Interaction of histone H4 with Cse4 facilitates conformational changes in Cse4 for its sumoylation and mislocalization.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an 'open' state, while Cse4 Y193A predominantly exhibits a 'closed' state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an 'open' state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.

A single sensor controls large variations in zinc quotas in a marine cyanobacterium.

Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans.

Therapeutic effects of mesenchymal stem cells and vitamin D on Bleomycin triggered lung damage in male adult albino rats.

Bleomycin is a cancer chemotherapeutic agent that induces pulmonary fibrosis. Vitamin D plays an immunomodulation role. Bone marrow-derived mesenchymal stem cells have a strong therapeutic effect in fatal pulmonary fibrosis. The objective of this study was to evaluate the significance of vitamin D and bone marrow mesenchymal stem cells as therapeutic agents on lung injuries caused by Bleomycin in adult male rats. Thirty-five adult male albino rats were allocated into five experimental groups. The control group was the group I. The group given a single intratracheal instillation of Bleomycin was group II. The group was given vitamin D3 for 2 days before Bleomycin administration was group III. Group IV was the group that was injected by a single dose of mesenchymal stem cells (MSCs) after 4 weeks of Bleomycin injection. Group V was the withdrawal group. Histological, immunohistochemical, and ultrastructural techniques were used to process and evaluate lung tissues. The lung of group 2 was demonstrated interalveolar septal thickening by RBCs, infiltration of mononuclear cells, deposition of collagen, and marked positive alpha-smooth muscle actin immunoreactivity. Mesenchymal stem cells derived from bone marrow can diminish Bleomycin-generated fibrosis of the lungs and inflammation in rats better than vitamin D treatment.

Bacterial zinc uptake regulator proteins and their regulons.

All organisms must regulate the cellular uptake, efflux, and intracellular trafficking of essential elements, including d-block metal ions. In bacteria, such regulation is achieved by the action of metal-responsive transcriptional regulators. Among several families of zinc-responsive transcription factors, the 'zinc uptake regulator' Zur is the most widespread. Zur normally represses transcription in its zinc-bound form, in which DNA-binding affinity is enhanced allosterically. Experimental and bioinformatic searches for Zur-regulated genes have revealed that in many cases, Zur proteins govern zinc homeostasis in a much more profound way than merely through the expression of uptake systems. Zur regulons also comprise biosynthetic clusters for metallophore synthesis, ribosomal proteins, enzymes, and virulence factors. In recognition of the importance of zinc homeostasis at the host-pathogen interface, studying Zur regulons of pathogenic bacteria is a particularly active current research area.

Electrophysiological effects of tamoxifen: mechanism of protection against reperfusion arrhythmias in isolated rat hearts.

Reperfusion arrhythmias are currently attributed to ionic imbalance and oxidative stress. Tamoxifen is a potent antioxidant that also modulates some ionic transport pathways. In this work, we tried to correlate the electrophysiological effects of 1, 2, and 5 µM of tamoxifen with the incidence and severity of arrhythmias appearing on reperfusion after 10 minutes of coronary occlusion in isolated hearts from female rats. All tamoxifen concentrations inhibited the action potential shortening observed in the control hearts during late ischemia (6-10 minutes), whereas 2 and 5 µM also reduced the resting membrane potential depolarization. The incidence of sustained ventricular tachycardia and/or ventricular fibrillation on reperfusion decreased from 10 of 12 (control group) to 5 of 10 (1 µM, P = 0.1718), 4 of 12 (2 µM, P = 0.0361), and 2 of 10 (5 µM, P = 0.0083). The possible role of chloride currents activated by cell swelling in these effects was explored in hearts submitted to a 10-minute hypotonic challenge, where tamoxifen (5 µM) blocked the action potential shortening and the late resting membrane potential depolarization produced by hypotonicity, mimicking its action in late ischemia. Tamoxifen produced a similar increase of the total antioxidant capacity of myocardial samples at all the concentration tested. In conclusion, our data strongly suggest that the antiarrhythmic action of this agent is mediated by its electrophysiological effect derived from modulation of chloride currents activated by cell swelling.

Alterations in biogenic amines levels associated with age-related muscular tissue impairment in Drosophila melanogaster.

