Comparative Studies on Generalized Transducing Bacteriophages of Proteus mirabilis, φm and π1

Comparative studies were made on the generalized transducing bacteriophages of Proteus mirabilis φm (Nakaya and Rownd), π1 (Böhme), and a clear plaque-forming mutant φm-c, derived from φm. Electron microscopic observations revealed that these phages were morphologically identical, indicating that they belonged to the group C of Bradley's classification, or to the type Cl of Ackermann's classification. Phages φm and π1 formed characteristic turbid plaques different from each other, and the plaques of π1 were smaller in size than those of φm. The plaques of phage φm-c were clear and also were the largest in size among those studied. Average latent periods of φm and π1 were 70 and 60 min, respectively. Average burst size was found to be 30 and 10 plaque-forming units per infected cell for φm and π1, respectively. It was confirmed by cross neutralization tests that φm and π1 differed serologically from each other. The host range of the two phages also differed, and phage φm was more sensitive to heat than π1. These results indicate that phages φm and π1 are different types of phages. Majority of the properties of phage φm-c were nearly identical with those of phage π1 except that the multiplication of φm-c was more strongly inhibited by methylene blue than that of φm and π1. Phage φm-c is considered to be a clear mutant of φm.     

Medicinal importance of fungal β-(1→3), (1→6)-glucans

Non-cellulosic β-glucans are now recognized as potent immunological activators, and some are used clinically in China and Japan. These β-glucans consist of a backbone of glucose residues linked by β-(1→3)-glycosidic bonds, often with attached side-chain glucose residues joined by β-(1→6) linkages. The frequency of branching varies. The literature suggests β-glucans are effective in treating diseases like cancer, a range of microbial infections, hypercholesterolaemia, and diabetes. Their mechanisms of action involve them being recognized as non-self molecules, so the immune system is stimulated by their presence. Several receptors have been identified, which include: dectin-1, located on macrophages, which mediates β-glucan activation of phagocytosis and production of cytokines, a response co-ordinated by the toll-like receptor-2. Activated complement receptors on natural killer cells, neutrophils, and lymphocytes, may also be associated with tumour cytotoxicity. Two other receptors, scavenger and lactosylceramide, bind β-glucans and mediate a series of signal pathways leading to immunological activation. Structurally different β-glucans appear to have different affinities toward these receptors and thus generate markedly different host responses. However, the published data are not always easy to interpret as many of the earlier studies used crude β-glucan preparations with, for the most part, unknown chemical structures. Careful choice of β-glucan products is essential if their benefits are to be optimized, and a better understanding of how β-glucans bind to receptors should enable more efficient use of their biological activities.     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Studies on (β,1→5) and (β,1→6) linked octyl Galf disaccharides as substrates for mycobacterial galactosyltransferase activity

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.     

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.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

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.     

Synthesis of methyl glycosides of β-(1→6)-linked -galactobiose, galactotriose, and galactotetraose having a 3-deoxy-3-fluoro-β- -galactopyranoside end-residue

Methyl 2,4-di-O-acetyl-3-deoxy-3-fluoro-β- -galactopyranoside was synthesized by sequential tritylation, acetylation, and detritylation of methyl 3-deoxy-3-fluoro-β- -galactopyranoside, and used as the initial nucleophile in the synthesis of methyl β-glycosides of (1→6)-β- -galacto-biose, -triose (20), and -tetraose (22) having a 3-deoxy-3-fluoro-β- -galactopyranoside end-residue. The extension of the oligosaccharide chais, to form the internal units in 20 and 22, was achieved by use of 2,3,4-tri-O-acetyl-6-O-bromoacetyl-α- -galactopyranosyl bromide as a glycosyl donor, and mercuric cyanide or silver triflate as the promotor. While fewer by-products were formed in the reactions involving mercuric cyanide, the reactions catalyzed by silver triflate were stereospecific and yielded only the desired β (trans) products.     

