Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4.

Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant beta-sheeted structures in water that convert progressively into alpha-helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes.     

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.     

Analyse comparative de 3 logiciels de reconstruction itérative de tomoscintigraphie monophotonique myocardique

ObjectiveThis study makes a comparative analysis of the quality reconstruction of three software: 2D ordered subset expectation maximisation (2D OSEM), 3D ordered subsets expectation maximisation (Flash 3D) and Wallis.Patients and methodsThe data from myocardial scintigraphy acquisitions of 50 patients (38 men and 12 women; average age 61 ± 9 years) were successively reconstructed using three myocardial perfusion SPECT algorithms (Flash 3D, OSEM 2D and Wallis). Different combinations of iterations and subsets were considered. For Wallis, only the cut-off frequency was considered. The image's quality was assessed by determining the maximum contrast and the signal to noise ratio.ResultsThe Wallis software provided a higher signal to noise ratio compared to Flash 3D and OSEM 2D at stress and rest. The Wallis signal to noise ratio increased by a factor 1.93 (P = 0.0010) and 2.28 at stress (P = 0.0009); 1.50 (P = 0.0011) and 2.84 at rest (P = 0.0024) compared to respectively Flash 3D and OSEM 2D. Flash 3D provided better signal to noise ratio than OSEM 2D at stress and at rest. The difference in medians and interquartile ranges of the signal-to-noise ratio in post-stress were 22 % and 54 %, respectively between Flash 3D and OSEM 2D. At rest, the difference between the two methods in signal to noise ratio was 32 % ± 0.,29.ConclusionWallis algorithm was improve image quality with better signal to noise ratio compared to the reference method of Siemens Flash 3D. For both Flash 3D and OSEM 2D methods, the combination with 8 subsets and 12 iterations provided the best contrast and signal to noise ratio.     

Seasonal Modulation of the 8‐and 24‐Hour Rhythms of Ondansetron Tolerance in Mice

Ondansetron (Zophren®) is a serotonin 5HT₃-receptor antagonist used primarily to control nausea and vomiting caused by cytotoxic chemo‐and radio‐therapy. Tolerance to this drug shows both 24 and 8 h periodicities. In this framework, this study aimed to determine whether these ondansetron tolerance rhythms are modulated by season. The chronotoxic effect of a fixed dose (3.5 mg/kg, i.p.) of the drug was investigated with reference to both time of the day and year dependencies. Season‐related studies were performed on 560 male Swiss mice, 10 to 12 wks old, synchronized with L:D=12:12 for three weeks. During a 1 yr span (2005), four 24 h studies were performed with a single dosing time at 1, 7, 13, and 19 hours after light onset (HALO), respectively. Tolerance was assessed daily during a 40‐day span after acute ondansetron treatment. Both χ² test and cosinor methods were used to analyze the time series data. Statistically significant dosing time‐dependent changes were validated in both yearly and daily time scales. The 24 h mean survival rate peaked in spring (92%) compared to fall (72%), the 20% difference being statistically significant (χ² test with p<0.05 and cosinor with p<0.0001 for seasonal rhythm detection and with a peak time, Ø,=April 3±6.6 days). A 24 h rhythm was also detected in each of the seasonal time points. However, the curve pattern was monophasic in fall as well as spring. In fall, a large amplitude (A) circadian rhythm was detected that peaked at 19 HALO, while in the spring, a small circadian rhythm was detected that peaked at 1 HALO. The curve pattern was biphasic in summer (with large A) and in winter (with a small A). The existence of two peaks of equal magnitude in winter (100% survival rate) and in summer (100% and 90%) suggests the presence of both circadian and ultradian rhythms rather than an ultradian component of the 24 h period. The seasonal modulation of ondansetron circadian chronotolerance seems to involve several rhythm parameters: season‐related changes in the 24 h mean (M), amplitude (A), acrophase location (Ø), as well as bimodal curve patterns including the coexistence of rhythms with respectively 24 and 8 h periods in winter and summer. In conclusion, tolerance to ondansetron varies not only according to the 24 and 8 h periods but also according to seasons, which suggests the complexity of ondansetron toxicity rhythms. Seasonal modulation of ondansetron tolerance may also influence the strategies of chemo‐and chrono‐therapy, and it is therefore necessary to take it into account in clinical drug‐delivery protocols to minimize side effects of cytotoxic anticancer and antiemetic agents.     

Histone Deacetylase Complex1 Expression Level Titrates Plant Growth and Abscisic Acid Sensitivity in Arabidopsis

Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.     

Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2–12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.     

Disentanglement of heterogeneous dynamics in mixed lipid systems

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of ³¹P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine.     

Metabolic and molecular action of <Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> (fenugreek) and trace metals in experimental diabetic tissues

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycaemia resulting in defective insulin secretion, resistance to insulin action or both. The use of biguanides, sulphonylurea and other drugs are valuable in the treatment of diabetes mellitus; their use, however, is restricted by their limited action, pharmaco-kinetic properties, secondary failure rates and side effects. Trigonella foenum-graecum, commonly known as fenugreek, is a plant that has been extensively used as a source of antidiabetic compounds from its seeds and leaf extracts. Preliminary human trials and animal experiments suggest possible hypoglycaemic and anti-hyperlipedemic properties of fenugreek seed powder taken orally. Our results show that the action of fenugreek in lowering blood glucose levels is almost comparable to the effect of insulin. Combination with trace metal showed that vanadium had additive effects and manganese had additive effects with insulin on in vitro system in control and diabetic animals of young and old ages using adipose tissue. The Trigonella and vanadium effects were studied in a number of tissues including liver, kidney, brain peripheral nerve, heart, red blood cells and skeletal muscle. Addition of Trigonella to vanadium significantly removed the toxicity of vanadium when used to reduce blood glucose levels. Administration of the various combinations of the antidiabetic compounds to diabetic animals was found to reverse most of the diabetic effects studied at physiological, biochemical, histochemical and molecular levels. Results of the key enzymes of metabolic pathways have been summarized together with glucose transporter, Glut-4 and insulin levels. Our findings illustrate and elucidate the antidiabetic/insulin mimetic effects of Trigonella, manganese and vanadium.