While holding on youth may be a universal wish, aging is a natural process associated with physical and physiological impairment in living organisms. Drosophila provides useful insights into aging-related events. Hence, this study was conducted to investigate the age-related changes in muscle function and architecture in relation to the biogenic amine titers. To achieve this aim, visceral and skeletal muscles performance was tested in newly-eclosed, sexually mature and old adult flies using climbing and gut motility assays. In addition, age-related ultrastructural alterations of muscular tissue were observed using transmission electron microscopy (TEM). The titer of selected biogenic amines was measured using high-performance liquid chromatography (HPLC). The results demonstrated that old flies were dramatically slower in upward movement than either newly-eclosed or sexually mature flies. Similarly, gut contraction rate was significantly lower in old flies than the sexually mature, although it was markedly higher than that in the newly-eclosed flies. In TEM examination, there were several ultrastructural changes in the midgut epithelium, legs and thorax muscles of old flies. Regarding biogenic amine titers, the old flies had significantly lower concentrations of octopamine, dopamine and serotonin than the sexually mature. We concluded that aging has adverse effects on muscular system function and ultrastructure, synchronized with biogenic amine titers changes. Our results highlighted the need for more researches on therapeutics that may balance the levels of age-related alterations in biogenic amines.

[Attitudes of teachers to tobacco smoking]. / Attitudes des enseignants à l'égard du tabagisme.

We aimed to evaluate the attitudes of teachers to tobacco smoking in Kalaa Kebira (a semi-urban region in the Tunisian Sahel). Data from 358 of 402 teachers surveyed were obtained using a self-completed questionnaire. The mean age of the teachers was 35.7 (SD 7.9) years. The prevalence of smoking was 29.3% (51.6% of men and 3.6% of women), and 79% of smokers were addicted to nicotine according to the Fagerström test. About 50% of the teachers were badly-informed about the dangers of smoking and 75.2% of smoking teachers did not refrain from smoking in front of their pupils. The lack of knowledge of teachers limits their role as a model and information source in the anti-tobacco struggle. It is essential to draw up an education programme for the teaching staff.

[Tobacco use among teachers in a district of the Tunisian Sahel]. / Le tabagisme des enseignants dans une commune du Sahel Tunisien.

INTRODUCTION: The aim of this study was to evaluate the scale of tobacco use among teachers in the district of Kalaa Kebira (a semi-urban region in the Tunisian Sahel). METHODS: The study design was descriptive and cross sectional, employing a self administered questionnaire given to 402 teachers. RESULTS: The response rate was 89%. The average age of respondents was 35.7 7.9 years. The global prevalence of smoking among teachers was 29.3% (men: 51.6%; women: 3.6%). 79% of smokers were addicted to nicotine according to the Fagerstrôm test. Half of the teachers were poorly informed about the hazards of smoking and 77.9% of smoking teachers didn't refrain from smoking in front of their pupils. CONCLUSIONS: The teachers' lack of knowledge limits their effectiveness both as a role model and information relay in the war against tobacco.

Pro- and anti-inflammatory cytokines in post-infarction left ventricular remodeling.

OBJECTIVES: Acute myocardial infarction (MI) leads to molecular, structural, geometric and functional changes in the heart during a process known as ventricular remodeling. Myocardial infarction is followed by an inflammatory response in which pro- and anti-inflammatory cytokines play a crucial role, particularly in left ventricular remodeling. This study aimed at evaluating serum concentrations of interleukin-8 (IL8), tumor-necrosis-factor-alpha (TNFα) and interleukin-10 (IL10), pro- and anti-inflammatory cytokines, and at correlating them with left ventricular remodeling as assessed by echocardiographic parameters. METHODS: In a case-control study 30 MI patients were compared with 30 healthy controls. Serum concentrations of IL8, TNFα and IL10 were measured on day 2 and day 30 post-MI by chemiluminescence immunoassay and correlated with echocardiographic parameters. RESULTS: There was an increase of IL8, and TNFα together with a decrease of IL10 at both time points. IL8 was negatively correlated with the left ventricular end-diastolic diameter (LVEDD) and positively with left ventricular systolic volume. IL10 was negatively correlated with LVEDD and left atrial volume 30days post-MI. CONCLUSION: The increase of pro-inflammatory cytokines TNFα and IL8 was accompanied by decreased anti-inflammatory IL10. This imbalance between pro- and anti-inflammatory cytokines might contribute to the progression of left ventricular remodeling and may lead to heart failure.

