts and tended to improve the 4th bout. SB alone significantly improved mean power in the 4th bout and tended to improve in the 2nd and 3rd bouts. BA+SB enhanced mean power in all four bouts. PL+PL did not elicit any alteration on mean and peak power. Post-exercise blood lactate increased with all treatments except with PL+PL. Only BA+SB resulted in lower ratings of perceived exertion (p = 0.05). Chronic BA and SB supplementation alone equally enhanced high-intensity intermittent upper-body performance in well-trained athletes. Combined BA and SB promoted a clear additive ergogenic effect.     

Second‐generation decline: Scenarios for the economic and ethnic futures of the post‐1965 American immigrants

‘Second‐generation decline’ questions the current American faith in the myth of nearly automatic immigrant success. In discussing economic scenarios, positive and negative, for the future of the children of the post‐1965 immigrants, the possibility is proposed that a significant number of the children of poor immigrants, especially dark‐skinned ones, might not obtain jobs in the mainstream economy. Neither will they be willing ‐ or even able ‐ to take low‐wage, long‐hour ‘immigrant’ jobs, as their parents did. As a result, they (and young males among them particularly) may join blacks and Hispanics among those already excluded, apparently permanently, from the mainstream economy. The article also deals with the relations between ethnicity and economic conditions in the USA and with the continued relevance of the assimilation and acculturation processes described by ‘straight‐line theory’. This issue, as well as most others discussed, may also be salient for European countries experiencing immigration, especially those countries with troubled economies.     

In Vivo and in Vitro Activity And Mechanism Of Action of the Multidrug Cytarabine-L-Glycerylyl-Fluorodeoxyuridine

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 μM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 μ M), but no activity in FM3A/TK^? (IC50: 18.3 μM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK^? (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7–70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.     

Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes

An extensive homology search based on the sequence of the yeast protein Brx1p (biogenesis of ribosomes in Xenopus, YOL077c) revealed that it is a member of a superfamily of proteins sharing remarkable sequence similarities. Previous work on Brx1p showed that this protein is involved in the process of ribosome biogenesis [Kaser et al., Biol. Chem. 382 (2001) 1637-1647]. Brx1p is the founding member of one of the five existing eukaryotic subfamilies which are all present in yeast. Four of them are represented by one essential gene each and one family is represented by two closely related genes which can functionally replace each other but are essential together for survival. We created conditional alleles of four of the five genes which allowed us to study the effect of depletion of the respective proteins on the ribosome profiles of the strains. In this study we show that not only Brx1p but also three additional superfamily members, namely YHR088w (Rpf1p), YKR081c (Rpf2p) and the homologous proteins Ssf1p (YHR066w)/Ssf2p (YDR312w) are all involved in the multistep process of the assembly of the large ribosomal subunit. This agrees well with the fact that these three proteins, like Brx1p, are located in the nucleolus. Moreover, all four proteins closely interact functionally, because all four mutants are suppressed by the same multicopy suppressor gene.     

Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

A comparative kinetic analysis of nitrate and ammonium influx in two early-successional tree species of temperate and boreal forest ecosystems

Root NO₃ ^? and NH₄ ⁺ influx systems of two early‐successional species of temperate (trembling aspen: Populus tremuloides Michx.) and boreal (lodgepole pine: Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) forest ecosystems were characterized. NO₃ ^? and NH₄ ⁺ influxes were biphasic, consisting of saturable high‐affinity (HATS) and constitutive non‐saturable low‐affinity transport systems (LATS) that were evident at low and relatively high N concentrations, respectively. NO₃ ^? influx via HATS was inducible (IHATS); nitrate pre‐treatment resulted in 8–10‐fold increases in the V_max for influx in both species. By contrast, HATS for NH₄ ⁺ were entirely constitutive. In both species, V_max values for NH₄ ⁺ influx were higher than those for NO₃ ^? uptake; the differences were larger in pine (6‐fold) than aspen (1·8‐fold). In aspen, the K_m for NH₄ ⁺ influx by HATS was approximately 3‐fold higher than for IHATS NO₃ ^? influx, while in pine the K_m for IHATS NO₃ ^? influx was approximately 3‐fold higher than for NH₄ ⁺ influx. The aspen IHATS for NO₃ ^? influx appeared to be more efficient than that of pine (V_max values for aspen being approximately 10‐fold higher and K_m values being approximately 13‐fold lower than for pine). By contrast, only small differences in values for the NH₄ ⁺ HATS were evident between the two species. The kinetic parameters observed here probably result from adaptations to the N availabilities in their respective natural habitats; these may contribute to the distribution and niche separation of these species.     

Uterine and eggshell structure and histochemistry in a lizard with prolonged uterine egg retention (Lacertilia,Scincidae, Saiphos)

The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts,and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1α-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1α-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1α in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1α showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1α also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1α increased tumor mass by ∼2-fold and tumor volume by ∼3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1α also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1α is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1α expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1α in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1α in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a three-fold reduction in tumor volume. We conclude that HIF1α activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly “feed” cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their “self-digestion.” Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other “classic” oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed “autophagic tumor stroma model of cancer.”Key words: caveolin-1, autophagy, mitophagy, the Warburg effect, tumor stroma, hypoxia, HIF1A, NFκB, compartment-specific oncogenesis, cancer-associated fibroblasts