Synthesis, and carbon-13 n.m.r. study, of O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose and O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl (1→3)- -rhamnopyranose, constituents of bacterial, cell-wall polysaccharides

O-α- -Rhamnopyranosyl-(1→3)- -rhamnopyranose (19) and O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α- -rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α- -rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β- -galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyl]-β- -galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-β- -rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β- -galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α- -rhamnopyranosylation at HO-2 or -3 of a free α- -rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α- -Rhamnopyranosylation of HO-6 of gb- -galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O₂,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

Conformational analysis and molecular dynamics simulation of α-(1 → 2) and α-(1 → 3) linked rhamnose oligosaccharides: Reconciliation with optical rotation and NMR experiments

Molecular mechanics and dynamics calculations were carried out on the disaccharides α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → OMe) (1) and α-L-Rhap-(1 → 3)-α-L-Rhap-(1 OMe) (2), and the trisaccharide α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → OMe) (3). The semiflexible conformational behavior of these molecules was characterized by the occupation of a combination of different glycosidic linkage and side-chain conformational positions whose relative occupations were sensitive to dielectric screening. Molecular dynamics simulations of the trisaccharide 3 showed little difference between the linkage conformations in the trisaccharide and the component disaccharides 1 and 2. Experimental optical rotation data of 1 and 2 were obtained as a function of temperature in varying solvents. The molecular models were combined with the semiempirical theory of Stevens and Sathyanarayana to yield calculated optical rotations. Interpretation of the data of both 1 and 2 implied that a combination of conformations, both in glycosidic and side-chain positions, could explain the experimental data. Solvents effects were important in influencing the conformational mix and averaged optical rotation. Three-bond heteronuclear coupling constants ³J_{C, H} were obtained for the glycosidic linkages of 1 and 2 in D₂O and DMSO. Analysis of the coupling constants with a Karplus curve showed that small reductions in the glycosidic torsion angles of the conformations of the models used here of ca. 10°–15° in ϕ and 5°–10° in ψ were required to give better agreement with experiment; a combination of conformations for both 1 and 2 was consistent with the data. There was a negligible influence on the coupling constants of 1 on changing the solvent from D₂O to DMSO. © 1997 John Wiley & Sons, Inc.     

Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomes

The interaction between the integrin α6β4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 Å resolution of the primary α6β4–plectin complex, formed by the first pair of fibronectin type III domains and the N‐terminal region of the connecting segment of β4 and the actin‐binding domain of plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 Å resolution of the β4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.     

Chemical synthesis of a dextran model,poly-α-(1 → 6)-anhydro-D-glucopyranose

Poly-α-(1 → 6)-anhydro-D -glucopyranose has been synthesized by the phosphorus pentafluoride-catalyzed polymerization of l,6-anhydro-2,3,4-tri-O-benzyl-β-D -glucopyranose and subsequent debenzylation. Physical characterization establishes its high polymeric character and stereoregularity.     

Physicochemical properties of (1 → 6)-branched (1 → 3)-β-D-glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data

The physical dimensions of several (1 → 6) branched (1 → 3) -β-D -glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm-like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, M_L = M_w/L_w for the macroscopically linear samples, was estimated to be 2100 ± 200 g mol^?1 nm^?1. The parameter m_L was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed M_L was consistent with the triple-helical structure reported from x-ray diffraction studies and observed degree of side-chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these β-D -glucans were determined to be 140 ± 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of M_L and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages. © 1993 John Wiley & Sons, Inc.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-3-deoxy-3-fluoro -β- -galactopyranoside and related n.m.r. studies

Sequential tritylation, benzoylation, and detritylation of methyl 3-deoxy-3-fluoro-β- -galactopyranoside gave crystalline methyl 2,4-di-O-benzoyl-3-deoxy-3-fluoro-β- -galactopyranoside (9), which was used as the initial nucleophile in the synthesis of the target oligosaccharide (16). Treatment of 9 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-α- -galactopyranosyl bromide gave the corresponding disaccharide derivative 13, having a selectively removable blocking group at O-6′. Debromoacetylation of 13 afforded the disaccharide nucleophile 14 which, when treated with 2,4,6-tri-O-benzoyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide, gave the fully protected trisaccharide 15. Debenzoylation of 15 gave the title glycoside 16. Condensation reactions were performed with silver trifluoromethane-sulfonate as a promoter in the presence of sym-collidine under base-deficient conditions, and gave excellent yields of the desired β-(trans)-products. Analyses of the ¹H- and ¹³C-n.m.r. spectra, as well as determination of the J_CF and J_HF coupling constants, were made by using various one- and two-dimensional n.m.r. techniques.     

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.