Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.     

Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor κB (NFκB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated that the initial increase in intracellular calcium ([Ca²⁺]_i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated 10,12 CLA–mediated production of reactive oxygen species (ROS), activation of ERK1/2 and cJun-NH₂-terminal kinase (JNK), and induction of inflammatory genes. 10,12 CLA–mediated binding of NFκB to the promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 and induction of calcium-calmodulin kinase II (CaMKII) β were attenuated by TMB-8. KN-62, a CaMKII inhibitor, also suppressed 10,12 CLA–mediated ROS production and ERK1/2 and JNK activation. Additionally, KN-62 attenuated 10,12 CLA induction of inflammatory and integrated stress response genes, increase in prostaglandin F_2α, and suppression of peroxisome proliferator activated receptor γ protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca²⁺]_i levels, particularly calcium from the ER.     

Preserved central memory and activated effector memory CD4+ T-cell subsets in human immunodeficiency virus controllers: an ANRS EP36 study

Human immunodeficiency virus (HIV) controllers are rare individuals who spontaneously control HIV type 1 replication for 10 years or more in the absence of antiretroviral treatment. In the present study, HIV controllers (n = 11) maintained potent HIV-specific CD4 responses in spite of very low antigenic loads. Their CD4⁺ central memory T (T_CM) cells were characterized by near-normal numbers and preserved interleukin-2 (IL-2) secretion in response to HIV antigens and uniformly high expression of the survival receptor IL-7 receptor α (IL-7Rα). Controllers expressed CCR7 at higher levels than uninfected controls, suggesting differences in T_CM-cell homing patterns. CD4⁺ effector memory T (T_EM)-cell responses were polyfunctional in HIV controllers, while IL-2 secretion was lost in viremic patients. Cytokine production was three times higher in controllers than in treated patients with undetectable viral loads, suggesting an intrinsically more efficient response in the former group. The total CD4⁺ T_EM-cell pool underwent immune activation in controllers, as indicated by increased HLA-DR expression, decreased IL-7Rα expression, a bias towards gamma interferon production upon polyclonal stimulation, and increased macrophage inflammatory protein 1β secretion associated with chronic CCR5 down-regulation. Thus, HIV controllers showed a preserved CD4⁺ T_CM-cell compartment and signs of potent functional activation in the CD4⁺ T_EM-cell compartment. While controllers did not show the generalized immune activation pattern associated with disease progression, they had signs of immune activation restricted to the effector compartment. These findings suggest the induction of an efficient, nondetrimental type of immune activation in patients who spontaneously control HIV.     

Expression of the Mucosal Homing Receptor α4β7 Correlates with the Ability of CD8+ Memory T Cells To Clear Rotavirus Infection

The integrin α₄β₇ plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with α₄β₇ expression. α₄β₇^hi memory phenotype (CD44^hi), α₄β₇⁻ memory phenotype, and presumptively naive (CD44^lo) CD8⁺ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. α₄β₇^hi memory phenotype CD8⁺ cells were highly efficient at clearing rotavirus infection, α₄β₇⁻ memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44^lo cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor α₄β₇.     

Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-α. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-α–activated aortic endothelium, ~80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti–L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by ~80% monocyte attachment to TNF-α–activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte–endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.     

Molecular and Haematological Characteristics of alpha-Thalassemia Deletions in Yogyakarta Special Region,Indonesia

