Regulation of an important glycolytic enzyme, pyruvate kinase, through phosphorylation in the larvae of a species of freeze-tolerant insect, Eurosta solidaginis.

Larvae of the goldenrod gall fly, Eurosta solidaginis, rely on a freeze tolerance strategy to survive the sub-zero temperatures of Canadian winter. Critical to their survival is the accumulation of polyol cryoprotectants and global metabolic rate depression, both of which require the regulation of glycolysis and reorganization of carbohydrate metabolism. This study explored the role that pyruvate kinase (PK) regulation plays in this metabolic reorganization. PK was purified from control (5 °C-acclimated) and frozen (-15 °C-acclimated) larvae and enzyme kinetic properties, structural stability, and post-translational modifications were examined in both enzyme forms. The Km phosphoenolpyruvate (PEP) of frozen PK was 20% higher than that of control PK, whereas the Vmax of frozen PK was up to 50% lower than that of control PK at the lowest assay temperature, suggesting inhibition of the enzyme during the winter. Additionally, the activity and substrate affinity of both forms of PK decreased significantly at low assay temperatures, and both forms were regulated allosterically by a number of metabolites. Pro-Q™ Diamond phosphoprotein staining and immunoblotting experiments demonstrated significantly higher threonine phosphorylation of PK from frozen animals while acetylation and methylation levels remained constant. Together, these results indicate that PK exists in two structurally distinct forms in E. solidaginis. In response to conditions mimicking the transition to winter, PK appears to be regulated to support metabolic rate depression, the accumulation of polyol cryoprotectants, and the need for extended periods of anaerobic carbohydrate metabolism to allow the animal to survive whole-body freezing.

Combination of sapacitabine and HDAC inhibitors stimulates cell death in AML and other tumour types.

BACKGROUND: Alternative treatments are needed for elderly patients with acute myeloid leukaemia, as the disease prognosis is poor and the current treatment is unsuitable for many patients. METHODS: In this study, we investigated whether combining the nucleoside analogue sapacitabine with histone deacetylase (HDAC) inhibitors could be an effective treatment. Synergy and mode-of-action analysis were studied in cultured cell lines and the efficacy of the combination was confirmed in a xenograft model. RESULTS: CNDAC (1-(2-C-cyano-2-deoxy-ß-D-arabino-pentofuranosyl)-cytosine), the active component of sapacitabine, synergised with vorinostat in cell lines derived from a range of tumour types. Synergy was not dependent on a specific sequence of drug administration and was also observed when CNDAC was combined with an alternative HDAC inhibitor, valproate. Flow cytometry and western blot analysis confirmed that the combination induced a significant increase in apoptosis. Mode-of-action analysis detected changes in Bcl-xl, Mcl-1, Noxa, Bid and Bim, which are all regulators of the apoptotic process. The sapacitabine/vorinostat combination demonstrated significant benefit compared with the single-agent treatments in an MV4-11 xenograft, in the absence of any observed toxicity. CONCLUSION: Sapacitabine and HDAC inhibitors are an effective drug combination that is worthy of clinical exploration.

Crosstalk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual exhaustion of Bcl-2 protection.

Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived Mcl-1 protein, but it is unknown how this leads to Bax/Bak activation that is required for most forms of cell death. Here, we studied the effects of seliciclib in B-cell chronic lymphocytic leukemia (B-CLL), a malignancy with aberrant expression of apoptosis regulators. Although seliciclib-induced Mcl-1 degradation within 4 h, Bax/Bak activation occurred between 16 and 20 h. During this period, no transcriptional changes in apoptosis-related genes occurred. In untreated cells, prosurvival Mcl-1 was engaged by the proapoptotic proteins Noxa and Bim. Upon drug treatment, Bim was quickly released. The contribution of Noxa and Bim as a specific mediator of seliciclib-induced apoptosis was demonstrated via RNAi. Significantly, 16 h after seliciclib treatment, there was accumulation of Bcl-2, Bim and Bax in the 'mitochondria-rich' insoluble fraction of the cell. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, until Bax forms large multimeric pores in the mitochondria. These data demonstrate in primary leukemic cells hierarchical binding and crosstalk among Bcl-2 members, and suggest that their functional interdependence can be exploited therapeutically.

