Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction

We have explored the role of mitochondrial function in aging by genetically and pharmacologically modifying yeast cellular respiration production during the exponential and/or stationary growth phases and determining how this affects chronological life span (CLS). Our results demonstrate that respiration is essential during both growth phases for standard CLS, but that yeast have a large respiratory capacity, and only deficiencies below a threshold (~40% of wild-type) significantly curtail CLS. Extension of CLS by caloric restriction also required respiration above a similar threshold during exponential growth and completely alleviated the need for respiration in the stationary phase. Finally, we show that supplementation of media with 1% trehalose, a storage carbohydrate, restores wild-type CLS to respiratory-null cells. We conclude that mitochondrial respiratory thresholds regulate yeast CLS and its extension by caloric restriction by increasing stress resistance, an important component of which is the optimal accumulation and mobilization of nutrient stores.     

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.     

Mutations in the Arabidopsis SWC6 gene, encoding a component of the SWR1 chromatin remodelling complex, accelerate flowering time and alter leaf and flower development

Mutations affecting the Arabidopsis SWC6 gene encoding a putativeorthologue of a component of the SWR1 chromatin remodellingcomplex in plants have been characterized. swc6 mutations causeearly flowering, shortened inflorescence internodes, and alteredleaf and flower development. These phenotypic defects resemblethose of the photoperiod independent early flowering 1 (pie1)and early in short days 1 (esd1) mutants, also affected in homologuesof the SWR1 complex subunits. SWC6 is a ubiquitously expressednuclear HIT-Zn finger-containing protein, with the highest levelsfound in pollen. Double mutant analyses suggest that swc6 abolishesthe FLC-mediated late-flowering phenotype of plants carryingactive alleles of FRI and of mutants of the autonomous pathway.It was found that SWC6 is required for the expression of theFLC repressor to levels that inhibit flowering. However, theeffect of swc6 in an flc null background and the down-regulationof other FLC-like/MAF genes in swc6 mutants suggest that floweringinhibition mediated by SWC6 occurs through both FLC- and FLC-likegene-dependent pathways. Both genetic and physical interactionsbetween SWC6 and ESD1 have been demonstrated, suggesting thatboth proteins act in the same complex. Using chromatin immunoprecipitation,it has been determined that SWC6, as previously shown for ESD1,is required for both histone H3 acetylation and H3K4 trimethylationof the FLC chromatin. Altogether, these results suggest thatSWC6 and ESD1 are part of an Arabidopsis SWR1 chromatin remodellingcomplex involved in the regulation of diverse aspects of plantdevelopment, including floral repression through the activationof FLC and FLC-like genes. Key words: Arabidopsis, chromatin remodelling, floral repression, HIT-Zn finger, phase transition, SWR1 complex     

Chromatin-remodelling mechanisms in cancer

Chromatin-remodelling mechanisms include DNA methylation, histone-tail acetylation, poly-ADP-ribosylation, and ATP-dependent chromatin-remodelling processes. Some epigenetic modifications among others have been observed in cancer cells, namely (1) local DNA hypermethylation and global hypomethylation, (2) alteration in histone acetylation/deacetylation balance, (3) increased or decreased poly-ADP-ribosylation, and (4) failures in ATP-dependent chromatin-remodelling mechanisms. Moreover, these alterations can influence the response to classical anti-tumour treatments. Drugs targeting epigenetic alterations are under development. Currently, DNA methylation and histone deacetylase inhibitors are in use in cancer therapy, and poly-ADP-ribosylation inhibitors are undergoing clinical trials. Epigenetic therapy is gaining in importance in pharmacology as a new tool to improve anti-cancer therapies.     

Inactivation of the polycomb group protein Ring1B unveils an antiproliferative role in hematopoietic cell expansion and cooperation with tumorigenesis associated with Ink4a deletion

Polycomb group (PcG) proteins act as positive regulators of cell proliferation. Ring1B is a PcG gene essential for embryonic development, but its contribution to cell turnover in regenerating tissues in not known. Here, we have generated a conditional mouse mutant line to study the Ring1B role in adult hematopoiesis. Mutant mice developed a hypocellular bone marrow that paradoxically contained an enlarged, hyperproliferating compartment of immature cells, with an intact differentiation potential. These alterations were associated with differential upregulation of cyclin D2, which occurred in all mutant bone marrow cells, and of p16^Ink4a, observed only in the differentiated compartment. Concurrent inactivation of Ink4a rescued the defective proliferation of maturing cells but did not affect the hyperproliferative activity of progenitors and resulted in a shortening of the onset of lymphomas induced by Ink4a inactivation. These data show that Ring1B restricts the progenitors' proliferation and promotes the proliferation of their maturing progeny by selectively altering the expression pattern of cell cycle regulators along hematopoietic differentiation. The novel antiproliferative role of Ring1B's downregulation of a cell cycle activator may play an important role in the tight control of hematopoietic cell turnover.     

Aluminium induces changes in organic acids metabolism in Coffea arabica suspension cells with differential Al-tolerance

The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al³⁺ under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 μM AlCl₃. HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl₃ (100 μM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.     