Background:alpha-Thalassemia is caused primarily by deletions of one to two alpha-globin genes and is characterized by absent or deficient production of alpha-globin protein. The South-East Asia (SEA) deletion, 3.7-kb and 4.2-kb deletions are the most common causes. The present study aimed to observe the molecular characteristics of this common alpha-Thalassemia deletions and analyse its haematological parameter.Methods:Blood samples from 173 healthy volunteers from thalassemia carrier screening in Yogyakarta Special Region were used. Haematological parameters were analysed and used to predict the carrier subjects. Genotype of suspected carriers was determined using multiplex gap-polymerase chain reaction and its haematological parameters were compared. The boundary site of each deletion was determined by analysing the DNA sequences.Results:Seventeen (9.8%) of the volunteers were confirmed to have alpha-Thalassemia trait. Of these, four genotypes were identified namely –α^3.7/αα (58.8%), –α^4.2/αα (5.9%), –α^3.7/–α^4.2 (5.9%) and – –^SEA/αα (29.4%). The 5′ and 3′ breakpoints of SEA deletion were located at nt165396 and nt184700 of chromosome 16, respectively. The breakpoint regions of 3.7-kb deletion were 176-bp long, whereas for 4.2-kb deletion were 321-bp long. The haematological comparison between normal and those with alpha-Thalassemia trait genotype indicated a significant difference in mean corpuscular volume (MCV) (p< 0.001) and mean corpuscular haemoglobin (MCH) (p< 0.001). As for identifying the number of defective genes, MCH parameter was more reliable (p= 0.003).Conclusion:The resultant molecular and haematological features provide insight and direction for future thalassemia screening program in the region.Key Words: Allelic Imbalance, Alpha-Thalassemia, Indonesia, Multiplex Polymerase Chain Reaction, Sequence Deletion     

Non-Eosinophilic Nasal Polyps Shows Increased Epithelial Proliferation and Localized Disease Pattern in the Early Stage

BackgroundNon-eosinophilic nasal polyps (NPs) show less inflammatory changes and are less commonly associated with lower airway inflammatory disorders such as asthma, compared with eosinophilic NPs. However, the development of non-eosinophilic NPs which is a predominant subtype in Asian population still remains unclear.MethodsA total of 81 patients (45 with non-eosinophilic NPs and 36 with eosinophilic NPs) were enrolled. Clinical information and computed tomography (CT), endoscopic, and histological findings were investigated. Tissue samples were analyzed for total IgE levels and for mRNA expression levels of interleukin (IL)-4, IL–5, IL–13, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17A, IL–22, IL-23p19, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, and periostin. Immunostaining assessment of Ki–67 as a proliferation marker was performed.ResultsWe found that epithelial in-growing patterns such as pseudocysts were more frequently observed in histological and endoscopic evaluations of non-eosinophilic NPs, which was linked to increase epithelial staining of Ki–67, a proliferating marker. Eosinophilic NPs were characterized by high infiltration of inflammatory cells, compared with non-eosinophilic NPs. To investigate the developmental course of each subtype, CT was analyzed according to CT scores and subtypes. Non-eosinophilic NPs showed more localized pattern and maxillary sinus involvement, but lesser olfactory involvement in early stage whereas eosinophilic NPs were characterized by diffuse ethmoidal and olfactory involvement. In addition, high ethmoidal/maxillary (E/M) CT scores, indicating ethmoidal dominant involvement, were one of surrogate markers for eosinophilic NP. E/M CT scores was positively correlated with levels of T_H2 inflammatory markers, including IL–4, IL–5, periostin mRNA expression and total IgE levels in NPs, whereas levels of the T_H1 cytokine, IFN- γ were inversely correlated. Moreover, if the combinatorial algorithm meet the three of the four markers, including IL–5 (<2.379), periostin (<3.889), IFN-γ (>0.316), and E/M ratio (<2.167), non-eosinophilic CRSwNP are diagnosed with a sensitivity of 84.4% and a specificity of 84.8%.ConclusionHistologic, immunologic and clinical data suggest that non-eosinophilic NPs showed enhanced epithelial alteration and more localized maxillary involvement. Combination of cutoff value on IL–5, periostin, IFN-γ, and E/M scores may be one of surrogate markers for non-eosinophil NP subtype.     

Molecular Plasticity Regulates Oligomerization and Cytotoxicity of the Multipeptide-length Amyloid-β Peptide Pool

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aβ_1–38 and Aβ_1–40 at the cost of longer Aβ peptides. Here, we consider the aggregation of Aβ_1–38 and Aβ_1–43 in addition to Aβ_1–40 and Aβ_1–42, in particular their behavior in mixtures representing the complex in vivo Aβ pool. We demonstrate that Aβ_1–38 and Aβ_1–43 aggregate similar to Aβ_1–40 and Aβ_1–42, respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aβ, Aβ_1–38 and Aβ_1–43 significantly affect the behaviors of Aβ_1–40 and Aβ_1–42. The short timescale conformational flexibility of Aβ_1–38 is suggested to be responsible for enhancing toxicity of Aβ_1–40 while exerting a cyto-protective effect on Aβ_1–42. Our results indicate that the complex in vivo Aβ peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.     

The Influence of Immunization Route,Tissue Microenvironment,and Cytokine Cell Milieu on HIV-Specific CD8+ T Cells Measured Using Fluidigm Dynamic Arrays

Thirty different genes including cytokines, chemokines, granzymes, perforin and specifically integrins were evaluated in Peyer''s patch-KdGag_197–205-specific CD8+ T cells (pools of 100 cells) using Fluidigm 48.48 Dynamic arrays following three different prime-boost immunization strategies. Data revealed that the route of prime or the booster immunization differentially influenced the integrin expression profile on gut KdGag_197–205-specific CD8+ T cells. Specifically, elevated numbers of integrin αE and αD expressing gut KdGag_197–205-specific CD8+ T cells were detected following mucosal but not systemic priming. Also, αE/β7 and αD/β2 heterodimerization were more noticeable in an intranasal (i.n.)/i.n. vaccination setting compared to i.n./intramuscular (i.m) or i.m./i.m. vaccinations. Moreover, in all vaccine groups tested α4 appeared to heterodimerize more closely with β7 then β1. Also MIP-1β, RANTES, CCR5, perforin and integrin α4 bio-markers were significantly elevated in i.n./i.m. and i.m./i.m. immunization groups compared to purely mucosal i.n./i.n. delivery. Furthermore, when wild type (WT) BALB/c and IL-13 knockout (KO) mice were immunized using i.n./i.m. strategy, MIP-1α, MIP-1β, RANTES, integrins α4, β1 and β7 mRNA expression levels were found to be significantly different, in mucosal verses systemic KdGag_197–205-specific CD8+ T cells. Interestingly, the numbers of gut KdGag_197–205-specific CD8+ T cells expressing gut-homing markers α4β7 and CCR9 protein were also significantly elevated in IL-13 KO compared to WT control. Collectively, our findings further corroborate that the route of vaccine delivery, tissue microenvironment and IL-13 depleted cytokine milieu can significantly alter the antigen-specific CD8+ T cell gene expression profiles and in turn modulate their functional avidities as well as homing capabilities.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

Determinants of muscle power and force as assessed by Jumping Mechanography in rural Indian children

Objectives:To: 1. Assess muscle function (MF) of rural Indian children (6-11y, n=232), using Jumping Mechanography (JM) and hand dynamometer, 2. Investigate gender differences, 3. Identify determinants of MF.Methods:Data on anthropometry, muscle mass%, diet, physical activity, sunlight exposure, MF (maximum relative power Pmax/mass, maximum relative force Fmax/BW by JM; relative grip strength (RGS) by hand dynamometer) were collected. Pearson’s correlation and hierarchical linear regression was performed.Results:Pmax/mass, Fmax/BW and RGS of the group were 31.7±5.0W/kg, 3.0±0.3 and 0.4±0.1 (mean±SD), respectively. The Pmax/mass Z-score was –1.1±0.9 and Fmax/BW Z-score was –0.9±1 (mean±SD) which was significantly lower than the machine reference data (p<0.05). Positive association of muscle mass% and protein intake was observed with all MF parameters and moderate+vigorous physical activity with Fmax/BW (p<0.05). Determinants of MF identified through regression for Pmax/mass were age (β=1.83,95% CI=0.973 – 2.686), muscle mass% (β=0.244,95% CI=0.131–0.358) and protein intake (β=3.211,95% CI=1.597–4.825) and for Fmax/BW was protein intake (β=0.130,95% CI=0.023–0.237) (p<0.05). Male gender was a positive predictor of having higher Pmax/mass (β=1.707,95% CI=0.040–3.373) (p<0.05).Conclusion:MF was lower than in western counterparts. To optimize MF of rural Indian children, focus should be on improving muscle mass, ensuring adequate dietary protein, and increasing physical activity, especially in girls.     

