The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.     

Human Exonuclease 5 Is a Novel Sliding Exonuclease Required for Genome Stability

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5′-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5′-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.     

Chromatin remodeling finds its place in the DNA double-strand break response

The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.     

Uses,traditional management,perception of variation and preferences in ackee (Blighia sapida K.D. Koenig) fruit traits in Benin: implications for domestication and conservation

Background

Changing lifestyles have recently caused a severe reduction of the gathering of wild food plants. Knowledge about wild food plants and the local environment becomes lost when plants are no longer gathered. In Central Europe popular scientific publications have tried to counter this trend. However, detailed and systematic scientific investigations in distinct regions are needed to understand and preserve wild food uses. This study aims to contribute to these investigations.

Methods

Research was conducted in the hill country east of Graz, Styria, in Austria. Fifteen farmers, most using organic methods, were interviewed in two distinct field research periods between July and November 2008. Data gathering was realized through freelisting and subsequent semi-structured interviews. The culinary use value (CUV) was developed to quantify the culinary importance of plant species. Hierarchical cluster analysis was performed on gathering and use variables to identify culture-specific logical entities of plants. The study presented was conducted within the framework of the master's thesis about wild plant gathering of the first author. Solely data on gathered wild food species is presented here.

Results

Thirty-nine wild food plant and mushroom species were identified as being gathered, whereas 11 species were mentioned by at least 40 percent of the respondents. Fruits and mushrooms are listed frequently, while wild leafy vegetables are gathered rarely. Wild foods are mainly eaten boiled, fried or raw. Three main clusters of wild gathered food species were identified: leaves (used in salads and soups), mushrooms (used in diverse ways) and fruits (eaten raw, with milk (products) or as a jam).

Conclusions

Knowledge about gathering and use of some wild food species is common among farmers in the hill country east of Graz. However, most uses are known by few farmers only. The CUV facilitates the evaluation of the culinary importance of species and makes comparisons between regions and over time possible. The classification following gathering and use variables can be used to better understand how people classify the elements of their environment. The findings of this study add to discussions about food heritage, popularized by organizations like Slow Food, and bear significant potential for organic farmers.     

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.      

Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles

Experiments were performed to localize the hepatic microsomal enzymes of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol biosynthesis to the cytoplasmic or lumenal surface of microsomal vesicles. Greater than 90 percent of the activities of fatty acid-CoA ligase (EC 6.2.1.3), sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15), lysophosphatidic acid acyltransferase, diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2), and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) was inactivated by proteolysis of intact microsomal vesicles. The phosphatidic acid phosphatase (EC 3.1.3.4) was not inactivated by any of the protease tested. Under conditions employed, <5 percent of the luminal mannose-6-phosphatase (EC 3.1.3.9) activity was lost. After microsomal integrity was disrupted with detergents, protease treatment resulted in a loss of >74 percent of the mannose-6-phosphatase activity. The latency of the mannose-6-phosphatase activity was not affected by protease treatment. Mannose-6-phosphatase latency was not decreased by the presence of the assay components of several of the lipid biosynthetic activities, indicating that those components did not disrupt the microsomal vesicles. None of the lipid biosynthetic activities appeared latent. The presence of a protease-sensitive component of these biosynthetic activities on the cytoplasmic surface of microsomal vesicles, and the absence of latency for any of these biosynthetic activities suggest that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. The location of biosynthetic activities within the transverse plane of the endoplasmic reticulum is of particular interest for enzymes whose products may be either secreted or retained within the cell. Phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol account for the vast majority of hepatic glycerolipid biosynthesis. The phospholipids are utilized for hepatic membrane biogenesis and for the formation of lipoproteins, and the triacylglycerols are incorporated into lipoproteins or accumulate within the hepatocyte in certain disease states (14). The enzymes responsible for the biosynthesis of these glycerolipids (Scheme I) from fatty acids and glycerol-3P have all been localized to the microsomal subcellular fraction (12, 16, 29, 30). Microsomes are derived from the endoplasmic reticulum and are sealed vesicles which maintain proper sidedness. (11, 22). The external surface of these vesicles corresponds to the cytoplasmic surface of the endoplasmic reticulum. Macromolecules destined for secretion must pass into the lumen of the endoplasmic reticulum (5, 23). Uncharged molecules of up to approximately 600 daltons are able to enter the lumen of rat liver microsomes, but macromolecules and charged molecules of low molecular weight do not cross the vesicle membrane (10, 11). Because proteases neither cross the microsomal membrane nor destroy the permeability barrier of the microsomal vesicles, only the enzymes and proteins located on the cytoplasmic surface of microsomal vesicles are susceptible to proteolysis unless membrane integrity is disrupted (10, 11). By use of this approach, several enzymes and proteins have been localized in the transverse plane of microsomal membranes (11). With the possible exception of cytochrome P 450, all of the enzymes and proteins investigated were localized asymmetrically by the proteolysis technique (11). By studies of this type, as well as by product localization, glucose-6-phosphate (EC 3.1.3.9) has been localized to the luminal surface of microsomal vesicles (11) and of the endoplasmic reticulum (18, 19). All microsomal vesicles contain glucose-6-phosphatase (18, 19) which can effectively utilize mannose-6-P as a substrate, provided the permeability barrier of the vesicles has been disrupted to allow the substrate access to the active site located on the lumenal surface (4). An exact correspondence between mannose- 6-phosphate activity and membrane permeability to EDTA has been established (4). The latency of mannose-6-phosphatase activity provides a quantitative index of microsomal integrity (4.) Few of the microsomal enzymes in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol have been solubilized and/or purified, and little is known about the topography of these enzymes in the transverse or lateral planes of the endoplasmic reticulum. An asymmetric location of these biosynthetic enzymes on the cytoplasmic or lumenal surface of microsomal vesicles may provide a mechanism for regulation of the glycerolipids to be retained or secreted by the cell, and for the biogenesis of asymmetric phospholipid bilayers. In this paper, we report investigations on the localization of all seven microsomal enzymes (Scheme I) in the biosynthesis of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, using the protease technique with mannose-6-phosphatase serving as luminal control activity. The latency of these lipid biosynthetic enzymes was also investigated, using the latency of mannose-6-phosphatase as an index of microsomal integrity.     

Telomerase activity as a measure for monitoring radiocurability of tumor cells.

Radiotherapy plays a key role in the treatment of many tumors. It is difficult to determine what fraction of tumor cells survives after treatment with ionizing radiation. A convenient and sensitive biochemical assay could be efficacious in determining the potential success of radiotherapy. Since telomerase activity is frequently associated with the malignant phenotype, we sought to determine whether a correlation existed between ionizing radiation-induced cell killing and telomerase activity. We evaluated telomerase activity in two telomerase-positive and one telomerase-negative human cell line exposed to ionizing radiation. Telomerase activity was determined using a PCR-based telomeric repeat amplification protocol coupled with ELISA. We found ionizing radiation treatment to decrease the telomerase activity (in plateau phase cells of RKO, HeLa; and growing cells of RKO) in a dose-dependent manner, which correlated with cell death in in vitro tests as well as during tumor regression in nude mice. In contrast, growing HeLa cells after 24 h postradiation treatment showed an increase in telomerase activity, but there was no increase in the levels of mRNA of hTERT. To assess the sensitivity of the telomerase activity assay, we performed mixing experiments of HeLa and AG1522 cell extracts. These studies showed that telomerase activity could be detected in lysate equal to a single HeLa cell when mixed with 10,000 AG1522 cells. Our results indicate that even a few surviving neoplastic cells can be detected by telomerase activity assay. Therefore, detection of telomerase activity may be a useful monitor of radiotherapeutic efficacy and an early predictor of outcome.     

Adding Content to Contacts: Measurement of High Quality Contacts for Maternal and Newborn Health in Ethiopia,North East Nigeria,and Uttar Pradesh,India

BackgroundFamilies in high mortality settings need regular contact with high quality services, but existing population-based measurements of contacts do not reflect quality. To address this, in 2012, we designed linked household and frontline worker surveys for Gombe State, Nigeria, Ethiopia, and Uttar Pradesh, India. Using reported frequency and content of contacts, we present a method for estimating the population level coverage of high quality contacts.ConclusionsMeasuring content of care to reflect the quality of contacts can reveal missed opportunities to deliver best possible health care.