Adenosine triphosphate induces amorphous aggregation of amyloid ß by increasing Aß dynamics.

Amyloid ß (Aß) aggregates into two distinct fibril and amorphous forms in the brains of patients with Alzheimer's disease. Adenosine triphosphate (ATP) is a biological hydrotrope that causes Aß to form amorphous aggregates and inhibit fibril formation at physiological concentrations. Based on diffracted X-ray blinking (DXB) analysis, the dynamics of Aß significantly increased immediately after ATP was added compared to those in the absence and presence of ADP and AMP, and the effect diminished after 30 min as the aggregates formed. In the presence of ATP, the ß-sheet content of Aß gradually increased from the beginning, and in the absence of ATP, the content increased rapidly after 180 min incubation, as revealed by a time-dependent thioflavin T fluorescence assay. Images of an atomic force microscope revealed that ATP induces the formation of amorphous aggregates with an average diameter of less than 100 nm, preventing fibrillar formation during 4 days of incubation at 37 °C. ATP may induce amorphous aggregation by increasing the dynamics of Aß, and as a result, the other aggregation pathway is omitted. Our results also suggest that DXB analysis is a useful method to evaluate the inhibitory effect of fibrillar formation.

Genetic diversity and declining norovirus prevalence in infants and children during Japan's COVID-19 pandemic: a three-year molecular surveillance.

Noroviruses (NoVs) are a global concern, causing widespread outbreaks and sporadic acute gastroenteritis (AGE) cases across all age groups. Recent research has shed light on the emergence of novel recombinant strains of NoV in various countries. To delve deeper into this phenomenon, we extensively analyzed 1,175 stool samples collected from Japanese infants and children with AGE from six different prefectures in Japan over three years, from July 2018 to June 2021. Our investigation aimed to determine the prevalence and genetic characteristics of NoV associated with sporadic AGE while exploring the possibility of detecting NoV recombination events. Among the analyzed samples, we identified 355 cases positive for NoV, 11 cases attributed to GI genotypes, and 344 associated with GII genotypes. Notably, we discovered four distinct GI genotypes (GI.2, GI.3, GI.4, and GI.6) and seven diverse GII genotypes (GII.2, GII.3, GII.4, GII.6, GII.7, GII.14, and GII.17). The predominant genotypes were GII.4 (56.4%; 194 out of 344), followed by GII.2 and GII.3. Through dual genotyping based on sequencing of the ORF1/ORF2 junction region, we identified a total of 14 different RdRp/capsid genotypes. Of particular interest were the prevalent recombinant genotypes GII.4[P31] and GII.2[P16]. Notably, our study revealed a decrease in the number of children infected with NoV during and after the COVID-19 pandemic. These findings underscore the importance of continuous NoV surveillance efforts.

Conformational changes in the catalytic region are responsible for heat-induced activation of hyperthermophilic homoserine dehydrogenase.

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.

2-[Bis(carboxymethyl)amino] propanoic acid-Chelated Copper Chelate Enhances Bacterial Elimination by Sodium Percarbonate.

Sodium percarbonate (SP) is a relatively low-cost and stable solid oxidizer with a small environmental burden. It is often included in cleansers for sanitizing circulating water pipes and as bleaching reagents in laundry, although the bactericidal effect of SP is lower than that of chlorine-based agents. 2-[Bis(carboxymethyl)amino] propanoic acid-chelated copper (MGDA-Cu) was added to increase the effect of SP. The addition of 12 µM MGDA-Cu increased the bactericidal effect of 0.5 wt% SP against Staphylococcus aureus even in the presence of 0.3 wt% BSA, which is an experimental model of organic stain to protect bacteria from SP. MGDA-Cu was effective against Escherichia coli only in the absence of BSA and showed little effect against Bacillus subtilis. It enhanced the effect of SP to decrease the viscosity of sodium alginate, which is one of the major components of biofilms. The effect of MGDA-Cu on sanitization was also evaluated by 16S rRNA amplicon sequencing of the bacterial flora of the biofilm on an experimental model of a circulating water pipe. The structure of the bacterial flora was more influenced by a cleanser containing both MGDA-Cu and SP than a cleanser with only SP, suggesting that MGDA-Cu increases the sanitization effect.