Human Innate Mycobacterium tuberculosis–Reactive αβTCR+ Thymocytes

The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-γ. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-γ to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4⁻ αβTCR⁺ innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-γ directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific αβTCR⁺ T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

Direct and Regulated Interaction of Integrin αEβ7 with E-Cadherin

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, α_Eβ₇, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant α_Eβ₇, and to α_Eβ₇ solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY′ cells expressing the α_Eβ₇ integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to α_Eβ₇ or E-cadherin.

The binding of α_Eβ₇ integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through α_Eβ₇ requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the α_E-chain is unique among integrins, the avidity of α_Eβ₇ for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of α_Eβ₇ for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the α_Eβ₇ integrin.

     

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1–3(GlcNAcβ1–6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation.     

Domain specific interaction in the XRCC1-DNA polymerase beta complex

XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase β (β-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain–domain interaction in the XRCC1-β-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I–linker–BRCT-II C-terminal fragment and the linker–BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact β-Pol and the β-Pol 31 kDa domain. The XRCC1-NTD_1–183 (residues 1–183) was found to bind β-Pol, the β-Pol 31 kDa domain and the β-Pol C-terminal palm-thumb (residues 140–335), and the interaction was further localized to XRCC1-NTD_1–157 (residues 1–157). The XRCC1-NTD_1–183-β-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36–355 and residues 1–159, were found to interact with β-Pol, the β-Pol 31 kDa domain, and the β-Pol C-terminal thumb-only domain polypeptides expressed from the respective β-Pol constructs. Neither the XRCC1-NTD_1–159, nor the XRCC1_36–355 polypeptide was found to interact with a β-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75–212) showed no interaction with β-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD_1–183 was found to bind β-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K_d of 0.4–2.4 µM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD_1–159 and β-Pol that is of an affinity comparable to other binding interactions involving β-Pol.     

Abeta42-Induced Neurodegeneration via an Age-Dependent Autophagic-Lysosomal Injury in Drosophila

The mechanism of widespread neuronal death occurring in Alzheimer''s disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Aβ_1–42) is believed to play a causative role in the development of AD. Here we expressed human Aβ_1–42 and amyloid beta 40 (Aβ_1–40) in Drosophila neurons. Aβ_1–42 but not Aβ_1–40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Aβ_1–42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Aβ_1–42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Aβ_1–42 and Aβ_1–40, and reveal an Aβ_1–42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.     

Cardiolipin interacts with beta‐2‐glycoprotein I and forms an open conformation–Mechanisms analyzed using hydrogen/deuterium exchange

Beta‐2‐glycoprotein I (β₂GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β₂GPI can be recognized by the anti‐β₂GPI antibody. Here, we prepared the anionic di‐oleoyl‐phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β₂GPI. The conformational changes of β₂GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21–27 significantly increased after β₂GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21–27 from the ring conformation. After β₂GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1–20, 21–27, 196–205, 273–279 and 297–306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70–86, 153–162, 191–198, 196–205 and 273–279 indicated β₂GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β₂GPI at sequences 21–27 and forms an intermediate conformation after 10 min of interaction. After a complete protein–lipid interaction, high negatively charged CL membrane induced a major conformation transition of β₂GPI.