DOUBLE-STRAND BREAK REPAIR PATHWAYS IN DNA STRUCTURE-INDUCED GENETIC INSTABILITY

Genetic instability in mammalian cells can occur by many different mechanisms. In the absence of exogenous sources of DNA damage, the DNA structure itself has been implicated in genetic instability. When the canonical B-DNA helix is naturally altered to form a non-canonical DNA structure such as a Z-DNA or H-DNA, this can lead to genetic instability in the form of DNA double-strand breaks (DSBs) (1, 2). Our laboratory found that the stability of these non-B DNA structures was different in mammals versus Escherichia coli (E.coli) bacteria (1, 2). One explanation for the difference between these species may be a result of how DSBs are repaired within each species. Non-homologous end-joining (NHEJ) is primed to repair DSBs in mammalian cells, while bacteria that lack NHEJ (such as E.coli), utilize homologous recombination (HR) to repair DSBs. To investigate the role of the error-prone NHEJ repair pathway in DNA structure-induced genetic instability, E.coli cells were modified to express genes to allow for a functional NHEJ system under different HR backgrounds. The Mycobacterium tuberculosis NHEJ sufficient system is composed of Ku and Ligase D (LigD) (3). These inducible NHEJ components were expressed individually and together in E.coli cells, with or without functional HR (RecA/RecB), and the Z-DNA and H-DNA-induced mutations were characterized. The Z-DNA structure gave rise to higher mutation frequencies compared to the controls, regardless of the DSB repair pathway(s) available; however, the type of mutants produced after repair was greatly dictated on the available DSB repair system, indicated by the shift from 2% large-scale deletions in the total mutant population to 24% large-scale deletions when NHEJ was present (4). This suggests that NHEJ has a role in the large deletions induced by Z-DNA-forming sequences. H-DNA structure, however, did not exhibit an increase in mutagenesis in the newly engineered E.coli environment, suggesting the involvement of other factors in regulating H-DNA formation/stability in bacterial cells. Accurate repair by established DNA DSB repair pathways is essential to maintain the stability of eukaryotic and prokaryotic genomes and our results suggest that an error-prone NHEJ pathway was involved in non-B DNA structure-induced mutagenesis in both prokaryotes and eukaryotes.

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Review of: Social Work Practice with Children Nancy Boyd Webb. New York: The Guilford Press. Reviewed by Lois Wright

The Use of Ships' Protests for Reconstruction of Synoptic-Scale Weather and Tropical Storm Identification in the Late Eighteenth Century

Ships’ protests have been used for centuries as legal documents to record and detail damages and indemnify Captains from fault. We use them in this article, along with data extracted through forensic synoptic analysis (McNally, 1994, 2004) to identify a tropical or subtropical system in the North Atlantic Ocean in 1785. They are shown to be viable sources of meteorological information. By comparing a damaging storm in New England in 1996, which included an offshore tropical system, with one reconstructed in 1785, we demonstrate that the tropical system identified in a ship’s protest played a significant role in the 1785 storm. With both forensic reconstruction and anecdotal evidence, we are able to assess that these storms are remarkably identical. The recurrence rate calculated in previous studies of the 1996 storm is 400–500 years. We suggest that reconstruction of additional years in the 1700s would provide the basis for a reanalysis of recurrence rates, with implications for future insurance and reinsurance rates. The application of the methodology to this new data source can also be used for extension of the hurricane database in the North Atlantic basin, and elsewhere, much further back into history than is currently available.

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K-m = 0.1-0.3 mM), lower for Pro (K-m = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K-m = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.

A novel family of transporters mediating the transport of glutathione derivatives in plants

Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes. Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa). The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5'-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein. Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source. The strain expressing OsGT1 mediated H-3]GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity. OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall.