Understanding auditory distance estimation by humpback whales: a computational approach.

Ranging, the ability to judge the distance to a sound source, depends on the presence of predictable patterns of attenuation. We measured long-range sound propagation in coastal waters to assess whether humpback whales might use frequency degradation cues to range singing whales. Two types of neural networks, a multi-layer and a single-layer perceptron, were trained to classify recorded sounds by distance traveled based on their frequency content. The multi-layer network successfully classified received sounds, demonstrating that the distorting effects of underwater propagation on frequency content provide sufficient cues to estimate source distance. Normalizing received sounds with respect to ambient noise levels increased the accuracy of distance estimates by single-layer perceptrons, indicating that familiarity with background noise can potentially improve a listening whale's ability to range. To assess whether frequency patterns predictive of source distance were likely to be perceived by whales, recordings were pre-processed using a computational model of the humpback whale's peripheral auditory system. Although signals processed with this model contained less information than the original recordings, neural networks trained with these physiologically based representations estimated source distance more accurately, suggesting that listening whales should be able to range singers using distance-dependent changes in frequency content.

Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator-activated receptor gamma.

The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator-activated receptor gamma (PPARgamma). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARgamma. Modified apoB had no such effect. Consistent with a participation of the PPARgamma signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARgamma in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

Interactions between double-stranded RNA regulators and the protein kinase DAI.

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

Protein serine/threonine phosphatase Ptc2p negatively regulates the unfolded-protein response by dephosphorylating Ire1p kinase.

Cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing the transcription of the genes encoding ER-resident chaperone proteins. Ire1p is a transmembrane protein kinase that transmits the signal from unfolded proteins in the lumen of the ER by a mechanism that requires oligomerization and trans-autophosphorylation of its cytoplasmic-nucleoplasmic kinase domain. Activation of Ire1p induces a novel spliced form of HAC1 mRNA that produces Hac1p, a transcription factor that is required for activation of the transcription of genes under the control of the unfolded-protein response (UPR) element. Searching for proteins that interact with Ire1p in Saccharomyces cerevisiae, we isolated PTC2, which encodes a serine/threonine phosphatase of type 2C. The Ptc2p interaction with Ire1p is specific, direct, dependent on Ire1p phosphorylation, and mediated through a kinase interaction domain within Ptc2p. Ptc2p dephosphorylates Ire1p efficiently in an Mg2+-dependent manner in vitro. PTC2 is nonessential for growth and negatively regulates the UPR pathway. Strains carrying null alleles of PTC2 have a three- to fourfold-increased UPR and increased levels of spliced HAC1 mRNA. Overexpression of wild-type Ptc2p but not catalytically inactive Ptc2p reduces levels of spliced HAC1 mRNA and attenuates the UPR, demonstrating that the phosphatase activity of Ptc2p is required for regulation of the UPR. These results demonstrate that Ptc2p downregulates the UPR by dephosphorylating Ire1p and reveal a novel mechanism of regulation in the UPR pathway upstream of the HAC1 mRNA splicing event.

Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.

Poplar for the phytomanagement of boron contaminated sites.

Boron (B) is a widespread environmental contaminant that is mobile relative to other trace elements. We investigated the potential of hybrid poplar (Populus sp.) for B phytomanagement using a lysimeter experiment and a field trial on B-contaminated wood-waste. In both studies, poplars enhanced evapotranspiration from the wood-waste, reduced B leaching, and accumulated B in the aerial portions of the tree. When grown in a substrate containing 30 mg/kg B, poplar leaves had an average B concentration of 845 mg/kg, while the stems contained 21 mg/kg B. Leaf B concentrations increased linearly with leaf age. A decomposition experiment revealed that abscised leaves released 14% of their B during the winter months. Fertiliser application enhanced tree growth without decreasing the leaf B concentrations. Harvesting alternate rows of trees on a contaminated site would reduce leaching from the site while removing B. Harvested plant material may provide bioenergy, stock fodder, or an amendment for B-deficient soils.

Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha.

Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha). The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha. Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa). Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA.

Influence of patient characteristics, socioeconomic factors, geography, and systemic risk on the use of breast-sparing treatment in women enrolled in adjuvant breast cancer studies: an analysis of two intergroup trials.

