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Diabetes mellitus is a metabolic disease spreading worldwide that has been reported to worsen the development and progression of other diseases (cancer, vascular diseases and dementia). To establish functional rice lines with anti-postprandial hyperglycaemic effects, we developed mutant rice lines, which lack one or two gene(s) related to starch synthesis, and evaluated their effects. Powder of mutant rice lines or other grains was loaded to rats fasted overnight (oral grain powder loading test). Incremental area under time-concentration curves (iAUC) were calculated with monitored blood glucose levels. Rice lines with anti-postprandial hyperglycaemic effects were separated by cluster analysis with calculated iAUC. A double mutant rice #4019 (starch synthase IIIa (ss3a)/branching enzyme IIb (be2b)), one of the screened mutant rice lines, was fed to Goto-Kakizaki (GK) rats, an animal model for type 2 diabetes, for 5 weeks. Plasma levels of C-peptide, a marker of pancreatic insulin secretion, were measured with ELISA. Forin vitrostudy, a rat pancreatic cell line was cultured with a medium containing rat serum which was sampled from rats fed #4019 diet for 2 d. After 24-h of incubation, an insulin secretion test was performed. Through the oral rice powder loading test, seven rice lines were identified as antidiabetic rice lines. The intake of #4019 diet increased plasma C-peptide levels of GK rats. This result was also observedin vitro.In rat serum added to cell medium, ornithine was significantly increased by the intake of #4019. In conclusion, the mutant rice #4019 promoted pancreatic insulin secretion via elevation of serum ornithine levels.

In order to identify potential unanticipated side reactions and immune responses, the evaluation of candidate vaccines should include immunization of the murine model of the disease in question and mutant animals, as well as normal laboratory animals. We employed WBB6F(1)-W/W(v) and WBB6F(1)-Sl/Sl(d) mutant mice, which are genetically mast cell deficient and lack intestinal pacemaker activity due to a severe deficiency in interstitial cells of Cajal. Antigen-specific mucosal and systemic immune responses in the mutant and congenic normal mice were induced by intranasal or intragastric immunization with ovalbumin (OVA) plus cholera toxin as an adjuvant. It was found that the levels of the OVA-specific humoral immune response in the mucosal and systemic tissues of the mutant mice immunized intranasally were roughly equivalent to those of the congenic normal mice. In contrast, the specific humoral immune response in the intragastrically immunized mutant mice was greater than that observed in the congenic normal mice. Unexpectedly, the titers of OVA-specific IgA antibodies and total IgA antibodies in the fecal extracts of both intranasally and intragastrically immunized mutant mice were significantly lower than in those of the congenic normal mice. Although the detailed mechanisms leading to these differences remain unclear, the unexpected immune responses observed in the gastrointestinal tracts of the mice in this study may be related to an abnormality of gastrointestinal motility. Our data therefore suggest that studies using mutant mice and physiological assessments should be carried out during mucosal vaccine development.

HIV infection is a major problem in Indonesia. The number of people living with HIV has been increasing from year to year, especially among injecting drug users (IDUs). Since there were only limited data about molecular epidemiology profiles of HIV/AIDS in Indonesia, a cross-sectional study involving 208 HIV-1-seropositive individuals was conducted in 2007 in Jakarta. The majority of participants were 16–30 years of age (64.9%) and 74.5% were male. The most frequent risk factor was injecting drug use (IDU) (45.7%) followed by heterosexual transmission (34.1%). Phylogenetic analysis of gag (p17 and p6) and env C2V3 regions showed 200 (96.2%) of 208 DNA samples were CRF01_AE and only 3 (1.4%) were subtype B. Five samples (2.4%) indicated discordant subtypes between the three aforementioned regions: three of them showed unique CRF01_AE/B recombination patterns in 2.3-kbp nucleotide sequences (from p17 to part of RT), including one sample showing similarity to CRF33_01B, reported previously in Malaysia. This study shows the current predominant subtype is CRF01_AE in every risk group, with a decreasing number of pure subtype B, and the first identification of CRF01_AE/B recombinant forms among HIV-1-seropositive Indonesians.

