Effects of inbreeding and rate of inbreeding in Drosophila melanogaster- Hsp70 expression and fitness

Induction of heat shock proteins (Hsp) is a well-known mechanism through which cells cope with stressful conditions. Hsp are induced by a variety of extrinsic stressors. However, recently intrinsic stressors (aging and inbreeding) have been shown to affect expression of Hsp. Increased homozygosity due to inbreeding may disrupt cellular homeostasis by causing increased expression of recessive deleterious mutations and breakdown of epistatic interactions. We investigated the effect of inbreeding and the rate of inbreeding on the expression of Hsp70, larval heat resistance and fecundity. In Drosophila melanogaster we found that inbred lines (F approximately 0.67) had significantly up-regulated expression of Hsp70, and reduced heat resistance and fecundity as compared with outbred control lines. A significant negative correlation was observed between Hsp70 expression and resistance to an extreme heat stress in inbred lines. We interpreted this as an increased requirement for Hsp70 in the lines suffering most from inbreeding depression. Inbreeding depression for fecundity was reduced with a slower rate of inbreeding compared with a fast rate of inbreeding. Thus, the effectiveness of purging seems to be improved with a slower rate of inbreeding.     

Heparan sulfate biosynthesis: a theoretical study of the initial sulfation step by N-deacetylase/N-sulfotransferase

Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N-acetyl-D-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (, J. Biol. Chem. 274:10673-10676). We have used the x-ray crystal structure of heparan sulfate N-sulfotransferase with 3'-phosphoadenosine 5'-phosphate to build a solution model with cofactor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu(642), Lys(614), and Lys(833), with the possible involvement of Thr(617) and Thr(618), in binding PAPS. Additionally, Lys(676) is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.     

QDist--quartet distance between evolutionary trees

SUMMARY: QDist is a program for computing the quartet distance between two unrooted trees, i.e. the number of quartet topology differences between the trees, where a quartet topology is the topological subtree induced by four species. The program is based on an algorithm with running time O(n log2 n), which makes it practical to compare large trees. Available under GNU license. AVAILABILITY: http://www.birc.dk/Software/QDist     

Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans

Heat shock protein (Hsp) 72 is a cytosolic stress protein that is highly inducible by several factors including exercise. Hsp60 is primarily mitochondrial in cellular location, plays a key role in the intracellular protein translocation and cytoprotection, is increased in skeletal muscle by exercise, and is found in the peripheral circulation of healthy humans. Glucose deprivation increases Hsp72 in cultured cells, whereas reduced glycogen availability elevates Hsp72 in contracting human skeletal muscle. To determine whether maintained blood glucose during exercise attenuates the exercise-induced increase in intramuscular and circulating Hsp72 and Hsp60, 6 males performed 120 minutes of semirecumbent cycling at approximately 65% maximal oxygen uptake on 2 occasions while ingesting either a 6.4% glucose (GLU) or sweet placebo (CON) beverage throughout exercise. Muscle biopsies, obtained before and immediately after exercise, were analyzed for Hsp72 and Hsp60 protein expression. Blood samples were simultaneously obtained from a brachial artery, a femoral vein, and the hepatic vein before and during exercise for the analysis of serum Hsp72 and Hsp60. Leg and hepatosplanchnic blood flow were measured to determine Hsp72-Hsp60 flux across these tissue beds. Neither exercise nor glucose ingestion affected the Hsp72 or Hsp60 protein expression in, or their release from, contracting skeletal muscle. Arterial serum Hsp72 increased (P < 0.05) throughout exercise in both trials but was attenuated (P < 0.05) in GLU. This may have been in part because of the increased (P < 0.05) hepatosplanchnic Hsp72 release in CON, being totally abolished (P < 0.05) in GLU. Serum Hsp60 increased (P < 0.05) after 60 minutes of exercise in CON before returning to resting levels at 120 minutes. In contrast, no exercise-induced increase in serum Hsp60 was observed in GLU. We detected neither hepatosplanchnic nor contracting limb Hsp60 release in either trial. In conclusion, maintaining glucose availability during exercise attenuates the circulating Hsp response in healthy humans.     

