Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-S-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-S,S′-Dihydroxy-2,2′-Bimorphine by Cylindrocarpon didymum

Whole-cell suspensions of Cylindrocarpon didymum were observed to transform 2,2′-bimorphine to the compounds 10-α-S-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-S,S′-dihydroxy-2,2′-bimorphine. Mass spectrometry and ¹H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.     

Degradation of the γ-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4′-Ethoxy-3′-Methoxyphenyl)-2-(4″-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4′-ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [¹⁴C]lignin to ¹⁴CO₂. 1-(4′-Ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by α, β cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under ¹⁸O₂, ¹⁸O was incorporated into the β position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

Exonic Sequences in the 5′ Untranslated Region of α-Tubulin mRNA Modulate trans Splicing in Trypanosoma brucei

Previous studies have identified a conserved AG dinucleotide at the 3′ splice site (3′SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei α-tubulin 3′SS region is required to specify accurate 3′-end formation of the upstream β-tubulin gene and trans splicing of the downstream α-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3′SS identification. Our results indicate that a minimal α-tubulin 3′SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the α-tubulin 3′SS is dependent upon the presence of exon sequences. Furthermore, β-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace α-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the α-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.     

Assessing potentiation of the (α4)3(β2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity “canonical” acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity “noncanonical” ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod–Wyman–Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.     

α- and β-Proteobacteria Control the Consumption and Release of Amino Acids on Lake Snow Aggregates

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4′,6′-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% ± 7.9% and 14.2% ± 10.2% of the DAPI cell counts were detected by probes specific for α- and β-Proteobacteria. These proportions increased to 12.0% ± 3.3% and 54.0% ± 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% ± 1.4% and 41.1% ± 8.4%, indicating a clear dominance of β-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. γ-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the α-Proteobacteria. In addition, with three probes highly specific for close relatives of the β-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the β-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of β-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of α- and β-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.     

The Protein Phosphatase 2A Regulatory Subunits B′β and B′δ Mediate Sustained TrkA Neurotrophin Receptor Autophosphorylation and Neuronal Differentiation

Nerve growth factor (NGF) is critical for the differentiation and maintenance of neurons in the peripheral and central nervous system. Sustained autophosphorylation of the TrkA receptor tyrosine kinase and long-lasting activation of downstream kinase cascades are hallmarks of NGF signaling, yet our knowledge of the molecular mechanisms underlying prolonged TrkA activity is incomplete. Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase composed of a scaffolding, catalytic, and regulatory subunit (B, B′, and B" gene families). Here, we employ a combination of pharmacological inhibitors, regulatory subunit overexpression, PP2A scaffold subunit exchange, and RNA interference to show that PP2A containing B′ family regulatory subunits participates in sustained NGF signaling in PC12 cells. Specifically, two neuron-enriched regulatory subunits, B′β and B′δ, recruit PP2A into a complex with TrkA to dephosphorylate the NGF receptor on Ser/Thr residues and to potentiate its intrinsic Tyr kinase activity. Acting at the receptor level, PP2A/ B′β and B′δ enhance NGF (but not epidermal growth factor or fibroblast growth factor) signaling through the Akt and Ras-mitogen-activated protein kinase cascades and promote neuritogenesis and differentiation of PC12 cells. Thus, select PP2A heterotrimers oppose desensitization of the TrkA receptor tyrosine kinase, perhaps through dephosphorylation of inhibitory Ser/Thr phosphorylation sites on the receptor itself, to maintain neurotrophin-mediated developmental and survival signaling.     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

Genistein-3′-sodium sulfonate Attenuates Neuroinflammation in Stroke Rats by Down-Regulating Microglial M1 Polarization through α7nAChR-NF-κB Signaling Pathway

