Historical view of schistosomiasis japonica in Japan: implementation and evaluation of disease-control strategies in Yamanashi Prefecture

We summarized historical aspects of disease control activities targeting schistosomiasis japonica in Kofu basin, Yamanashi Prefecture, Japan. Kofu Basin was one of the biggest endemic foci of schistosomiasis japonica in Japan, and the last place where transmission of Schistosoma japonicum was confirmed in Japan. Because of the most severe endemic situations in Yamanashi, intensive control measures had been implemented by the central as well as the local government. The last human case in Japan was in 1977, which is just before praziquantel being available. Therefore, the main efforts were focused on snail control. Mass examination and mass chemotherapy were implemented, however, the compliance was not so good because of the severe side effects due to the available therapeutics, Stibnal. Along with socioeconomic development after World War II, big changes in land use, life style, and farming led drastic reduction in the disease prevalence in Kofu Basin in the 1960s. A large amount of budget was also used for disease control. Cementing water canals covered more than 95% of paddy fields in Kofu Basin, and this resulted in ecological changes. After elimination of schistosomiasis, environmental repair is the urgent subject in Kofu Basin. Our experiences in Yamanashi contain both good influences and also a lot of reflection. It is important to evaluate each activity in our history before we give intensive cooperation with countries where endemic foci is still present.     

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.     

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

ABSTRACT: BACKGROUND: Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation. METHODS: Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay. RESULTS: Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms. CONCLUSION: The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.     

The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (~200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (K(D) = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.     

Association of Genetic Variants Influencing Lipid Levels with Coronary Artery Disease in Japanese Individuals

Background/Objective

In Japanese populations, we performed a replication study of genetic loci previously identified in European-descent populations as being associated with lipid levels and risk of coronary artery disease (CAD).

Methods

We genotyped 48 single nucleotide polymorphisms (SNPs) from 22 candidate loci that had previously been identified by genome-wide association (GWA) meta-analyses for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and/or triglycerides in Europeans. We selected 22 loci with 2 parallel tracks from 95 reported loci: 16 significant loci (p<1×10⁻³⁰ in Europeans) and 6 other loci including those with suggestive evidence of lipid associations in 1292 GWA-scanned Japanese samples. Genotyping was done in 4990 general population samples, and 1347 CAD cases and 1337 controls. For 9 SNPs, we further examined CAD associations in an additional panel of 3052 CAD cases and 6335 controls.

Principal Findings

Significant lipid associations (one-tailed p<0.05) were replicated for 18 of 22 loci in Japanese samples, with significant inter-ethnic heterogeneity at 4 loci–APOB, APOE-C1, CETP, and APOA5–and allelic heterogeneity. The strongest association was detected at APOE rs7412 for LDL-C (p = 1.3×10⁻⁴¹), CETP rs3764261 for HDL-C (p = 5.2×10⁻²⁴), and APOA5 rs662799 for triglycerides (p = 5.8×10⁻⁵⁴). CAD association was replicated and/or verified for 4 loci: SORT1 rs611917 (p = 1.7×10⁻⁸), APOA5 rs662799 (p = 0.0014), LDLR rs1433099 (p = 2.1×10⁻⁷), and APOE rs7412 (p = 6.1×10⁻¹³).

Conclusions

Our results confirm that most of the tested lipid loci are associated with lipid traits in the Japanese, further indicating that in genetic susceptibility to lipid levels and CAD, the related metabolic pathways are largely common across the populations, while causal variants at individual loci can be population-specific.     

Deletion of CDKAL1 Affects High-Fat Diet–Induced Fat Accumulation and Glucose-Stimulated Insulin Secretion in Mice,Indicating Relevance to Diabetes

Background/Objective

The CDKAL1 gene is among the best-replicated susceptibility loci for type 2 diabetes, originally identified by genome-wide association studies in humans. To clarify a physiological importance of CDKAL1, we examined effects of a global Cdkal1-null mutation in mice and also evaluated the influence of a CDKAL1 risk allele on body mass index (BMI) in Japanese subjects.

Methods

In Cdkal1-deficient (Cdkal1 ^−/−) mice, we performed oral glucose tolerance test, insulin tolerance test, and perfusion experiments with and without high-fat feeding. Based on the findings in mice, we tested genetic association of CDKAL1 variants with BMI, as a measure of adiposity, and type 2 diabetes in Japanese.

Principal Findings

On a standard diet, Cdkal1 ^−/− mice were modestly lighter in weight than wild-type littermates without major alterations in glucose metabolism. On a high fat diet, Cdkal1 ^−/− mice showed significant reduction in fat accumulation (17% reduction in %intraabdominal fat, P = 0.023 vs. wild-type littermates) with less impaired insulin sensitivity at an early stage. High fat feeding did not potentiate insulin secretion in Cdkal1 ^−/− mice (1.0-fold), contrary to the results in wild-type littermates (1.6-fold, P<0.01). Inversely, at a later stage, Cdkal1 ^−/− mice showed more prominent impairment of insulin sensitivity and glucose tolerance. mRNA expression analysis indicated that Scd1 might function as a critical mediator of the altered metabolism in Cdkal1 ^−/− mice. In accordance with the findings in mice, a nominally significant (P<0.05) association between CDKAL1 rs4712523 and BMI was replicated in 2 Japanese general populations comprising 5,695 and 12,569 samples; the risk allele for type 2 diabetes was also associated with decreased BMI.

Conclusions

Cdkal1 gene deletion is accompanied by modestly impaired insulin secretion and longitudinal fluctuations in insulin sensitivity during high-fat feeding in mice. CDKAL1 may affect such compensatory mechanisms regulating glucose homeostasis through interaction with diet.     

Extracellular Superoxide Dismutase in Cultured Astrocytes: Decrease in Cell-Surface Activity and Increase in Medium Activity by Lipopolysaccharide-Stimulation

Under pathological conditions such as ischemia/reperfusion, a large amount of superoxide anion (O(2) (-)) is produced and released in brain. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD, known to be excreted outside cells and bound to extracellular matrix, should play a role to detoxify O(2) (-) in extracellular space; however, a little is known about EC-SOD in brain. In order to evaluate the SOD activity in extracellular space of CNS as direct as possible, we attempted to measure the cell-surface SOD activity on primary cultured rat brain cells by the inhibition of color development of a water-soluble tetrazolium due to O(2) (-) generation by xanthine oxidase/hypoxanthine added into extracellular medium of intact cells. The cell-surface SOD activity on cultured neuron and microglia was below the detection limit; however, that on cultured astrocyte was high enough to measure. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected in the three types of the cells examined; however, the semi-quantitative analysis revealed that the level of EC-SOD mRNA in astrocytes was significantly higher than that in neurons and microglia. When astrocytes were stimulated with lipopolysaccharide (LPS) for 12-24?h, the cell-surface SOD activity decreased to a half, whereas the activity recovered after 36-48?h. The decrease in the activity was dependent on the LPS concentration. On the other hand, the SOD activity in the medium increased by the LPS-stimulation in a dose dependent manner; suggesting that the SOD protein localized on cell-surface, probably EC-SOD, was released into the medium. These results suggest that EC-SOD of astrocyte play a role for detoxification of extracellular O(2) (-) and the regulation of EC-SOD in astrocytes may contribute to the defensive mechanism against oxidative stress in brain.     

Muscle contraction increases carnitine uptake via translocation of OCTN2

Since carnitine plays an important role in fat oxidation, influx of carnitine could be crucial for muscle metabolism. OCTN2 (SLC22A5), a sodium-dependent solute carrier, is assumed to transport carnitine into skeletal muscle cells. Acute regulation of OCTN2 activity in rat hindlimb muscles was investigated in response to electrically induced contractile activity. The tissue uptake clearance (CL(uptake)) of l-[(3)H]carnitine during muscle contraction was examined in vivo using integration plot analysis. The CL(uptake) of [(14)C]iodoantipyrine (IAP) was also determined as an index of tissue blood flow. To test the hypothesis that increased carnitine uptake involves the translocation of OCTN2, contraction-induced alteration in the subcellular localization of OCTN2 was examined. The CL(uptake) of l-[(3)H]carnitine in the contracting muscles increased 1.4-1.7-fold as compared to that in the contralateral resting muscles (p<0.05). The CL(uptake) of [(14)C]IAP was much higher than that of l-[(3)H]carnitine, but no association between the increase in carnitine uptake and blood flow was obtained. Co-immunostaining of OCTN2 and dystrophin (a muscle plasma membrane marker) showed an increase in OCTN2 signal in the plasma membrane after muscle contraction. Western blotting showed that the level of sarcolemmal OCTN2 was greater in contracting muscles than in resting muscles (p<0.05). The present study showed that muscle contraction facilitated carnitine uptake in skeletal muscles, possibly via the contraction-induced translocation of its specific transporter OCTN2 to the plasma membrane.