Mitochondrial p38 Mitogen-Activated Protein Kinase: Insights into Its Regulation of and Role in LONP1-Deficient Nematodes.

p38 Mitogen-Activated Protein Kinase (MAPK) cascades are central regulators of numerous physiological cellular processes, including stress response signaling. In C. elegans, mitochondrial dysfunction activates a PMK-3/p38 MAPK signaling pathway (MAPKmt), but its functional role still remains elusive. Here, we demonstrate the induction of MAPKmt in worms deficient in the lonp-1 gene, which encodes the worm ortholog of mammalian mitochondrial LonP1. This induction is subjected to negative regulation by the ATFS-1 transcription factor through the CREB-binding protein (CBP) ortholog CBP-3, indicating an interplay between both activated MAPKmt and mitochondrial Unfolded Protein Response (UPRmt) surveillance pathways. Our results also reveal a genetic interaction in lonp-1 mutants between PMK-3 kinase and the ZIP-2 transcription factor. ZIP-2 has an established role in innate immunity but can also modulate the lifespan by maintaining mitochondrial homeostasis during ageing. We show that in lonp-1 animals, ZIP-2 is activated in a PMK-3-dependent manner but does not confer increased survival to pathogenic bacteria. However, deletion of zip-2 or pmk-3 shortens the lifespan of lonp-1 mutants, suggesting a possible crosstalk under conditions of mitochondrial perturbation that influences the ageing process. Furthermore, loss of pmk-3 specifically diminished the extreme heat tolerance of lonp-1 worms, highlighting the crucial role of PMK-3 in the heat shock response upon mitochondrial LONP-1 inactivation.

The Thermal Stress Coping Network of the Nematode Caenorhabditis elegans.

Response to hyperthermia, highly conserved from bacteria to humans, involves transcriptional upregulation of genes involved in battling the cytotoxicity caused by misfolded and denatured proteins, with the aim of proteostasis restoration. C. elegans senses and responds to changes in growth temperature or noxious thermal stress by well-defined signaling pathways. Under adverse conditions, regulation of the heat shock response (HSR) in C. elegans is controlled by a single transcription factor, heat-shock factor 1 (HSF-1). HSR and HSF-1 in particular are proven to be central to survival under proteotoxic stress, with additional roles in normal physiological processes. For years, it was a common belief that upregulation of heat shock proteins (HSPs) by HSF-1 was the main and most important step toward thermotolerance. However, an ever-growing number of studies have shown that targets of HSF-1 involved in cytoskeletal and exoskeletal integrity preservation as well as other HSF-1 dependent and independent pathways are equally important. In this review, we follow the thermal stimulus from reception by the nematode nerve endings till the activation of cellular response programs. We analyze the different HSF-1 functions in HSR as well as all the recently discovered mechanisms that add to the knowledge of the heat stress coping network of C. elegans.

Fluorescent blood-brain barrier tracing shows intact leptin transport in obese mice.

BACKGROUND/OBJECTIVES: Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance. METHODS: We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting. RESULTS: Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in microdissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment. CONCLUSIONS: Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.

Interplay between endogenous and exogenous human retroviruses.

In humans, retroviruses thrive more as symbionts than as parasites. Apart from the only two modern exogenous human retroviruses (human T-cell lymphotropic and immunodeficiency viruses; HTLV and HIV, respectively), ~8% of the human genome is occupied by ancient retroviral DNA [human endogenous retroviruses (HERVs)]. Here, we review the recent discoveries about the interactions between the two groups, the impact of infection by exogenous retroviruses on the expression of HERVs, the effect of HERVs on the pathogenicity of HIV and HTLV and on the severity of the diseases caused by them, and the antiviral protection that HERVs can allegedly provide to the host. Tracing the crosstalk between contemporary retroviruses and their endogenized ancestors will provide better understanding of the retroviral world.

Unexpected enzyme-catalyzed regioselective acylation of flavonoid aglycones and rapid product screening.

Unprecedented regioselective acylation of flavonoid aglycones was achieved using Candida antarctica lipase B (CALB). The rapid screening of product formation was performed by the use of the high resolution phenol-type OH (1)H NMR spectral region recorded after the addition of picric acid.

