Characterization of the metabolic effects of irisin on skeletal muscle in vitro.

AIMS: This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. METHODS: C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. RESULTS: Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. CONCLUSIONS: Our observations are the first to document increased metabolism in myocytes through irisin-mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.

Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.

DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex.

Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50.Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3' to 5' exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50.Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3' and 5' single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3' single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5' strand at secondary structures formed upon DNA strand separation.

Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy.

The RAD50 gene of Saccharomyces cerevisiae is one of several genes required for recombinational repair of double-strand DNA breaks during vegetative growth and for initiation of meiotic recombination. Rad50 forms a complex with two other proteins, Mre11 and Xrs2, and this complex is involved in double-strand break formation and processing. Rad50 has limited sequence homology to the structural maintenance of chromosomes (SMC) family of proteins and shares the same domain structure as SMCs: N- and C-terminal globular domains separated by two long coiled-coils. However, a notable difference is the much smaller non-coil hinge region between the two coiled-coils. We report here a structural analysis of full-length S. cerevisiae Rad50, alone and in a complex with yeast Mre11 by electron microscopy. Our results confirm that yeast Rad50 does have the same antiparallel coiled-coil structure as SMC proteins, but with no detectable globular hinge domain. However, the molecule is still able to bend sharply in the middle to bring the two catalytic domains together, indicating that the small hinge domain is flexible. We also demonstrate that Mre11 binds as a dimer between the catalytic domains of Rad50, bringing the nuclease activities of Mre11 in close proximity to the ATPase and DNA binding activities of Rad50.

Basis for avid homologous DNA strand exchange by human Rad51 and RPA.

Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown here to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors.

Are NMDA receptors involved in opiate-induced neural and behavioral plasticity? A review of preclinical studies.

RATIONALE: Research over the past decade demonstrating that NMDA receptor antagonists have the ability to inhibit opiate tolerance, sensitization and physical dependence has led to the suggestion that NMDA receptors may have a critical role in opiate-induced neural and behavioral plasticity. However, there have been suggestions that the effects of NMDA receptor antagonists on these phenomena result from non-specific behavioral or pharmacological effects, rather than from a specific inhibition of plasticity. OBJECTIVES: To review the literature in order to explore whether the effects of NMDA receptor antagonists on opiate-induced changes in behavior are best accounted for by an inhibition of neural and behavioral plasticity, or if alternative explanations might better account for the results. RESULTS: The effects of NMDA receptor antagonists on the development of tolerance to opiate analgesia and the development of opiate physical dependence do not appear to be due to confounding behavioral effects produced by high doses of NMDA receptor antagonists, "side-effects" of a particular drug or drug class, blockade of associative learning processes, or state-dependency. Results on tolerance and sensitization to the locomotor effects of morphine are more mixed and controversial; however, there is evidence suggesting that NMDA receptor antagonists may inhibit these phenomena in a similar manner. CONCLUSIONS: NMDA receptor antagonists appear to inhibit the neural plasticity underlying some forms of opiate tolerance, sensitization and physical dependence, suggesting that NMDA receptors are involved in the development of these drug-induced changes in behavior. Further research will help to determine the neural mechanisms responsible for these phenomena, and the therapeutic potential for drugs acting on the NMDA receptor complex in the treatment of pain and addiction.

Prospective, randomized, single-blind comparison of two preparations for screening flexible sigmoidoscopy.

