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.

Follow-up Results of HCV GT2 Patients After Sofosbuvir/Ribavirin Therapy: Careful Attention to Occurrence of HCC.

BACKGROUND: We examined treatment the efficacy and data on long-term outcomes in real-world Japanese patients infected with hepatitis C virus (HCV) genotype 2 treated with 12-week sofosbuvir/ribavirin combination therapy. PATIENTS AND METHODS: In a total of 86 patients who were treated with sofosbuvir/ribavirin, sustained virological response (SVR) rates and long-term-outcomes were retrospectively analyzed. RESULTS: The adherence to this combination therapy was 98.8%. The rates of SVR at week 24 (SVR24) achieved with this treatment according to the 'intention-to-treat' and 'per-protocol' analyses were 89.5% and 96.2%, respectively. Two patients who experienced relapse did not have any previously reported resistance-associated substitutions in the HCV non-structural protein 5B (NS5B) polymerase region. We did not observe any patients who experienced late relapse but did observe that 50% and 1.3% of patients with and without a previous history of hepatocellular carcinoma (HCC), respectively, developed HCC after achieving SVR24 (with a mean follow-up period of 2.7±0.8 years). CONCLUSION: Patients with SVR should be carefully followed-up to screen for the occurrence of HCC, although it is infrequent.

Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children.

We studied stool specimens from 33 autistic children aged 2-9 years with gastrointestinal (GI) abnormalities and 13 control children without autism and without GI symptoms. We performed quantitative comparison of all Clostridium species and Clostridium perfringens strains from the fecal microbiota by conventional, selective anaerobic culture methods. We isolated C. perfringens strains and performed PCR analysis for the main C. perfringens toxin genes, alpha, beta, beta2, epsilon, iota and C. perfringens enterotoxin gene. Our results indicate that autistic subjects with gastrointestinal disease harbor statistically significantly (p = 0.031) higher counts of C. perfringens in their gut compared to control children. Autistic subjects also harbor statistically significantly (p = 0.015) higher counts of beta2-toxin gene-producing C. perfringens in their gut compared to control children, and the incidence of beta2-toxin gene-producing C. perfringens is significantly higher in autistic subjects compared to control children (p = 0.014). Alpha toxin gene was detected in all C. perfringens strains studied. C. perfringens enterotoxin gene was detected from three autistic and one control subject. Beta, epsilon, and iota toxin genes were not detected from autistic or control subjects.

In vitro study of the prebiotic xylooligosaccharide (XOS) on the growth of Bifidobacterium spp and Lactobacillus spp.

We recently demonstrated that XOS increased the counts of Bifidobacterium in vivo without increasing Lactobacillus in healthy adults. In the current study, we evaluated the effect of XOS on the growth of 35 Bifidobacterium and 29 Lactobacillus strains in in vitro conditions. Bacteria were identified by 16S rRNA sequence analysis. The growth stimulation was determined by agar dilution technique on plates containing two-fold serial dilutions of XOS (100-0.1 mg/ml). The growth of 86% of Bifidobacterium strains was stimulated at 1.56 mg/ml XOS and 100% at 6.25 mg/ml XOS. The growth of 38% of Lactobacillus strains was stimulated at 1.56 mg/ml XOS and 62% at 6.25 mg/ml XOS; 31% of Lactobacillus were not stimulated by XOS. Our results further suggest that XOS may be beneficial in stimulating intestinal Bifidobacterium without having much effect on Lactobacillus. The potential role for XOS in managing obesity should be investigated further.

Pomegranate ellagitannins stimulate growth of gut bacteria in vitro: Implications for prebiotic and metabolic effects.

The present study investigated the effect of pomegranate extract (POMx) and pomegranate juice (POM juice) on the growth of major groups of intestinal bacteria: Enterobacteriaceae, Bacteroides fragilis group, clostridia, bifidobacteria, and lactobacilli, and the utilization of pomegranate polyphenols by Bifidobacterium and Lactobacillus. The total phenolic content of the pomegranate extract and juice was determined using the Folin-Ciocalteau colorimetric method and reported as gallic acid equivalent (GAE). The polyphenol composition was determined by HPLC. Stool specimens were incubated with 400, 100, and 25 µg/ml GAE POMx and POM juice and subjected to selective culture. Bifidobacterium and Lactobacillus strains were incubated with 400 µg/ml GAE POMx and POM juice and metabolites were analyzed. POMx and POM juice increased the mean counts of Bifidobacterium and Lactobacillus and significantly inhibited the growth of B. fragilis group, clostridia, and Enterobacteriaceae in a dose-response manner. Bifidobacterium and Lactobacillus utilized ellagic acid and glycosyl ellagic acid but little or no punicalin was utilized. Neither POMx nor POM juice was converted to urolithins by the test bacteria or the in vitro stool cultures. The effect of pomegranate on the gut bacteria considered to be beneficial (Bifidobacterium and Lactobacillus) suggests that pomegranate may potentially work as a prebiotic. The concept that polyphenols such as those in pomegranate impact gut microbiota populations may establish a new role for polyphenols in human health.

Antimicrobial Activity of Pomegranate and Green Tea Extract on Propionibacterium Acnes, Propionibacterium Granulosum, Staphylococcus Aureus and Staphylococcus Epidermidis.

We used pomegranate extract (POMx), pomegranate juice (POM juice) and green tea extract (GT) to establish in vitro activities against bacteria implicated in the pathogenesis of acne. Minimum inhibitory concentrations (MIC) of 94 Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus aureus, and Staphylococcus epidermidis strains were determined by Clinical and Laboratory Standards Institute-approved agar dilution technique. Total phenolics content of the phytochemicals was determined using the Folin-Ciocalteu method and the polyphenol composition by HPLC. Bacteria were identified by 16S rRNA sequence analysis. GT MIC of 400 µg/ml or less was obtained for 98% of the strains tested. 64% of P. acnes strains had POMx MICs at 50 µg/ml whereas 36% had MIC >400 µg/ml. POMx, POM juice, and GT showed inhibitory activity against all the P. granulosum strains at ≤100 µg/ml. POMx and GT inhibited all the S. aureus strains at 400 µg/ml or below, and POM juice had an MIC of 200 µg/ml against 17 S. aureus strains. POMx inhibited S. epidermidis strains at 25 µg/ml, whereas POM juice MICs were ≥200 µg/ml. The antibacterial properties of POMx and GT on the most common bacteria associated with the development and progression of acne suggest that these extracts may offer a better preventative/therapeutic regimen with fewer side effects than those currently available.

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.

Virucidal effects of bleach activators, sodium alkyl acyloxybenzene sulfonate and acyloxybenzoic acid, against Feline calicivirus.

Noroviruses (NVs) are major causative pathogens of gastroenteritis. The disinfection of contaminated clothing during common household washing is desirable. The virucidal effects of 2 bleach activators, sodium alkyl acyloxybenzene sulfonate (OBS) and alkyl acyloxybenzoic acid (OBC), were studied using Feline calicivirus (FCV) as a surrogate for NVs. FCV was added to solutions containing either OBS or OBC and sodium percarbonate at various temperatures and for varying lengths of time. OBS and OBC, which generate long carbon chain peroxy acids, enhanced the virucidal effect of sodium percarbonate (PC). In particular, sodium lauroyloxybenzene sulfonate (OBS-12) and decanoyloxybenzoic acid (OBC-10) showed superior virucidal effects. Although the virucidal effect of 38-200 mg/L OBS-12 was maintained with 2-5% (v/v) horse serum, there was less of an effect with the same concentration of available chlorine. OBS and OBC have been used as ingredients in some laundry products to increase bleaching activity. It is expected that the use of OBS and OBC is also effective for the inactivation of NVs under common household washing conditions.

Development of a high-sensitivity latex reagent for the detection of C-reactive protein.

Various convenient and high-sensitivity immunoassays based on luminescent oxygen channeling and chromatographic techniques have been developed in recent years. This study focused on the latex agglutination immunoassay because it is a simple, homogenous immunoassay, which is also cost effective. We developed a highly sensitive latex reagent and examined the method of antibody conjugation on the latex particle surface. We introduced spacer amino acids in the latex surface to investigate the relationship between the amino acid spacer and the binding of an anti-C-reactive protein (anti-CRP) antibody as well as to investigate the resulting reactivity of the latex reagent to antigen. Because the distance between the latex particle and the antibody is equal in each case, differences in immunoreactivity are attributed to the structure of the amino acid side chain (R). Thus, reactivity of the latex reagent depends on the inorganicity and organicity of R. We suggest that a useful amino acid spacer has an inorganicity-to-organicity ratio of approximately 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.

Development of a highly sensitive latex reagent directed against C-reactive protein (CRP) using epitope analysis with monoclonal antibodies.

C-Reactive protein (CRP) is an acute-phase protein that increases during systemic inflammation and is currently one of the most frequently studied inflammatory markers in epidemiology. We have determined CRP concentration using novel latex reagent with polyclonal antibody. In the present study, we determined the concentration of CRP using monoclonal antibodies, and evaluated the interaction of antigen-antibody reactive sites and latex agglutination to detect low CRP concentrations. We developed four novel monoclonal antibodies that we classified into two major groups, and that were used to prepare the latex reagents. The latex reagents prepared using a cocktail of monoclonal antibodies for different epitopes appeared highly sensitive. The lower limit of CRP detection, which was defined using the mean 3 SD method, was calculated to be 5 ng/ml for the latex reagents when oligoclonal antibodies were utilized. Furthermore, the latex reagents were found to react specifically with CRP in clinical samples.

Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.

Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin using ESI/MS has not been reported. In this study, we report the successful cloning, expression, secretion, identification and sequence determination of the C. perfringens alpha-toxin.

Development of latex turbidimetric immunoassay for rapid and sensitive detection of influenza virus.

For the rapid and sensitive detection of influenza A and B viruses, a latex turbidimetric immunoassay (LTIA) was developed using latex reagents prepared by the sensitization of anti-influenza A or B monoclonal antibodies on latex particles. We measured the immunoreactivity of these latex reagents to influenza A and B viral antigens. The sensitivity and specificity of LTIA and reverse transcription-polymerase chain reaction (RT-PCR) for the detection of these viruses in clinical specimens (96 nasal swabs) were compared. The absorbance change in the latex agglutination reaction increased for each latex reagent with increasing concentration of the viral antigens. Reaction curves were obtained with each concentration of viral antigens for 5 min. The effective concentration ranges were 0-10 microg/ml for influenza A and 0-20 microg/ml for influenza B. The LTIA using clinical specimens revealed 8 positive and 73 negative results for influenza A and 15 positive and 52 negative results for influenza B. The sensitivities and specificities were 89% (8/9) and 84% (73/87), respectively, for influenza A and 100% (15/15) and 64% (52/81), respectively, for influenza B. The corresponding positive predictive values (PPV) were 36% (8/22) for influenza A and 34% (15/44) for influenza B. The negative predictive values (NPV) were approximately 99% (73/74) for influenza A and 100% (52/52) for influenza B. The LTIA is a rapid and sensitive method for detection of the influenza virus; It can be used for high throughput assay by automatic measurement and can potentially be used during influenza pandemics.

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

The development of sensitive latex agglutination tests for detecting astroviruses (serotypes 1 and 3) from clinical stool specimen.

Astrovirus is a small single strand RNA virus, that are associated with pediatric gastroenteritis. In order to detect astrovirus, latex agglutination test (LA) is simpler, more convenient and rapid diagnostic method for detection of viruses in stool specimens than that of electron microscopy (EM) or reverse transcriptase-polymerase chain reaction (RT-PCR). Latex reagents was developed with cultured astroviruses serotypes 1 and 3, and tested on rotaviruses- and adenoviruses-negative stool specimens between 1996 and 1998. Out of the 220 specimens, 23 were positive by both LA of serotype 1 and RT-PCR in 26 positive by RT-PCR. The sensitivity was 88.5%, the specificity was 97.9%. Four were positive by both LA of serotype 3 and RT-PCR. Though seroptype 3 LA needs to be improved, LA for screenings astrovirus is useful in rapidly detecting as a large number of screening test.