Organismal and Cellular Stress Responses upon Disruption of Mitochondrial Lonp1 Protease.

Cells engage complex surveillance mechanisms to maintain mitochondrial function and protein homeostasis. LonP1 protease is a key component of mitochondrial quality control and has been implicated in human malignancies and other pathological disorders. Here, we employed two experimental systems, the worm Caenorhabditis elegans and human cancer cells, to investigate and compare the effects of LONP-1/LonP1 deficiency at the molecular, cellular, and organismal levels. Deletion of the lonp-1 gene in worms disturbed mitochondrial function, provoked reactive oxygen species accumulation, and impaired normal processes, such as growth, behavior, and lifespan. The viability of lonp-1 mutants was dependent on the activity of the ATFS-1 transcription factor, and loss of LONP-1 evoked retrograde signaling that involved both the mitochondrial and cytoplasmic unfolded protein response (UPRmt and UPRcyt) pathways and ensuing diverse organismal stress responses. Exposure of worms to triterpenoid CDDO-Me, an inhibitor of human LonP1, stimulated only UPRcyt responses. In cancer cells, CDDO-Me induced key components of the integrated stress response (ISR), the UPRmt and UPRcyt pathways, and the redox machinery. However, genetic knockdown of LonP1 revealed a genotype-specific cellular response and induced apoptosis similar to CDDO-Me treatment. Overall, the mitochondrial dysfunction ensued by disruption of LonP1 elicits adaptive cytoprotective mechanisms that can inhibit cancer cell survival but diversely modulate organismal stress response and aging.

DNA damage independent inhibition of NF-κB transcription by anthracyclines.

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.

Detection and quantification of the anti-obesity drug celastrol in murine liver and brain.

Celastrol is a natural pentacyclic triterpene extracted from the roots of Tripterygium wilfordi (thunder god vine). Celastrol was reported as a powerful anti-obesity drug with leptin sensitizing properties that decreases food consumption and mediates body weight loss when administered to diet-induced obese mice at 100 µg/kg body weight. The weight lowering properties of celastrol are likely mediated by the CNS, in particular, by the hypothalamus, but the final proof for the accumulation of celastrol in the brain and hypothalamus remains to be established. Here, we aimed to demonstrate that intraperitoneal celastrol administration at 100 µg/kg can rapidly reach the brain and, in particular, the hypothalamus of mice. We developed and validated a sensitive liquid chromatography mass spectrometry method for the quantitative determination of celastrol in murine tissues, namely liver, brain and hypothalamus. Chow-fed lean mice were randomly assigned to the vehicle vs. celastrol groups, injected with saline or 100 µg/kg body weight of celastrol, and sacrificed 30 min or 120 min post injection. Celastrol was extracted from homogenized tissue using ethyl acetate as organic solvent, and quantified using a matrix-matched calibration curve with glycyrrhetinic acid as internal standard. Liver celastrol concentrations were 32.60 ± 8.21 pg/mg and 40.52 ± 15.6 pg/mg, 30 and 120 min after injection, respectively. We found 4.70 ± 0.31 pg/mg celastrol after 30 min, and 16.22 ± 3.33 pg/mg after 120 min in whole brain lysates, and detectable amounts in the hypothalamus. These results corroborate the validity of our methodology, demonstrate the accumulation of celastrol in the brain of mice injected intraperitoneally with a dose of 100 µg/kg, and confirm the CNS as possible site of action for the weight lowering properties of celastrol.

Biodegradation and toxicity of emerging contaminants: Isolation of an exopolysaccharide-producing Sphingomonas sp. for ionic liquids bioremediation.

Ionic liquids (ILs) have been characterized as contaminants of emerging concern (CEC) that often resist biodegradation and impose toxicity upon environmental release. Sphingomonas sp. MKIV has been isolated as an extreme microorganism capable for biodegradation of major classes of ILs. Six imidazolium-, pyridinium- and ammonium-based ILs (pyridinium trifluoromethanesulfonate [Py][CF3SO3], 1-(4-pyridyl)pyridinium chloride [1-4PPy][Cl], 1-butyl-3-methylimidazolium bromide [BMIM][Br], 1-butyl-3-methylimidazolium methanesulfonate [BMIM][MeSO4], tetrabutylammonium iodide [n-Bu4N][I] and tetrabutylammonium hexafluorophosphate [n-Bu4N][PF6]) were used for microbial growth. The strain achieved 91% and 87% removal efficiency for cultures supplemented with 100 mg L-1 of [BMIM][MeSO4] and [n-Bu4N][I] respectively. The metabolic activity of MKIV was inhibited following preliminary stages of cultures conducted using [BMIM][MeSO4], [BMIM][Br], [Py][CF3SO3] and [n-Bu4N][PF6], indicating potential accumulation of inhibitory metabolites. Thus, a comprehensive toxicological study of the six ILs on Aliivibrio fischeri, Daphnia magna and Raphidocelis subcapitata was conducted demonstrating that the compounds impose moderate and low toxicity. The end-products from [BMIM][MeSO4] and [n-Bu4N][I] biodegradation were assessed using Aliivibrio fischeri, exhibiting increased environmental impact of the latter following biotreatment. MKIV produced 19.29 g L-1 of biopolymer, comprising mainly glucose and galacturonic acid, from 25 g L-1 of glucose indicating high industrial significance for bioremediation and exopolysaccharide production.

Celastrol Promotes Weight Loss in Diet-Induced Obesity by Inhibiting the Protein Tyrosine Phosphatases PTP1B and TCPTP in the Hypothalamus.

Celastrol is a natural pentacyclic triterpene used in traditional Chinese medicine with significant weight-lowering effects. Celastrol-administered mice at 100 µg/kg decrease food consumption and body weight via a leptin-dependent mechanism, yet its molecular targets in this pathway remain elusive. Here, we demonstrate in vivo that celastrol-induced weight loss is largely mediated by the inhibition of leptin negative regulators protein tyrosine phosphatase (PTP) 1B (PTP1B) and T-cell PTP (TCPTP) in the arcuate nucleus (ARC) of the hypothalamus. We show in vitro that celastrol binds reversibly and inhibits noncompetitively PTP1B and TCPTP. NMR data map the binding site to an allosteric site in the catalytic domain that is in proximity of the active site. By using a panel of PTPs implicated in hypothalamic leptin signaling, we show that celastrol additionally inhibited PTEN and SHP2 but had no activity toward other phosphatases of the PTP family. These results suggest that PTP1B and TCPTP in the ARC are essential for celastrol's weight lowering effects in adult obese mice.

Celastrol-Induced Weight Loss Is Driven by Hypophagia and Independent From UCP1.

Celastrol, a plant-derived constituent of traditional Chinese medicine, has been proposed to offer significant potential as an antiobesity drug. However, the molecular mechanism for this activity is unknown. We show that the weight-lowering effects of celastrol are driven by decreased food consumption. Although young Lep ob mice respond with a decrease in food intake and body weight, adult Lep db and Lep ob mice are unresponsive to celastrol, suggesting that functional leptin signaling in adult mice is required to elicit celastrol's catabolic actions. Protein tyrosine phosphatase 1 (PTP1B), a leptin negative-feedback regulator, has been previously reported to be one of celastrol's targets. However, we found that global PTP1B knockout (KO) and wild-type (WT) mice have comparable weight loss and hypophagia when treated with celastrol. Increased levels of uncoupling protein 1 (UCP1) in subcutaneous white and brown adipose tissue suggest celastrol-induced thermogenesis as a further mechanism. However, diet-induced obese UCP1 WT and KO mice have comparable weight loss upon celastrol treatment, and celastrol treatment has no effect on energy expenditure under ambient housing or thermoneutral conditions. Overall, our results suggest that celastrol-induced weight loss is hypophagia driven and age-dependently mediated by functional leptin signaling. Our data encourage reconsideration of therapeutic antiobesity strategies built on leptin sensitization.

A protein binding site in the M mitochondrial genome of Mytilus galloprovincialis may be responsible for its paternal transmission.

