Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

Background

Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/Principal Findings

Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females'' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/Significance

Herein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.     

Integrin-β1, not integrin-β5, mediates osteoblastic differentiation and ECM formation promoted by mechanical tensile strain

Background

Mechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-β (β1, β5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-β in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear.

Results

After transfection with integrin-β1 siRNA or integrin-β5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-β1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-β5 siRNA had little effect on the osteoblastic differentiation induced by the strain. At the same time, the result of ECM formation promoted by the strain, was similar to the osteoblastic differentiation.

Conclusion

Integrin-β1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-β5 is not involved in the osteoblasts response to the tensile strain.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0014-y) contains supplementary material, which is available to authorized users.     

The N-terminal Helix Controls the Transition between the Soluble and Amyloid States of an FF Domain

Background

Protein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer’s and Parkinson’s diseases.

Methods

Here, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system.

Results

We show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein.

Conclusions

The data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments.

Significance

The results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.     

Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β

Background

Altered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β over-expression has not yet been evaluated in a mouse model.

Methodology/Principal Findings

We have recently developed a novel transgenic mouse model that over-expresses Pol β. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner''s gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β expression in esophageal adenocarcinomas and squamous carcinomas.

Conclusions/Significance

These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.     

Development of β-Lactoglobulin-Specific Chimeric Human IgEκ Monoclonal Antibodies for In Vitro Safety Assessment of Whey Hydrolysates

Background

Cow’s milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow’s milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.

Objective

An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.

Methods

Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains.

Results

After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow’s milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow’s milk allergic children.

Conclusion

Usage of our ‘unlimited’ source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow’s milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.     

RNA interference for CFTR attenuates lung fluid absorption at birth in rats

Background

Small interfering RNA (siRNA) against αENaC (α-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs.

Methods

Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C₂), we measured CFTR and ENaC expression, extravascular lung water, and mortality.

Results

αENaC and CFTR mRNA and protein decreased by ~80% and ~85%, respectively, following αENaC and CFTR silencing. Extravascular lung water and mortality increased after αENaC and CFTR-silencing. In pSi-C₂-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells αENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced αENaC mRNA and protein. αENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein.

Conclusion

Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.     

Ginsenoside Rg1 Decreases Aβ1–42 Level by Upregulating PPARγ and IDE Expression in the Hippocampus of a Rat Model of Alzheimer's Disease

Background and Purpose

The present study was designed to examine the effects of ginsenoside Rg1 on expression of peroxisome proliferator-activated receptor γ (PPARγ) and insulin-degrading enzyme (IDE) in the hippocampus of rat model of Alzheimer''s disease (AD) to determine how ginsenoside Rg1 (Rg1) decreases Aβ levels in AD.

Experimental Approach

Experimental AD was induced in rats by a bilateral injection of 10 µg soluble beta-amyloid peptide 1–42 (Aβ_1–42) into the CA1 region of the hippocampus, and the rats were treated with Rg1 (10 mg·kg⁻¹, intraperitoneally) for 28 days. The Morris water maze was used to test spatial learning and memory performance. Hematoxylin-eosin staining was performed to analyze the hippocampal histopathological damage. Immunohistochemistry, western blotting, and real-time PCR were used to detect Aβ_1–42, PPARγ, and insulin-degrading enzyme (IDE) expression in the hippocampus.

Key Results

Injection of soluble Aβ_1–42 into the hippocampus led to significant dysfunction of learning and memory, hippocampal histopathological abnormalities and increased Aβ_1–42 levels in the hippocampus. Rg1 treatment significantly improved learning and memory function, attenuated hippocampal histopathological abnormalities, reduced Aβ_1–42 levels and increased PPARγ and IDE expression in the hippocampus; these effects of Rg1 could be effectively inhibited by GW9662, a PPARγ antagonist.

Conclusions and Implications

Given that PPARγ can upregulate IDE expression and IDE can degrade Aβ_1–42, these results indicate that Rg1 can increase IDE expression in the hippocampus by upregulating PPARγ, leading to decreased Aβ levels, attenuated hippocampal histopathological abnormalities and improved learning and memory in a rat model of AD.     

Cell Type-Dependent Induction of DNA Damage by 1800 MHz Radiofrequency Electromagnetic Fields Does Not Result in Significant Cellular Dysfunctions

Background

Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF) as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity.

Objectives

To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation.

Methods

Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay.

Results

Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs), but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level.

Conclusions

RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.     

Transmembrane Protein Alignment and Fold Recognition Based on Predicted Topology

Background

Although Transmembrane Proteins (TMPs) are highly important in various biological processes and pharmaceutical developments, general prediction of TMP structures is still far from satisfactory. Because TMPs have significantly different physicochemical properties from soluble proteins, current protein structure prediction tools for soluble proteins may not work well for TMPs. With the increasing number of experimental TMP structures available, template-based methods have the potential to become broadly applicable for TMP structure prediction. However, the current fold recognition methods for TMPs are not as well developed as they are for soluble proteins.

Methodology

We developed a novel TMP Fold Recognition method, TMFR, to recognize TMP folds based on sequence-to-structure pairwise alignment. The method utilizes topology-based features in alignment together with sequence profile and solvent accessibility. It also incorporates a gap penalty that depends on predicted topology structure segments. Given the difference between α-helical transmembrane protein (αTMP) and β-strands transmembrane protein (βTMP), parameters of scoring functions are trained respectively for these two protein categories using 58 αTMPs and 17 βTMPs in a non-redundant training dataset.

Results

We compared our method with HHalign, a leading alignment tool using a non-redundant testing dataset including 72 αTMPs and 30 βTMPs. Our method achieved 10% and 9% better accuracies than HHalign in αTMPs and βTMPs, respectively. The raw score generated by TMFR is negatively correlated with the structure similarity between the target and the template, which indicates its effectiveness for fold recognition. The result demonstrates TMFR provides an effective TMP-specific fold recognition and alignment method.     

Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain

Background

To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC β₁ and α subunits.

Methodology/Principal Findings

Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged α subunits with the carboxy-terminally tagged β₁ subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the α subunits is close to the carboxy-terminus of the β₁ subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO.

Conclusions/Significance

Based on the ability of an amino-terminal fragment of the β₁ subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (α_catβ_cat), Winger and Marletta (Biochemistry 2005, 44:4083–90) have proposed a direct interaction of the amino-terminal region of β₁ with the catalytic domains. In support of such a concept of “trans” regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed “fluorescent-conjoined” sGC''s by fusion of the α amino-terminus to the β₁ carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.     

Proinflammatory Cytokines Are Elevated in Serum of Patients with Multiple System Atrophy

Background

Despite several lines of evidence from preclinical and post-mortem studies suggesting that inflammation is involved in Multiple System Atrophy (MSA), no previous studies have measured peripheral indices of inflammation in MSA patients.

Methods

We measured C-reactive protein, interleukin (IL)-6, soluble IL-2 receptor and tumor necrosis factor (TNF)-α in blood samples from MSA patients (n = 14) and healthy controls (n = 40).

Results

IL-6 and TNF-α were significantly elevated in MSA patients compared to healthy controls. After controlling for the potentially confounding effects of age, gender, and somatic co-morbidities, a diagnosis of MSA was still significantly associated with high levels of TNF-α. Higher TNF-α levels were associated with less severe motor symptoms and earlier disease stage.

Conclusions

Our findings are in line with the hypothesis that inflammation might be involved at an early stage of MSA pathophysiology.     

Structure of the Altitude Adapted Hemoglobin of Guinea Pig in the R2-State

Background

Guinea pigs are considered to be genetically adapted to a high altitude environment based on the consistent finding of a high oxygen affinity of their blood.

Methodology/Principal Findings

The crystal structure of guinea pig hemoglobin at 1.8 Å resolution suggests that the increased oxygen affinity of guinea pig hemoglobin can be explained by two factors, namely a decreased stability of the T-state and an increased stability of the R2-state. The destabilization of the T-state can be related to the substitution of a highly conserved proline (P44) to histidine (H44) in the α-subunit, which causes a steric hindrance with H97 of the β-subunit in the switch region. The stabilization of the R2-state is caused by two additional salt bridges at the β1/β2 interface.

Conclusions/Significance

Both factors together are supposed to serve to shift the equilibrium between the conformational states towards the high affinity relaxed states resulting in an increased oxygen affinity.     

NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis.

[Methods]

Primary skeletal muscle cells were isolated from the gastrocnemius in C57BL/6 mice. The muscle cells were stimulated by electrical current at 1Hz for 3 minutes in conditions of normal or NAD metabolism related inhibitor treatment. NAD/NADH level, Sirt1 and mitochondria biogenesis related signal factor’s changes were examined in normal or NAD metabolism related inhibitor treated cells.

[Results]

Electrical stimulation (ES) induced muscle contractions significantly increased NAD/NADH levels, NAMPT inhibitor FK-866 inhibited ES-induced NAD formation, which caused SIRT1 expression and PGC-1α deacetylation to decrease. Moreover, NAMPT inhibition decreased mitochondrial biogenesis related mRNA, COX-1 and Tfam levels. Along with AMPK inhibitor, compound C decreases SIRT1 expression, PGC-1α deacetylation and muscle contraction induced mitochondrial biogenesis related mRNA increment. These results indicated that the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis. Inhibition of the AMPK upregulator, Camkkβ, STO-609 decreased AMPK phosphorylation and SIRT1 expression but did not decrease PGC-1α deacetylation. However, CAMKII inhibition via AIP decreased PGC-1α deacetylation.

[Conclusion]

In conclusion, the results indicate that NAMPT plays an important role in NAD metabolism and mitochondrial biogenesis. However, mitochondrial biogenesis is also controlled by different calcium binding protein signals including Camkkβ and CAMKII. [Keyword] Muscle contraction, NAD metabolism, SIRT1, PGC-1 α, mitochondria biogenesis.     

Membrane Bound Monomer of Staphylococcal α-Hemolysin Induces Caspase Activation and Apoptotic Cell Death despite Initiation of Membrane Repair Pathway

Background

Wild type Staphylococcal α-hemolysin (α-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by α-HL are unknown till date.

Results

Cells treated with H35N (a mutant of α-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of α-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of ‘kiss and run’ dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1.

Conclusions

This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal α-hemolysin.     

Persistent Inflammation and Endothelial Activation in HIV-1 Infected Patients after 12 Years of Antiretroviral Therapy

Objective

The study investigated markers of inflammation and endothelial activation in HIV infected patients after 12 years of successful combination antiretroviral treatment (cART).

Methods

Inflammation and endothelial activation were assessed by measuring levels of immunoglobulins, β2-microglobulin, interleukin (IL) 8, tumor necrosis factor α (TNFα), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), sE-Selectin, and sP-Selectin.

Results

HIV infected patients had higher levels of β2-microglobulin, IL-8, TNFα, and sICAM-1 than uninfected controls, and HIV infected patients lacked correlation between platelet counts and sP-Selectin levels found in uninfected controls.

Conclusion

Discrete signs of systemic and vascular inflammation persist even after very long term cART.     

HIF-1α Inhibition Reduces Nasal Inflammation in a Murine Allergic Rhinitis Model

Background

Hypoxia-inducible factor 1α (HIF-1α) is an important regulator of immune and inflammatory responses. We hypothesized that nasal allergic inflammation is attenuated by HIF-1α inhibition and strengthened by HIF-1α stabilization.

Objective

To elucidate the role of HIF-1α in a murine model of allergic rhinitis (AR).

Methods

Mice were pretreated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2) or the HIF-1α inducer cobalt chloride (CoCl₂) in an established AR murine model using ovalbumin (OVA)-sensitized BALB/c mice. HIF-1α and vascular endothelial growth factor (VEGF) expression in nasal mucosa was measured and multiple parameters of allergic responses were evaluated.

Results

HIF-1α and VEGF levels were locally up-regulated in nasal mucosa during AR. Inflammatory responses to OVA challenge, including nasal symptoms, inflammatory cell infiltration, eosinophil recruitment, up-regulation of T-helper type 2 cytokines in nasal lavage fluid, and serum OVA-specific IgE levels were present in the OVA-challenged mice. 2ME2 effectively inhibited HIF-1α and VEGF expression and attenuated the inflammatory responses. Stabilization of HIF-1α by CoCl₂ facilitated nasal allergic inflammation. HIF-1α protein levels in nasal airways correlated with the severity of AR in mice.

