Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB, PrtB-BLG, and PrtB-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB-BLG yielded up to 170 μg per 10⁹ CFU in the culture supernatant and 9 μg per 10⁹ CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.     

Inhibitory Impact of Bifidobacteria on the Transfer of β-Lactam Resistance among Enterobacteriaceae in the Gnotobiotic Mouse Digestive Tract

While looking for new means to limit the dissemination of antibiotic resistance, we evaluated the role of potentially probiotic bifidobacteria on the transfer of resistance genes between enterobacteria. Transfers of bla genes encoding extended-spectrum β-lactamases (SHV-5 and CTX-M-15) were studied in the absence or presence of bifidobacteria. In vitro, transfer frequencies of these bla genes decreased significantly in the presence of three of five tested strains, i.e., Bifidobacterium longum CUETM-89-215, Bifidobacterium bifidum CIP-56.7T, and Bifidobacterium pseudocatenulatum CIP-104168T. Four transfer experiments were conducted in the digestive tract of gnotobiotic mice, the first three observing the effect of B. longum CUETM-89-215, B. bifidum CIP-56.7T, and B. pseudocatenulatum CIP-104168T on bla_SHV-5 transfer and the fourth experiment studying the effect of B. bifidum CIP-56.7T on bla_CTX-M-15 transfer. These experiments revealed significant decreases in the transconjugant levels (up to 3 logs) in mice having received B. bifidum CIP-56.7T or B. pseudocatenulatum CIP-104168T compared to control mice. Bifidobacteria appear to have an inhibitory impact on the transfer of antibiotic resistance genes. The inhibitory effect is associated to specific bifidobacterial strains and may be related to the production of thermostable metabolites by these strains.     

Effect of Ligands on HP-Induced Unfolding and Oligomerization of β-Lactoglobulin

To probe intermediate states during unfolding and oligomerization of proteins remains a major challenge. High pressure (HP) is a powerful tool for studying these problems, revealing subtle structural changes in proteins not accessible by other means of denaturation. Bovine β-lactoglobulin (BLG), the main whey protein, has a strong propensity to bind various bioactive molecules such as retinol and resveratrol, two ligands with different affinity and binding sites. By combining in situ HP-small-angle neutron scattering (SANS) and HP-ultraviolet/visible absorption spectroscopy, we report the specific effects of these ligands on three-dimensional conformational and local changes in BLG induced by HP. Depending on BLG concentration, two different unfolding mechanisms are observed in situ under pressures up to ∼300 MPa: either a complete protein unfolding, from native dimers to Gaussian chains, or a partial unfolding with oligomerization in tetramers mediated by disulfide bridges. Retinol, which has a high affinity for the BLG hydrophobic cavity, significantly stabilizes BLG both in three-dimensional and local environments by shifting the onset of protein unfolding by ∼100 MPa. Increasing temperature from 30 to 37°C enhances the hydrophobic stabilization effects of retinol. In contrast, resveratrol, which has a low binding affinity for site(s) on the surface of the BLG, does not induce any significant effect on the structural changes of BLG due to pressure. HP treatment back and forth up to ∼300 MPa causes irreversible covalent oligomerization of BLG. Ab initio modeling of SANS shows that the oligomers formed from the BLG-retinol complex are smaller and more elongated compared to BLG without ligand or in the presence of resveratrol. By combining HP-SANS and HP-ultraviolet/visible absorption spectroscopy, our strategy highlights the crucial role of BLG hydrophobic cavity and opens up new possibilities for the structural determination of HP-induced protein folding intermediates and irreversible oligomerization.     

Microbial Transformation of β-Ionone and β-Methylionone

Aspergillus niger JTS 191 was selected from many microorganisms tested as capable of converting ionones to other compounds having aromas. The individual transformation products from β-ionone were isolated and identified by comparison with synthetically derived compounds. The major products were (R)-4-hydroxy-β-ionone and (S)-2-hydroxy-β-ionone. 2-Oxo-, 4-oxo-, 3,4-dehydro-, 2,3-dehydro-4-oxo-, 3,4-dehydro-2-oxo-, (S)-2-acetoxy-, (R)-4-acetoxy-, and 5,6-epoxy-β-ionone and 4-(2,3,6-trimethylphenyl)-but-3-en-2-one were also identified. Analogous transformation products of β-methylionone also were identified. Based on gas-liquid chromatographic analysis during the fermentation, we propose two main oxidative pathways of β-ionone. The results of this study suggest that these transformations of β-ionones may be useful as tobacco-flavoring compounds.     

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Free beta-tubulin not in heterodimers with alpha-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free beta-tubulin. This study focused specifically on the function of Rbl2p, which, like alpha-tubulin, can rescue cells from free beta-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of beta-tubulin relative to alpha-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of beta-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to beta-tubulin. The data suggest that Rbl2p binds transiently to free beta-tubulin, which then passes into an aggregated form that is not toxic.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Staphylococcal β-Hemolysin I. Purification of β-Hemolysin

The purification of staphylococcal β-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic β-hemolysin yielded one line of precipitate when tested against the original crude β-hemolysin. The purified cationic β-hemolysin was stable in the lyophilized state. Crude β-hemolysin was dermonecrotic, whereas purified cationic β-hemolysin was not dermonecrotic even after Mg⁺⁺ activation.     

Molten globule state of equine β-Lactoglobulin

The acid-unfolded state of equine β-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine β-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedimentation experiments, and it has the exposed hydrophobic surface as indicated by its tendency to aggregate. All of these characteristics indicate that the acid-unfolded state of equine β-lactoglobulin is a molten globule state. The α helix content in the acid-unfolded state, which has been estimated from the circular dichroism spectrum, is larger than that in the native state, suggesting the presence of nonnative α helices in the molten globule state. This result suggests the generality of the intermediate with nonnative α helices during the folding of proteins having the β-clam fold. © 1997 Wiley-Liss Inc.     

Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human ^Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin ^Aγ-globin (βsp/^Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/^Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the ^Aγ-globin gene promoter showed developmental switching and expressed ^Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.     

A model for how Gβγ couples Gα to GPCR

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine–proline–phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR–G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ–rhodopsin interactions may facilitate GPCR dimer transactivation.     

Purification of β-acetylglucosaminase and β-galactosidase from ram testis

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.     

Resistance of Interleukin-1β-Deficient Mice to Fatal Sindbis Virus Encephalitis

Interleukin-1β (IL-1β) concentrations are frequently elevated in central nervous system (CNS) viral infections, but the pathophysiologic significance of such elevations is not known. To examine the role of IL-1β in CNS viral pathogenesis, we compared the natural histories of IL-1β-deficient and wild-type 129 SV(ev) mice infected with a neurovirulent viral strain, neuroadapted Sindbis virus (NSV). We found that the incidence of severe paralysis and death was markedly decreased in NSV-infected IL-1β^−/− mice compared to NSV-infected wild-type mice (4 versus 88%, P < 0.001). Despite this marked difference in clinical outcome, no differences in numbers of apoptotic cells or presence of histopathologic lesions in the brains of moribund wild-type mice and those of clinically healthy IL-1β^−/− mice could be detected. These results suggest that IL-1β deficiency is protective against fatal Sindbis virus infection by a mechanism that does not involve resistance to CNS virus-induced apoptosis or histopathology.     

Production of Epoxides from α,β-Halohydrins by Flavobacterium sp

The relative activity of Flavobacterium whole cells on the enzymatic synthesis of epoxides from α,β-chlorohydrins, -bromohydrins, and -iodohydrins is described.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.