Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor.

Humanin is a secreted bioactive peptide that is protective in a variety of death models, including cell-based neuronal death models related to Alzheimer's disease (AD). To mediate the protective effect in AD-related death models, Humanin signals via a cell-surface receptor that is generally composed of three subunits: ciliary neurotrophic factor receptor α, WSX-1 and gp130 (heterotrimeric Humanin receptor; htHNR). However, the protective effect of Humanin via the htHNR is weak (EC50=1-10 µM); therefore, it is possible that another physiological agonist for this receptor exists in vivo. In the current study, calmodulin-like skin protein (CLSP), a calmodulin relative with an undefined function, was shown to be secreted and inhibit neuronal death via the htHNR with an EC50 of 10-100 pM. CLSP was highly expressed in the skin, and the concentration in circulating normal human blood was ~5 nM. When administered intraperitoneally in mice, recombinant CLSP was transported across the blood-cerebrospinal fluid (CSF)-barrier and its concentration in the CSF reaches 1/100 of its serum concentration at 1 h after injection. These findings suggest that CLSP is a physiological htHNR agonist.

Reduced expression of BTBD10, an Akt activator, leads to motor neuron death.

BTBD10, an Akt interactor, activates Akt by decreasing the protein phosphatase 2A-mediated dephosphorylation and inactivation of Akt. Overexpression of BTBD10 suppresses motor neuron death that is induced by a familial amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutant, G93A-SOD1 in vitro. In this study, we further investigated the BTBD10-mediated suppression of motor neuron death. We found that the small interfering RNA-mediated inhibition of BTBD10 expression led to the death of cultured motor neurons. In Caenorhabditis elegans (C. elegans), disruption of the btbd-10 gene caused not only loss of neurons, including both motor and touch-receptor neurons, but also a locomotion defect. In addition, we found that the expression of BTBD10 was generally decreased in the motor neurons from patients of sporadic ALS and transgenic mice overexpressing G93A-SOD1 (G93A-SOD1-transgenic mice). Collectively, these results suggest that the reduced expression of BTBD10 leads to motor neuron death both in vitro and in vivo.

Immunoglobulin A antibody responses in dengue patients: a useful marker for serodiagnosis of dengue virus infection.

We determined the usefulness of an immunoglobulin A (IgA) antibody-capture enzyme-linked immunosorbent assay for serodiagnosis of dengue virus infections. The results indicate that the presence of IgA and IgM in serum samples assures recent primary dengue virus infection even with a single serum sample.

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody.

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.

Increase in the sensitivity of dengue diagnosis by combination of reverse transcriptase-polymerase chain reaction and passage on cell cultures.

We passaged 52 serum samples from dengue patients on C6/36 cells for 7 days and checked the culture fluids by RT-PCR. Two serum samples, which were negative by direct RT-PCR, became positive. One sample was collected on fever day 1 and the other on fever day 2. Results indicate that combination of reverse transcriptase-polymerase chain reaction (RT-PCR) with passage of serum samples on C6/36 cells increases the sensitivity of dengue diagnosis.

Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay.

We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.

Repression of TNF-alpha-induced E-selectin expression by PPAR activators: involvement of transcriptional repressor LRF-1/ATF3.

Peroxisome proliferator-activated receptor (PPAR) activators were shown to inhibit the expression of E-selectin of human vascular endothelial cells in response to tumor necrosis factor-alpha (TNF-alpha). Troglitazone, pioglitazone, alpha-clofibrate, and 15-deoxy-Delta12,14-prostaglandin J2 all inhibited the TNF-alpha-stimulated E-selectin gene transcription in reporter assay. To further clarify the underlying transcriptional regulation, nuclear factor(s) that binds to the nuclear factor-endothelial leukocyte adhesion molecule 1 (NF-ELAM1) site of the E-selectin gene promoter was investigated. The activators caused a significant induction of liver regenerating factor 1 (LRF1)/activating transcription factor 3 (ATF3), which bound to the NF-ELAM1 site and repressed the TNF-alpha-induced E-selectin gene expression. From these data, the effect of PPAR activators was mediated, in part, through the induction of LRF1/ATF3. This might provide a novel molecular mechanism of anti-inflammatory effect of PPAR activators.