BACKGROUND: The best and most cost-effective bowel cleansing regimen for patients undergoing flexible sigmoidoscopy is not known. The aim of this study was to compare patient tolerance, quality of preparation, and cost of 2 bowel cleansing regimens for flexible sigmoidoscopy. METHODS: Two hundred fifty consecutive patients referred for screening flexible sigmoidoscopy were randomized to receive an oral preparation (45 mL oral sodium phosphate and 10 mg bisacodyl) or an enema preparation (2 Fleet enemas and 10 mg bisacodyl). Tolerance of the preparation was graded as easy, tolerable, slightly difficult, extremely difficult, or intolerable. The endoscopist was blinded to which preparation the patient received and graded the quality of the preparation as poor, fair, good, or excellent. Cost was calculated by adding the cost of the medications and the cost for the nursing time required to prepare the patient for endoscopy. RESULTS: Patients in the oral preparation group were more likely to grade the preparation as easy or tolerable when compared with the enema group (96.8% vs. 56.4%, p < 0.001). The endoscopist graded the quality of the preparation as good or excellent in 86.5% of the patients in the oral preparation group compared with 57.3% in the enema group (p < 0.001). In the oral preparation group, the mean nursing time (34.6 vs. 65.3 minutes, p < 0.001) and cost ($16.39 vs. $31.13, p < 0.001) were significantly less than in the enema group. CONCLUSIONS: An oral sodium phosphate preparation results in a superior quality endoscopic examination that is better tolerated and more cost-effective than enemas in patients undergoing screening flexible sigmoidoscopy.

Recombination factors of Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has been an excellent genetic and biochemical model for our understanding of homologous recombination. Central to the process of homologous recombination are the products of the RAD52 epistasis group of genes, whose functions we now know include the nucleolytic processing of DNA double-stand breaks, the ability to conduct a DNA homology search, and the capacity to promote the exchange of genetic information between homologous regions on recombining chromosomes. It is also clear that the basic functions of the RAD52 group of genes have been highly conserved among eukaryotes. Disruption of this important process causes genomic instability, which can result in a number of unsavory consequences, including tumorigenesis and cell death.

Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK), consisting of Ku and the DNA-PK catalytic subunit (DNA-PKcs), and the DNA ligase IV-XRCC4 complex function together in the repair of DNA double-strand breaks by non-homologous end joining. These protein complexes are also required for the completion of V(D)J recombination events in immune cells. Here we demonstrate that the DNA ligase IV-XRCC4 complex binds specifically to the ends of duplex DNA molecules and can act as a bridging factor, linking together duplex DNA molecules with complementary but non-ligatable ends. Although the DNA end-binding protein Ku inhibited DNA joining by DNA ligase IV-XRCC4, it did not prevent this complex from binding to DNA. Instead, DNA ligase IV-XRCC4 and Ku bound simultaneously to the ends of duplex DNA molecules. DNA ligase IV-XRCC4 and DNA-PKcs also formed complexes at the ends of DNA molecules, but DNA-PKcs did not inhibit ligation. Interestingly, DNA-PKcs stimulated intermolecular ligation by DNA ligase IV-XRCC4. In the presence of DNA-PK, the majority of the joining events catalyzed by DNA ligase IV-XRCC4 were intermolecular because Ku inhibited intramolecular ligation, but DNA-PKcs still stimulated intramolecular ligation. We suggest that DNA-PKcs-containing complexes formed at DNA ends enhance the association of DNA ends via protein-protein interactions, thereby stimulating intermolecular ligation.

Modulation of terminal deoxynucleotidyltransferase activity by the DNA-dependent protein kinase.

Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86-/- animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions.

Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl.

Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts. Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo. In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair. After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R.

Plasmodium berghei: identification of an mdr-like gene associated with drug resistance.

Amplification, mutations, or overexpression of the pfmdr1 gene have been associated with multiple drug resistance in some strains of Plasmodium falciparum. In order to better understand this potential mechanism of drug resistance, we are currently investigating putative mdr homologues in vivo in the rodent malaria Plasmodium berghei. We have identified and partially sequenced a gene that is amplified in a MFQ-resistant (MFQr) line. Using degenerate primers, a 579-bp fragment was amplified by PCR using P. berghei genomic DNA as template. The predicted amino acid sequence shares 66% identity with the previously reported pfmdr1 gene product (Pgh1) of P. falciparum. Southern blots and slot blots of genomic DNA suggest that this gene is amplified two- to threefold in a MFQr line (N/1100), as has been previously reported in some MFQr strains of P. falciparum. The P. berghei gene was mapped to chromosome 12 in all of the lines analyzed. Furthermore, the cloned PCR product also hybridizes to chromosome 5 of the MFQr strain.

Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95.

Genetic studies in yeast have indicated a role of the RAD50 and MRE11 genes in homologous recombination, telomere length maintenance, and DNA repair processes. Here, we purify from nuclear extract of Raji cells a complex consisting of human Rad50, Mre11, and another protein factor with a size of about 95 kDa (p95), which is likely to be Nibrin, the protein encoded by the gene mutated in Nijmegen breakage syndrome. We show that the Rad50-Mre11-p95 complex possesses manganese-dependent single-stranded DNA endonuclease and 3' to 5' exonuclease activities. These nuclease activities are likely to be important for recombination, repair, and genomic stability.

Motivational properties of oxytocin in the conditioned place preference paradigm.

We hypothesized that oxytocin might have intrinsic reinforcing properties and studied it using a conditioned place preference. Three studies examining motivational properties of oxytocin in nonpreferred, preferred, and balance designs were performed utilizing two compartment apparatus. On alternate days, compartments were paired with subcutaneously injected oxytocin (6 mg/kg) or saline, and animal pre- and post-conditioning place preference was compared. Whereas in animals paired with saline there was a shift to a lack of preference, oxytocin-treated animals reversed their preference, spending more time in a previously unpreferred, compartment. In preferred compartment design, oxytocin-treated animals further increased their preference, whereas saline-treated animals decreased their preference toward a nonpreference for either compartment. Our results demonstrate that oxytocin produces a reliable and robust preference for the environment with which it is repeatedly associated, and has rewarding or potentially anti-aversive properties. Future studies are needed to distinguish among these possibilities.

Effects of noncompetitive N-methyl-D-aspartate receptor antagonists on opiate tolerance and physical dependence.

Recent research has demonstrated that N-methyl-D-aspartate (NMDA) receptors, a class of excitatory amino acid receptors, may have an important role in opiate tolerance and physical dependence. Much of the evidence for this has arisen from studies that have examined the effects of NMDA receptor antagonists on these phenomena. This article summarizes research from our laboratory on the effects of NMDA receptor antagonists on opiate tolerance and dependence in rats. Noncompetitive NMDA antagonists, including MK-801, ketamine, phencyclidine, and dextrorphan have been found at low doses to inhibit the development, or acquisition, of opiate tolerance and dependence but not the expression. The results suggest that NMDA receptors have a role in the neural plasticity responsible for tolerance and dependence. Selected theoretical and therapeutic implications of these findings are discussed.

Effects of chronic opiate and opioid antagonist treatment on striatal opioid peptides.

It has long been speculated that feedback inhibition of endogenous opioid neurons may have a role in opiate tolerance and dependence. However, in studies in which opiates or opioid antagonists have been administered to animals, mixed results have been obtained on the ability of these drugs to regulate endogenous opioids. The present studies were undertaken to determine the effects of chronic administration of opiate drugs on opioid peptides. These studies focused on the regulation of prodynorphin (Prodyn) and proenkephalin (Proenk) peptides in striatal tissue. Morphine, whether administered by chronic infusion or repeated injection, was found to increase the concentration of Prodyn peptides in striatum. Increases were statistically significant in the sensorimotor dorsal striatum (caudate-putamen) but not in the limbic-motor ventral striatum (nucleus accumbens-olfactory tubercle). No changes in Prodyn peptides were found following chronic administration of the opioid antagonist naltrexone. No changes in the Proenk peptide MERGL were found following chronic treatment with morphine or naltrexone. These studies are consistent with the suggestion that Prodyn neurons may have a role in the consequences of long-term opiate administration.