Alternate AChE-R variants facilitate cellular metabolic activity and resistance to genotoxic stress through enolase and RACK1 interactions

Tumorogenic transformation is a multifaceted cellular process involving combinatorial protein-protein interactions that modulate different cellular functions. Here, we report apparent involvement in two independent tumorogenic processes by distinct partner protein interactions of the stress-induced acetylcholinesterase AChE-R and N-AChE-R variants. Human testicular tumors showed elevated levels of N-terminally extended N-AChE-R compared with healthy tissue, indicating alternate promoter usage in the transformed cells. Two-hybrid screens demonstrate that the C-terminus common to both N-AChE-R and AChE-R interacts either with the glycolytic enzyme enolase or with the scaffold protein RACK1. In vitro, the AChE-R C-terminal peptide ARP elevated enolase's activity by 12%, suggesting physiological relevance for this interaction. Correspondingly, CHO cells expressing either human AChE-R or N-AChE-R but not AChE-S showed a 25% increase in cellular ATP levels, indicating metabolic significance for this upregulation of enolase activity. ATP levels could be reduced by AChE-targeted siRNA in CHO cells expressing AChE-R but not AChE-S, attributing this elevation to the AChE-R C-terminus. Additionally, transfected CHO cells expressing AChE-R but not N-AChE-R showed resistance to up to 60muM of the common chemotherapeutic agent, cis-platinum, indicating AChE-R involvement in another molecular pathway. cis-Platinum elevates the expression of the apoptosis-regulator p53-like protein, p73, which is inactivated by interaction with the scaffold protein RACK1. In co-transfected cells, AChE-R competed with endogenous RACK1 for p73 interaction. Moreover, AChE-R-transfected CHO cells presented higher levels than control cells of the pro-apoptotic TAp73 as well as the anti-apoptotic dominant negative DeltaNp73 protein, leading to an overall decrease in the proportion of pro-apoptotic p73. Together, these findings are compatible with the hypothesis that in cancer cells, both AChE-R and N-AChE-R elevate cellular ATP levels and that AChE-R modifies p73 gene expression by facilitating two independent cellular pathways, thus conferring both a selective metabolic advantage and a genotoxic resistance.     

Cholinergic location of delta-opioid receptors in canine atria and SA node

Delta-opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and <8% were adrenergic. DORs colocalized 75-85% with synapsin in tissue slices from both atria and SA node. The colocalization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Colocalization between DOR and VAChT was 75-85% regardless of the source. Overlap between DOR and TH was uniformly low, ranging from 8% to 17%. Western blots with synaptosomal extracts confirmed two DOR-positive bands at molecular masses corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes, largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals support the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.     

Gamma-lactones alpha,beta- and beta,gamma-fused to carbocycles as novel antiproliferative drugs

A set of gamma-lactones alpha,beta-fused and beta,gamma-fused to carbocycles have been synthesized and evaluated for their in vitro antiproliferative activities using the human cancer cell lines SW1573 (lung), T-47D (breast) and WiDr (colon). The compounds are obtained by intramolecular ring closing metathesis of the corresponding dienes. Active compounds exhibited GI(50) values in the range 8-18 microM. A structure-activity relationship is also discussed.     

Evaluation in a Dog Model of Three Antimicrobial Glassy Coatings: Prevention of Bone Loss around Implants and Microbial Assessments

ObjectivesThe aim of the present study is to evaluate, in a ligature-induced peri-implantitis model, the efficacy of three antimicrobial glassy coatings in the prevention of biofilm formation, intrasulcular bacterial growth and the resulting peri-implant bone loss.MethodsMandibular premolars were bilaterally extracted from five beagle dogs. Four dental implants were inserted on each hemiarch. Eight weeks after, one control zirconia abutment and three with different bactericidal coatings (G1n-Ag, ZnO35, G3) were connected. After a plaque control period, bacterial accumulation was allowed and biofilm formation on abutments was observed by Scanning Electron Microscopy (SEM). Peri-implantitis was induced by cotton ligatures. Microbial samples and peri-implant crestal bone levels of all implant sites were obtained before, during and after the breakdown period.ResultsDuring experimental induce peri-implantitis: colony forming units counts from intrasulcular microbial samples at implants with G1n-Ag coated abutment remained close to the basal inoculum; G3 and ZnO35 coatings showed similar low counts; and anaerobic bacterias counts at control abutments exhibited a logarithmic increase by more than 2. Bone loss during passive breakdown period was no statistically significant. Additional bone loss occurred during ligature-induce breakdown: 0.71 (SD 0.48) at G3 coating, 0.57 (SD 0.36) at ZnO35 coating, 0.74 (SD 0.47) at G1n-Ag coating, and 1.29 (SD 0.45) at control abutments; and statistically significant differences (p<0.001) were found. The lowest bone loss at the end of the experiment was exhibited by implants dressing G3 coated abutments (mean 2.1; SD 0.42).SignificanceAntimicrobial glassy coatings could be a useful tool to ward off, diminish or delay peri-implantitis progression.