Formation of Proton Motive Force Under Low-Aeration Alkaline Conditions in Alkaliphilic Bacteria.

In Mitchell's chemiosmotic theory, a proton (H+) motive force across the membrane (Δp), generated by the respiratory chain, drives F1Fo-ATPase for ATP production in various organisms. The bulk-base chemiosmotic theory cannot account for ATP production in alkaliphilic bacteria. However, alkaliphiles thrive in environments with a H+ concentrations that are one-thousandth (ca. pH 10) the concentration required by neutralophiles. This situation is similar to the production of electricity by hydroelectric turbines under conditions of very limited water. Alkaliphiles manage their metabolism via various strategies involving the cell wall structure, solute transport systems and molecular mechanisms on the outer surface membrane. Our experimental results indicate that efficient ATP production in alkaliphilic Bacillus spp. is attributable to a high membrane electrical potential (ΔΨ) generated for an attractive force for H+ on the outer surface membrane. In addition, the enhanced F1Fo-ATPase driving force per H+ is derived from the high ΔΨ. However, it is difficult to explain the reasons for high ΔΨ formation based on the respiratory rate. The Donnan effect (which is observed when charged particles that are unable to pass through a semipermeable membrane create an uneven electrical charge) likely contributes to the formation of the high ΔΨ because the intracellular negative ion capacities of alkaliphiles are much higher than those of neutralophiles. There are several variations in the adaptation to alkaline environments by bacteria. However, it could be difficult to utilize high ΔΨ in the low aeration condition due to the low activity of respiration. To explain the efficient ATP production occurring in H+-less and air-limited environments in alkaliphilic bacteria, we propose a cytochrome c-associated "H+ capacitor mechanism" as an alkaline adaptation strategy. As an outer surface protein, cytochrome c-550 from Bacillus clarkii possesses an extra Asn-rich segment between the region anchored to the membrane and the main body of the cytochrome c. This structure may contribute to the formation of the proton-binding network to transfer H+ at the outer surface membrane in obligate alkaliphiles. The H+ capacitor mechanism is further enhanced under low-aeration conditions in both alkaliphilic Bacillus spp. and the Gram-negative alkaliphile Pseudomonas alcaliphila.

Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex.

Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 µM and uncompetitive an inhibitor of NAD and NADP with Ki's of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD first binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption-difference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring.

Assay of hemoglobin A1c using lectin from Aleuria aurantia.

Hemoglobin A1c (HbA1c) has an N-terminal fructosyl valine on the ß-chain, and this modification is caused by the non-enzymatic glycosylation of hemoglobin (Hb). The relative concentration ratio of HbA1c to total Hb is an important biomarker for the diagnosis of diabetes. HbA1c-binding lectins were screened from 29 sources of lectin, and the lectin from Aleuria aurantia (AAL) was revealed to have higher affinity to HbA1c than to Hb. The concentration of HbA1c was determined by lectin-based enzyme-linked immunosorbent assay (ELISA) using the AAL lectin. Higher reproducibility of the assay was observed at 4 °C than at 25 and 37 °C. This observation is consistent with the known temperature-dependent behavior of lectins. Preincubation of HbA1c with an anti-HbA1c antibody inhibited the binding, suggesting that AAL binds to the N-terminal fructosyl valine epitope of HbA1c. Higher inhibitory effect was observed for 10 mM D-fructose than for the same concentrations of L-fucose, D-fucose, or D-glucose.

Immunohistochemical detection of the delayed formation of nonfibrillar large amyloid-ß aggregates.