Laboratory diagnosis of dengue virus infection by reverse transcriptase polymerase chain reaction (RT-PCR) and IgM-capture enzyme-linked immunosorbent assay (ELISA).

Dengue virus infections are a major public health problem in most tropical and sub-tropical countries of the world. Dengue is occasionally imported by travelers who visit tropical areas and become infected with dengue virus. Laboratory diagnosis is essential for confirming the diagnosis of this virus. For purposes of confirmation, detection of specific IgM by IgM-capture enzyme-linked immunosorbent assay (ELISA) and of dengue virus genome by reverse transcriptase polymerase chain reaction (RT-PCR) have recently been used. In the present study, we tested serum specimens from dengue-suspected Japanese cases, by IgM-capture ELISA, RT-PCR, HI, and virus isolation. Serum samples collected before or on the day of defervescence were positive by RT-PCR, though no PCR-positive samples were obtained after fever day 1. IgM-capture ELISA was positive as early as disease day 4, and all samples but one were IgM-positive when collected on disease day 5 or later. In light of these findings, we recommend that both RT-PCR and IgM-capture ELISA be performed, irrespective of the stage of dengue illness. Combination of RT-PCR and IgM-capture ELISA increases the ability to diagnose dengue virus infection, even in the only that a single serum specimen from the patient is available.

Effects of bafilomycin A1 on Japanese encephalitis virus in C6/36 mosquito cells.

Involvement of intracellular acidic compartments in the early phase of Japanese encephalitis (JE) virus infection of C6/36 mosquito cells was examined by bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase (V-ATPase). Dose dependent reduction of viral envelope protein (E) produced into the infected culture fluid was observed by pretreating the cells with 0.25 to 1.0 microM bafilomycin A1. In synchronized infection, cell surface-bound virions were internalized immediately by heating at 31 degrees C, followed by the release of nucleocapsid into the cytosol within a short lag period. Subcellular distribution of infecting 3H-uridine-labeled viral RNA (V-RNA) and its RNase sensitivity were analyzed by fractionation in Percoll density gradient centrifugation. At a 10 min chasing period, an RNase resistant V-RNA peak was found in fractions with a mean density of 1.05 g/ml corresponding to the endosome, while an RNase sensitive V-RNA peak was detected at density range of 1.052-1.054 g/ml corresponding to the ribosome in C6/36 cell homogenate. The results indicate that JE virus infection in C6/36 cells proceeded through the endocytic pathway involving intracellular acidic compartments which was affected by bafilomycin A1.

Japanese encephalitis virus infection in Vero cells: the involvement of intracellular acidic vesicles in the early phase of viral infection was observed with the treatment of a specific vacuolar type H+-ATPase inhibitor, bafilomycin A1.

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H+-ATPase (V-ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and 3H-uridine-labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1-treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1-treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.

Stability of hemagglutinating activity of extracellular and intracellular forms of Japanese encephalitis virus exposed to acidic pH.

The hemagglutinating (HA) activity of extracellular and intracellular forms of Japanese encephalitis (JE) virus was comparatively titrated by exposure to acidic pH below 7.0. A pH-dependent irreversible loss in titer was observed with the virus grown in both C6/36 and BHK 21 (BHK) cells maintained in the pH range of 5.8 to 7.0 for 10 min at 37 C. The HA activity of intracellular virus was relatively more stable than that of extracellular virus in the pH range of 5.8 to 6.4. Virion structural components, envelope glycoprotein (E), capsid (C), and membrane (M) proteins in extracellular virus and E, C, and the precursor form of M (prM) proteins in intracellular virus were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A panel of monoclonal antibody (mAb) directed for nine antigenic epitopes on the JE virus E protein molecule was used for the analysis of antigenic reactivity of E protein after treatment at pH 6.0. The reaction between the extracellular virus and three HA-inhibiting (HI) mAbs was significantly reduced after acid treatment; however, the antigenic reactivity of intracellular virus was much more stable with a 100- to 1,000-fold difference. Infectivity titers of extracellular and intracellular viruses in Vero cells were reduced by 1/24,100 and 1/21,666 after acidic treatment at pH 6.0. In contrast, the infectivity of intracellular viruses was more stable, with residual infectivity of 1/182 and 1/340 for BHK and C6/36 cell-grown virus, respectively. Acidic treatment of JE virus not only resulted in the irreversible loss of its HA activity but also affected the antigenic reactivity of HI epitopes on its E protein molecule.