Excitatory amino acids and drugs of abuse: a role for N-methyl-D-aspartate receptors in drug tolerance, sensitization and physical dependence.

N-methyl-D-aspartate (NMDA) receptors have been implicated in several types of neural and behavioral plasticity ranging from development to learning. The present paper reviews evidence suggesting that these receptors might also be involved in the neural and behavioral changes resulting from chronic administration of drugs of abuse. NMDA receptor antagonists have been found to interfere with tolerance, sensitization, physical dependence and conditioning to a variety of self-administered drugs, including psychomotor stimulants, opiates, ethanol and nicotine. The results indicate a broad role for NMDA receptors in drug-induced neural and behavioral plasticity, including changes in the brain and behavior that may lead to compulsive drug use, and suggest that drugs acting at the NMDA receptor complex may be clinically useful.

Does chronic nociceptive stimulation alter the development of morphine tolerance?

Conflicting results exist concerning the issues of whether chronic nociceptive stimulation (a) increases or decreases the effectiveness of morphine analgesia, and (b) facilitates or inhibits the development of narcotic tolerance. We carried out a series of experiments with appropriate controls in order to examine these two issues and their possible relationship. In experiment 1, rats received complete Freund's adjuvant (CFA), a chronic nociceptor, injected into a single hind paw or anesthesia without injection, together with morphine or placebo pellets in a 2 x 2 study design. The data indicate that the presence of the chronic nociceptive stimulus significantly facilitated the development of tolerance to morphine analgesia as measured using tail-flick latency (TFL) testing. Experiment 2 was designed to compare the analgetic effectiveness of an acute injection of morphine in rats experiencing chronic nociceptive stimulation and in controls. CFA was injected in the right hindpaw, and nine days later TFLs were tested after morphine doses of 1 and 2 mg/kg s.c. The data obtained showed that chronic nociceptive stimulation significantly reduced the effectiveness of morphine at the 1 mg/kg dose. However, baseline TFLs appeared to be shorter in rats treated with CFA, suggesting that the decrease in morphine effectiveness could be due to a general increase in pain sensitivity. Therefore, a third experiment was performed, using a less intense thermal stimulus to prolong baseline TFLs and accentuate any potential differences. Sixteen rats either received CFA or served as controls. TFLs were then measured at baseline and one hour after a 0.5 mg/kg dose of morphine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of opiate tolerance by non-competitive N-methyl-D-aspartate receptor antagonists.

Our laboratory and others have previously reported that the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, interferes with the development of tolerance to the analgesic effects of morphine. The present studies were performed in order to further characterize the role of NMDA receptors in opiate tolerance. The results demonstrate that opiate tolerance is inhibited rapidly, and at low doses, by four different non-competitive NMDA receptor antagonists (MK-801, ketamine, dextrorphan and phencyclidine), suggesting that this inhibition results from blockade of NMDA receptors rather than from the 'side-effect' of a particular drug. The NMDA antagonists were found to inhibit the development but not the expression of opiate tolerance; i.e. they were able to prevent but not reverse tolerance. Finally, the results suggest that NMDA receptor antagonists do not interfere with associative tolerance; instead it appears that these drugs may specifically inhibit non-associative tolerance. It thus appears that NMDA receptors may have a fundamental role in the development of opiate tolerance, and that non-competitive NMDA receptor antagonists may be effective adjuncts to opiates in the treatment of chronic pain.

MK-801 inhibits the development of morphine tolerance at spinal sites.

The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 has been shown to attenuate tolerance development in rats. In this study, we show that MK-801 inhibits tolerance to the antinociceptive effects of morphine, as assessed by the tail-flick test, in spinalized rats. These results suggest that NMDA receptor antagonists inhibit opiate tolerance at spinal sites, and also provide strong evidence that the effects of MK-801 are not due to its ability to interfere with associative learning, but instead to inhibition of non-associative mechanisms of opiate tolerance.