NADH: Nitrate reductase activity restoration by rhodanese

The NADH: nitrate reductase from durum wheat leaves was inactivated by cyanide and its activity restored by thiosulphate and beef kidney rhodanese. Rhodanese and thiosulphate, added to NADH-nitrate reductase before cyanide treatment protected NADH-nitrate reductase activity. No oxidizing agent was required for the protection or restoration of cyanide treated NADH-nitrate reductase.     

Decrease in maximum consumption of oxygen after blockage of beta-andrenergic receptors]

VO2 max, maximum oxygen uptake, has been measured in 4 normal young men, before and after beta-adrenergic blockade (0.5 to 5 mg Pindolol by mouth). Pindolol induces bradycardia and reduces VO2 max. A statistically significant positive correlation appears between posology of Pindolol and bradycardia, this posology and reduction of VO2 max, and finally between bradycardia and reduction of VO2 max. These correlations indicate that the reduction of VO2 max is best explained by a circulatory limitation of oxygen supply to active muscles.     

Electrophoretic demonstration and initial characterization of human red cell NAD(P)+ase

A method for electrophoretic detection of NAD(P)+ase solubilized from red cell membranes is described. The method reveals both NAD+ase and NADP+ase and can be applied to a screening procedure for the detection of electrophoretic variants. The initial chracterization of the solubilized enzyme is also described.     

Sequence dependence of transient Hoogsteen base pairing in DNA

Hoogsteen (HG) base pairing is characterized by a 180° rotation of the purine base with respect to the Watson-Crick-Franklin (WCF) motif. Recently, it has been found that both conformations coexist in a dynamical equilibrium and that several biological functions require HG pairs. This relevance has motivated experimental and computational investigations of the base-pairing transition. However, a systematic simulation of sequence variations has remained out of reach. Here, we employ advanced path-based methods to perform unprecedented free-energy calculations. Our methodology enables us to study the different mechanisms of purine rotation, either remaining inside or after flipping outside of the double helix. We study seven different sequences, which are neighbor variations of a well-studied A⋅T pair in A₆-DNA. We observe the known effect of A⋅T steps favoring HG stability, and find evidence of triple-hydrogen-bonded neighbors hindering the inside transition. More importantly, we identify a dominant factor: the direction of the A rotation, with the 6-ring pointing either towards the longer or shorter segment of the chain, respectively relating to a lower or higher barrier. This highlights the role of DNA’s relative flexibility as a modulator of the WCF/HG dynamic equilibrium. Additionally, we provide a robust methodology for future HG proclivity studies.     

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase,POLG1)

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.     

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.     

Substance P and acetylcholine are co-localized in the pathway mediating mucociliary activity in Rana pipiens

Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play a key role in the stimulation of the mucociliary transport in the frog palate. In this study, retrograde neuronal tracing was combined with immunocytochemistry for SP and choline acetyl transferase (ChAT) in the trigeminal ganglion and for neurokinin-1 receptor (NK1R) in the palate of Rana pipiens. The cells of origin of the palatine nerve were identified in the trigeminal ganglion using the retrograde tracer Fluorogold (FG). Optimal labeling of FG cells in the trigeminal ganglion was obtained at 96 h of exposure. Immunoflorescent shows that SP and acetylcholine are co-localized in 92% of the cells labeled with FG in the trigeminal ganglion. NK1 receptors were found in the membrane of epithelial and goblet cells of the palate. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.