The occurrence of senile plaques consisting of amyloid-ß protein (Aß) is a major neuropathological hallmark of Alzheimer's disease (AD). We previously developed and characterized monoclonal antibodies 31-2 and 75-2 that specifically bind to nonfibrillar Aß1-42 aggregates with diameters of more than 220 and 50 nm, respectively. Here, we report the use of these antibodies to examine the aggregation of exogenous Aß1-42 in cultured rat hippocampal neurons. From 6 to 24 h after transfection of Aß1-42, antibody 75-2 immunolabeled almost all transfected neurons, whereas 31-2-positive cells were restricted to a part of the transfected neurons and gradually increased in number. Expression of the F19S/L34P-mutant Aß1-42, which showed less of a tendency to aggregate, resulted in clearly reduced immunoreactivity to both antibodies. We also immunohistochemically investigated the temporal cortices of patients with AD and found that 31-2 preferentially labeled the cores of a subpopulation of large amyloid plaques. The relative number of 31-2-immunoreactive plaques was found to correlate with the Braak stages of neurofibrillary tangles, but not with that of amyloid plaques. These results suggest that 31-2-reactive Aß aggregates develop with a delayed time course in cultured neurons and amyloid plaques of AD brains.

Enzyme assay for pyridoxal 5'-phosphate by apo-D-amino acid aminotransferase.

D-amino acid aminotransferase (D-AAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme. D-AAT from thermophilic Bacillus sp. YM-1 was inactivated by ultraviolet irradiation. The activity was restored by the addition of PLP and showed a linear correlation with respect to PLP concentrations in the range of 10-40 nM.

Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction.

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.

The combination of functional metagenomics and an oil-fed enrichment strategy revealed the phylogenetic diversity of lipolytic bacteria overlooked by the cultivation-based method.

Metagenomic screening and conventional cultivation have been used to exploit microbial lipolytic enzymes in nature. We used an indigenous forest soil (NS) and oil-fed enriched soil (OS) as microbial and genetic resources. Thirty-four strains (17 each) of lipolytic bacteria were isolated from the NS and OS microcosms. These isolates were classified into the (sub)phyla Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria, all of which are known to be the main microbial resources of commercially available lipolytic enzymes. Seven and 39 lipolytic enzymes were successfully retrieved from the metagenomic libraries of the NS and OS microcosms, respectively. The screening efficiency (a ratio of positive lipolytic clones to the total number of environmental clones) was markedly higher in the OS microcosm than in the NS microcosm. Moreover, metagenomic clones encoding the lipolytic enzymes associated with Alphaproteobacteria, Deltaproteobacteria, Acidobacteria, Armatimonadetes, and Planctomycetes and hitherto-uncultivated microbes were recovered from these libraries. The results of the present study indicate that functional metagenomics can be effectively used to capture as yet undiscovered lipolytic enzymes that have eluded the cultivation-based method, and these combined approaches may be able to provide an overview of lipolytic organisms potentially present in nature.

Monoclonal antibodies against large oval aggregates of Aß1-42.

Abnormal cerebral accumulation of amyloid beta protein(1-42) (Aß(1-42)) is one of the hallmarks of Alzheimer's disease (AD). Aß(1-42) aggregates exist in two distinct forms: fibrils that are composed of highly ordered ß-sheets and amorphous aggregates that differ in size and toxicity. Here, we generated large oval aggregates (LOA) 369 ± 81 nm and 224 ± 92 nm in size on their major and minor axes, respectively, as measured by tapping-mode atomic force microscopy. LOA were produced by slow rotation of high concentrations (0.22 mM, 1.0 mg/mL) of Aß(1-42) for 16 h at 37°C in the presence of 2.2 mM Aß(16-20), which prevents the fibril formation, and purified with 0.22-µm filters. Analysis with thioflavin T showed that LOA have little ß-sheet structure on their surfaces. Monoclonal antibodies that react with LOA, but not the fibril forms, were screened from 960 mouse hybridoma cell lines, and seven antibodies consisting of four IgG and three IgM antibodies were obtained. Four IgG monoclonal antibodies showed cross-reactivity of <10% against the monomer and fibril forms and amorphous aggregates that passed through 0.22-µm filters. Among the four antibodies, the antibody that was designated as 31-2 exhibited the highest reactivity against LOA and showed the lowest reactivity against the fibril forms. On the basis of these results, a unique epitope on the surface of LOA was suggested. The 31-2 antibody may be useful for future basic research and therapeutic applications for AD.

Use of a highly sensitive latex reagent with amino acid spacer for determination of C-reactive protein concentration in a variety of liver diseases.