O2 -independent demethylation of trimethylamine N-oxide by Tdm of Methylocella silvestris.

Bacterial trimethylamine N-oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen-independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, site-directed mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn2+ and 1 molar equivalent of non-haem Fe2+ . We further investigated Zn2+ - and Fe2+ -binding sites through homology modelling and site-directed mutagenesis and found that Zn2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe2+ and Zn2+ centres, either directly or indirectly via H-bonding through a neighbouring H2 O molecule. H276 contributes to Fe2+ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation-π interaction. Furthermore, a crossover experiment using a substrate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn2+ and Fe2+ in the catalysis of TMAO demethylation by this unique oxygen-independent enzyme.

Neurogenesis and growth factors expression after complete spinal cord transection in Pleurodeles waltlii.

Following spinal lesion, connections between the supra-spinal centers and spinal neuronal networks can be disturbed, which causes the deterioration or even the complete absence of sublesional locomotor activity. In mammals, possibilities of locomotion restoration are much reduced since descending tracts either have very poor regenerative ability or do not regenerate at all. However, in lower vertebrates, there is spontaneous locomotion recuperation after complete spinal cord transection at the mid-trunk level. This phenomenon depends on a translesional descending axon re-growth originating from the brainstem. On the other hand, cellular and molecular mechanisms underlying spinal cord regeneration and in parallel, locomotion restoration of the animal, are not well known. Fibroblast growth factor 2 (FGF-2) plays an important role in different processes such as neural induction, neuronal progenitor proliferation and their differentiation. Studies had shown an over expression of this growth factor after tail amputation. Nestin, a protein specific for intermediate filaments, is considered an early marker for neuronal precursors. It has been recently shown that its expression increases after tail transection in urodeles. Using this marker and western blots, our results show that the number of FGF-2 and FGFR2 mRNAs increases and is correlated with an increase in neurogenesis especially in the central canal lining cells immediately after lesion. This study also confirms that spinal cord re-growth through the lesion site initially follows a rostrocaudal direction. In addition to its role known in neuronal differentiation, FGF-2 could be implicated in the differentiation of ependymal cells into neuronal progenitors.

Mining genomes of marine cyanobacteria for elements of zinc homeostasis.

Zinc is a recognized essential element for the majority of organisms, and is indispensable for the correct function of hundreds of enzymes and thousands of regulatory proteins. In aquatic photoautotrophs including cyanobacteria, zinc is thought to be required for carbonic anhydrase and alkaline phosphatase, although there is evidence that at least some carbonic anhydrases can be cambialistic, i.e., are able to acquire in vivo and function with different metal cofactors such as Co(2+) and Cd(2+). Given the global importance of marine phytoplankton, zinc availability in the oceans is likely to have an impact on both carbon and phosphorus cycles. Zinc concentrations in seawater vary over several orders of magnitude, and in the open oceans adopt a nutrient-like profile. Most studies on zinc handling by cyanobacteria have focused on freshwater strains and zinc toxicity; much less information is available on marine strains and zinc limitation. Several systems for zinc homeostasis have been characterized in the freshwater species Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, but little is known about zinc requirements or zinc handling by marine species. Comparative metallo-genomics has begun to explore not only the putative zinc proteome, but also specific protein families predicted to have an involvement in zinc homeostasis, including sensors for excess and limitation (SmtB and its homologs as well as Zur), uptake systems (ZnuABC), putative intracellular zinc chaperones (COG0523) and metallothioneins (BmtA), and efflux pumps (ZiaA and its homologs).

Modulation of AP-endonuclease1 levels associated with hepatic cirrhosis in rat model treated with human umbilical cord blood mononuclear stem cells.

