Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro

Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.     

Conformational analysis of an EP24.15 inhibitor by NMR and molecular modelling

The enzyme thimet oligopeptidase (EC3.4.24.15, EP24.15) is responsible for the hydrolysis of a number of neuropeptides. Despite much research examining its substrate specificity, little is known about the conformational requirements of its active site. We have used 1D ¹H and 2D TOCSY NMR experiments to assign the proton resonances of the EP24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), and 2D ROESY NMR to investigate whether cFP exhibits any conformational preferences in CD₃OD and in aqueous CD₃OD. Molecular modelling of charged cFP in the gaseous phase generated a number of conformations that were consistent with the NMR data obtained in CD₃OD. Analogous modelling on the uncharged cFP did not result in conformations consistent with any of the NMR data, but did suggest that, under non-polar conditions, cFP could adopt a hairpin conformation which would allow simultaneous coordination of the two carboxyl groups of cFP to the zinc ion in the active site of EP24.15.     

The First Extracellular Domain Plays an Important Role in Unitary Channel Conductance of Cx50 Gap Junction Channels

Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as V_j-gating) and display a wide range of unitary channel conductance (γ_j), yet the domains responsible for V_j-gating and γ_j are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in V_j-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct V_j-gating and γ_j from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γ_j, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in V_j-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in V_j-gating and γ_j. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.     

Neurotransmitter Specific,Cellular-Resolution Functional Brain Mapping Using Receptor Coated Nanoparticles: Assessment of the Possibility

Receptor coated resonant nanoparticles and quantum dots are proposed to provide a cellular-level resolution image of neural activities inside the brain. The functionalized nanoparticles and quantum dots in this approach will selectively bind to different neurotransmitters in the extra-synaptic regions of neurons. This allows us to detect neural activities in real time by monitoring the nanoparticles and quantum dots optically. Gold nanoparticles (GNPs) with two different geometries (sphere and rod) and quantum dots (QDs) with different sizes were studied along with three different neurotransmitters: dopamine, gamma-Aminobutyric acid (GABA), and glycine. The absorption/emission spectra of GNPs and QDs before and after binding of neurotransmitters and their corresponding receptors are reported. The results using QDs and nanorods with diameter 25nm and aspect rations larger than three were promising for the development of the proposed functional brain mapping approach.     

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation ( Ds ), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep⁺) and non-replicating (rep⁻) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep⁺) or mutant, inactive derivatives of these genes (rep⁻). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds -vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 × 10⁵-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.