C-reactive protein (CRP) is a major acute-phase protein, which is extremely important in inflammatory disease diagnosis. CRP is rapidly elevated in various diseases as a result of tissue injury, infection and inflammation. Recently, many reports have shown its usefulness as a risk marker for arteriosclerosis and metabolic syndrome. However, the lack of sensitivity of existing CRP assays has hampered CRP testing in conditions associated with viral infections, where CRP levels typically elevate only marginally. In this report, we prepared a novel, ultra-sensitive latex-based CRP test using amino acid spacers with a high sensitivity and a wider assay range. Our method of conjugating latex beads enabled us to measure CRP in the range of 5-500 ng/mL in patient sera. Furthermore, we studied CRP levels in patients with various liver diseases, such as chronic hepatitis, liver cirrhosis and hepatic carcinoma, in order to examine the correlation between severity of liver dysfunction and CRP levels, and to examine the likelihood of recurrence of liver dysfunction. The reagent was simple to prepare and sensitive during clinical investigation, where it discriminated clearly between normal subjects and those with liver diseases. Therefore, we conclude that our ultra-sensitive CRP assay will contribute greatly to the clinical study of hepatic disorders.

Relationship between rates of respiratory proton extrusion and ATP synthesis in obligately alkaliphilic Bacillus clarkii DSM 8720(T).

To elucidate the energy production mechanism of alkaliphiles, the relationship between the rate of proton extrusion via the respiratory chain and the corresponding ATP synthesis rate was examined in obligately alkaliphilic Bacillus clarkii DSM 8720(T) and neutralophilic Bacillus subtilis IAM 1026. The oxygen consumption rate of B. subtilis IAM 1026 cells at pH 7 was approximately 2.5 times higher than that of B. clarkii DSM 8720(T) cells at pH 10. The H⁺/O ratio of B. clarkii DSM 8720(T) cells was approximately 1.8 times higher than that of B. subtilis IAM 1026 cells. On the basis of oxygen consumption rate and H⁺/O ratio, the rate of proton translocation via the respiratory chain in B. subtilis IAM 1026 is expected to be approximately 1.4 times higher than that in B. clarkii DSM 8720(T). Conversely, the rate of ATP synthesis in B. clarkii DSM 8720(T) at pH 10 was approximately 7.5 times higher than that in B. subtilis IAM 1026 at pH 7. It can be predicted that the difference in rate of ATP synthesis is due to the effect of transmembrane electrical potential (Δψ) on protons translocated via the respiratory chain. The Δψ values of B. clarkii DSM 8720(T) and B. subtilis IAM 1026 were estimated as -192 mV (pH 10) and -122 mV (pH 7), respectively. It is considered that the discrepancy between the rates of proton translocation and ATP synthesis between the strains used in this study is due to the difference in ATP production efficiency per translocated proton between the two strains caused by the difference in Δψ.

Characterization of catalase from psychrotolerant Psychrobacter piscatorii T-3 exhibiting high catalase activity.

A psychrotolerant bacterium, strain T-3 (identified as Psychrobacter piscatorii), that exhibited an extraordinarily high catalase activity was isolated from the drain pool of a plant that uses H(2)O(2) as a bleaching agent. Its cell extract exhibited a catalase activity (19,700 U·mg protein(-1)) that was higher than that of Micrococcus luteus used for industrial catalase production. Catalase was approximately 10% of the total proteins in the cell extract of the strain. The catalase (PktA) was purified homogeneously by only two purification steps, anion exchange and hydrophobic chromatographies. The purified catalase exhibited higher catalytic efficiency and higher sensitivity of activity at high temperatures than M. luteus catalase. The deduced amino acid sequence showed the highest homology with catalase of Psycrobacter cryohalolentis, a psychrotolelant bacterium obtained from Siberian permafrost. These findings suggest that the characteristics of the PktA molecule reflected the taxonomic relationship of the isolate as well as the environmental conditions (low temperatures and high concentrations of H(2)O(2)) under which the bacterium survives. Strain T-3 efficiently produces a catalase (PktA) at a higher rate than Exiguobacterium oxidotolerans, which produces a very strong activity of catalase (EktA) at a moderate rate, in order to adapt to high concentration of H(2)O(2).

