Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles

Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with β1–3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62 000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of -(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.     

Functionally stable and phylogenetically diverse microbial enrichments from microbial fuel cells during wastewater treatment

Microbial fuel cells (MFCs) are devices that exploit microorganisms as biocatalysts to recover energy from organic matter in the form of electricity. One of the goals of MFC research is to develop the technology for cost-effective wastewater treatment. However, before practical MFC applications are implemented it is important to gain fundamental knowledge about long-term system performance, reproducibility, and the formation and maintenance of functionally-stable microbial communities. Here we report findings from a MFC operated for over 300 days using only primary clarifier effluent collected from a municipal wastewater treatment plant as the microbial resource and substrate. The system was operated in a repeat-batch mode, where the reactor solution was replaced once every two weeks with new primary effluent that consisted of different microbial and chemical compositions with every batch exchange. The turbidity of the primary clarifier effluent solution notably decreased, and 97% of biological oxygen demand (BOD) was removed after an 8-13 day residence time for each batch cycle. On average, the limiting current density was 1000 mA/m(2), the maximum power density was 13 mW/m(2), and coulombic efficiency was 25%. Interestingly, the electrochemical performance and BOD removal rates were very reproducible throughout MFC operation regardless of the sample variability associated with each wastewater exchange. While MFC performance was very reproducible, the phylogenetic analyses of anode-associated electricity-generating biofilms showed that the microbial populations temporally fluctuated and maintained a high biodiversity throughout the year-long experiment. These results suggest that MFC communities are both self-selecting and self-optimizing, thereby able to develop and maintain functional stability regardless of fluctuations in carbon source(s) and regular introduction of microbial competitors. These results contribute significantly toward the practical application of MFC systems for long-term wastewater treatment as well as demonstrating MFC technology as a useful device to enrich for functionally stable microbial populations.     

CD2AP/SHIP1 complex positively regulates plasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitin ligase Cbl

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.     

Induction of Adult T-Cell Leukemia-Like Lymphoproliferative Disease and Its Inhibition by Adoptive Immunotherapy in T-Cell-Deficient Nude Rats Inoculated with Syngeneic Human T-Cell Leukemia Virus Type 1-Immortalized Cells

Human T-cell leukemia virus type 1 (HTLV-1) has been shown to be the etiologic agent of adult T-cell leukemia (ATL), but the in vivo mechanism by which the virus causes the malignant transformation is largely unknown. In order to investigate the mechanisms of HTLV-1 leukemogenesis, we developed a rat model system in which ATL-like disease was reproducibly observed, following inoculation of various rat HTLV-1-immortalized cell lines. When previously established cell lines, F344-S1 and TARS-1, but not TART-1 or W7TM-1, were inoculated, systemic multiple tumor development was observed in adult nude (nu/nu) rats. FPM1 cells, newly established from a heterozygous (nu/+) rat syngeneic to nu/nu rats, caused transient tumors only at the injection site in adult nu/nu rats, but could progressively grow in newborn nu/nu rats and metastasize in lymph nodes. The derivative cell line (FPM1-V1AX) serially passed through newborn nu/nu rats acquired the potency to grow in adult nu/nu rats. These results indicated that only some with additional changes but not all of the in vitro HTLV-1-immortalized cell lines possessed in vivo tumorigenicity. Using the syngeneic system, we further showed the inhibition of tumor development by transferring splenic T cells from immunized rats, suggesting the involvement of T cells in the regression of tumors. This novel and reproducible nude rat model of human ATL would be useful for investigation of leukemogenesis and antitumor immune responses in HTLV-1 infection.     

Comparison of Macular Integrity Assessment (MAIA ?), MP-3, and the Humphrey Field Analyzer in the Evaluation of the Relationship between the Structure and Function of the Macula

Purpose

This study was conducted in order to compare relationships between the macular visual field (VF) mean sensitivity measured by MAIA^TM (Macular Integrity Assessment), MP-3, or Humphry field analyzer (HFA) and the ganglion cell and inner plexiform layer (GCA) thicknesses.

Methods

This cross-sectional study examined 73 glaucoma patients and 19 normal subjects. All subjects underwent measurements for GCA thickness by Cirrus HD-OCT and static threshold perimetry using MAIA^TM, MP-3, or HFA. VF and OCT in the retinal view were used to examine both the global relationship between the VF sensitivity and GCA thickness, and the superior hemiretina and inferior hemiretina. The relationship between the GCA thickness and macular sensitivity was examined by Spearman correlation analysis.

Results

For each instrument, statistically significant macular VF sensitivity (dB) and GCA thickness relationships were observed using the decibel scale (R = 0.547–0.687, all P < 0.001). The highest correlation for the global (R = 0.682) and the superior hemiretina (R = 0.594) GCA thickness-VF mean sensitivity was observed by the HFA. The highest correlation for the inferior hemiretina (R = 0.687) GCA thickness-VF mean sensitivity was observed by the MP-3. Among the three VF measurement instruments, however, no significant differences were found for the structure-function relationships.

Conclusions

All three VF measurement instruments found similar structure-function relationships in the central VF.     

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.     

Low-Carbohydrate Diet and Type 2 Diabetes Risk in Japanese Men and Women: The Japan Public Health Center-Based Prospective Study

ObjectiveEvidence is sparse and contradictory regarding the association between low-carbohydrate diet score and type 2 diabetes risk, and no prospective study examined the association among Asians, who consume greater amount of carbohydrate. We prospectively investigated the association of low-carbohydrate diet score with type 2 diabetes risk.MethodsParticipants were 27,799 men and 36,875 women aged 45–75 years who participated in the second survey of the Japan Public Health Center-Based Prospective Study and who had no history of diabetes. Dietary intake was ascertained by using a validated food-frequency questionnaire, and low-carbohydrate diet score was calculated from total carbohydrate, fat, and protein intake. The scores for high animal protein and fat or for high plant protein and fat were also calculated. Odds ratios of self-reported, physician-diagnosed type 2 diabetes over 5-year were estimated by using logistic regression.ResultsDuring the 5-year period, 1191 new cases of type 2 diabetes were self-reported. Low-carbohydrate diet score for high total protein and fat was significantly associated with a decreased risk of type 2 diabetes in women (P for trend <0.001); the multivariable-adjusted odds ratio of type 2 diabetes for the highest quintile of the score were 0.63 (95% confidence interval 0.46–0.84), compared with those for the lowest quintile. Additional adjustment for dietary glycemic load attenuated the association (odds ratio 0.75, 95% confidence interval 0.45–1.25). When the score separated for animal and for plant protein and fat, the score for high animal protein and fat was inversely associated with type 2 diabetes in women, whereas the score for high plant protein and fat was not associated in both men and women.DiscussionLow-carbohydrate diet was associated with decreased risk of type 2 diabetes in Japanese women and this association may be partly attributable to high intake of white rice. The association for animal-based and plant-based low-carbohydrate diet warrants further investigation.     

Construction and in vitro properties of chimeric simian and human immunodeficiency virus with the human TNF-alpha gene

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be involved in the development and progression of acquired immunodeficiency syndrome (AIDS). To study the role of this cytokine in AIDS pathogenesis, we constructed a chimeric simian and human immunodeficiency virus (SHIV) having the human TNF-alpha gene (SHIV-TNF) and characterized its properties in vitro. SHIV-TNF replicated both in M8166, a human T cell line, and in monkey peripheral blood mononuclear cells (PBMCs). Along with SHIV-TNF replication, TNF-alpha was detected in the culture supernatant by ELISA. The maximum expression level of TNF-alpha reached 120 ng/ml in M8166 cells, and 2.5 ng/ml in monkey PBMCs. The expressed TNF was biologically active, as shown by a cytotoxic assay using TNF-sensitive L929 mouse fibroblasts. This activity was detected at least until 10 passages of SHIV-TNF (74 days after the initial infection). In monkey PBMCs, SHIV-TNF replicated much better than the parental SHIV-NI. Flow cytometric analysis showed that the death of monkey PBMCs infected with SHIV-TNF was severer than that caused by the parental SHIV-NI. These results suggest that SHIV-TNF would be useful for inducing the disease in a monkey model, which may contribute to a better understanding of the role of TNF-alpha in AIDS etiology.     

Cryopreservation of spermatozoa from closed colonies, and inbred, spontaneous mutant, and transgenic strains of rats

We attempted to cryopreserve spermatozoa from closed colonies (Jcl:SD and Jcl:Wistar), and inbred (BN/Crj, F3441 DuCrj, LEW/Crj, Long-Evans and WKY/NCrj), mutant (Zitter [WTC.ZI-zi] and Tremor [TRM]), transgenic (human A-transferase [A], and green fluorescent protein [GFP]) strains of rats. Rat epididymal spermatozoa suspended in cryopreservation solution (23% egg yolk, 8% lactose monohydrate, and 0.7% Equex Stm, pH 7.4, adjusted with 10% Tris [hydroxymethy] aminomethane) were frozen and stored at -196 degrees C. After thawing at 37 degrees C, the spermatozoa were instilled into the tip of each uterine horn of the recipients. A total of five recipient females for each strain were inseminated with cryopreserved spermatozoa, and normal live offspring of all strains (Jcl:SD: 11, Jcl:Wistar: 13, BN/Crj: 9, F344/DuCrj: 28, LEW/Crj: 4, Long-Evans: 6, WKY/NCrj: 8, TRM: 24, WTC.ZI-zi: 27, A: 30 and GFP: 20) were obtained.