Inhibitory mechanism of the Qβ lysis protein A2

The lysis protein A₂, present as a single copy on the surface of Qβ virion particles, was previously shown to inhibit the activity of MurA, an enzyme that catalyses the first committed step of murein biosynthesis. Here we report experiments with a two‐hybrid study that indicates A₂ and MurA interact directly. Moreover, experiments with a soluble MBP–A₂ fusion indicate that the interaction between MurA and A₂ is dependent on a substrate‐induced conformational change featured in the UDP‐NAG‐liganded state of MurA but not the tetrahedral intermediate state. Moreover, based on the location of L138Q, the original dominant A₂‐resistant mutant that identified MurA as the target, a directed mutagenesis strategy has identified a continuous surface required for A₂ binding. This surface spans the catalytic loop/cleft and encompasses both the catalytic and C‐terminal domains. These data support a model in which A₂ preferentially binds MurA liganded with UDP‐NAG, thereby preventing catalysis by occluding PEP from accessing the active site.     

Real-Time Fluorescence Detection of RNA Amplified by Qβ Replicase

Amplification of RNA probes by Qβ replicase can be used to detect a wide range of analytes with a potential sensitivity of a single molecule. A system has been developed in which Qβ amplification of midivariant-(MDV)-based RNA is measured in real time by fluorescence. This was accomplished by including a fluorescent intercalating dye, propidium iodide, in the reactions and monitoring the fluorescence change using a custom fluorometer. The time at which fluorescence is detectable above background is referred to as the "response time" and is calculated using curve-fitting algorithms. A response time is inversely and linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permits an unknown amount of input RNA probe to be quantified through 11 orders of magnitude when compared to a standard curve. Under the described conditions with MDV RNA, the response time occurs when about 3 × 10¹¹ RNA molecules are synthesized and occurs within the exponential phase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine the replication properties of MDV RNA reporter molecules bearing specific probe sequences and to develop hybridization assays for the clinical diagnostic field.     

Alteration of a Host Character by Infection with RNA Phage Qβ

An F + strain of E. coli K 12, W-2241 has a genetic constitution lac^– (i⁺ z⁺ y^–), str-r. The inability of this strain to utilize lactose is due to a deletion at the y locus controlling formation of an enzyme permease and therefore, a true lac⁺ revertant can not be expected. The strain, however, produces many colonies on minimal lactose agar medium, when it is plated after infection with RNA phage Qβ, while much fewer colonies are observed on the same medium seeded with uninfected cells. In analyzing this phenomenon, we discovered that these LAC⁺ colonies were not the result of a mechanism similar to transduction but produced by a mechanism related to phage liberation, and that the colonies originated from Qβ⁺, const cells. The data suggest the following working hypothesis for the mechanism by which LAC⁺ colonies are produced in a population of W-2241 cells infected with phage Qβ. Constitutive cells carrying Qβ produce intracellular β-galactosidase and at the stage of phage liberation, the enzyme is liberated into the medium, hydrolyzing lactose to form glucose. Since glucose could be utilized by all of the cells on the medium, the Qβ⁺, const cell acts as a colony forming center. The phenomenon described in this paper shows a new type of virus-host relationship which may have some bearing on the influence of animal or plant viruses on host tissues.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

噬菌体Qβ病毒样颗粒的制备、纯化及鉴定

目的: 利用大肠杆菌原核表达系统制备噬菌体Qβ VLPs,验证其自组装能力,纯化后免疫动物以确定其免疫原性,同时制备兔多克隆抗体验证其在哺乳动物细胞中内化能力。方法: 合成pET-28-Qβ-CP质粒,利用大肠杆菌原核表达系统制备Qβ VLPs,通过蔗糖密度梯度离心纯化VLPs,将经凝胶层析柱Sephacryl S-400纯化的Qβ VLPs用透射电镜观察颗粒形态;纯化后的Qβ VLPs加入或不加入佐剂分别免疫新西兰兔,获得血清后用Protein G纯化得到兔多克隆抗体,通过Western blot确定制备的兔多克隆抗体特异性,并利用间接免疫荧光法对Qβ VLPs的细胞内化能力进行鉴定。结果: 制备并获得纯度较高的Qβ VLPs,通过透射电镜观察到大量直径约为28 nm的颗粒;Western blot结果表明制备的兔多克隆抗体能特异性识别Qβ VLPs,且在免疫实验中加入佐剂与不加入佐剂分别免疫动物,对抗体水平的影响不显著。间接免疫荧光法结果表明Qβ VLPs在哺乳动物细胞中具有内化能力。结论: 成功制备Qβ VLPs为后续研发以噬菌体Qβ VLPs为载体的相关疫苗奠定基础。     

