Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.     

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified.

Objectives

The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice.

Methods

Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.

Results

Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis.

Conclusion

Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.     

Ecological niche breadth and microhabitat guild structure in temperate Australian reptiles: Implications for natural resource management in endangered grassy woodland ecosystems

Ecological theory predicts that species with narrow niche requirements (habitat specialists) are more vulnerable to anthropocentric disturbances than those with broad niche requirements (habitat generalists). Hence, understanding a species ecological niche and guild membership would serve as a valuable management tool for providing a priori assessments of a species extinction risk. It also would help to forecast a species capacity to respond to land use change, as what might be expected to occur under financial incentive schemes to improve threatened ecological vegetation communities. However, basic natural history information is lacking for many terrestrial species, particularly reptiles in temperate regions of the world. To overcome this limitation, we collated 3527 reptile observations from 52 species across an endangered woodland ecoregion in south‐eastern Australia and examined ecological niche breadth and microhabitat guild structure. We found 30% of species had low ecological niche values and were classified as habitat specialists associated with large eucalypt trees, woody debris, surface rock or rocky outcrops. Cluster analysis separated species into six broad guilds based on microhabitat similarity. Approximately 80% of species belonged to guilds associated with old growth vegetation attributes or non‐renewable litho‐resources such as surface rock or rocky outcrops. Our results suggest that agri‐environment schemes that focus purely on grazing management are unlikely to provide immediate benefits to broad suites of reptiles associated with old growth vegetation and litho‐resources. Our classification scheme will be useful for identifying reptile species that are potentially vulnerable to anthropocentric disturbances and may require alternative strategies for improving habitat suitability and reptile conservation outcomes in grassy woodland ecosystems.     

Diabetes screening intervals based on risk stratification

Background

Guidelines for frequency of Type 2 diabetes mellitus (DM) screening remain unclear, with proposed screening intervals typically based on expert opinion. This study aims to demonstrate that HbA1c screening intervals may differ substantially when considering individual risk for diabetes.

Methods

This was a multi-institutional retrospective open cohort study. Data were collected between April 1999 to March 2014 from one urban and one rural cohort in Japan. After categorization by age, we stratified individuals based on cardiovascular disease risk (Framingham 10-year cardiovascular risk score) and body mass index (BMI). We adapted a signal-to-noise method for distinguishing true HbA1c change from measurement error by constructing a linear random effect model to calculate signal and noise of HbA1c. Screening interval for HbA1c was defined as informative when the signal-to-noise ratio exceeded 1.

Results

Among 96,456 healthy adults, 46,284 (48.0%) were male; age (range) and mean HbA1c (SD) were 48 (30–74) years old and 5.4 (0.4)%, respectively. As risk increased among those 30–44 years old, HbA1c screening intervals for detecting Type 2 DM consistently decreased: from 10.5 (BMI <18.5) to 2.4 (BMI?>?30) years, and from 8.0 (Framingham Risk Score <10%) to 2.0 (Framingham Risk Score ≥20%) years. This trend was consistent in other age and risk groups as well; among obese 30–44 year olds, we found substantially shorter intervals compared to other groups.

Conclusion

HbA1c screening intervals for identification of DM vary substantially by risk factors. Risk stratification should be applied when deciding an optimal HbA1c screening interval in the general population to minimize overdiagnosis and overtreatment.

     

Detection of HIV-Gag p24-Specific Antibodies in Sera and Saliva of HIV-1-Infected Adults and in Sera of Infants Born to HIV-1-Infected Mothers

Secretory immunoglobulin A (IgA) is known to play an important role in the mucosal defense against a variety of pathogens. Although the role of IgA antibodies during sexual transmission of HIV is not clear, HIV-specific IgA antibodies have been detected in various mucosal secretions of HIV-infected individuals. Using a monoclonal antibody against human IgA, we established an ELISA system to detect anti-HIV p24 IgA antibodies in sera and saliva. We have analyzed the levels of anti-HIV p24 IgG and IgA antibodies in sera and saliva of 107 and 119 adults, respectively, with HIV infection at different clinical stages, and in the sera of 13 infants born to HIV-infected mothers. The level of anti-HIV p24 IgA antibodies was lower in sera and higher in saliva as compared to that of anti-HIV p24 IgG antibodies. Where the percentage of HIV-specific serum antibody-positive cases decreased with disease progression, that of saliva antibody-positive cases increased in AIDS patients. Among the 13 infants born to HIV-infected mothers, 7 infants were HIV-p24-specific serum IgA positive. These sera were negative for anti-HIV p24 secretory IgA, suggesting that some infants develop their own immune responses against HIV infection. Thus, the detection of HIV-specific IgA antibodies, especially in saliva, could be a simple and reliable test for the diagnosis of HIV infection.     

Substrate specificity and gene expression of two Penicillium chrysogenum α-l-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54

We previously isolated two α-l-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an “Alpha-L-AF_C” domain in AFQ1 and “ArabFuran-catal” and “AbfB” domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-l-arabinofuranoside and polysaccharides with different specificities. ¹H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and l-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.     

Status of Systemic Oxidative Stresses in Patients with Primary Open-Angle Glaucoma and Pseudoexfoliation Syndrome

Background

The involvement of local and systemic oxidative stress in intraocular pressure (IOP) elevation and optic nerve damage has been hypothesized in the pathogenesis of glaucoma. To test this, we measured the systemic levels of prooxidants and antioxidants by analyzing the blood biochemistry in patients with glaucoma.

Methods

Peripheral blood samples were collected from Japanese patients with primary open-angle glaucoma (PG) (n = 206), exfoliation syndrome (EX) (n = 199), and controls (n = 126). Plasma levels of lipid peroxides, ferric-reducing activity, and thiol antioxidant activity were measured by diacron reactive oxygen metabolites (dROM), biological antioxidant potential (BAP), and sulfhydryl (SH) tests, respectively, using a free radical analyzer.

Results

In the PG, EX, and control groups, the mean ± standard deviation values were 355±63, 357±69, and 348±56 (U. Carr), respectively, for dROM; 1,951±282, 1,969±252, and 2,033±252 (µmol/L), respectively, for BAP (µmol/L); and 614±98, 584±91, and 617±99 (µmol/L), respectively, for SH. The differences in the BAP values were significant between the PG and control groups (p = 0.0062), for SH between the EX and control groups (p = 0.0017), and for SH between the PG and EX groups (p = 0.0026). After adjustment for differences in age and sex among groups using multiple regression analysis, lower BAP values were correlated significantly with PG (p = 0.0155) and EX (p = 0.0049). Higher dROM values with and without glaucoma were correlated with female gender, and lower SH values with older age. There were no significant differences between the higher (≥21 mmHg) and lower (<21 mmHg) baseline IOPs in the PG group or between the presence or absence of glaucoma in the EX group.

Conclusions

Lower systemic antioxidant capacity that measured by ferric-reducing activity is involved in the pathogenesis of PG and EX.     

Expression of NADH-Dependent Glutamate Synthase in Response to the Supply of Nitrogen in Rice Cells in Suspension Culture

The effects of inorganic and organic nitrogen on the levelsof mRNA for NADH-dependent glutamate synthase (GOGAT) and theprotein were examined in rice cells in suspension culture. Asupply of NH⁺₄, NO^-₃, glutamine, or asparagine induced the accumulationof the protein and mRNA, but levels of mRNA for ferredoxin-GOGATwere hardly affected. ¹Present address: P.C. Center Wakuya-cho, Toda-gun, Miyagi,Japan.     

Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor-specific inhibition of autoreactive Th1 responses in vivo

Prior studies have revealed that the sympathetic nervous system regulates the clinical and pathological manifestations of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model mediated by Th1 T cells. Although the regulatory role of catecholamines has been indicated in the previous works, it remained possible that other sympathetic neurotransmitters like neuropeptide Y (NPY) may also be involved in the regulation of EAE. Here we examined the effect of NPY and NPY receptor subtype-specific compounds on EAE, actively induced with myelin oligodendrocyte glycoprotein 35-55 in C57BL/6 mice. Our results revealed that exogenous NPY as well as NPY Y(1) receptor agonists significantly inhibited the induction of EAE, whereas a Y(5) receptor agonist or a combined treatment of NPY with a Y(1) receptor antagonist did not inhibit signs of EAE. These results indicate that the suppression of EAE by NPY is mediated via Y(1) receptors. Furthermore, treatment with the Y(1) receptor antagonist induced a significantly earlier onset of EAE, indicating a protective role of endogenous NPY in the induction phase of EAE. We also revealed a significant inhibition of myelin oligodendrocyte glycoprotein 35-55-specific Th1 response as well as a Th2 bias of the autoimmune T cells in mice treated with the Y(1) receptor agonist. Ex vivo analysis further demonstrated that autoimmune T cells are directly affected by NPY via Y(1) receptors. Taken together, we conclude that NPY is a potent immunomodulator involved in the regulation of the Th1-mediated autoimmune disease EAE.