Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.     

PT-ACRAMTU, A Platinum–Acridine Anticancer Agent, Lengthens and Aggregates, but does not Stiffen or Soften DNA

We used atomic force microscopy (AFM) to study the dose-dependent change in conformational and mechanical properties of DNA treated with PT-ACRAMTU ([PtCl(en)(ACRAMTU-S)](NO₃)₂, (en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea. PT-ACRAMTU is the parent drug of a family of non-classical platinum-based agents that show potent activity in non-small cell lung cancer in vitro and in vivo. Its acridine moiety intercalates between DNA bases, while the platinum group forms mono-adducts with DNA bases. AFM images show that PT-ACRAMTU causes some DNA looping and aggregation at drug-to-base pair ratio (r _b) of 0.1 and higher. Very significant lengthening of the DNA was observed with increasing doses of PT-ACRAMTU, and reached saturation at an r _b of 0.15. At r _b of 0.1, lengthening was 0.6 nm per drug molecule, which is more than one fully stretched base pair stack can accommodate, indicating that ACRAMTU also disturbs the stacking of neighboring base pair stacks. Analysis of the AFM images based on the worm-like chain (WLC) model showed that PT-ACRAMTU did not change the flexibility of (non-aggregated) DNA, despite the extreme lengthening. The persistence length of untreated DNA and DNA treated with PT-ACRAMTU was in the range of 49–65 nm. Potential consequences of the perturbations caused by this agent for the recognition and processing of the DNA adducts it forms are discussed.     

Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct

Glutathione reacts with orthophthalaldehyde to form a stable, highly fluorescent tricyclic derivative which is easily separated and quantitated by high-performance liquid chromatography. Separation of the glutathione adduct is achieved by isocratic elution over a reverse-phase column with 7.5% methanol/92.5% 0.15 M sodium acetate, pH 7.00. The adduct is detected fluorometrically and quantitated by integration of peak area. Detection of 0.1 to 200 pmol glutathione produces a linear response and the recovery of reduced and oxidized glutathione from rat liver homogenate, bile, and plasma is quantitative. The chemical identity of the adduct was confirmed by mass spectrometry.     

A comparison of univariate and multivariate gene selection techniques for classification of cancer datasets

Background  

Gene selection is an important step when building predictors of disease state based on gene expression data. Gene selection generally improves performance and identifies a relevant subset of genes. Many univariate and multivariate gene selection approaches have been proposed. Frequently the claim is made that genes are co-regulated (due to pathway dependencies) and that multivariate approaches are therefore per definition more desirable than univariate selection approaches. Based on the published performances of all these approaches a fair comparison of the available results can not be made. This mainly stems from two factors. First, the results are often biased, since the validation set is in one way or another involved in training the predictor, resulting in optimistically biased performance estimates. Second, the published results are often based on a small number of relatively simple datasets. Consequently no generally applicable conclusions can be drawn.     

Acupuncture for migraine prophylaxis: a randomized controlled trial

Background:

Acupuncture is commonly used to treat migraine. We assessed the efficacy of acupuncture at migraine-specific acupuncture points compared with other acupuncture points and sham acupuncture.

Methods:

We performed a multicentre, single-blind randomized controlled trial. In total, 480 patients with migraine were randomly assigned to one of four groups (Shaoyang-specific acupuncture, Shaoyang-nonspecific acupuncture, Yangming-specific acupuncture or sham acupuncture [control]). All groups received 20 treatments, which included electrical stimulation, over a period of four weeks. The primary outcome was the number of days with a migraine experienced during weeks 5–8 after randomization. Our secondary outcomes included the frequency of migraine attack, migraine intensity and migraine-specific quality of life.

Results:

Compared with patients in the control group, patients in the acupuncture groups reported fewer days with a migraine during weeks 5–8, however the differences between treatments were not significant (p > 0.05). There was a significant reduction in the number of days with a migraine during weeks 13–16 in all acupuncture groups compared with control (Shaoyang-specific acupuncture v. control: difference –1.06 [95% confidence interval (CI) –1.77 to –0.5], p = 0.003; Shaoyang-nonspecific acupuncture v. control: difference –1.22 [95% CI –1.92 to –0.52], p < 0.001; Yangming-specific acupuncture v. control: difference –0.91 [95% CI –1.61 to –0.21], p = 0.011). We found that there was a significant, but not clinically relevant, benefit for almost all secondary outcomes in the three acupuncture groups compared with the control group. We found no relevant differences between the three acupuncture groups.

Interpretation:

Acupuncture tested appeared to have a clinically minor effect on migraine prophylaxis compared with sham acupuncture.

Trial Registration:

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00599586","term_id":"NCT00599586"}}NCT00599586About 6%–8% of men and 16%–18% of women in the United States and England experience migraines, with or without an aura.^1,2 A prevalence of 1% has been reported in mainland China,³ compared with 4.7% in Hong Kong and 9.1% in Taiwan.^4,5 A recent Cochrane meta-analysis suggests that acupuncture as migraine prophylaxis is safe and effective and should be considered as a treatment option for willing patients.⁶Although the specific effects acupuncture are controversial, acupuncture, as it is currently practised, clearly differentiates between real acupuncture points and nonacupuncture points. The Chinese Government launched the National Basic Research Program to obtain more data about the specificity of acupuncture points.⁷Trials from Italy and Brazil^8,9 showed that acupuncture was more effective than sham acupuncture in preventing migraines, but other trials have reported no differences.^10–13 There is no evidence that one acupuncture strategy is more effective than another for treating migraines. According to acupuncture theory, a headache on the lateral side is usually defined as a Shaoyang headache. In Jinkuiyi,¹⁴ migraines are said to affect the yang meridians (including the Taiyang, Yangming and Shaoyang meridians). In Lingshu,¹⁵ the Shaoyang meridians are said to go through the lateral side of the body, therefore the Shaoyang meridians are thought to be superior for treating migraines. Some points on the Shaoyang meridians are regarded as being more specific for migraines than other points.¹⁶Our aim was to investigate whether acupuncture at specific acupuncture points was more efficacious in preventing migraine than sham acupuncture at nonacupuncture points. We also investigated whether the efficacy varied when acupuncture points along different meridians or points along the same meridian were used.     

Specific dechlorinase activity in lindane degradation by Streptomyces sp. M7

Synthesis of dechlorinase in Streptomyces sp. M7 was induced when the microorganism was grown in the presence of lindane (γ-hexachlorocyclohexane) as the only carbon source. Activity of cells grown with lindane was about four and half times higher compared to cells grown with glucose. Maximum dechlorinase activity was observed at 30°C in alkaline conditions pH (7.9) and the enzyme did not show cation dependency. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed one differential band with a molecular weight similar to serum albumin (M _r 66,200), which corresponded to polynucleotide phosphorylase, an enzyme that plays an important role in the regulation system and could be involved in the regulation of the dechlorinase gene. Detected in cell-free extracts were γ-pentachlorocyclohexene and 1,3,4,6-tetrachloro-1,4-cyclohexadiene, both being products of the dechlorinase activity. This is the first time that the presence of an enzyme with dechlorinase activity has been demonstrated in an actinomycete strain isolated in Tucumán, Argentina. Characteristics of this enzyme revealed that Streptomyces sp. M7 could be useful in the future in bioremediation of soil or as a biosensor.