Sea mussels (genus Mytilus) have two mitochondrial genomes in obligatory co-existence, one that is transmitted through the egg and the other through the sperm. The phenomenon, known as Doubly Uniparental Inheritance (DUI) of mitochondrial DNA (mtDNA), is presently known to occur in more than 40 molluscan bivalve species. Females and the somatic tissues of males contain mainly the maternal (F) genome. In contrast, the sperm contains only the paternal (M) genome. Through electrophoretic mobility shift assay (EMSA) experiments we have identified a sequence element in the control region (CR) of the M genome that acts as a binding site for the formation of a complex with a protein factor that occurs in the male gonad. An adenine tract upstream to the element is also essential for the formation of the complex. The reaction is highly specific. It does not occur with protein extracts from the female gonad or from a male or female somatic tissue. Further experiments showed that the interaction takes place in mitochondria surrounding the nucleus of the cells of male gonads, suggesting a distinct role of perinuclear mitochondria. We propose that at a certain point during spermatogenesis mitochondria are subject to degradation and that perinuclear mitochondria with the M mtDNA-protein complex are protected from this degradation with the result that mature spermatozoa contain only the paternal mitochondrial genome.

Regioselective chemical and rapid enzymatic synthesis of a novel redox ­ Antiproliferative molecular hybrid.

Recent science evidenced the interlinkage of oxidative stress and cancer. Due to the inherent complexity of cancer and its accompanying effect of oxidative stress, novel molecules, containing combinatorial functionalities should be targeted. Herein, we synthesized gemcitabine-5'-O-lipoate derived from a regioselective coupling of the chemotherapeutic drug gemcitabine (GEM), a first-line agent for cancer therapy and α-lipoic acid (LA), a potent antioxidant. gemcitabine-5'-O-lipoate was obtained in 4 chemical steps. To avoid the tedious and laborious chemical steps we also utilized biocatalysis using immobilized Candida antarctica lipase B (CALB), and the optimum conditions for the regioselective and one-pot synthesis of gemcitabine-5'-O-lipoate were established by exploiting different solvents (organic solvents and ionic liquids) and enzyme immobilization (acrylic resin and carbon nanotubes). Cytotoxic activity of co-administrating GEM and LA was proven to be synergistic against non-small cell lung cancer cells whereas antagonistic for bladder cancer cells. In contrast, the gemcitabine-5'-O-lipoate hybrid was found to be superior to the parent compounds against both non-small cell lung cancer and bladder cancer cells as also was found to preserve the redox activity of the parent compound LA. The regioselective biotransformation mediated synthesis of the anticancer-antioxidant hybrid illustrates the capacity of biocatalysis to act as an asset in molecular hybridization techniques.

The rRNA and tRNA transcripts of maternally and paternally inherited mitochondrial DNAs of Mytilus galloprovincialis suggest presence of a "degradosome" in mussel mitochondria and necessitate the re-annotation of the l-rRNA/CR boundary.

Species of the genus Mytilus carry two mitochondrial genomes in obligatory coexistence; one transmitted though the eggs (the F type) and one through the sperm (the M type). We have studied the 3' and 5' ends of rRNA and tRNA transcripts using RT-PCR and RNA circularization techniques in both the F and M genomes of Mytilus galloprovincialis. We have found polyadenylated and non-adenylated transcripts for both ribosomal and transfer RNAs. In all these genes the 5' ends of the transcripts coincided with the first nucleotide of the annotated genes, but the 3' ends were heterogeneous. The l-rRNA 3' end is 47 or 48 nucleotides upstream from the one assigned by a previous annotation, which makes the adjacent first domain (variable domain one, VD1) of the main control region (CR) correspondingly longer. We have observed s-rRNA and l-rRNA transcripts with truncated 3' end and polyadenylated tRNA transcripts carrying the CCA trinucleotide. We have also detected polyadenylated RNA remnants carrying the sequences of the control region, which strongly suggests RNA degradation activity and thus presence of degradosomes in Mytilus mitochondria.

Does the ORF in the control region of Mytilus mtDNA code for a protein product?

The control region of the mtDNA of Mytilus is known to contain sequences that determine whether the genome will be paternally or maternally transmitted. An open reading frame (ORF) in this region raised suspicion that it may code for a protein involved in this mechanism. An analysis of the mtDNA transcriptome failed to produce evidence for this hypothesis.

