Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation

Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema et al., 2004, Plant Mol. Biol. 54: 853–863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the β-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 87.     

Dynamics of hydrogen-deuterium exchange in Chlamydomonas centrin

Chlamydomonas reinhardtii centrin is a 169-amino acid residue calcium binding protein belonging to the EF-hand protein superfamily. Centrin is associated with the microtubule organizing center (MTOC) in all eukaryotes, and in Chlamydomonas, centrin is a component of the flagellar basal body apparatus. Recombinant full-length centrin, calmodulin, and terminal domain fragments [Ccen-N (residues 1-94) and Ccen-C (residues 99-169)] were used to examine hydrogen-deuterium (H --> D) exchange dynamics using combined attenuated total reflectance (ATR) Fourier transform-infrared (FT-IR) spectroscopy, curve fit, and two-dimensional correlation analysis. Analysis of the Ccen-N and Ccen-C fragments allowed separation of domain specific solvent exchange events and together with analysis of the full-length proteins provides novel insight into domain accessibility to the aqueous environment and the internal dynamics of the protein.     

Beta-catenin regulates differentiation of respiratory epithelial cells in vivo

An activated form of beta-catenin [Catnb(Delta(ex3))] was expressed in respiratory epithelial cells of the developing lung. Although morphogenesis was not altered at birth, air space enlargement and epithelial cell dysplasia were observed in the early postnatal period and persisted into adulthood. The Catnb(Delta(ex3)) protein caused squamous, cuboidal, and goblet cell dysplasia in intrapulmonary conducting airways. Atypical epithelial cells that stained for surfactant pro protein C (pro-SP-C) and had morphological characteristics of alveolar type II cells were observed in bronchioles of the transgenic mice. Catnb(Delta(ex3)) inhibited expression of Foxa2 and caused goblet cell hyperplasia associated with increased staining for mucins and the MUC5A/C protein. In vitro, both wild type and activated beta-catenin negatively regulated the expression of the Foxa2 promoter. Catnb(Delta(ex3)) also caused pulmonary tumors in adult mice. Activation of beta-catenin caused ectopic differentiation of alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, demonstrating a critical role for the Wnt/beta-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung.     

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.     

Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant

Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r²) were routinely achieved for a broad range of antigen doses from 0 to 150 μg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen–adjuvant desorption methods.     

Correlates of dental visits among community‐residing Latino elders: a public health alert

doi:10.1111/j.1741‐2358.2009.00335.x
Correlates of dental visits among community‐residing Latino elders: a public health alert Objectives: To examine oral service utilisation in a probability sample of community‐residing Latino elders. Background: Older Latinos are at a potential increased risk of oral diseases, given their higher prevalence of co‐morbidities and lower rate of dental service utilisation. Methods: A prevalence survey was conducted among a random sample of Latino (largely Puerto Rican) elders (n = 205; mean age = 75.8; SD ± 5.3) in New York City during 2001–2002. A systematic random sample was drawn from the Centers for Medicare and Medicaid Services Beneficiary tape files. Current use of oral health services and self‐reported health conditions was obtained. Functional and cognitive impairment were assessed. Results: Less than half of the sample reported a dental visit in the previous year. The average time since the last dental visit was 54 months (SD ± 84.5). Last year dental visit compliers were more likely to be unmarried, living alone, with higher levels of education, fewer health conditions and less impairment with activities of daily living. In multivariate analyses, problem‐oriented behaviour, Medicaid beneficiary, education, living alone, chronic health conditions and mobility impairment explained 14% of the ‘time since last dental visit’ variance. Conclusions: Given that socio‐demographic and level of functioning determinants appear to influence the frequency of dental visits, a multilevel approach to oral health promotion is imperative.     

Cutting edge: NLRC5-dependent activation of the inflammasome

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1β, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1β, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.     

Mycophenolic acid differentially impacts B cell function depending on the stage of differentiation

Production of pathogenic Abs contributes to disease progression in many autoimmune disorders. The immunosuppressant agent mycophenolic acid (MPA) has shown clinical efficacy for patients with autoimmunity. The goal of these studies was to elucidate the mechanisms of action of MPA on B cells isolated from healthy individuals and autoimmune patients. In this study, we show that MPA significantly inhibited both proliferation and differentiation of primary human B cells stimulated under various conditions. Importantly, MPA did not globally suppress B cell responsiveness or simply induce cell death, but rather selectively inhibited early activation events and arrested cells in the G0/G1 phase of the cell cycle. Furthermore, MPA blocked expansion of both naive and memory B cells and prevented plasma cell (PC) differentiation and Ab production from healthy controls and individuals with rheumatoid arthritis. Finally, whereas MPA potently suppressed Ig secretion from activated primary B cells, terminally differentiated PCs were not susceptible to inhibition by MPA. The target of MPA, IMPDH2, was found to be downregulated in PCs, likely explaining the resistance of these cells to MPA. These results suggest that MPA provides benefit in settings of autoimmunity by directly preventing activation and PC differentiation of B cells; however, MPA is unlikely to impact autoantibody production by preexisting, long-lived PCs.     

Cytomegalovirus e127 protein interacts with the inhibitory CD200 receptor

CD200 is a cell surface glycoprotein that binds an inhibitory receptor (CD200R) on myeloid cells. CD200 orthologues are present in many species of virus, and we show that the rat cytomegalovirus CD200 orthologue (e127) is expressed at the cell surface on infected cells. It binds the host CD200R with the same affinity as that of the host protein, and thus this protein acts as a close mimic of the host protein and has the potential to downregulate immune responses to the virus.     

The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with K(d) values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.