Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.     

Uncoupling protein 2: a novel player in neuroprotection

A recent report provides exciting new evidence that suggests that uncoupling protein 2 (UCP2), a mitochondrial protein expressed in specific cells of numerous tissues, might be neuroprotective by reducing mitochondrial Ca²⁺ uptake and preventing mitochondrial accumulation of reactive oxygen species (ROS) following cerebral ischemia. The mitochondrial sequestration of Ca²⁺ and ROS, which depends on the mitochondrial membrane potential (ΔΨ_m), is a deleterious consequence of excitotoxicity. A neuroprotective role for Ucp2 is consistent with the already proposed property of this gene in mitigating cellular damage caused by ROS.     

Structural chemistry and In vitro antitubercular activity of acetylpyridine benzoyl hydrazone and its copper complex against Mycobacterium smegmatis

Acetylpyridine benzoyl hydrazone (APBH) 1 and its copper complex [[(APBH)CuCl](2)].(EtOH) 2 were structurally characterized by elemental analysis, magnetic measurements, spectroscopy, electrochemistry and single crystal X-ray diffraction studies. The ligand assumes Z-isomeric form and planar geometry in solid state, coordinating through pyridyl nitrogen, azomethine nitrogen and the carbonyl oxygen of the benzoyl group. The copper complex is dimeric and has a distorted octahedral geometry in which the two copper atoms are bridged by two chloride atoms. Antimycobacterial screening of ligand and its copper compound against Mycobacterium smegmatis shows clear enhancement in the antitubercular activity upon copper complexation.     

Life table characteristics of Macrolophus caliginosus preying upon Tetranychus urticae

The life table characteristics of the polyphagous mirid Macrolophus caliginosus Wagner (Heteroptera: Miridae) preying on various stages of Tetranychus urticae Koch (Acari: Tetranychidae) with tomato as host plant were described at 22 °C. The following average parameters were obtained: Female longevity: 28.7 days; fecundity: 0.7 eggs/female/day; egg mortality: 2.6%; pre-oviposition period: 5.5 days; oviposition period: 18.1 days; post-oviposition period: 3.2 days; juvenile development time: 26.8 days; juvenile mortality: 34.9%; and sex ratio (/(+): 0.46. Life table parameters were estimated as net reproduction rate (R ₀): 6.15; intrinsic rate of increase (r _m): 0.031 day^–1; finite rate of increase (): 1.032; mean generation time (T _c): 58.17 days; and doubling time (T ₂) 22.2 days. The parameters obtained were in accordance with those reported for M. caliginosus fed on another mite species (T. turkestani Ugarov & Nikolski (Acari: Tetranychidae)). However, compared to the performance of M. caliginosus fed on common glasshouse insect pests, a diet consisting of only mites appeared to be inferior. However, being a voracious predator, M. caliginosus may be a valuable addition to existing methods of mite control.     

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι^−/− embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.     

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

Summary In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron ) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron , was detected in five senescent subclones which all showed strong amplification of the intronic sequence (Sen DNA ).     

Two phases of seasonal stem radius variations of Sabina przewalskii Kom. in northwestern China inferred from sub-diurnal shrinkage and expansion patterns

In the National Natural Reserve of the Qilian Mountains, northwest China, automated dendrometers were installed on Qilian juniper (Sabina przewalskii Kom.) in 2010. Here, we present a complete 1-year data series of dendrometer measurements and synchronous meteorological records. The mean daily radius change index curve obtained by first-difference standardization was analyzed. The results indicate strong similarities of stem radius variations among the studied trees. By graphical inspection of sub-diurnal shrinkage and expansion patterns, seasonal stem radius variations could be divided into two general phases. During early winter to spring, daily amplitudes show strong fluctuations, and meteorological data indicate that both air and soil temperatures are positively related to stem radius swelling. From late March to early November 2011, stem radius expansion is negatively related to both air and soil temperatures, as well as to vapor pressure deficit, and positively related to precipitation and soil moisture. We found that the dividing lines between the two phases were strongly linked to the occurrence of a 0?°C daily mean air temperature, indicating that air temperature played an important effect on stem radius variations in this cold and arid environment. During the growing season in 2011, May and June are the most productive months.