The mRNAs of maternally and paternally inherited mtDNAs of the mussel Mytilus galloprovincialis: start/end points and polycistronic transcripts.

Transcription of the mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis has been studied by RT-PCR and RNA circularization. This species has an egg-transmitted (F) and a sperm-transmitted (M) mitochondrial genome, in accordance with the doubly uniparental inheritance (DUI) pattern of mtDNA transmission. The primary transcript is cleaved into ten transcripts, eight of which are monocistronic, one is tricistronic and one is most likely, but not certainly, bicistronic. The start/end points of these transcripts have been determined. In the majority of cases cleavage is mediated according to the "tRNA punctuation" model. However, we have identified four cases of cleavage that do not coincide with the presence of a tRNA. In these cases transcription starts immediately or only a few bases from the end point of the preceding gene and cleavage is, most likely, mediated by a stem-loop structure formed at the start point of the gene. The identification of a tricistronic transcript is a novel finding for metazoan mtDNA. We propose that its evolution has been facilitated by the fact that all coding genes are transcribed from the same DNA strand and that co-transcription is sustained by selection emanating from the fact that proteins derived from all three co-transcribed genes participate in the formation of the same oxidative phosphorylation complex.

Enzymatic hybridization of α-lipoic acid with bioactive compounds in ionic solvents.

The lipase-catalyzed molecular hybridization of α-lipoic acid (LA) with bioactive compounds pyridoxine, tyrosol and tyramine was performed in ionic solvents and deep eutectic solvents. The biocatalytic reactions were catalyzed by Candida antarctica lipase B immobilized onto various functionalized multi-walled carbon nanotubes (f-CNTs-CaLB), as well as by commercial Novozym 435. The use of f-CNTs-CaLB leads, in most cases, to higher conversion yields as compared to Novozym 435. The nature and ion composition of ionic solvents affect the performance of the biocatalytic process. The highest conversion yield was observed in (mtoa)NTf2. The high enzyme stability and the relatively low solubility of substrates in specific media account for the improved biocatalytic synthesis of molecular hybrids of LA. Principal component analysis was used to screen for potential lipoxygenase inhibitors. In vitro studies showed that the synthesized compounds exhibit up to 10-fold increased inhibitory activity on lipoxygenase mediated lipid peroxidation as compared to parent molecules.

Homocysteine levels and MTHFR polymorphisms in young patients with acute myocardial infarction: a case control study.

INTRODUCTION: Increased levels of homocysteine are known to be associated with coronary artery disease (CAD). The most common form of genetic hyperhomocysteinemia results from MTHFR polymorphisms. To examine the role of homocysteine levels and MTHFR polymorphisms in premature CAD and acute myocardial infarction (MI) in the Cypriot population, a case control study was performed in Nicosia General Hospital. METHODS: Sixty-three male patients less than 50 years old who presented with MI in Nicosia General Hospital were compared with 54 controls without CAD. Fasting homocysteine and lipids were tested within 24 hrs from admission, while MTHFR C677T and A1298C polymorphisms were also tested. RESULTS: Mean homocysteine levels were 14.5 mol/L in patients and 12.3 mol/L in controls (p=0.017). Mutant homozygous MTHFR C677T was present in 17.7% of the patients and 19.2% of the controls (p=0.838), while mutant homozygous MTHFR A1298C was found in 16.1% of patients and 13.5% of controls (p=0.690). Mean homocysteine levels were 12.6 mol/L in patients with single-vessel CAD and 15.5 mol/L in patients with multi-vessel CAD (p=0.025). Lower HDL appeared to be associated with higher levels of homocysteine with an odds ratio of 0.901, indicating that for each unit increase in HDL, the expected odds of having high homocysteine levels decreased by approximately 10%. CONCLUSIONS: Higher levels of homocysteine are associated with acute MI and multi-vessel disease in Cypriot patients under the age of 50. The existence and extent of disease are not associated with MTHFR polymorphisms. Lower HDL is associated with higher levels of homocysteine.

Exploration of the antiplatelet activity profile of betulinic acid on human platelets.

Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation.