Chain Precursor Research Articles

VanX, a bacterial D-alanyl-D-alanine dipeptidase: resistance, immunity, or survival function?

The zinc-containing D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in D-alanyl-D-lactate (D-Ala-D-lactate) rather than D-Ala-D-Ala. The modified peptidoglycan exhibits a 1, 000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to D-Ala-D-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (sigma(s)). The combined action of DdpX and the permease would permit hydrolysis of D-Ala-D-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The D-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

Reinforcement of Poly(dimethylsiloxane) Elastomers by Chain-End Anchoring to Clay Particles

Difunctional hydroxyl-terminated and vinyl-terminated poly(dimethylsiloxane) (PDMS) precursors with similar molecular weight distributions were used to synthesize end-linked networks in bulk with appropriate tetrafunctional cross-linkers. Composite PDMS elastomers from the same precursors were also synthesized with low concentrations of montmorillonite nanosize clay particles. For unfilled networks, larger amounts of tetraethyl orthosilicate (TEOS) cross-linkers than conventionally used led to optimal networks with higher moduli and lower soluble fractions. In the montmorillonite−PDMS elastomeric composites, enhancement of the modulus was obtained only for nonoptimal networks formed with the hydroxyl-terminated precursor chains but not with the vinyl-terminated chains. These results indicate that the reinforcement in these elastomers can be attributed to the anchoring of the hydroxyl end group to the silicate filler that dramatically reduces the soluble fraction and binds pendent chain ends. The modulus o...

Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII.

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.

An enantiocontrolled synthesis of the masked taxol C-13 side chain, oxazoline carboxylic acid

Oxazoline carboxylic acid 6 as the taxol side chain precursor has been efficiently synthesized via the intramolecular iodoamidation of allylic trichloroacetimidate derived from trans-olefinic diol 1.

One-step synthesis of paclitaxel side-chain precursor: benzamide-based asymmetric aminohydroxylation of isopropyl trans-cinnamate

A highly enantioselective (up to 97% ee) one-step synthesis of paclitaxel side chain precursor, (2 R,3 S)-isopropyl 3-benzamido-2-hydroxy-3-phenylpropionate, has been achieved by osmium-catalyzed asymmetric aminohydroxylation of isopropyl trans-cinnamate with N-bromobenzamide as an oxidant/nitrogen source in the presence of (DHQ) 2PHAL as a chiral ligand. Simple recrystallization of crude product (containing regioisomer and diol) from ethyl acetate gave the enantiomerically pure product.

Anticoagulant Heparan Sulfate Precursor Structures in F9 Embryonal Carcinoma Cells

To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. In this study, HSact precursor structures have been studied by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by 3'-phosphoadenosine 5'-phospho-35S and purified 3-O-sulfotransferase-1. This later in vitro labeling allows the regions of HS destined to become the antithrombin (AT)-binding sites to be tagged for subsequent structural studies. It was shown that six 3-O-sulfation sites exist per HSact precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HSact precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HSinact precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and HSinact precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HSact hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged HSact hexasaccharides exhibited the following structures, DeltaUA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS3(35)S+/-6S-++ +IdceA2S-[6-3H]Glc NS6S. The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the 3-O-sulfate moiety. The presence of five potential AT-binding precursor hexasaccharides in all HSact precursor chains demonstrates for the first time the processive assembly of specific sequence in HS. The difference in structures around potential 3-O-sulfate acceptor sites in HSact and HSinact precursors suggests that these precursors might be generated by different concerted assembly mechanisms in the same cell. This study permits us to understand better the nature of the HS biosynthetic pathway that leads to the generation of specific saccharide sequences.

A Single-Step Purification of Assembled Insulin on a Resource RPC Column

ABSTRACT We have designed an efficient single step purification process using Resource RPC column in conjunction with triethyl amine/acetonitrile solvent system at similar pH at which the in vitro assembly of insulin is carried out from its two chains. The conditions have been optimized to separate native insulin from its single chain precursors as well as from other by-products. This method obviates the need of acidification, centrifugation, gel filtration, and concentration steps before HPLC purification of insulin from assembly mixture. The system offers wide pH stability, rapid regenerability, excellent pressure/flow characteristics, and high loading capacity. The authenticity and purity of human insulin purified by this procedure was evaluated by analytical reverse phase HPLC, peptide mapping, and isoelectric focusing.

Isolation and Characterization of a Gene Expressed during Early Embryo Sac Development in Apomictic Guinea Grass ( Panicum maximum)

A novel classification method, based on ovary length, for sampling different developmental stages of embryo sac formation in obligate sexual and facultatively apomictic genotypes of guinea grass (Panicum maximum) has been employed to make a cDNA library and to isolate a stage-specific cDNA clone, probably representing a full-length gene. This A2-134 cDNA, designated ASG-1 (Apomixis Specific Gene), was found to be expressed in flower buds of the apomictic but not of the sexual accession. Furthermore, the gene was also not expressed in buds of apomicts until completion of megasporogenesis but could only be found during a phase characterized by the. appearance of aposporous initial cells (AICs) of the embryo sac. The appearance of these cells is strictly limited to apomictic genotypes. Sequence analysis revealed that the A2-134 cDNA (1,177bp) codes for a protein of 305 amino acids with a molecular mass of 34.2 kDa. The amino acid sequence of A2-134 is related to RD22, a seed-specific and drought-induced gene of Arabidopsis thaliana, to USP, an unknown seed protein precursor of Vicia faba, to a polygalaturonase 1 beta chain precursor (Polygl) of Lycopersicon esculentum and to ADR6, an auxin down-regulated gene of Glycine max. Southern blot analysis showed a different hybridization pattern at the genomic level when A2134 cDNA was hybridized with total DNAs isolated from leaves of sexual as well as apomictic plants, indicating that the gene (or related family members) exists in both types. The cDNA library and the ASG-1 gene should be valuable tools toward understanding the molecular mechanism of apomixis, particularly apospory, and toward the transfer of apomixis to important crops. To our knowledge, A2-134 represents the first case of the isolation and characterization of a cDNA related to apomictic embryo sac development.

Characterization of two monoclonal antibodies against the RON tyrosine kinase receptor.

RON is a receptor protein tyrosine kinase belonging to the hepatocyte growth factor (HGF) receptor family. Using Récepteur d'Origine Nantais (RON) transfected cell lines, Macrophage Stimulating Protein (MSP) was identified as the ligand of RON. RON is synthesized as a single chain precursor, which subsequently is cleaved to yield a disulfide-linked heterodimer, with a 40-kDa alpha chain and a 150-kDa beta chain. Activation of RON by MSP results in cell migration, shape change, and proliferation. The present work centers on the production and characterization of two monoclonal antibodies (MAbs) to RON called ID-1 and ID-2. Antibodies were generated by immunization of mice with Madin-Darby Canine Kidney (MDCK) cells expressing human RON (clone RE7). Both antibodies recognized the mature and precursor form of RON. The specificity of the anti-RON antibodies was confirmed using a hepatocarcinoma cell line HepG2 expressing both task MET and RON receptors. Specific immunoprecipitation with ID-1 and ID-2 or anti-MET antibody followed by Western blotting under reducing conditions with rabbit polyclonal antibodies against RON and MET showed that our anti-RON antibodies recognize specifically the RON receptor. Ligand binding experiments showed that both antibodies are able to block the binding of radiolabeled MSP to RON and showed also that the antibodies recognize two different epitopes in the molecule. The blocking of MSP binding to RON by the anti-RON antibodies was confirmed by inhibition of cell migration induced by MSP in HT-29-D4 cells. Significant immunostaining was not observed in any subpopulation of whole blood with either ID-1 or ID-2. We analyzed the expression of RON receptor in a number of human hematopoietic and nonhematopoietic cells lines by flow cytometry. We found a strong mean of fluorescence intensity (MFI) in colon adenocarcinoma cells SW620 and HT-29-D4, low MFI in SVK14 and HepG2 cells, and no immunostaining in melanoma, lymphoma, and leukemia cells. Immunohistochemistry revealed that RON was expressed in germinal centers of tonsil, in skin, small intestine, and colon. These antibodies defined RON as CDw136 during the last leucocyte typing VI.

