Proteomic analysis of the monkey hippocampus for elucidating ischemic resistance.

It is well-known that the cornu Ammonis 1 (CA1) sector of hippocampus is vulnerable for the ischemic insult, whereas the dentate gyrus (DG) is resistant. Here, to elucidate its underlying mechanism, alternations of protein oxidation and expression of DG in the monkey hippocampus after ischemia-reperfusion by the proteomic analysis were studied by comparing CA1 data. Oxidative damage to proteins such as protein carbonylation interrupt the protein function. Carbonyl modification of molecular chaperone, heat shock 70 kDa protein 1 (Hsp70.1) was increased remarkably in CA1, but slightly in DG. In addition, expression levels of nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-2 (SIRT2) was significantly increased in DG after ischemia, but decreased in CA1. Accordingly, it is likely that SIRT2 upregulation and negligible changes of carbonylation of Hsp70.1 exert its neuroprotective effect in DG. On the contrary, carbonylation level of dihydropyrimidinase related protein 2 (DRP-2) and l-lactate dehydrogenase B chain (LDHB) were slightly increased in CA1 as shown previously, but remarkably increased in DG after ischemia. It is considered that DRP-2 and LDHB are specific targets of oxidative stress by ischemia insult and high carbonylation levels of DRP-2 may play an important role in modulating ischemic neuronal death.

PEG-OligoRNA Hybridization of mRNA for Developing Sterically Stable Lipid Nanoparticles toward In Vivo Administration.

Lipid nanoparticles (LNPs) exhibit high potential as carriers of messenger RNA (mRNA). However, the arduous preparation process of mRNA-loaded LNPs remains a huge obstacle for their widespread clinical application. Herein, we tackled this issue by mRNA PEGylation through hybridization with polyethylene glycol (PEG)-conjugated RNA oligonucleotides (PEG-OligoRNAs). Importantly, mRNA translational activity was preserved even after hybridization of 20 PEG-OligoRNAs per mRNA. The straightforward mixing of the PEGylated mRNA with lipofectamine LTX, a commercial lipid-based carrier, just by pipetting in aqueous solution, allowed the successful preparation of mRNA-loaded LNPs with a diameter below 100 nm, whereas the use of non-PEGylated mRNA provided large aggregates above 100- and 1000-nm. In vivo, LNPs prepared from PEG-OligoRNA-hybridized mRNA exhibited high structural stability in biological milieu, without forming detectable aggregates in mouse blood after intravenous injection. In contrast, LNPs from non-PEGylated mRNA formed several micrometer-sized aggregates in blood, leading to rapid clearance from blood circulation and deposition of the aggregates in lung capillaries. Our strategy of mRNA PEGylation was also versatile to prevent aggregation of another type of mRNA-loaded LNP, DOTAP/Chol liposomes. Together, our approach provides a simple and robust preparation method to LNPs for in vivo application.

Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeast Candida boidinii.

In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (ρ0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins.