Visualization of polypeptides including fragmented α-amylase in rice koji grains using mass spectrometry imaging.

The quality of rice koji greatly affects the quality of sake. To accurately evaluate the quality of rice koji, various approaches for the evaluation of rice koji are required. In this study, we directly and simultaneously visualized the distribution of polypeptides in rice koji using mass spectrometry imaging. We demonstrated four koji-specific polypeptides at m/z 4660, 6140, 8170, and 11,840 and one rice-derived polypeptide at m/z 5330. To identify the koji-specific polypeptides, extracts from rice koji were separated using tricine SDS-PAGE, and the band appeared to coincide with the polypeptide at m/z 11,840 was identified to be the N-terminal fragment of α-amylase. The polypeptide seemed to have no hydrolytic activity based on the primary structure of α-amylase. The polypeptide at m/z 11,840 seemed to coincide with the fragmented α-amylase was detected at the later stage of koji making (after 42 h). At the same period during koji making, the increasing rate of α-amylase activity decreased compared to that of glucoamylase activity, suggesting that α-amylase fragmentation possibly leads to the deceleration of the increase in α-amylase activity at the later stage of koji making. This is the first study to directly and simultaneously demonstrate the distribution of polypeptides in rice koji using mass spectrometry imaging and imply the relationship between α-amylase fragmentation and activity in rice koji.

Relationship between the occurrence of falls by season and physical functions of community-dwelling old-old people living in cold, snowy areas.

AIM: To investigate the functional characteristics of older adults who experienced a fall in the winter season and other seasons. METHODS: Participants were 403 older adults enrolled in the project "Population-Based and Inspiring Potential Activity for Old-old Inhabitants," and were living in cold, snowy regions in Japan. They were aged ≥75 years, and 41.9% (n = 169) were men. Sociodemographic characteristics, and physical, psycho-cognitive and social factors were surveyed. By experiences of falls, they were divided into three groups: the non-fall group, the fall in non-winter group and the fall in winter group. Each factor was compared with a χ2 -test, Student's t-test and Mann-Whitney U-test. Logistic regression analysis was carried out. spss version 25 was used for the statistical analysis. The level of significance was set at 5%. RESULTS: No differences were confirmed between the non-fall and fall in winter groups. In contrast, the maximum walking speed in the fall in non-winter group was significantly slower than the non-fall group, even with adjustment by variables, such as age, sex and self-efficacy. CONCLUSIONS: When considering intervention methods for health promotion, it is necessary to consider not only the presence or absence of falls, but also the seasons of falls. Geriatr Gerontol Int 2019; 19: 124-129.

Structural analysis of impact of physical, cognitive and social status on the incidence of disability in community-dwelling people aged ≥75 years.

AIM: The present study aimed to propose a structural model to explain the interaction of physical, cognitive and social domains of health status in the incidence of disability in community-dwelling people aged ≥75 years. METHODS: We analyzed 185 older adults (mean age 79.4 years, 58.4% female) who participated in a baseline assessment from 2012 to 2013. They were followed for incident certification of care needs in the national long-term care insurance certification system during the 2 years. Baseline assessments included several measurements related to the physical, cognitive and social domains of health status. We compared the model fit index between two hypothesis models - the parallel model and the hierarchical model - using structural equation modeling. RESULTS: During the follow-up period, 15 participants (8.1%) were newly certified as requiring personal support from the long-term care insurance system. The structural equation modeling showed that the hierarchical model, indicating that cognitive and social status were indirectly associated with disability through physical status, had a better fit with the data than the parallel model, indicating that physical, cognitive and social status each were directly associated with disability. CONCLUSIONS: The present results suggest that cognitive and social status might indirectly affect disability incidence through physical aging. Further research is required to examine the temporal relationship between physical, cognitive and social change using data over several time-periods. Geriatr Gerontol Int 2018; 18: 1614-1619.

Participation of cysteine 30 residue in the folding process of ovalbumin evaluated in a refolding experiment using cysteine mutants.

To understand the role of cysteine (SH) residues in the folding of hen ovalbumin (OVA), SH-mutated OVAs, in which each SH residue was replaced by alanine (C11A, C30A, C367A, and C382A), were prepared. SDS-PAGE analysis under non-reducing conditions showed that the C11A and C30A mutants produced a disulfide (SS) isomer in addition to a protein with a native SS bond (Cys73-Cys120). The susceptibility to elastase digestion suggested that the Cys73 residue in the SS isomer participates as a counterpart of the SS bond. Upon refolding of the SH-mutated OVAs under the denatured and SS-reduced states, only C30A failed to refold into an intact form. This indicated that the Cys30 residue plays an important role in correct refolding. To confirm this, each of the four SH-mutated OVAs, in which the original SS-forming sites (C11/73/120A, C30/73/120A, C73/120/367A, and C73/120/382A) were deleted, was constructed and expressed. The C11/73/120A and C30/73/120A mutants formed no SS form, in contrast to C73/120A as a control. Thus, we concluded that Cys30 participates in the correct folding of OVA, and that its SS bond (Cys11-Cys30) is transiently generated during the early folding stage to avoid misfolding, and then the native SS form of OVA is regenerated through SH-SS exchanges.

Quantitative evaluation of haze formation of koji and progression of internal haze by drying of koji during koji making.

The construction of an experimental system that can mimic koji making in the manufacturing setting of a sake brewery is initially required for the quantitative evaluation of mycelia grown on/in koji pellets (haze formation). Koji making with rice was investigated with a solid-state fermentation (SSF) system using a non-airflow box (NAB), which produced uniform conditions in the culture substrate with high reproducibility and allowed for the control of favorable conditions in the substrate during culture. The SSF system using NAB accurately reproduced koji making in a manufacturing setting. To evaluate haze formation during koji making, surfaces and cross sections of koji pellets obtained from koji making tests were observed using a digital microscope. Image analysis was used to distinguish between haze and non-haze sections of koji pellets, enabling the evaluation of haze formation in a batch by measuring the haze rate of a specific number of koji pellets. This method allowed us to obtain continuous and quantitative data on the time course of haze formation. Moreover, drying koji during the late stage of koji making was revealed to cause further penetration of mycelia into koji pellets (internal haze). The koji making test with the SSF system using NAB and quantitative evaluation of haze formation in a batch by image analysis is a useful method for understanding the relations between haze formation and koji making conditions.

Change in enzyme production by gradually drying culture substrate during solid-state fermentation.

The influence of drying the culture substrate during solid-state fermentation on enzyme production was investigated using a non-airflow box. The drying caused a significant increase in enzyme production, while the mycelium content decreased slightly. This suggests that changes in the water content in the substrate during culture affect enzyme production in fungi.

Rapid enzyme production and mycelial growth in solid-state fermentation using the non-airflow box.

Solid-state fermentation (SSF) has become an attractive alternative to submerged fermentation (SMF) for the production of enzymes, organic acids, and secondary metabolites, while there are many problems during the culture of SSF. We recently created a SSF system using a non-airflow box (NAB) in order to resolve the problems, which enabled the uniform culture in the whole substrate and high yield of many enzymes. In this paper, further characterization of SSF using the NAB was carried out to obtain other advantages. The NAB culture under the fixed environmental condition exhibited a rapid increase in enzyme production at earlier phase during the culture compared with conventional SSF. Total mycelial growth also exhibited the same trend as enzyme production. Thus, the increase in the rate of the enzyme production was thought to mainly be attributed to that of the growth. To support it, it was suggested that the NAB culture resulted in most optimal water activity for the growth just at the log phase. In addition, the NAB culture was able to achieve high reproducibility of enzyme production, derived from uniform condition of the substrate during the culture. The results indicate that the NAB culture has many benefits for SSF.

The role of the disulfide bridge in the stability and structural integrity of ovalbumin evaluated by site-directed mutagenesis.

To provide a molecular explanation of the role of the disulfide (SS) bridge in the thermostability and structural integrity of ovalbumin (OVA), we prepared SS-mutated OVAs in which SS-forming residues were replaced by Ala or Ser (C73A, C73S, C120A, and C73/120A), and compared the conformation, thermostability, susceptibility to elastase, and formation of heat-stable OVA (S-OVA) with those of the wild-type. The circular dichroism (CD) and tryptophan fluorescence spectra revealed that the SS-mutated OVAs assumed a native-like conformation similar to the wild-type. The thermal denaturation temperature for the SS-mutated OVAs was significantly lower than that for the wild-type. C73S, C120A, and C73/120A mutants converted to S-OVA on alkaline treatment. Analyses for elastase digestion fragments showed that a non-native SS bridge was generated in all SS-mutated OVAs, but non-native SS-pairing did not contribute to thermostability. Hence, we concluded that the presence of the original SS bridge in OVA contributes to conformational stability but is not directly related to the conversion to S-OVA.

Uniform culture in solid-state fermentation with fungi and its efficient enzyme production.

Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.

Thermostabilization of ovalbumin by alkaline treatment: Examination of the possible roles of D-serine residues.

It was revealed from the crystal structure analysis of S-ovalbumin (S-OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the D-amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524-35530). To address the implications of a D-configuration for these Ser residues in S-OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline treatment. Following alkaline treatment, S236A showed a marked increase in melting temperature similar to the wild type (DeltaT(m), +9 degrees C) which corresponded to the formation of S-OVA, whereas the increment in T(m) for both S164A and S320A was only 4.5 degrees C. Furthermore, the T(m) value of the double mutant S164/320A remained unchanged after alkaline treatment, supporting the relevance of Ser164 and Ser320 for thermostabilization of OVA. As Arg142 was predicted to interact with D-Ser164 upon S-OVA formation, it was substituted to Ala to generate R142A. The resulting increment in T(m) of mutant R142A after alkaline treatment was 5.8 degrees C. The double mutant R142/S320A was therefore prepared to eliminate the participation of Ser320 in thermostabilization, and its T(m) value was compared before and after alkaline treatment. As expected, the increase in T(m) for the double mutant was only 1.2 degrees C. Taken together, the data suggest that D-configuration of Ser164 caused by alkaline treatment favors interaction with Arg142 through conformational changes of the side chain. These results strongly supported the participation of the configurational inversion of both Ser164 and Ser320 residues in the formation of S-OVA.

Identification of ZAG1, a novel protein expressed in mouse preimplantation, and its putative roles in zygotic genome activation.

We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.

Importance of N-glycosylation positioning for secretion and folding of ovalbumin.

To investigate the role of the carbohydrate chain of hen egg ovalbumin (OVA), potential N-glycosylation site-deletion OVA mutants were expressed in yeast. The secretion level of the N292Q and N292/311Q mutants was greatly reduced compared with the wild-type OVA. Furthermore, secretion of the mutants without a carbohydrate chain on Asn-292 could hardly be detected in the culture medium, even if an additional N-glycosylation site was introduced to the OVA molecule. The reduction in secretion level seems to be due to incorrectly folded protein. Moreover, the secretion levels of the wild-type and N311Q mutant reduced in a similar extent as those of the mutants without a carbohydrate chain on Asn-292 in calnexin-disrupted yeast. These results indicate that the carbohydrate chain attached to Asn-292 of OVA has an important role for the secretion and folding in the cells.

Site-specific glycosylation at Asn-292 in ovalbumin is essential to efficient secretion in yeast.

Chicken ovalbumin (OVA) exists as mono-N-glycosylated form with a carbohydrate chain on Asn-292 in egg white, despite the possession of two potential N-glycosylation sites. To investigate the roles of N-glycosylation of OVA, we constructed a series of N-glycosylation mutants deleted N-glycosylation site and compared the secretion level of the mutants in Pichia pastoris. N292Q and N292/311Q mutants resulted in greater lowering of the secretion level as compared with wild-type, whereas N311Q mutant was secreted in approximately equal amounts to wild-type. However, secretion of wild-type and N311Q mutant was inhibited completely by tunicamycin treatment. All the N-glycosylation mutants have been expressed in the cells, as well as wild-type. Circular dichroism and fluorescence spectra of secreted N311Q mutant were almost identical to those of wild-type, while those of N292Q and N292/311Q mutants were different from wild-type; and, N292Q and N292/311Q mutants showed considerably lower denaturation temperature than wild-type. The results indicate that N-glycosylation at Asn-292 of OVA is required for the folding and secretion.

Preventive effect of egg yolk phosvitin on heat-insolubilization of egg white protein and its application to heat-induced egg white gel.

The effects of phosvitin (PV) on insolubilization of egg white protein (EWP) and ovotransferrin (OT) were examined by measuring turbidity after heating at 80 degrees C in a pH range of 5 to 8. PV showed preventive ability against heat-insolubilization of EWP, especially heat-labile OT. The preventive ability of PV was reduced by adding NaCl to a PV-OT mixture on heating. Native PAGE and gel filtration analyses showed that PV prevented an insolubilization of heat-denatured OT through ionic interactions. The preventive effects of PV on insolubilization of EWP and OT resulted in the formation of a firm, transparent gel from EWP in coexistence with PV on heating. The addition of PV might make possible the preparation of liquid egg white without insoluble products even on heat-treatment at high temperatures.

Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris.

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 degrees C as compared with that of hen egg OVA (77.3 degrees C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.