Peptide and amino acid transporters are differentially regulated during seed development and germination in faba bean

Two peptide transporter (PTR) homologs have been isolated from developing seeds of faba bear, (Vicia faba). VfPTR1 was shown to be a functional peptide transporter through complementation of a yeast mutant. Expression patterns of VfPTR1 and VfPTR2 as well as of the amino acid permease VfAAP1 (Miranda et al., 2001) were compared throughout seed development and germination. In developing seeds, the highest levels of VfPTR1 transcripts were reached during midcotyledon development, whereas VfAAP1 transcripts were most abundant during early cotyledon development, before the appearance of storage protein gene transcripts, and were detectable until late cotyledon development. During early germination, VfPTR1 mRNA appeared first in cotyledons and later, during seedling growth, also in axes and roots. Expression of VfPTR2 and VfAAP1 was delayed compared with VfPTR1, and was restricted to the nascent organs of the seedlings. Localization of VfPTR1 transcripts showed that this FTR is temporally and spatially regulated during cotyledon development. In germinating seeds, VfPTR1 mRNA was localized in root hairs and root epidermal cells, suggesting a role in nutrient uptake from the soil. In seedling roots, VfPTR1 was repressed by a dipeptide and by an amino acid, whereas nitrate was without influence.

The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells

Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection. Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms. Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways. Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum). ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not. Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues. On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif. Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed. We suggest that ORF13 confers meristematic competence to cells infected by A. rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.

The Pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.

Influence of increment thickness on microhardness and dentin bond strength of bulk fill resin composites

OBJECTIVES To investigate the influence of increment thickness on Vickers microhardness (HV) and shear bond strength (SBS) to dentin of a conventional and four bulk fill resin composites. METHODS HV and SBS were determined on specimens of the conventional resin composite Filtek Supreme XTE (XTE) and the bulk fill resin composites SDR (SDR), Filtek Bulk Fill (FBF), x-tra fil (XFIL), and Tetric EvoCeram Bulk Fill (TEBF) after 24h storage. HV was measured either as profiles at depths up to 6mm or at the bottom of 2mm/4mm/6mm thick resin composite specimens. SBS of 2mm/4mm/6mm thick resin composite increments was measured to dentin surfaces of extracted human molars treated with the adhesive system OptiBond FL, and the failure mode was stereomicroscopically determined at 40× magnification. HV profiles and failure modes were descriptively analysed whereas HV at the bottom of resin composite specimens and SBS were statistically analysed with nonparametric ANOVA followed by Wilcoxon rank sum tests (α=0.05). RESULTS HV profiles (medians at 2mm/4mm/6mm): XTE 105.6/88.8/38.3, SDR 34.0/35.5/36.9, FBF 36.4/38.7/37.1, XFIL 103.4/103.9/101.9, TEBF 63.5/59.7/51.9. HV at the bottom of resin composite specimens (medians at 2mm/4mm/6mm): XTE (p<0.0001) 105.5>85.5>31.1, SDR (p=0.10) 25.8=21.9=26.0, FBF (p=0.16) 26.6=25.3=28.9, XFIL (p=0.18) 110.5=107.2=101.9, TEBF (p<0.0001) 63.0>54.9>48.2. SBS (MPa, medians at 2mm/4mm/6mm): XTE (p<0.0001) 23.9>18.9=16.7, SDR (p=0.26) 24.6=22.7=23.4, FBF (p=0.11) 21.4=20.3=22.0, x-tra fil (p=0.55) 27.0=24.0=23.6, TEBF (p=0.11) 21.0=20.7=19.0. The predominant SBS failure mode was cohesive failure in dentin. SIGNIFICANCE At increasing increment thickness, HV and SBS decreased for the conventional resin composite but generally remained constant for the bulk fill resin composites.

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.

Resisting the power of temptations: the right prefrontal cortex and self-control.

Imagine you are overweight and you spot your favorite pastry in the storefront of a bakery. How do you manage to resist this temptation? Or to give other examples, how do you manage to restrain yourself from overspending or succumbing to sexual temptations? The present article summarizes two recent studies stressing the fundamental importance of inhibition in the process of decision making. Based on the results of these studies, we dare to claim that the capacity to resist temptation depends on the activity level of the right prefrontal cortex (PFC).

Qeren tûšiyyā : eine tzaiṭšrifṭ fir di raifere jugend tzur beferderung der ziṭṭlichqeiṭ und moraliṭeṭ

fon Heiman Šwabacher