Microglial M1 depolarization mediated prolonged inflammation contributing to brain injury in ischemic stroke. Our previous study revealed that Genistein-3′-sodium sulfonate (GSS) exerted neuroprotective effects in ischemic stroke. This study aimed to explore whether GSS protected against brain injury in ischemic stroke by regulating microglial M1 depolarization and its underlying mechanisms. We established transient middle cerebral artery occlusion and reperfusion (tMCAO) model in rats and used lipopolysaccharide (LPS)-stimulated BV2 microglial cells as in vitro model. Our results showed that GSS treatment significantly reduced the brain infarcted volume and improved the neurological function in tMCAO rats. Meanwhile, GSS treatment also dramatically reduced microglia M1 depolarization and IL-1β level, reversed α7nAChR expression, and inhibited the activation of NF-κB signaling in the ischemic penumbra brain regions. These effects of GSS were further verified in LPS-induced M1 depolarization of BV2 cells. Furthermore, pretreatment of α7nAChR inhibitor (α-BTX) significantly restrained the neuroprotective effect of GSS treatment in tMCAO rats. α-BTX also blunted the regulating effects of GSS on neuroinflammation, M1 depolarization and NF-κB signaling activation. This study demonstrates that GSS protects against brain injury in ischemic stroke by reducing microglia M1 depolarization to suppress neuroinflammation in peri-infarcted brain regions through upregulating α7nAChR and thereby inhibition of NF-κB signaling. Our findings uncover a potential molecular mechanism for GSS treatment in ischemic stroke.     

Construction and Properties of Escherichia coli Strains Exhibiting α-Complementation of β-Galactosidase Fragments In Vivo

In vivo α-complementation of β-galactosidase was demonstrated in 16 Z gene terminator (nonsense) mutant strains of Escherichia coli upon introduction of the episome F′M15 which specifies production of a mutant Z gene polypeptide containing a small deletion in the N-terminal region of the enzyme monomer. Genetic and biochemical analyses of the merodiploids showed that restoration of enzyme activity was due to their terminator/F′M15 genetic constitution resulting in the production of two enzymatically inactive polypeptides which associate in vivo to reconstitute active, stable β-galactosidase. The prematurely terminated polypeptide fragments known to be rapidly degraded in haploid cells were shown by phenotypic and biochemical studies to be stabilized (i.e., protected) in merodiploids by formation of complemented enzyme complexes with the M15 protein. Phenotypic properties of complementing diploids are described and are discussed in relation to in vitro determination of β-galactosidase activity.     

Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA

The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).     

A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite''s glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)_≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.     

Arylglycerol-γ-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic β-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-γ-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-β-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a ¹³C-labeled compound (compound II′) indicated that the formyl group of compound IV was derived from the β-phenoxyl group of β-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (compound III). γ-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-β,γ-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with ¹³C-labeled compounds II′ and III′ indicated that the carbonate carbon of compound V was derived from the β-phenoxyl groups of β-O-4 substructure.     

The stiffness‐controlled release of interleukin‐6 by cardiac fibroblasts is dependent on integrin α2β1

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin‐6 (IL‐6), interleukin‐11 (IL‐11) and soluble receptor of IL‐6 (sIL‐6R). It also examines the roles of integrin α2β1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2β1 integrin count and released higher levels of IL‐6 and sIL‐6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL‐11 content. The silencing of the α2 integrin subunit decreased the release of IL‐6. Similar effects were induced by TC‐I 15 (an α2β1 integrin inhibitor). The IL‐6 levels in the serum and heart were markedly lower in α2 integrin‐deficient mice B6.Cg‐Itga2^{tm1.1Tkun/tm1.1Tkun} than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL‐6 level. sIL‐6R secretion is not dependent on α2β1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL‐6 by cardiac fibroblasts, and this effect is dependent on α2β1 integrin and kinase Src activation.     

Force Measurements of the α5β1 Integrin–Fibronectin Interaction

The interaction of the α₅β₁ integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α₅β₁/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α₅β₁ and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α₅β₁ expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α₅β₁/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α₅β₁/FN7-10 interactions. The dynamic rupture force of the α₅β₁/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α₅β₁/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.     

Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISP_BPH), a ferredoxin (FER_BPH), and a ferredoxin reductase (RED_BPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISP_BPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISP_BPH of strain LB400 and the β subunit of ISP_BPH of strain B-356 (the α_LB400β_B-356 chimera) and the α_B-356β_LB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP_BPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α_B-356β_LB400 chimera should have behaved like B-356 ISP_BPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP_BPH. On the other hand, the α_LB400β_B-356 chimera showed features of both B-356 and LB400 ISP_BPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.     

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.