The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum,a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

Car Crashes and Central Disorders of Hypersomnolence: A French Study

Background

Drowsiness compromises driving ability by reducing alertness and attentiveness, and delayed reaction times. Sleep-related car crashes account for a considerable proportion of accident at the wheel. Narcolepsy type 1 (NT1), narcolepsy type 2 (NT2) and idiopathic hypersomnia (IH) are rare central disorders of hypersomnolence, the most severe causes of sleepiness thus being potential dangerous conditions for both personal and public safety with increasing scientific, social, and political attention. Our main objective was to assess the frequency of recent car crashes in a large cohort of patients affected with well-defined central disorders of hypersomnolence versus subjects from the general population.

Methods

We performed a cross-sectional study in French reference centres for rare hypersomnia diseases and included 527 patients and 781 healthy subjects. All participants included needed to have a driving license, information available on potential accident events during the last 5 years, and on potential confounders; thus analyses were performed on 282 cases (71 IH, 82 NT2, 129 NT1) and 470 healthy subjects.

Results

Patients reported more frequently than healthy subjects the occurrence of recent car crashes (in the previous five years), a risk that was confirmed in both treated and untreated subjects at study inclusion (Untreated, OR = 2.21 95%CI = [1.30–3.76], Treated OR = 2.04 95%CI = [1.26–3.30]), as well as in all disease categories, and was modulated by subjective sleepiness level (Epworth scale and naps). Conversely, the risk of car accidents of patients treated for at least 5 years was not different to healthy subjects (OR = 1.23 95%CI = [0.56–2.69]). Main risk factors were analogous in patients and healthy subjects.

Conclusion

Patients affected with central disorders of hypersomnolence had increased risk of recent car crashes compared to subjects from the general population, a finding potentially reversed by long-term treatment.     

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth,respiration and protein biosynthesis

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O₂ consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.Abbreviations DNP dinitrophenol - PG propylgallate - SHAM salicylhydroxamic acid     

Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo

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Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.     

On-Going Frontal Alpha Rhythms Are Dominant in Passive State and Desynchronize in Active State in Adult Gray Mouse Lemurs

The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8–12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7–9 Hz) during passive state. During active state, there was a reduction in alpha power density (8–12 Hz) and an increase of power density at slow frequencies (1–4 Hz). Relative EMG activity was related to EEG power density at 2–4 Hz (positive correlation) and at 8–12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology.     

Membranes undergoing phase transitions are preferentially hydrolyzed by beta-bungarotoxin

β-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited.     

Development of a cyclosporin A derivative with excellent anti-hepatitis C virus potency

Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional “slow release” concept.     

Some 3-(4-Aminophenyl)pyrrolidine-2,5-diones as All- trans -retinoic Acid Metabolising Enzyme Inhibitors (RAMBAs)

A series of (±) -3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3- position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC 50 = 98.8 μM, ketoconazole, 22.15 μM) showed that it was not stereoselective in its inhibition. (±) - (1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC 50 = 20.9 μM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC 50 = 211.6 μM respectively; ketoconazole, 38.8% and 85.95 μM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (±) - (1) was a weak inhibitor (c. 53% at 200 μM) whereas ketoconazole showed high potency (c. 65% at 0.625 μM and 0.25 μM respectively). The nature of the induced target enzyme is discussed.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.