NetMHCpan,a method for MHC class I binding prediction beyond humans

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide–MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method’s ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at . Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The cancer cell’s “power plants” as promising therapeutic targets: An overview

This introductory article to the review series entitled “The Cancer Cell’s Power Plants as Promising Therapeutic Targets” is written while more than 20 million people suffer from cancer. It summarizes strategies to destroy or prevent cancers by targeting their energy production factories, i.e., “power plants.” All nucleated animal/human cells have two types of power plants, i.e., systems that make the “high energy” compound ATP from ADP and P _i . One type is “glycolysis,” the other the “mitochondria.” In contrast to most normal cells where the mitochondria are the major ATP producers (>90%) in fueling growth, human cancers detected via Positron Emission Tomography (PET) rely on both types of power plants. In such cancers, glycolysis may contribute nearly half the ATP even in the presence of oxygen (“Warburg effect”). Based solely on cell energetics, this presents a challenge to identify curative agents that destroy only cancer cells as they must destroy both of their power plants causing “necrotic cell death” and leave normal cells alone. One such agent, 3-bromopyruvate (3-BrPA), a lactic acid analog, has been shown to inhibit both glycolytic and mitochondrial ATP production in rapidly growing cancers (Ko et al., Cancer Letts., 173, 83–91, 2001), leave normal cells alone, and eradicate advanced cancers (19 of 19) in a rodent model (Ko et al., Biochem. Biophys. Res. Commun., 324, 269–275, 2004). A second approach is to induce only cancer cells to undergo “apoptotic cell death.” Here, mitochondria release cell death inducing factors (e.g., cytochrome c). In a third approach, cancer cells are induced to die by both apoptotic and necrotic events. In summary, much effort is being focused on identifying agents that induce “necrotic,” “apoptotic” or apoptotic plus necrotic cell death only in cancer cells. Regardless how death is inflicted, every cancer cell must die, be it fast or slow.     

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.     

Insulin stimulates interleukin-6 and tumor necrosis factor-alpha gene expression in human subcutaneous adipose tissue

High circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are found in patients with hyperinsulinemia. Insulin stimulates release of IL-6 from adipocyte cultures, and it stimulates IL-6 gene expression in insulin-resistant, but not control, rat skeletal muscle. In addition, TNF-alpha may be involved in the pathogenesis of insulin resistance. Therefore, we studied the effect of insulin on IL-6 and TNF-alpha gene expression in human skeletal muscle and adipose tissue. Nine healthy young volunteers participated in the study. They underwent a 6-h hyperinsulinemic euglycemic clamp at a fixed insulin infusion rate, with blood glucose clamped at fasting level. Blood samples drawn at 0, 1, 2, 3, 4, 5, and 6 h were analyzed for IL-6 and TNF-alpha. Muscle and fat biopsies, obtained at 0, 2, 4, and 6 h, were analyzed for IL-6 and TNF-alpha mRNA with real-time PCR. IL-6 mRNA increased 11-, 3-, and 5-fold at 2, 4, and 6 h, respectively, in adipose tissue (ANOVA P = 0.027), whereas there was no significant effect of insulin on skeletal muscles. Plasma IL-6 increased during insulin stimulation. TNF-alpha mRNA increased 2.4-, 1.4-, and 2.2-fold in adipose tissue (ANOVA P = 0.001) and decreased 0.74-, 0.64-, and 0.68-fold in muscle tissue (ANOVA P = 0.04). Plasma levels of TNF-alpha were constant. In conclusion, the finding that insulin stimulates IL-6 and TNF-alpha gene expression in adipose tissue only and inhibits the TNF-alpha production in skeletal muscles suggests a differential regulation of muscle- and adipose tissue-derived IL-6 and TNF-alpha.