Effects of mutation at a conserved N-glycosylation site in the bovine retinal cyclic nucleotide-gated ion channel

AbstractBovine retinal cyclic nucleotide-gated (CNG) ion channel contains an evolutionary conserved N-glycosylation site in the external loop between the fifth transmembrane segment and the pore-forming region. The effect of tunicamycin treatment and the site-specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N-glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N-glycosylation site did not affect expression and functionality of the CNG channel in oocytes

Imaging evaluation of the liver using multi-detector row computed tomography in micropigs as potential living liver donors

The shortage of organ donors has stimulated interest in the possibility of using animal organs for transplantation into humans. In addition, pigs are now considered to be the most likely source animals for human xenotransplantation because of their advantages over non-human primates. However, the appropriate standard values for estimations of the liver of micropigs have not been established. The determination of standard values for the micropig liver using multi-detector row computed tomography (MDCT) would help to select a suitable donor for an individual patient, determine the condition of the liver of the micropigs and help predict patient prognosis. Therefore, we determined the standard values for the livers of micropigs using MDCT. The liver parenchyma showed homogenous enhancement and had no space-occupying lesions. The total and right lobe volumes of the liver were 698.57 ± 47.81 ml and 420.14 ± 26.70 ml, which are 51.74% and 49.35% of the human liver volume, respectively. In micropigs, the percentage of liver volume to body weight was approximately 2.05%. The diameters of the common hepatic artery and proper hepatic artery were 6.24 ± 0.20 mm and 4.68 ± 0.13 mm, respectively. The hepatic vascular system of the micropigs was similar to that of humans, except for the variation in the length of the proper hepatic artery. In addition, the diameter of the portal vein was 11.27 ± 0.38 mm. In conclusion, imaging evaluation using the MDCT was a reliable method for liver evaluation and its vascular anatomy for xenotransplantation using micropigs

An Engineered Viral Protease Exhibiting Substrate Specificity for a Polyglutamine Stretch Prevents Polyglutamine-Induced Neuronal Cell Death

BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases

Associate Editor: Golan Yona

doi:10.1093/bioinformatics/btl33