PURPOSE: To investigate the frequency of breast-sparing treatment among breast cancer patients subsequently enrolled in national cooperative group studies of adjuvant chemotherapy. PATIENTS AND METHODS: A data base was formed of 5,172 patients randomized onto two intergroup trials. Lumpectomy rates were analyzed within study-defined risk strata and across geographic regions. Significant predictors of lower lumpectomy usage were determined in multivariate analyses with variables that described patient and disease characteristics, systemic risk strata, geographic region, and socioeconomic indicators based on zip code of residence. RESULTS: Breast-conservation rates were 30% in the node-negative and 15% in the node-positive trials, with a wide geographic variation within each study (range, 14% to 49% and 9% to 31%, respectively). Lumpectomy use declined with increasing tumor size and did not exceed 40% even for tumors < or = 1 cm with negative nodes. With increasing risk of systemic relapse, frequency of lumpectomy declined (rates for five strata in order of increasing systemic risk: 41%, 33%, 24%, 18%, and 11%), even though these strata were not known at the time of the surgical decision. A logistic model confirmed the joint significance of geographic region and systemic risk. An exploratory model that adjusted for all important variables identified the following significant predictors of lower lumpectomy use: positive nodes; many positive nodes, increased systemic risk; tumor size > or = 2.0 cm; older age; South, Central or non-New England regions; and either lack of college degree or lower income levels. CONCLUSION: Breast-sparing therapy was used in the minority of women subsequently accrued to two national adjuvant breast cancer studies, even though this cohort and their referring surgeons represented a select population. Although multiple concrete factors were independent predictors of lower lumpectomy rates, prospective research is needed into how patients and their physicians approach the mastectomy versus lumpectomy decision.

Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR).

The double-stranded (ds) RNA-activated protein kinase, DAI (also known as PKR), contains an RNA-binding domain comprising two tandem repeats of a motif, the dsRBM, which is shared with a number of other proteins that interact with structured RNAs. We have expressed the entire domain and the first copy of the motif in Escherichia coli and purified the two proteins, p20 and p10, to apparent homogeneity in order to study their interactions with RNA and with the intact kinase enzyme. Both p20 and p10 bound preferentially to structured RNA molecules. Competition assays showed that in both cases the order of affinity is dsRNA > VA RNA > tRNA, but the isolated motif bound much less tightly than the entire domain. Measurement of the dissociation constants for dsRNA by quantitative gel mobility shift analysis gave apparent Kd values of 4 x 10(-9) M and 3.8 x 10(-7) M for p20 and p10, respectively. The binding of p20 molecules to dsRNA appeared to be cooperative. Multiple complexes were formed between the intact domain and dsRNA, saturating at a density of about one p20 molecule/11.25 base-pairs (or one turn) of duplex, whereas p10 achieved only about half of this packing density. The apparent Kd for the p20-VA RNA interaction was estimated as 3.5 x 10(-7) M and at least three complexes were detected, but no distinct complexes were visualized for the interaction between p10 and VA RNA. Both p20 and p10 inhibited autophosphorylation of intact DAI, probably by binding the dsRNA activator. Once activated, DAI could phosphorylate both p10 and p20, suggesting that intermolecular phosphorylation can occur.

Cancer cachexia.

Cancer cachexia is a severe debilitating disorder for which there are currently few therapeutic options. It is driven by the release of pro-inflammatory cytokines and cachectic factors by both host and tumour. Over the past few years, basic science advances have begun to reveal the breadth and complexity of the immunological mechanisms involved, and in the process have uncovered some novel potential therapeutic targets. The effectiveness of thalidomide and eicosapentaenoic acid at attenuating weight loss in clinical trials also provides a further rationale for modulating the immune response. We are now entering an exciting period in cachexia research, and it is likely that the next few years will see effective new biological therapies reach clinical practice.

Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.

A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.

Positive and negative symptoms and social competence in adolescents at risk for schizophrenia and affective disorder.

The authors compared adolescents at risk for schizophrenia and affective disorder and normal adolescents. The subjects at risk for schizophrenia had significantly poorer social competence, and formal thought disorder was greater in both high-risk groups. There were no group differences in negative symptoms.

Solute movement through an allophanic soil.

