Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Novel α + β Zr Alloys with Enhanced Strength

Low-alloyed zirconium alloys are widely used in nuclear applications due to their low neutron absorption cross-section. These alloys, however, suffer from limited strength. Well-established guidelines for the development of Ti alloys were applied to design new two-phase ternary Zr alloys with improved mechanical properties. Zr-4Sn-4Nb and Zr-8Sn-4Nb alloys have been manufactured by vacuum arc melting, thermo-mechanically processed by annealing, forging, and aging to various microstructural conditions and thoroughly characterized. Detailed Scanning electron microscopy (SEM) analysis showed that the microstructural response of the alloys is rather similar to alpha + beta Ti alloys. Duplex microstructure containing primary alpha phase particles surrounded by lamellar alpha + beta microstructure can be achieved by thermal processing. Mechanical properties strongly depend on the previous treatment. Ultimate tensile strength exceeding 700 MPa was achieved exceeding the strength of commercial Zr alloys for nuclear applications by more than 50%. Such an improvement in strength more than compensates for the increased neutron absorption cross-section. This study aims to exploit the potential of alpha + beta Zr alloys for nuclear applications.     

α2-macroglobulin associates with β-amyloid peptide and prevents fibril formation

We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa β-amyloid peptide (Aβ), a major constituent of senile plaques in Alzheimer’s disease. The carboxy terminus of α₂-macroglobulin (α2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Aβ, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of α2M with Aβ from the yeast cell, and by formation of SDS-resistant Aβ complexes in polyacrylamide gels by using synthetic Aβ and purified α2M. The association of Aβ with α2M and various purified amyloid binding proteins was assessed by employing a method measuring protein–protein interactions in liquid phase. The dissociation constant by this technique for the α2M–Aβ association using labeled purified proteins was measured (K_d = 350 nM). Electron microscopy showed that a 1:8 ratio of α2M to Aβ prevented fibril formation in solution; the same ratio to Aβ of another acute phase protein, α₁-antichymotrypsin, was not active in preventing fibril formation in vitro. These results were corroborated by data obtained from an in vitro aggregation assay employing Thioflavine T. The interaction of α2M with Aβ suggests new pathway(s) for the clearance of the soluble amyloid peptide.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Intraneuronal β-Amyloid Accumulation: Aging HIV-1 Human and HIV-1 Transgenic Rat Brain

The prevalence of HIV-1 associated neurocognitive disorders (HAND) is significantly greater in older, relative to younger, HIV-1 seropositive individuals; the neural pathogenesis of HAND in older HIV-1 seropositive individuals, however, remains elusive. To address this knowledge gap, abnormal protein aggregates (i.e., β-amyloid) were investigated in the brains of aging (>12 months of age) HIV-1 transgenic (Tg) rats. In aging HIV-1 Tg rats, double immunohistochemistry staining revealed abnormal intraneuronal β-amyloid accumulation in the prefrontal cortex (PFC) and hippocampus, relative to F344/N control rats. Notably, in HIV-1 Tg animals, increased β-amyloid accumulation occurred in the absence of any genotypic changes in amyloid precursor protein (APP). Furthermore, no clear amyloid plaque deposition was observed in HIV-1 Tg animals. Critically, β-amyloid was co-localized with neurons in the cortex and hippocampus, supporting a potential mechanism underlying synaptic dysfunction in the HIV-1 Tg rat. Consistent with these neuropathological findings, HIV-1 Tg rats exhibited prominent alterations in the progression of temporal processing relative to control animals; temporal processing relies, at least in part, on the integrity of the PFC and hippocampus. In addition, in post-mortem HIV-1 seropositive individuals with HAND, intraneuronal β-amyloid accumulation was observed in the dorsolateral PFC and hippocampal dentate gyrus. Consistent with observations in the HIV-1 Tg rat, no amyloid plaques were found in these post-mortem HIV-1 seropositive individuals with HAND. Collectively, intraneuronal β-amyloid aggregation observed in the PFC and hippocampus of HIV-1 Tg rats supports a potential factor underlying HIV-1 associated synaptodendritic damage. Further, the HIV-1 Tg rat provides a biological system to model HAND in older HIV-1 seropositive individuals.     

Interferon α2–Thymosin α1 Fusion Protein (IFNα2–Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2–thymosin α1 fusion protein (IFNα2–Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2–Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2–Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2–Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2–Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

Functional Nonequivalence of α and β Hemes in Human Adult Hemoglobin

Nuclear magnetic resonance studies of the contact-shifted spectra of heme protons in deoxyhemoglobin A from human adults show conclusively that oxygen binds to the alpha hemes in preference to the beta hemes. The preferential binding is produced in 10% hemoglobin solution at neutral pH by either a 15-fold molar excess of 2,3-diphosphoglycerate or a 5-fold molar excess of inositol hexaphosphate. Preferential binding is not observable in the absence of the organic phosphates. The results indicate that the oxygenation of hemoglobin may be described by a sequential model, or by a concerted model that allows the alpha hemes to bind ligand first.     