Conclusions

HIF-1α is intimately involved in the pathogenesis of nasal allergies, and the inhibition of HIF-1α may be useful as a novel therapeutic approach for AR.     

Activation of Invariant NKT Cells with Glycolipid Ligand α-Galactosylceramide Ameliorates Glucose-6-Phosphate Isomerase Peptide-Induced Arthritis

Objective

Invariant natural killer T (iNKT) cells regulate collagen-induced arthritis (CIA) when activated by their potent glycolipid ligand, alpha-galactosylceramide (α-GalCer). Glucose-6-phosphate isomerase (GPI)-induced arthritis is a closer model of human rheumatoid arthritis based on its association with CD4+ T cells and cytokines such as TNF-α and IL-6 than CIA. Dominant T cell epitope peptide of GPI (GPI325-339) can induce arthritis similar to GPI-induced arthritis. In this study, we investigated the roles of activation of iNKT cells by α-GalCer in GPI peptide-induced arthritis.

Methods

Arthritis was induced in susceptible DBA1 mice with GPI peptide and its severity was assessed clinically. The arthritic mice were treated with either the vehicle (DMSO) or α-GalCer. iNKT cells were detected in draining lymph nodes (dLNs) by flow cytometry, while serum anti-GPI antibody levels were measured by enzyme-linked immunosorbent assay. To evaluate GPI peptide-specific cytokine production from CD4+ T cells, immunized mice were euthanized and dLN CD4+ cells were re-stimulated by GPI-peptide in the presence of antigen-presenting cells.

Results

α-GalCer induced iNKT cell expansion in dLNs and significantly decreased the severity of GPI peptide-induced arthritis. In α-GalCer-treated mice, anti-GPI antibody production (total IgG, IgG1, IgG2b) and IL-17, IFN-γ, IL-2, and TNF-α produced by GPI peptide-specific T cells were significantly suppressed at day 10. Moreover, GPI-reactive T cells from mice immunized with GPI and α-GalCer did not generate any cytokines even when these cells were co-cultured with APC from mice immunized with GPI alone. In vitro depletion of iNKT cells did not alter the suppressive effect of α-GalCer on CD4+ T cells.

Conclusion

α-GalCer significantly suppressed GPI peptide-induced arthritis through the suppression of GPI-specific CD4+ T cells.     

CCL2 Accelerates Microglia-Mediated Aβ Oligomer Formation and Progression of Neurocognitive Dysfunction

Background

The linkages between neuroinflammation and Alzheimer''s disease (AD) pathogenesis are well established. What is not, however, is how specific immune pathways and proteins affect the disease. To this end, we previously demonstrated that transgenic over-expression of CCL2 enhanced microgliosis and induced diffuse amyloid plaque deposition in Tg2576 mice. This rodent model of AD expresses a Swedish β-amyloid (Aβ) precursor protein mutant.

Methodology/Principal Findings

We now report that CCL2 transgene expression accelerates deficits in spatial and working memory and hippocampal synaptic transmission in β-amyloid precursor protein (APP) mice as early as 2–3 months of age. This is followed by increased numbers of microglia that are seen surrounding Aβ oligomers. CCL2 does not suppress Aβ degradation. Rather, CCL2 and tumor necrosis factor-α directly facilitated Aβ uptake, intracellular Aβ oligomerization, and protein secretion.

Conclusions/Significance

We posit that CCL2 facilitates Aβ oligomer formation in microglia and propose that such events accelerate memory dysfunction by affecting Aβ seeding in the brain.     

17-AAG Induces Cytoplasmic α-Synuclein Aggregate Clearance by Induction of Autophagy

Background

The accumulation and aggregation of α-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. α-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved.

Methodology/Principal Findings

In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human α-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of α-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH₄Cl the aggregate clearing effects of 17-AAG were abolished and α-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of α-synuclein aggregates in OLN-A53T cells.

Conclusions

Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which α-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.     

Functions of Phenylalanine Residues within the β-Barrel Stem of the Anthrax Toxin Pore

Background

A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA) moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2β2–2β3 loops of the heptameric precursor to generate a 14-strand transmembrane β barrel.

Methodology/Principal Findings

We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the β barrel, and a third one, F324, that lies part way up the barrel.

Conclusions/Significance

Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.