Allophanic soils are widespread around the world, but little research has been done on their transport properties. This study reveals the effect of two soil water potential heads and two water-flow regimes of continuous and intermittent flow on solute transport through undisturbed soil columns of Horotiu silt loam (Typic Hapludand), an allophanic soil. Two different methods--breakthrough curves (BTCs) and time domain reflectometry (TDR)--were employed to determine the extent of preferential solute transport in the topsoil. The TDR data were also used to look at the depth dependence of the transport properties. The convection-dispersion equation (CDE) with the appropriate boundary conditions adequately described the movement of both Br and Cl under the various flow conditions. Although no preferential flow was found under the imposed unsaturated flow conditions, the flow of water and transport of solute became more uniform with depth. The results show that both Br and Cl are retarded in this allophanic soil. Retardation values range from 1.5 to 1.9, and, as the TDR data showed, increase from the depth of 5.0 to 10.0 cm. Intermittent leaching results showed that there was no effect on solute concentrations in the leachate following no-flow periods. This suggests that water and solute transport in this soil were either relatively uniform or that transverse mixing during flow was already fast enough to eliminate concentration gradients between regions of different "mobility."

Tree species for recovering nitrogen from dairy-farm effluent in New Zealand.

Land treatment of dairy-farm effluent is being widely adopted as an alternative to disposal into surface waters in New Zealand. This study investigated water balances and associated N leaching from short-rotation forest (SRF) species irrigated with dairy-farm effluent. Single trees were grown in lysimeters filled with Manawatu fine sandy loam (mixed mesic Dystric Eutrochrept). Dairy-farm effluent was applied during two irrigation periods at 21.5 mm wk(-1) with a total loading equivalent to 870 kg N ha(-1) occurring over 17 mo. Following tree harvest in April 1997, measurements continued until August 1997 to monitor tree reestablishment. Cumulative N leached did not differ between lysimeters in which evergreen Sydney blue gum (Eucalyptus saligna Sm.) and shining gum [Eucalyptus nitens (H. Deane & Maiden) Maiden] and deciduous kinu-yanagi (Salix kinuyanagi Kimura) were grown. Leachate N concentrations of all treatments were on average higher than the New Zealand drinking water standard of 11.3 mg N L(-1). The E. nitens and S. kinuyanagi treatments leached 33 and 35 kg N ha(-1) yr(-1) in 1996 following application of 236 kg N ha(-1) during the first irrigation season. Leaf area was strongly correlated to evapotranspiration, drainage volume, and nitrogen leached. The majority of leaching in the tree treatments occurred after harvest. Reducing the leaching in the regrowth phase may be achieved through timing harvest in the spring when growth rates are higher and leaching potential is lower. Based on N uptake rates observed in this study and average pond discharge, a plantation of 5.4 ha would be required for N recovery on a typical dairy farm in New Zealand.

Antiproliferative effects of sapacitabine (CYC682), a novel 2'-deoxycytidine-derivative, in human cancer cells.

This study assessed the antiproliferative activity of sapacitabine (CYC682, CS-682) in a panel of 10 human cancer cell lines with varying degrees of resistance or sensitivity to the commonly used nucleoside analogues ara-C and gemcitabine. Growth inhibition studies using sapacitabine and CNDAC were performed in the panel of cell lines and compared with both nucleoside analogues and other anticancer compounds including oxaliplatin, doxorubicin, docetaxel and seliciclib. Sapacitabine displayed antiproliferative activity across a range of concentrations in a variety of cell lines, including those shown to be resistant to several anticancer drugs. Sapacitabine is biotransformed by plasma, gut and liver amidases into CNDAC and causes cell cycle arrest predominantly in the G(2)/M phase. No clear correlation was observed between sensitivity to sapacitabine and the expression of critical factors involved in resistance to nucleoside analogues such as deoxycytidine kinase (dCK), human equilibrative nucleoside transporter 1, cytosolic 5'-nucleotidase and DNA polymerase-alpha. However, sapacitabine showed cytotoxic activity against dCK-deficient L1210 cells indicating that in some cells, a dCK-independent mechanism of action may be involved. In addition, sapacitabine showed a synergistic effect when combined with gemcitabine and sequence-specific synergy with doxorubicin and oxaliplatin. Sapacitabine is therefore a good candidate for further evaluation in combination with currently used anticancer agents in tumour types with unmet needs.