Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

Immunotherapy of renal cell carcinoma

Carcinomas of the kidney generally have a poor prognosis and respond minimally to classical radiotherapy or chemotherapy. Immunotherapy constitutes an interesting alternative to these established forms of treatment, and indeed, cytokine-based therapies have been used for many years, leading to favorable clinical responses in a small subset of patients. During the past few years, immunotherapeutical trials targeting renal cell tumor-associated antigens have also been reported, with diverse passive or active approaches using antibodies or aimed at activating tumor-directed T lymphocytes. The following review presents the results and the progress made in the field, including classical cytokine treatments, non-myeloablative stem cell transplantation and antigen specific-based trials, with special focus on T-cell studies. In consideration of the few specific molecular targets described so far for this tumor entity, current strategies which can lead to the identification of new relevant antigens will be discussed. Hopefully these will very soon contribute to an improvement in renal cell carcinoma specific immunotherapy and its evaluation.     

Weak base dispiro-1,2,4-trioxolanes: potent antimalarial ozonides

Thirty weak base 1,2,4-dispiro trioxolanes (secondary ozonides) were synthesized. Amino amide trioxolanes had the best combination of antimalarial and biopharmaceutical properties. Guanidine, aminoxy, and amino acid trioxolanes had poor antimalarial activity. Lipophilic trioxolanes were less stable metabolically than their more polar counterparts.     

SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.     

Propyl paraben inhibits voltage-dependent sodium channels and protects cardiomyocytes from ischemia-reperfusion injury

The effects of propyl paraben, an antimicrobial preservative, on voltage-dependent sodium current and myocardial ischemia-reperfusion injury were investigated in isolated adult rat cardiomyocytes. Whole cell voltage-clamp recording showed that propyl paraben reversibly blocked the voltage-gated sodium channel both in concentration- and voltage-dependent manners. Propyl paraben (500 microM but not 100 microM) significantly shifted the steady-state inactivation of the sodium channel toward the hyperpolarizing direction at the V(1/2) point. Consistent with the above result, the propidium iodide (PI) uptake test revealed that pretreatment with 500 microM but not 100 microM of propyl paraben significantly reduced cell death induced by 45 min of sustained ischemia followed by 15 h of reperfusion (42.37 +/- 7.01% of cell viability in control and 71.05 +/- 7.06% in the propyl paraben group), suggesting that propyl paraben can protect myocytes from ischemia-reperfusion injury. These results indicate a possible correlation between the inhibition of sodium current and cardioprotection against ischemia-reperfusion injury.     

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.     

Availability, not respiratory capacity governs oxygen consumption of solid tumors

Contrary to conventional belief, the mitochondria of most cancer cells usually function normally, i.e., their respiratory capacity is not fundamentally impaired as compared to normal cells. Strong evidence against the misconception of mitochondrial dysfunction is provided by in vivo data clearly showing that O(2) availability is the major determinant of the O(2) consumption rate of cancer cells, independent of the means for increasing availability (e.g., by increasing blood flow or by elevating arterial O(2) content, the latter being accomplished either by an increase in the hemoglobin level and/or arterial hyperoxia). Additional support against the Warburg effect in its original concept comes from normal temperature coefficients (Q(10)) for O(2) consumption rates of malignant cells. Thus, the Warburg hypothesis postulating that mitochondrial dysfunction in cancer cells forces them to generate energy with a poor ATP yield through glycolysis appears to be elusive. Instead, due to a "reprogrammed" cancer cell metabolism, glycolysis is used to produce intermediates as building blocks for various biosynthetic pathways of cancer cells.     

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.     

The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain

The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 microm cGMP for half-maximal effective concentration (EC(50)) of a cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we examine whether modifications in the N-terminus of PDE11A4 affect cGMP signalling. The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases (Ser117, Ser168). Phosphorylation of both by cAMP-dependent protein kinase decreased the EC(50) value for cGMP from 72 to 23 microm. Phosphomimetic point mutations (S117D/S167D), which project complete phosphorylation, lowered the EC(50) value to 16 microm. Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC(50) value of 7.6 microm. Truncation by 176 amino acids was required for cGMP EC(50) values to decrease to below 10 microm; a construct truncated by 168 amino acids had an EC(50) value of 224 microm. The decrease in EC(50) values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however. We conclude that N-terminal modifications strongly affect cGMP regulation of hPDE11A4.