Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis

The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.     

Association of spermatogenic failure with the b2/b3 partial AZFc deletion

Infertility affects around 1 in 10 men and in most cases the cause is unknown. The Y chromosome plays an important role in spermatogenesis and specific deletions of this chromosome, the AZF deletions, are associated with spermatogenic failure. Recently partial AZF deletions have been described but their association with spermatogenic failure is unclear. Here we screened a total of 339 men with idiopathic spermatogenic failure, and 256 normozoospermic ancestry-matched men for chromosome microdeletions including AZFa, AZFb, AZFc, and the AZFc partial deletions (gr/gr, b1/b3 and b2/b3).AZFa and AZFc deletions were identified in men with severe spermatogenic failure at similar frequencies to those reported elsewhere. Gr/gr deletions were identified in case and control populations at 5.83% and 6.25% respectively suggesting that these deletions are not associated with spermatogenic failure. However, b2/b3 deletions were detected only in men with spermatogenic failure and not in the normospermic individuals. Combined with our previous data this shows an association of the b2/b3 deletion (p = 0.0318) with spermatogenic failure in some populations. We recommend screening for this deletion in men with unexplained spermatogenic failure.     

Ranolazine inhibits shear sensitivity of endogenous Na+ current and spontaneous action potentials in HL-1 cells

Na_V1.5 is a mechanosensitive voltage-gated Na⁺ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na⁺ current and delayed rectifier (I_Kr) currents. Recently, ranolazine was also shown to be an inhibitor of Na_V1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na⁺ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na⁺ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.     

Breeding of Brassica rapa for Biogas Production: Heterosis and Combining Ability of Biomass Yield

The use of plant biomass as substrate for biogas production has recently become of major interest in Europe. Winter Brassica rapa produces high early biomass and could be grown as a pre-crop harvested early in the year followed by a second crop such as maize. The objectives of this study were to estimate heterosis and combining ability of 15 European winter B. rapa cultivars for biomass yield at end of flowering. A half-diallel without reciprocals was conducted among cultivars to produce 105 crosses. These crosses and their parents were evaluated in two years at two locations in Northern Germany. Data collected were days to flowering (DTF), fresh biomass yield (FBY), dry matter content (DMC) and dry biomass yield (DBY). The mean DBY was 5.3 t/ha for the parental cultivars and 5.6 t/ha for their crosses. The crosses surpassed on average their parents by 7.6% for FBY and 5.9% for DBY whereas DMC was 1.4% higher in the parents. Maximum mid parent heterosis was 21.0% for FBY and 30.4% for DBY. Analysis of variance showed that genetic variance was mainly due to specific combining ability (SCA). The correlation between parental performance and general combining ability (GCA) was 0.42** for FBY and 0.53** for DBY. In conclusion, the amount of heterosis in crosses between European winter B. rapa cultivars is not very high on average, but can be up to 30% in the best crosses. Selection of parental combinations with high specific combining ability to produce synthetic cultivars can rapidly improve biomass yield.     

DNA repair and free radicals, new insights into the mechanism of spore photoproduct lyase revealed by single amino acid substitution

The major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(alpha-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed.     

Aminolevulinic acid and nitric oxide regulate oxidative defense and secondary metabolisms in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under drought stress

To minimize the damaging effects of stresses, plant growth regulators (PGRs) are widely used to sustain the plant life under stress-prone environments. So, a study was carried out to evaluate the response of two canola (Brassica napus L.) cultivars, Dunkeld and Cyclone, to foliar-applied two potential PGRs, nitric oxide (NO) and 5-aminolevulinic acid, under water deficit conditions. In this study, the levels of NO and ALA used were 0.02 and 0.895 mM, respectively. Plants of both canola cultivars were subjected to control (100% field capacity) and water deficit (60% field capacity). Drought stress significantly decreased growth, chlorophyll pigments, relative water contents (RWC), and soluble proteins, while it increased relative membrane permeability (RMP), proline, glycinebetaine (GB), malondialdehyde (MDA), total phenolics, and activities of catalase (CAT) and peroxidase (POD) enzymes in both cultivars. Foliar application of PGRs improved growth, chlorophyll a, GB, total phenolics, CAT activity, and total soluble proteins, while it decreased RMP, MDA, and POD activity in both canola cultivars. Other physio-biochemical attributes such as chlorophyll b, RWC, hydrogen peroxide (H₂O₂) and proline contents as well as superoxide dismutase (SOD) activity remained unaffected due to application of PGRs. So, the results of the present study suggest that exogenous application of NO and ALA could be useful to enhance the drought tolerance of canola plants by up-regulating the oxidative defense system, osmoprotectant accumulation, and minimizing the lipid peroxidation.     

Length–weight and length–length relationships for three species of Squalius (Cyprinidae; Leuciscinae) from the Caspian Sea,Namak and Tigris basins of Iran

Length‐weight and length‐length relationships were estimated for three species of the genus Squalius. These fish were sampled from the Caspian Sea as well as the Namak and Tigris basins of Iran. Sampling took place between July 2009 and August 2010 using a seine net (15 m long, 2 m width and 5 mm mesh size). The values of the b parameter in the length‐weight relationship equations were determined as 3.37 for S. turcicus, 3.25 for S. namak and 3.14 for S. berak (b > 3).     

Respiratory symptoms and lung function in taxi drivers and manual workers

The aim of this study, was to determine the prevalence of some respiratory symptoms and possible diseases among taxi drivers and manual workers. This prospective study was performed on 165 Pakistani male drivers, (mean age: 34.5±7.8 years) and 165 Pakistani male manual workers not exposed to dust or fumes, without occupational exposure to driving employed in the Water and Electricity Department and recruited as controls (mean age: 34.6±7.6 years and mean height and weight 169.8±6.0 cm and 71.9±10.9 kg). The data on chronic respiratory symptoms showed that taxi drivers had higher prevalence of symptoms than manual workers, being significantly greater for asthma (RR=1.72; 95% CI=1.00–2.88,P=0.037); allergic rhinitis (RR=2.41; 95% CI=1.46–3.94,P=0.0006); dyspnea (RR=2.13; 95% CI=1.22–3.71,P=0.009); and nasal catarrh (RR=2.19; 95% CI=1.22–3.91,P=0.0106). Thirty percent of taxi drivers and 27% of manual workers were smokers, there was no significant differences in the prevalence of chronic respiratory symptoms between smokers and non-smokers. Lung function parameters in the taxi drivers were significantly lower than in manual workers group (P<0.0001) except PEF parameter. When comparing the measured mean values of lung function parameters in the drivers among smokers and nonsmokers, there was no significant differences between smokers and nonsmokers. Also, a comparison of ventilatory capacity of paired predicted values with measured normal values showed statistically significant differences between predicted and measured values for taxi drivers and manual workers for FVC, FEV₁, FEF_25–75 and PEF parameters except for FEV₁/FVC test in manual workers. In conclusion, the results of the present study provide evidence regarding effects of such as carbon monoxide, nitrogen dioxide, sulfur dioxide and gases exposures on taxi drivers and long-term driving, which may be associated with the development of chronic respiratory symptoms and lung function impairment.