Inhibition of bacterial cell wall mucopeptide synthesis: A new function of RNA bacteriophage Qβ

Studies of the morphology of Escherichia coli showed that these bacilli when infected with RNA phage Qβ in broth containing hypertonic sucrose and Mg²⁺ formed osmotically labile spherical cells or spheroplasts. Phage-induced spheroplasts readily released their burst of phage when diluted into ordinary culture broth without sucrose. Investigation of the mechanism of host cell lysis revealed that incorporation of [³H]diaminopimelic acid (DAP) into the mucopeptide layer of the cell wall was markedly inhibited starting at about the midpoint of the phage replication cycle. The major site of inhibition is the DAP-containing mucopeptide layer since the synthesis of the lipoprotein-lipopolysaccharide layer, making up the bulk of the cell wall of E. coli, was not affected. A model for Qβ-mediated cell lysis is discussed which is analogous to penicillin-induced cell rupture.     

In vitro site-directed mutagenesis: Generation and properties of an infectious extracistronic mutant of bacteriophage Qβ

An infectious extracistronic mutant of phage Qβ has been prepared by site-directed mutagenesis. Qβ RNA minus strands containing the mutagenic base analog N⁴-hydroxy-CMP instead of UMP at position 39 from the 5′ end were synthesized in vitro and used as template for Qβ replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A → G transition at position 40 from the 3′-end (which corresponds to position 39 of the minus strand). Thus, the viability of phage Qβ does not depend on a unique nucleotide sequence in the 3′-extracistronic RNA segment.Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that Qβ RNA containing an A → G transition in nucleotide −40 of Qβ RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo.The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA.     

Piezoelectric properties of poly-β-hydroxybutyrate and copolymers of β-hydroxybutyrate and β-hydroxyvalerate

The shear piezoelectricity was observed in oriented films of poly-β-hydroxybutyrate (PHB) and copolymers of β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV). The piezoelectric stress constant 3₁₄ = e′₁₄ − ie″₁₄ (polarization/strain), the piezoelectric strain constant d₁₄ = d′₁₄ − id″₁₄ (polarization/stress), the elastic constant c = c′ + ic″ and the dielectric constant = ′ − i″ were determined at a frequency of 10 Hz over a temperature range from −150° to +150°C. Piezoelectric relaxations as well as elastic and dielectric relaxations were clearly observed at the glass transition temperature of about 15°C. In order to evaluate the piezoelectric constants (e₂ and d₂) for the piezoelectric phase which consists of the crystalline region and the oriented non-crystalline region, a spherical dispersion two phase model was utilized. Assuming the appropriate fixed values for the elastic and dielectric constants in the piezoelectric phase, d₂ and d₂ were calculated as a function of temperature. For a PHB and a copolymer (17 HV/83 HB), e₂ and d₂ showed relaxations, leading to a conclusion that the instantaneous piezoelectric constant in the crystalline phase is constant independent of temperature but the piezoelectric constant in the oriented non-crystalline phase is relaxational and has the opposite sign. For a copolymer (25 HV/75 HB) and a chloroform treated copolymer (17 HV/83 HB), e₂ and d₂ were constant independent of temperature, indicating that the oriented non-crystalline phase has disappeared owing to the increased molecular flexibility due to copolymerization or annealing in chloroform vapour.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

N-Nitroso-β-lactams as β-lactamase inhibitor

N-Nitroso-β-phenyl-β-lactam has been found to be a specific inhibitor of β-lactamase. N-Nitroso--phenyl-β-lactam, by contrast, was virtually ineffective although a transient inhibition of short duration was observed. The acyl enzyme derived from the β-phenyl isomer is presumably involved in a cross-linking reaction, whereas that from the -phenyl isomer was quenched by spontaneous hydrolysis without formation of a covalent bond. No inhibitory effect of the β-phenyl isomer on chymotrypsin has been observed.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca²⁻ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.     

Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β₂‐glycoprotein I (β₂GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen‐activated protein kinase (MAPK) pathway plays an important role in aPL‐induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β₂GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β₂GPI interacts with plasma gelsolin, which binds to integrin a₅β₁ through fibronectin. The tethering of β₂GPI to monoclonal anti‐β₂GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti‐β₂GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti‐integrin a₅β₁ antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin‐integrin signalling pathway, was phosphorylated by anti‐β₂GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti‐β₂GPI antibody‐induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.     

Divalent cations regulate the organization of integrins αvβ3 and αvβ5 on the cell surface

Extracellular divalent cations are important regulators of integrin ligand binding activity. In this study we evaluated how divalent cations affect the organization of integrins into focal adhesion sites. Integrins αvβ3 and αvβ5 were compared because they share a high degree of structural homology and because both integrins mediate cell adhesion to vitronectin. On MG-63 osteosarcoma cells, we found that both the extent and pattern of integrin organization was regulated by the type of extracellular divalent ion. Integrin αvβ3 organized in focal contacts when Mn²⁺ or Mg²⁺ was present, but not in Ca²⁺. In contrast, αvβ5 organized in focal contacts only when Ca²⁺ or Mg²⁺ was present. Integrin αvβ5 clustered in a centrally located punctate field on the ventral surface of the cell in the presence of Mn²⁺. These observations reveal a previously unappreciated role for divalent ions in regulating the organization of integrins into focal adhesion sites. © 1996 Wiley-Liss, Inc.     

LYMPHOTOXIN-α (TNF-β) DURING SEPSIS

T cell release of lymphotoxin-α (LT-α, or TNF-β) is stimulated by pyrogenic exotoxins of Gram-positive bacteria and mitogens. In contrast to TNF-α, it is unknown whether LT-α plays any role in the pathogenesis of sepsis and, in particular, the pathogenesis of Gram-positive sepsis. Sera from patients with sepsis were examined for LT-α and compared with normal volunteers and pregnant women. LT-α was detected in 33% of sepsis sera (mean 608.4 pg/ml SE 306), 16% of normal sera (mean 167 pg/ml SE 87) and 23% of sera from pregnant women (mean 714 pg/ml SE 191). These differences were not significant and there were no differences within species sera when grouped by the type of causative organism, or disease severity. LT-α detected by immunoassay in serum was not bioactive, in contrast to that produced in cell culture. Recombinant soluble TNF receptors (rSTNFR) neutralized the bioactivity of recombinant LT-α at rSTNFR concentrations which did not interfere with immunoreactivity and which are known to prevailin vivo. Hence, LT-α is unlikely to have a critical role in the pathogenesis of sepsis. Much of the potential bioactivity of this lymphokine may be abrogated by TNFR in serum.     

Δ-β-Sitosten-3-one from β-sitosterol by leaf homogenates

β-Sitosterol-4-¹⁴C is metabolized to Δ⁴-β-sitosten-3-one by Cheiranthus cheiri leaf homogenates. Greater than 60% conversion occurs within 2 hr. Under identical conditions, leaf homogenates of Strophanthus kombé fail to metabolize β-sitosterol, while Digitalis purpurea leaf homogenates yield only very small amounts of the metabolite.     

Modeling the αIIbβ3 integrin solution conformation

The α_IIbβ₃ platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of α and β subunits. Electron microscopic (EM) images of the α_IIbβ₃ complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the α_IIbβ₃ complex. We have studied the shape of detergent-solubilized α_IIbβ₃ by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100). Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized α_IIbβ₃ complex, where the relative spatial location of specific domains the size of 5–10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.