Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein.

Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms.

In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans.

Human neuraminidase 1 (NEU1) is a lysosomal glycosidase that cleaves the terminal sialic acids of sialylglycoconjugates. NEU1 is biosynthesized in the endoplasmic reticulum (ER) lumen as an N-glycosylated protein. NEU1 also associates with cathepsin A (CTSA) in ER, migrates to lysosomes, and exerts catalytic activity. Extraordinary in cellulo crystallization of NEU1 protein in ER despite carrying three N-glycans per molecule at N186, N343, and N352, respectively, were observed when the single human NEU1 gene was overexpressed in mammalian cells. In this study, we first purified the NEU1 from the isolated crystals produced by the HEK293 NEU1-KO cell transiently overexpressing the normal NEU1 and found that the N-glycans were high-mannose or complex types carrying terminal sialic acids. The result suggests that a part of NEU1 crystals were formed or transported to the Golgi apparatus. Second, we compared the effects of single amino acid substitution at the N-sequons, including N186Q, N343Q, and N352Q, each one N-glycan reduction from one NEU1 molecule. We demonstrated that N186Q mutant protein with low enzyme activity and formed a few amounts of smaller crystals. The N343Q mutant exhibited half of the normal intracellular activity, but the numbers and sizes of crystals were almost the same as those of normal NEU1. The N352Q mutant exhibited almost the same activity as the normal enzyme. The numbers of the N352Q crystals were smaller than those of normal NEU1. According to these findings, the N186Q NEU1 protein should have lower stability in ER due to abnormal folding. The second N-glycan at the N343-sequon has little effect on self-aggregation of NEU1. The third N-glycan at the N352-sequon contributes to the self-aggregation of NEU1. We also demonstrated that the three NEU1 mutants associate with the relatively excessive CTSA and migrate to lysosomes.

Local-Level Genetic Diversity and Structure of Matsutake Mushroom (Tricholoma matsutake) Populations in Nagano Prefecture, Japan, Revealed by 15 Microsatellite Markers.

The annual yield of matsutake mushrooms (Tricholoma matsutake) has consistently decreased in Japan over the past few decades. We used 15 polymorphic and codominant simple sequence repeat (SSR) markers, developed using next-generation sequencing, to carry out genetic analyses of 10 populations in Nagano, Japan. Using the SSRs, we identified 223 genotypes, none of which was observed in more than one population. The mean expected heterozygosity and standardized allelic richness values were 0.67 and 4.05, respectively. Many alleles appeared in only one of the 10 populations; 34 of these private alleles were detected with a mean number per population of 3.4. The fixation index (FST) and standardized genetic differentiation (G'ST) values were 0.019 and 0.028, respectively. Analysis of molecular variance (AMOVA) showed that the contribution of among population, among genets within a population, and within genets variation to the total variation was 2.91%, 11.62%, and 85.47%, respectively, with genetic differentiation being detected for all sources. Twenty-eight of 45 pairwise FST values were significantly larger than zero, and no pattern of isolation by distance was detected among the 10 populations. Bayesian-based clustering did not show clear differences among populations. These results suggest that reestablishment of a colony would be best accomplished by transplantation within a field; if this is not possible, then transplantation from within several dozen kilometers will cause little damage to the original population genetic structure.

Effect of dentures wearing on motor reaction time and balance function in elderly people.

OBJECTIVE: To investigate the influence of dentures wearing on the parameters of physical fitness, particularly on agility and balance function in elderly people. DESIGN: A case control study. SETTING: Motohachiohjimachi, Hachiohji, Tokyo, Japan. METHODS: Motor reaction time was measured in the presence and absence of dentures in the subjects who were 1) in a sitting position and lifted the lower limbs as fast as possible in response to a stimulus (Sitting Group) and those who were 2) in a standing position and jumped upright as fast as possible in response to a light stimulus (Jumping Group). The effects of dentures wearing on balance function were investigated by comparing the measured values of static and dynamic body sway. RESULTS AND CONCLUSIONS: Light-reaction time was not significantly influenced by dentures wearing in Sitting Group performing a light body movement that required little muscular force. In a relatively heavy body movement that required agility (i.e., jumping from the standing position), the reactivity changed depending on the muscular force; which might result in the difference of the reactivity due to dentures wearing (i.e., t-test showed a significant difference in the light-reaction time under clenching posture between with and without wearing dentures (p < 0.01)). No significant difference was observed in body sway under clenching posture between with and without wearing dentures.Therefore, we assumed that reaction speed varied depending upon dentures wearing.