Efficient hydrogen production from acetate through isolated Rhodobacter sphaeroides.

Photosynthetic bacteria produce hydrogen from lactate and acetate that are products of hydrogen producing bacteria in the dark. Thus, their coculture is a promising method for hydrogen production. However, the hydrogen production yield from acetate of Rhodobacter sphaeroides RV, which has been shown to possess the highest yield and hydrogen production rate, is low as compared to that from lactate. Photosynthetic bacteria that produce hydrogen from acetate as well as lactate were screened from lakes and swamps in the Tokyo and Chiba areas in Japan. Seventy-six strains of photosynthetic bacteria were obtained and the analysis of their 16S rRNA gene sequences revealed that they belong to R. sphaeroides. Among the isolated bacteria, R. sphaeroides HJ produced the highest amount of hydrogen from acetate and lactate. The HJ strain produced a 2300±93ml/L-broth of hydrogen from 75mM acetate consumed during for 120h of fermentation. The amount of hydrogen and the yield from acetate were 1.9 and 2.1 times higher, respectively, than those of R. sphaeroides RV. The amount and yield of hydrogen, produced by R. sphaeroides HJ from lactate were similar to those produced by R. sphaeroides RV. Since the amount and yield of produced hydrogen by the HJ strain were similar regardless of the substrate (acetate or lactate), its metabolic pathway could have a key to increasing hydrogen production from acetate.

Physiological function of soluble cytochrome c-552 from alkaliphilic Pseudomonas alcaliphila AL15-21(T).

It has been found that the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) produces a larger amount of soluble c-type cytochromes at pH 10.0 under air-limited condition than at pH 7.0 under high aeration. Cytochrome c-552 was confirmed as the major c-type cytochrome among three soluble c-type cytochromes in the strain. To understand the physiological function of cytochrome c-552, a P. alcaliphila AL15-21(T) cytochrome c-552 gene deletion mutant without a marker gene was constructed by electrotransformation adjusted in this study for the strain. The maximum specific growth rate and maximum cell turbidity of cells grown at pHs 7.0 and 10.0 under the high-aeration condition did not differ significantly between the wild-type and cytochrome c-552 deletion mutant strains. In the mutant grown at pH 10.0 under low-aeration condition, marked decreases in the maximum specific growth rate (40%) and maximum cell turbidity (25%) compared with the wild type were observed. On the other hand, the oxygen consumption rates of cell suspensions of the mutant obtained by the growth at pH 10 under low-aeration condition were slightly higher than that of the wild type. Considering the high electron-retaining ability of cytochrome c-552, the above observations could be accounted for by cytochrome c-552 acting as an electron sink in the periplasmic space. This may facilitate terminal oxidation in the respiratory system at high pH under air-limited conditions.

Bacterial community characterization and dynamics of indigo fermentation.

Indigo fermentation has been traditionally performed for dyeing textiles in Japan. Limited information is available on the microbiota involved and the succession of the bacterial community structure during indigo reduction. We investigated the bacterial community structure associated with indigo fermentation using denaturing gradient gel electrophoresis and clone library analyses of a PCR-amplified 16S rRNA gene in the early phase of fermentation carried out in our laboratory. A marked substitution of Halomonas spp. by Amphibacillus spp. was observed corresponding to the marked change in the state of indigo reduction. Although the reported indigo-reducing bacteria, Alkalibacterium spp., were not predominant in the early phase of fermentation, they were predominant in fermentation liquor aged for 10 months obtained from Date City, Japan, as determined by culture-dependent and culture-independent analyses. Novel indigo-reducing strains, Amphibacillus spp. strain C40 and Oceanobacillus spp. strain A21, were isolated from fermentation liquor aged for 10 months and from liquor aged for 4 days, respectively. It is considered that, in addition to the strains belonging to the genus Alkalibacterium, strains belonging to genera Amphibacillus and Oceanobacillus play important roles in sustaining the reduced state of indigo during fermentation.

The obligate alkaliphile Bacillus clarkii K24-1U retains extruded protons at the beginning of respiration.

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (Delta pH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Delta psi), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the Delta pH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na(+) channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Delta psi-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient Delta pH under alkaline conditions.