Rice bran oil is a byproduct of the milling of rice (Oryza sativa L.). It offers various health benefits and has a beneficial fatty acid composition. To increase the amount of rice bran as a sink for triacylglycerol (TAG), we developed and characterized new breeding materials with giant embryos. To induce mutants, we treated fertilized egg cells of the high-yielding cultivar ‘Mizuhochikara’ with N-methyl-N-nitrosourea (MNU). By screening M2 seeds, we isolated four giant embryo mutant lines. Genetic analysis revealed that the causative loci in lines MGE12 and MGE13 were allelic to giant embryo (ge) on chromosome 7, and had base changes in the causal gene Os07g0603700. On the other hand, the causative loci in lines MGE8 and MGE14 were not allelic to ge, and both were newly mapped on chromosome 3. The TAG contents of all four mutant lines increased relative to their wild type, ‘Mizuhochikara’. MGE13 was agronomically similar to ‘Mizuhochikara’ and would be useful for breeding for improved oil content.

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)-beta-D-glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.

It has been reported that subtypes A throughout H of HIV-1 are circulating in the former Soviet Union. In this sequence note, we analyzed the genetic prevalence of HIV-1 among injecting drug users (IDUs) in Ukraine. The subjects studied included two individuals from Kiev and six individuals from Simferopol', the latter located in the Crimean Peninsula. We found that one of eight IDUs was infected with a CRF03 gagA/envB recombinant HIV-1 and was from Simferopol', whereas the others were infected with HIV-1 subtype A. The recombinant was closely related to other A/B recombinants reported previously, and had silent mutations in the V3 region, the same as other envB strains of HIV-1 circulating among IDUs in the former Soviet Union. The data supported reports that the Russian A/B recombinant HIV-1 was probably from Ukraine. This is the first report of a CRF03 gagA/envB recombinant HIV-1 found in Ukraine.

Neurologic diseases are important complications of measles. The role of virus infection of the central nervous system as well as the route of virus entry has been unclear. Five autopsied cases of individuals who died with severe acute measles 3-10 d after the onset of the rash were studied for evidence of viral involvement of the central nervous system. In all cases, in situ hybridization and RT-PCR in situ hybridization techniques showed endothelial cell infection. Immunoperoxidase staining with an anti-ferritin antibody revealed a reactive microgliosis. These data suggest that endothelial cells in the brain are frequently infected during acute fatal measles. This site of infection may provide a portal of entry for virus in individuals who subsequently develop subacute sclerosing panencephalitis or measles inclusion body encephalitis and a target for immunologic reactions in post-measles encephalomyelitis.

Cocirculation of subtype B and CRF01_AE in Southeast Asia has led to the establishment of new recombinant forms. In our previous study, we found five samples suspected of being recombinants between subtype B and CRF01_AE, and here, we analyzed near full-length sequences of two samples and compared them to known CRFs_01B, subtype B, and CRF01_AE. Five overlapped segments were amplified with nested PCR from PBMC DNA, sequenced, and analyzed for genome mosaicism. The two Indonesian samples, 07IDJKT189 and 07IDJKT194, showed genome-mosaic patterns similar to CRF33_01B references from Malaysia, with one short segment in the 3' end of the p31 integrase-coding region, which was rather more similar to subtype B than CRF01_AE, consisting of unclassified sequences. These results suggest gene-specific continuous diversification and spread of the CRF33_01B genomes in Southeast Asia.