An enzyme-linked immunosorbent assay using a chaotropic agent (sodium thiocyanate) for serotype specific reaction between crude dengue viral antigen and anti-dengue mouse antibody.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti-dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement-fixing antigen (SCF) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted serotype specifically to anti-dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti-type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti-type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.

[A case report of a patient repeatedly infected with Ancylostoma duodenale, probably from himself through his artificial anus, and resistant against a single dose of pyrantel pamoate].

A 88 year old Japanese male was repeatedly infected with Ancyclostoma duodenale. He underwent an artificial anus operation about 55 years ago. He appeared to be infected with hookworm earlier than in 1977 and developed severe anemia. Though he was treated with pyrantel pamoate and mebendazole several times, reinfections developed in each time. A possible origin for his reinfections was his own feces defecated through his artificial anus. Unsanitary handling of the anus and the feces exposed himself to oral or percutaneous infection. Besides, a single dose of pyrantel pamoate, usually very effective against Ancylostoma duodenale, was not so effective in this patient. Therefore, we prescribed multiple doses of pyrantel pamoate, and followed by a single dose of mebendazole. However, reinfections still persisted because of his unsanitary behavior.

Development of a practicable method for isolation and identification of dengue viruses in developing countries.

We sought a practicable method for isolation and identification of dengue viruses in South-East Asia. We compared two mosquito cell lines, C6/36 and TRA-284-SFG, for virus isolation and two identification methods, immunofluorescent staining of infected cells with serotype-specific mouse monoclonal antibodies and a sandwich-type enzyme-linked immunosorbent assay with conventional mouse hyperimmune ascitic fluids. We found that the combination of TRA-284-SFG cells and ELISA is a useful and feasible method in developing countries.

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay.

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.

In vitro effect of estrogens on the peroxidase activity of human endometrium.

The effect of estrogen on the induction of human endometrial peroxidase was studied in vitro. When 17 beta-estradiol was added to the incubation medium, endometrial peroxidase activity was markedly increased, and the highest enzymatic activities were reached at about 12 h of incubation. The ability of estrogen and related compounds to increase peroxidase activity at a concentration of 10(-7) M was 17 beta-estradiol greater than estrone greater than 17 alpha-ethynylestradiol greater than estriol = diethylstilbestrol. Mestranol, progesterone, and testosterone had almost no activity. The induction of peroxidase by 17 beta-estradiol was inhibited by actinomycin D or cycloheximide. This method may be useful for determining the direct estrogenic effect of compounds in human endometrium.

Development of a new cell system for the infectivity assay of dengue viruses: plaque formation and virus growth of prototype and wild-type dengue virus strains in a newly established cell line, GK.

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild-type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures. Virus yields in GK cells of all the 14 dengue virus strains tested, including four prototype and ten wild-type viruses, were 5 to 1,000-times lower than those in C6/36 cells. After five serial passages in GK cells, types 2, 3, and 4 prototype viruses and type 2 wild strain increased virus yields, and one strain of prototype virus and one strain of wild-type virus decreased mouse neurovirulence.

A continuous cell line, GK, derived from the kidney tissue of Mongolian gerbil (Meriones unguiculatus) and its virological application.

A fibroblast-like continuous cell line was established from the kidney tissue of the female Mongolian gerbil (Meriones unguiculatus) and designated as GK cell line. The cells were susceptible to the infection with several DNA and RNA animal viruses, particularly with four prototype strains of dengue viruses and the HEP-Flury strain of rabies virus.