Participation of ATP in the binding of a yeast replicative complex to DNA.

The activity that replicates yeast DNA in vitro can be isolated from cells of the budding yeast Saccharomyces in a high-Mr (approximately 2 X 10(6] form. Several lines of evidence indicate that this fraction contains a multiprotein replicative complex. A functional assay has been developed for the analysis of the interaction of the replicating activity with DNA. Binding of the activity required Mg2+, but did not require the addition of ATP or the other ribo- or deoxynucleoside triphosphates. However, the ATP analogues adenosine 5'-[gamma-thio]triphosphate and adenosine 5'-[beta gamma-imido]triphosphate blocked the binding, suggesting that ATP participates in the interaction at some stage. The binding was template (origin)-specific in either the presence or the absence of ATP and the other nucleoside triphosphates; however, ATP stabilized the replicating activity. The preferential inhibition of binding that was observed in the presence of the DNA topoisomerase II inhibitor coumermycin suggests that the requirement for ATP may be at least partially accounted for by the involvement of this enzyme in the initial interaction of the replicating activity with DNA. Finally, the binding was rapid. In contrast, DNA synthesis displayed a lag when assayed directly without first allowing a period for the replicating activity to bind to the DNA. In addition, binding was 'tight', as judged by the resistance of the protein--DNA complexes to salt in comparison with the relative sensitivity of binding. The replicating activity was not readily displaced from the complexes by exogenous DNAs, either possessing or lacking yeast origins of replication. The results suggest that the interaction of the replicating activity with the DNA occurs in more than one stage.     

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.     

Telomere length constancy during aging of Saccharomyces cerevisiae.

It has been proposed that a decrease in the length of telomeres with the successive rounds of DNA replication that accompany mitotic division could play a causal role in the aging process. To investigate this possibility, telomeres from cells of the budding yeast Saccharomyces cerevisiae that varied in replicative age were examined. No change in the lengths of the telomeres was observed in cells that had completed up to 83% of the mean life span. We conclude that the length of the telomeres is not a contributing factor in the natural aging process in individual yeast cells.     

Aging and senescence of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has a limited life span, defined by the number of times an individual cell divides. Longevity in this organism involves a genetic component. Several morphological and physiological changes are associated with yeast aging and senescence. One of these, an increase in generation time with age, provides a 'biomarker' for the aging process. This increase in generation time has revealed the operation of a 'senescence factor(s)', which is likely to be a product of age-specific gene expression. The Cell Spiral Model indicates coordination of successive cell cycles to be inherent in the determination of life span. It is proposed that life expectancy depends on the function of a stochastic trigger during aging that sets in motion a programme leading to cell senescence and death.     

The retrograde response: when mitochondrial quality control is not enough

Mitochondria are responsible for generating adenosine triphosphate (ATP) and metabolic intermediates for biosynthesis. These dual functions require the activity of the electron transport chain in the mitochondrial inner membrane. The performance of these electron carriers is imperfect, resulting in release of damaging reactive oxygen species. Thus, continued mitochondrial activity requires maintenance. There are numerous means by which this quality control is ensured. Autophagy and selective mitophagy are among them. However, the cell inevitably must compensate for declining quality control by activating a variety of adaptations that entail the signaling of the presence of mitochondrial dysfunction to the nucleus. The best known of these is the retrograde response. This signaling pathway is triggered by the loss of mitochondrial membrane potential, which engages a series of signal transduction proteins, and it culminates in the induction of a broad array of nuclear target genes. One of the hallmarks of the retrograde response is its capacity to extend the replicative life span of the cell. The retrograde signaling pathway interacts with several other signaling pathways, such as target of rapamycin (TOR) and ceramide signaling. All of these pathways respond to stress, including metabolic stress. The retrograde response is also linked to both autophagy and mitophagy at the gene and protein activation levels. Another quality control mechanism involves age-asymmetry in the segregation of dysfunctional mitochondria. One of the processes that impinge on this age-asymmetry is related to biogenesis of the organelle. Altogether, it is apparent that mitochondrial quality control constitutes a complex network of processes, whose full understanding will require a systems approach. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.     

Risk factors of peripheral arterial disease: a case control study in Sri Lanka

Background

Peripheral artery disease (PAD) is an important global health problem and contributes to notable proportion of morbidity and mortality. This particular manifestation of systemic atherosclerosis is largely under diagnosed and undertreated. For sustainable preventive strategies in a country, it is mandatory to identify country-specific risk factors. We intended to assess the risk factors of PAD among adults aged 40–74 years.

Methods

This case control study was conducted in 2012–2013 in Sri Lanka. Seventy-nine cases and 158 controls in the age group of 40–74 years were selected for the study in order to have case to control ratio 1:2. The criterion for selecting cases and control was based on Ankle brachial pressure index (ABPI). Cases were selected from those who had ABPI 0.85 or less (ABPI ≤0.85) in either lower limb. Controls were selected from those ABPI score between 1.18 and 1.28 in both lower limbs. Only newly identified individuals with PAD were selected as cases. Controls were selected from the same geographical location and within the 5 year age group as cases.

Results

The history of diabetes mellitus more than 10 years (OR 5.8, 95% CI 2.2–14.2), history of dyslipidemia for more than 10 years (OR 4.9, 95% CI 2.1–16.2), history of hypertension for more than 10 years (OR 3.8, 95% CI 1.8–12.7) and smoking (OR 2.9, 95% CI 1.2–6.9), elevated HsCRP (OR 3.7, 95% CI 1.2–12.0) and hyperhomocysteinemia (OR 3.0, 95% CI 1.1–8.1) were revealed as country specific significant risk factor of PAD.

Conclusions

Diabetes mellitus, hypertension, dyslipidemia, smoking as well as elevated homocysteine and HsCRP found as risk factors of PAD. Longer the duration or higher level exposure to these risk factors has increased the risk of PAD. These findings emphasis the need for routine screening of PAD among patients with the identified risk factors.

     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.