The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

Crustacean and rotifer composition of temporary ponds in the Doñana National Park (SW Spain) during floods

The zooplankton of 18 temporary ponds in the Doñana National Park (SW Spain) was studied during floods in February and May 1997. A total of 37 rotifer taxa and 34 crustaceans species were identified (17 cladocerans, 2 ostracans, 4 diaptomids, 7 cyclopodids, 1 harpacticoid, 1 anostracan, 1 notostracan and 1 conchostracan). Zooplankton samples were collected separately from the littoral and the open-water of 12 different ponds. Commonly distributed zooplankton species (frequency of appearance 50%) were not segregated to either the littoral or the open-water according to a chi-square test (P>0.005). The study ponds were divided into seasonal, intermediate and ephemeral ponds according to the length of their hydroperiod. The total numbers of both crustacean and rotifer taxa were highest in the intermediate-hydroperiod ponds (26 and 32 taxa, respectively). The total number of zooplankton taxa collected in both February and May was not significantly correlated to the hydroperiod of the temporary ponds of Doñana during the study period (r=0.165, P=0.526). 21 rotifer taxa and 20 crustacean species were found in the ephemeral ponds; the number of restricted species was also relatively high (3 rotifers and 4 crustaceans). Therefore, the ephemeral ponds held a relatively rich community during floods compared to other temporary ponds of Doñana despite their small size and short wet phase.     

Technical communication: Determination of cuprous ions in bacterial leachates and for environmental monitoring

A sensitive and precise spectrophotometric method has been developed for the determination of copper(I) in bacterial leach liquors produced by the action of Thiobacillus ferrooxidans and T. thiooxidans on copper ores. In this method bicinchoninic acid (BCA) has been used as the chromogenic reagent which produces a stable purple complex with Cu(I) which was found to obey Beer's Law and with _max at 560 nm. The coloured complex has a molar extinction coefficient () value of 6.6 × 10³ l mol^–1 cm^–1; specific absorptivity () value of 0.104 ml^–1 g cm^–1 and the Sandell sensitivity (S) value was 0.0096 g cm². Optimal conditions for development of coloration/sensitivity were determined. Interferences due to cations and anions were investigated and various masking agents for alleviating their inhibition were studied. The method has been found very useful in determining ratios of Cu(I) to Cu(II) in bacterial leach liquors and should play a significant role in determining the reaction mechanisms of biological leaching and for environmental monitoring.     

Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (eg logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [³⁵S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunoflurescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.     

16.1% Efficient Hysteresis‐Free Mesostructured Perovskite Solar Cells Based on Synergistically Improved ZnO Nanorod Arrays

Significant efficiency improvements are reported in mesoscopic perovskite solar cells based on the development of a low‐temperature solution‐processed ZnO nanorod (NR) array exhibiting higher NR aspect ratio, enhanced electron density, and substantially reduced work function than conventional ZnO NRs. These features synergistically result in hysteresis‐free, scan‐independent, and stabilized devices with an efficiency of 16.1%. Electron‐rich, nitrogen‐doped ZnO (N:ZnO) NR‐based electron transporting materials (ETMs) with enhanced electron mobility produced using ammonium acetate show consistently higher efficiencies by one to three power points than undoped ZnO NRs. Additionally, the preferential electrostatic interaction between the ­nonpolar facets of N:ZnO and the conjugated polyelectrolyte polyethylenimine (PEI) has been relied on to promote the hydrothermal growth of high aspect ratio NR arrays and substantially improve the infiltration of the perovskite light absorber into the ETM. Using the same interactions, a conformal PEI coating on the electron‐rich high aspect ratio N:ZnO NR arrays is ­successfully applied, resulting in a favorable work function shift and altogether leading to the significant boost in efficiency from <10% up to >16%. These results largely surpass the state‐of‐the‐art PCE of ZnO‐based perovskite solar cells and highlight the benefits of synergistically combining mesoscale control with doping and surface modification.     

The alteration in the architecture of a T‐DNA insertion rice mutant osmtd1 is caused by up‐regulation of MicroRNA156f

Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter‐Binding Protein‐Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T‐DNA insertion mutant Osmtd1 (Oryza sativa multi‐tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T‐DNA insertion in Osmtd1. Further analysis revealed that the T‐DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild‐type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.     

Loss of Prolyl Carboxypeptidase in Two-Kidney,One-Clip Goldblatt Hypertensive Mice

It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP.