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.

New wearable walking-type continuous passive motion device for postsurgery walking rehabilitation.

While total knee arthroplasty is useful for treating osteoarthritis of the knee, the success of this treatment depends on effective rehabilitation. The goal of this study was to develop an assistive device for post-total knee arthroplasty patients for walking rehabilitation and for shortening the hospitalization period. We developed a brace electronic assist system termed the knee assistive instrument for walking rehabilitation (KAI-R) to illustrate the need for training during postoperative rehabilitation. Sixteen osteoarthritis patients (1 male and 15 females; average age 68.9 years) who underwent total knee arthroplasty were analyzed before operation and 2-4 weeks after operation, and 25 healthy individuals (14 males and 11 females; average age 26.2 years) formed the control group. Based on the pre- and postoperative data on peak knee flexion angle, foot height, and walking velocity, we developed the KAI-R, which consists of an assistive mechanism for the knee joint, a hip joint support system, and a foot pressure sensor system and is driven by a CPU board that generates the walking pattern. We then tested the walking gait in seven healthy volunteers with and without KAI-R assistance. KAI-R increased the peak flexion angle of the knee and foot height in all seven volunteers; their range of motion of the knee joint was increased. However, KAI-R also decreased the walking velocity of subjects, which was explained by reaction delay and slightly compromised physical balance, which was caused by wearing the KAI-R. KAI-R is useful for gait improvement. In future studies, KAI-R will be investigated in a clinical trial for its ability for walking rehabilitation in post-total knee arthroplasty patients.

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.

Anti-inflammatory and anti-tumor-promoting effects of 5-deprenyllupulonol C and other compounds from Hop (Humulus lupulus L.).

A new phloroglucinol derivative, 5-deprenyllupulonol C (1), along with four other phloroglucinol derivatives, 2-5, five chalcones, 6-10, four flavanones, 11-14, two flavonol glycosides, 15 and 16, and five triterpenoids, 17-21, were isolated from the female inflorescence pellet extracts of hop (Humulus lupulus L.). Upon evaluation of these compounds against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells, twelve compounds, i.e., 1-4, 11-14, 17-19, and 21, showed potent inhibitory effects on EBV-EA induction, with IC50 values in the range of 215-393 mol ratio/32 pmol TPA. In addition, eleven compounds, i.e., 1-4, 6, 11, 12, 14, 17, 18, and 20, were found to inhibit TPA-induced inflammation (1 µg/ear) in mice, with ID50 values in the range of 0.13-1.06 µmol per ear. Further, lupulone C (2) and 6-prenylnaringenin (14) exhibited inhibitory effects on skin-tumor promotion in an in vivo two-stage mouse-skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter.

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.

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 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.

Chemiluminescent enzyme immunoassay for measuring leptin.

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1-1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.

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.

Isobavachalcone, a chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma.

Six chalcones from Angelica keiskei KOIDZUMI (Ashitaba in Japanese) and two chalcones from Humulus lupulus L. (hop) were examined for their cytotoxicity in two human neuroblastoma cell lines (IMR-32 and NB-39) and normal cells (primary culture of rat cerebellar granule cells) by [3-(4,5)-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. All chalcones exhibited cytotoxicity against neuroblastoma cells, and two of them (isobavachalcone and xanthoangelol H) had no effect on normal cells even at high concentration (10(-4) M) exposure. Typical morphologic features of apoptosis, including cell shrinkage, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies, were observed in isobavachalcone-treated cells by Hoechst 33342 staining. Western blot analysis showed that isobavachalcone significantly reduced pro-caspase-3 and pro-caspase-9, and subsequently increased the level of cleaved caspase-3 and cleaved caspase-9 in both neuroblastoma cell lines. Moreover, Bax was markedly induced by isobavachalcone application. These results suggest that isobavachalcone induces apoptotic cell death in neuroblastoma via the mitochondrial pathway and has no cytotoxicity against normal cells. Therefore, isobavachalcone may be applicable as an efficacious and safe drug for the treatment of neuroblastoma.

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope.

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.

Anti-norovirus polyclonal antibody and its potential for development of an antigen-ELISA.

Norovirus (NoV) capsid proteins were expressed as virus-like particles (VLPs) by using recombinant baculovirus in insect cells, which had 5 genotypes in genogroup I and 11 genotypes in genogroup II, and the VLPs were used as immunogens. Polyclonal antibody against the VLP of GII/3 genotype showed broad-range cross-reactivity, reacting not only with intra-genogroup strains, but also inter-genogroup strains, by antibody-ELISA using 16 kinds of VLPs. Furthermore, antigen-ELISA was conducted in sandwich enzyme-linked immunosorbent assay (ELISA) using the polyclonal antibody for capturing antigens, and three kinds of monoclonal antibodies against the VLP of GII/4 genotype for detecting antigens. This format successfully detected eight genotypes of NoV from clinical specimens and proved that polyclonal antibody, which has broad-range cross-reactivity, was capable of detecting various types of genotypes from clinical specimens.

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.

Mobile submuscular mass resembling a hard-boiled egg: case report.

A 68-year-old woman had a rare mobile mass on her back. The mass was similar to encapsulated fat necrosis except for its unusual stalked appearance, large size, and high degree of mobility underneath the anterior serratus muscle on the back. Based on previous descriptions of encapsulated fat necrosis, the clinical presentation of the mass might be regarded as an extreme example of this rare condition.