Multivalent structure of an αβT cell receptor

Whether there is one or multiple αβT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRβ chains incorporate at least two αβTCRs in a single TCR/CD3 complex. Evidence for bispecific αβTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRβ-variable regions. Such (αβ)₂TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (αβ)₂TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.     

Association of calcium channel α1S and β1a subunits is required for the targeting of β1a but not of α1S into skeletal muscle triads

The skeletal muscle L-type Ca²⁺ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca²⁺ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α_1S–β_1a interactions in the incorporation of the functional channel complex into the triad, α_1S and β_1a [or a β_1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α_1S subunit was diffusely distributed in the cytoplasm. On coexpression with the α_1S subunit βGFP distribution became clustered and colocalized with α_1S immunofluorescence. Based on the colocalization of βGFP and α_1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α_1S with and without β_1a restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α_1S mutants in which the β interaction domain had been altered (α_1S-Y366S) or deleted (α_1S-Δ351–380). Although α_1S-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. Thus, β_1a requires the association with the β interaction domain in the I–II cytoplasmic loop of α_1S for its own incorporation into triad junctions, but stable α_1S–β_1a association is not necessary for the targeting of α_1S into the triads or for its normal function in Ca²⁺ conductance and excitation-contraction coupling.     

Noncooperativity of the αβ Dimer in the Reaction of Hemoglobin with Oxygen

The theory that the alphabeta dimer is the functional unit of cooperativity in hemoglobin has been tested by determination of the oxygen equilibrium curve of stable deoxy dimers, obtained by the addition of 0.9 M MgCl(2) to human des-Arg 141alpha-hemoglobin. Cooperativity was absent in this medium, but was regained on transfer of the hemoglobin to a dilute phosphate buffer, where tetramers reformed. X-ray analysis of crystals of oxy- and deoxy-des-Arg hemoglobins showed that the removal of Arg 141alpha would leave the structure of alphabeta dimers unchanged. Nonreactivity of the sulfhydryl groups at 112beta proved that the subunits in deoxy dimers form the same contact as in oxy dimers, namely alpha(1)beta(1), and that no significant dissociation into free subunits occurs in 0.9 M MgCl(2). The absorption spectrum of the deoxy dimers corresponded to the sum of the spectra of the free deoxy alpha and beta subunits, and was different from that of the deoxy tetramer, showing the constraining salt bridges formed by the C-terminal residues in the tetramer to be necessary for the spectral changes normally observed on association of the deoxy subunits.     

Interferon γ induces differential upregulation of α and β chemokine secretion in colonic epithelial cell lines

Background—Production of chemoattractant factorsby the intestinal epithelium may contribute to mucosal infiltration byinflammatory cells in inflammatory bowel disease. Secretion of the α chemokine interleukin 8 (IL-8), a neutrophil chemoattractant, has beenwidely studied, but little is known about epithelial secretion of β chemokines, which are preferentially involved in recruiting monocytes.
Aims—To investigate the profiles of α and β chemokine secretion in colonic cell lines and their differentialmodulation by interferon γ (IFN-γ), a product of activated Tlymphocytes and natural killer cells.
Methods and results—HT29-19A, a model of theCl secretory crypt cell, exhibited a parallel secretionof the α chemokines IL-8 and GROα, which could be markedlyupregulated by tumour necrosis factor α (TNF-α) and IL-1β. Thesecells showed no significant expression of the β chemokines RANTES(regulated upon activation T cell expressed and secreted), MIP-1α(macrophage inflammatory protein 1α), and MCP-1 (monocyte chemotacticprotein 1) under these conditions, but IFN-γ in combination withTNF-α caused a dose dependent induction of RANTES and MCP-1secretion. This was accompanied by a marked increase of RANTES mRNA. Incontrast, IFN-γ had no significant effect on TNF-α stimulated IL-8secretion. Caco-2 cells, with features more typical of villusabsorptive cells, were relatively poor secretors of α chemokines butsecreted high levels of MCP-1 in response to IL-1β. IFN-γ did notinfluence α or β chemokine secretion in these cells.
Conclusions—These studies suggest that intestinalepithelial cells may produce chemokines capable of attracting bothneutrophils and monocytes. The ability of IFN-γ to activate theexpression of β chemokines preferentially could facilitate thedevelopment of chronic inflammatory infiltrates.

Keywords:inflammatory bowel disease; RANTES; interferongamma; chemokine

     

Integrin αvβ1 promotes infection by human metapneumovirus

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for αvβ1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with αv or β1 cDNAs confers hMPV infectivity, whereas reduction of αv and β1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that αvβ1 integrin is a functional receptor for hMPV.     

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na⁺ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na⁺] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na⁺ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.