Herpes simplex virus type 1 (HSV1) and type 2 (HSV2) were differentiated on the basis of different plaque appearance on semicontinuous rabbit lens epithelial (RLE) cells. Plaques produced by HSV1 strains were small; the mean diameter was 1.29 +/- 0.37 mm 3 days after inoculation. HSV2 strains produced large and small plaques, with the ratio of large to small about 20:1. The mean diameters of the large and the small plaques of HSV2 were 3.34 +/- 0.56 mm and 0.97 +/- 0.31 mm respectively 3 days after inoculation. The clones from the large plaques consistently produced large and small plaques and the small-plaque clones produced only small plaques. Round cells plus heterokaryotes were characteristic of the CPE of HSV1. Large plaques of HSV2 were produced by a large membranous syncytium that was liable to lyse. Small round cells were characteristic of the CPE of the small-plaque clones of HSV2. Glycoprotein C-negative (gC-) strains produced intermediate-sized plaques and a few pin point ones that consisted of membranous syncytia and round cells, respectively. Except for the HF strain (a reference strain of HSV1 producing a membranous syncytium on RLE cells), the result of the differentiation of HSV1 (179 strains) and HSV2 (40 strains) with the RLE plaque assay system was consistent with that of Syva's monoclonal antibody assay system and the restriction endonuclease digestion method.

Background Shiga toxins (Stxs) are the major agents responsible for hemorrhagic colitis and hemolytic-uremic syndrome (HUS) during infections caused by Stx-producing Escherichia coli (STEC) such as serotype O157:H7. Central nervous system (CNS) involvement is an important determinant of mortality in diarrhea associated-HUS. It has been suggested that vascular endothelial injuries caused by Stxs play a crucial role in the development of the disease. The current study investigates the relationship between the cytotoxic effects of Stxs and inflammatory responses in a rabbit brain treated with Stx2. Methods In a rabbit model treated with purified Stx2 or PBS(-), we examined the expression of the Stx receptor globotriaosylceramide (Gb3)/CD77 in the CNS and microglial activation using immunohistochemistry. The relationship between inflammatory responses and neuronal cell death was analyzed by the following methods: real time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) to determine the expression levels of pro-inflammatory cytokines, and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method to detect apoptotic changes. Results Gb3/CD77 expression was detected in endothelial cells but not in neurons or glial cells. In the spinal cord gray matter, significant levels of Gb3/CD77 expression were observed. Severe endothelial injury and microvascular thrombosis resulted in extensive necrotic infarction, which led to acute neuronal damage. Conversely, in the brain, Stx receptor expression was much lower. The observed neuropathology was less severe. However, neuronal apoptosis was observed at the onset of neurological symptoms, and the number of apoptotic cells significantly increased in the brain at a later stage, several days after onset. Microglial activation was observed, and tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the CNS parenchyma was significantly up-regulated. There was significant overexpression of TNF-α transcripts in the brain. Conclusion This study indicates that Stx2 may not directly damage neural cells, but rather inflammatory responses occur in the brain parenchyma in response to primary injury by Stx2 in vascular endothelial cells expressing Gb3/CD77. These findings suggest that neuroinflammation may play a critical role in neurodegenerative processes during STEC infection and that anti-inflammatory intervention may have therapeutic potential.

Although there have been many reports of the usefulness of serodiagnosis of enterohemorrhagic Escherichia coli (EHEC) O157, the serotype of the bacteria detected and the increase in anti-LPS antibody have not always been consistent. In this study we investigated the diagnostic significance of measurements of anti-LPS antibody by ELISA in an outbreak of O157 infection among schoolchildren in whom the bacteriological test findings were clarified and the age groups were uniform. The anti-LPS antibody titer was measured in 31 patients (77 serum samples) in an outbreak of EHEC O157 : H7 infection (220 children infected) that occurred in a primary school in Morioka in 1996. The anti-O157 LPS antibody positivity rates of IgM, IgG, and IgA were 98.7%, 85.7%, and 98.7%, respectively. Between the time the meal that caused the outbreak and 19 days later, anti-O157 LPS IgM antibody and IgA antibody were detected in all patients. The specificity was investigated using control serum, and the specificity of IgM, IgG, and IgA was 93.5%, 93.5%, and 97.2%, respectively. Some samples contained antibodies against O111 and O26 LPS, but the titers were lower than the anti-O157 antibody titer. The anti-O111 antibody titer and anti-O26 antibody titer were highly correlated, suggesting that they were crossreactive antibodies for O157 LPS. No significant correlation was found between differences in clinical manifestations and the anti-O157 LPS antibody titer in this O157 outbreak in schoolchildren. It was clarified that an increase in anti-LPS antibody was found to support the diagnosis of mild cases of 0157 infection infection as well as severe cases.

HIV-induced neurological disease is postulated to be caused by indirect mechanisms. Tumor necrosis factor (TNF)alpha is increased in the brains in human immunodeficiency virus (HIV)-associated dementia and in the spinal cord in vacuolar myelopathy and may play a pathogenetic role in these diseases. Microglia, astrocytes and infiltrating macrophages can be induced to produce TNF alpha and each has been identified as a source of TNF alpha in neurological disease. Reverse transcriptase synthesis of cDNA and polymerase chain reaction amplification of the cDNA was combined with immunocytochemistry to identify the cellular source of TNF alpha in HIV-induced neurological disease. Cells positive for TNF alpha mRNA were more abundant in white matter than gray matter of the brain from demented individuals. TNF alpha mRNA-positive cells in brains and spinal cords were almost exclusively macrophage-lineage cells. Only rare TNF alpha mRNA-positive cells were astrocytes. We conclude that macrophage-lineage cells are the primary source of elevated central nervous system TNF alpha mRNA in providing further evidence that macrophage activation is an important element in the pathogenesis of HIV-associated neurological disease.

Human immunodeficiency virus type 1 (HIV-1) infects the brains of a majority of patients with the acquired immunodeficiency syndrome (AIDS), and has been linked to the development of a progressive dementia termed “HIV-associated dementia.” This disorder results in severe cognitive, behavioral, and motor deficits. Despite this neurological dysfunction, HIV-1 infection of brain cells does not occur significantly in neurons, astrocytes, or oligodendrocytes, but is restricted to brain macrophages and microglia. To identify possible low-level or latent infection of other brain cells, we combined the techniques of the polymerase chain reaction with in situ hybridization for the detection of HIV DNA, and used immunocytochemistry to identify the HIV-expressing cells. In the 21 adult brains studied (15 AIDS and 6 seronegative control brains), we found that polymerase chain reaction/in situ hybridization was both sensitive and specific for identifying HIV-infected cells. In all brains, the majority of infected cells were macrophages and microglia. In several brains, however, a substantial minority of cells harboring HIV DNA were identified as astrocytes. Neurons, oligodendrocytes, and endothelial cells were not infected with HIV, even in cases with HIV-associated dementia. These findings confirm previous data regarding the importance of macrophage/microglial infection, and essentially exclude neuronal infection in pathogenetic models of HIV-associated neurological disease. These data also demonstrate that latent or low-level infection of astrocytes occurs in AIDS, a finding that may be of importance in understanding HIV neuropathogenesis.

There have been several reports that lymphocytes of human cord blood have poor functional properties because of their immaturity and/or the presence of suppressor cells. We studied here the in vitro effects of thymopoietin pentapeptide (TP-5) and interleukin 2 (IL-2) on cord blood T cells. Lymphocyte subsets were analyzed by flow cytometry through the use of monoclonal antibodies after a 48 h culture period in TP-5 (optimal dose; 1μg/ml) and/or IL-2 (non-mitogenic dose used; 1% vol/vol). OKT 8-positive cells were significantly decreased after treatment in combination with TP-5 and IL-2. But there were no changes in the propotion of OKT 3-, T4-, T11- and Ial-positive cells. Poor helper activity of cord blood T cells, which was examined by enhancing activity of immunoglobulin-production of normal adult non-T cells stimulated by pokeweed mitogen, was enhanced to a significant level of IgG production after treatment in combination with two agents. Crude IL-2 activity was reevaluated through the use of recombinant IL-2.