Oxidative stress in liver cells may contribute to the etiology of hepatic diseases, as in liver cirrhosis. AP-Endonuclease1 (APE1/Ref-1) is essential for cell protection toward oxidative stress by acting as a transcriptional regulator of pro-survival genes and as a redox sensitive protein. The aim of this study was to critically analyze the various parameters governing the success of human umbilical cord blood mononuclear stem cell-based (MNCs) therapy without the use of an immunosuppressant and to investigate for the first time the expression of APE1 during thioacetamide (TAA)-induced cirrhosis and MNCs therapy in a rat model. Umbilical cord blood samples from full-term deliveries were collected. Lethal fulminant hepatic cirrhosis in rats was induced by intraperitoneal injection of thio-acetamide. MNCs were then intrahepatically transplanted. We measured APE1 expression at mRNA and protein levels, mRNA expression of TGF-ß, α-SMA, STAP, CTGF, MMP-9 and TIMP-1 in a follow up study. Histopathological and immunohistochemical analyses were performed 10 weeks after intrahepatic injection of the cells. Transdifferentiated cells could be efficiently stained with antihuman hepatocytes. Interestingly, human hepatocyte-specific markers, human albumin, cytokeratin-18 and cytokeratin-19 mRNAs were detected in rat liver after 10 days of MNCs infusion. MNC transplanted by intrahepatic route, could engraft recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Moreover up regulation of APE1 expression confirmed by marked immunohistochemical staining may be involved in MNCs-induced hepatocytes regeneration suggesting that maintaining high level of APE1 has protective effect as pro-survival signal.

Efectos de la hipoxia aguda y cronica sobre el electrocardiograma de la rata. / Effects of acute and chronic hypoxia on electrocardiogram of rats

Se compararon los valores de los parametros electrocardiograficos, obtenidos en ratas sometidas previamente a hipoxia hipobarica cronica (GII), con los de los animales del grupo control (GI), mantenidos a presion ambiente. Durante los registros ambos grupos de ratas eran ventilados primero con aire y luego con una mezcla pobre en oxigeno. El grupo GII, durante la respiracion con aire, mostro un incremento de los intervalos QT y QaT y una disminucion de la frecuencia cardiaca espontanea. Durante la ventilacion con la mezcla pobre en oxigeno, en ambos grupos se observo una prolongacion del intervalo PR que condujo a un bloqueo AV de 2o grado, de aparicion mas tardia en GII. La hipoxia aguda produjo una disminucion del QT en todos los animales y un aumento del QRS solamente en los adaptados. Estos resultados muestran que la hipoxia hipobarica cronica prolonga la repolarizacion ventricular. La hipoxia aguda aumenta la velocidad de repolarizacion en ambos grupos, pero en el GII permanece prolongada respecto al valor alcanzado en GI. Este ultimo hecho podria interpretarse como el desarrollo de un mecanismo de adaptacion inducido por la hipoxia hipobarica cronica

A modified technique for canine sinoatrial node autoperfusion

En el presente trabajo se propone un nuevo modelo experimental para la autoperfusión del nódulo sinusal canino, basado en el originalmente descripto por James y Nadeau, con modificaciones que, a nuestro juicio, lo hacen más ventajoso. Se realizó un cortocircuito entre las arterias mamaria interna y auricular dorsal derecha. Esta última, en un 82% de los perros, es la arteria dominante en la irrigación del nódulo. Midiendo la velocidad de avance del colorante se calculó el flujo sanguíneo del cortocircuito, que fue de 2.5ñ0.15 ml/m. Cuando se inyectaron en el "by-pass" tanto isoproterenol como verapamil, se produjeron rápidos cambios en la frecuencia cardíaca, sin que se observaran modificaciones apreciables en el comportamiento eléctrico del resto del miocardio. Ello se puede constatar a través del electrocardiograma y de los registros endocavitarios. Este modelo de autoperfusión sinusal deja la circulación coronaria intacta, a diferencia de James y Nadeau, que introduce el catéter en la arteria coronaria derecha. El cortocircuito intratorácico reduce la pérdida de calor y disminuye la posibilidad de aparición de coágulos dentro del catéter. El modelo es apropiado tambíen para estudios experimentales crónicos

[Attitudes of teachers to tobacco smoking]

We aimed to evaluate the attitudes of teachers to tobacco smoking in Kalaa Kebira [a semi-urban region in the Tunisian Sahel]. Data from 358 of 402 teachers surveyed were obtained using a self-completed questionnaire. The mean age of the teachers was 35.7 [SD 7.9] years. The prevalence of smoking was 29.3% [51.6% of men and 3.6% of women], and 79% of smokers were addicted to nicotine according to the Fagerstrom test. About 50% of the teachers were badly-informed about the dangers of smoking and 75.2% of smoking teachers did not refrain from smoking in front of their pupils. The lack of knowledge of teachers limits their role as a model and information source in the anti-tobacco struggle. It is essential to draw up an education programme for the teaching staff