ChemInform Abstract: A New Synthetic Route to (3R,4S)‐3‐Hydroxy‐4‐phenylazetidin‐2‐one as a Taxol Side Chain Precursor.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Crystal Structures of FlavobacteriumGlycosylasparaginase: AN N-TERMINAL NUCLEOPHILE HYDROLASE ACTIVATED BY INTRAMOLECULAR PROTEOLYSIS

Glycosylasparaginase (GA) is a member of a novel family of N-terminal nucleophile hydrolases that catalytically use an N-terminal residue as both a polarizing base and a nucleophile. These enzymes are activated from a single chain precursor by intramolecular autoproteolysis to yield the N-terminal nucleophile. A deficiency of GA results in the human genetic disorder known as aspartylglycosaminuria. In this study, we report the crystal structure of recombinant GA from Flavobacterium meningosepticum. Similar to the human structure, the bacterial GA forms an alphabetabetaalpha sandwich. However, some significant differences are observed between the Flavobacterium and human structures. The active site of Flavobacterium glycosylasparaginase is in an open conformation when compared with the human structure. We also describe the structure of a mutant wherein the N-terminal nucleophile Thr152 is substituted by a cysteine. In the bacterial GA crystals, we observe a heterotetrameric structure similar to that found in the human structure, as well as that observed in solution for eukaryotic glycosylasparaginases. The results confirm the suitability of the bacterial enzyme as a model to study the consequences of mutations in aspartylglycosaminuria patients. They also suggest that further studies are necessary to understand the detail mechanism of this enzyme. The presence of the heterotetrameric structure in the crystals is significant because dimerization of precursors has been suggested in the human enzyme to be a prerequisite to trigger autoproteolysis.

SAXS study on poly(dimethylsiloxane) networks with controlled distributions of chain lengths between crosslinks

Supermolecular structures for poly(dimethylsiloxane) (PDMS) gels with unimodal and bimodal distributions of the chain lengths between the crosslinks are investigated by a small angle X-ray scattering (SAXS) technique. The unimodal and bimodal networks are prepared by the end-linking reaction of oligomeric and polymeric dimethylsiloxane mixtures with a series of compositions, and they are fully swollen in toluene. The scattering profiles for unimodal networks primarily depend on whether the precursor chains are highly entangled or not in the uncrosslinked state. For bimodal networks, the correlation lengths Ξ for the supermolecular structures do not strongly depend on the mixing ratio of the precursor chains, and the values are close to Ξ for the unimodal network of oligomeric chains. The value of Ξ for the unimodal network of polymeric chains is larger than for the unimodal network of oligomeric chains, while it is considerably reduced by adding a small amount of the oligomeric chain. The experimental results are interpreted on the basis of the shortest closed circuits concept describing spatial non-uniformities formed by end-linking, and the consideration that the elementary meshes in the networks composed of long precursor chains are mainly formed by entanglements.

Simultaneous Expression of Type 1 and Type 2 Lewis Blood Group Antigens by Helicobacter pyloriLipopolysaccharides: MOLECULAR MIMICRY BETWEEN H. PYLORILIPOPOLYSACCHARIDES AND HUMAN GASTRIC EPITHELIAL CELL SURFACE GLYCOFORMS

Previous structural investigations performed on the lipopolysaccharides (LPSs) from the human gastric pathogen Helicobacter pylori have revealed that these cell surface glycan molecules express type 2 partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine O antigen chains (O-chains) of various lengths, which may or may not be terminated at the nonreducing end by Lewis X (Lex) and/or Ley blood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with commercially available Lewis-specific monoclonal antibodies also have recognized the presence of Lex and Ley blood group antigens in H. pylori but, in addition, have indicated the presence of type 1 chain Lea, Leb, and Led (H-type 1) blood group epitopes in some H. pylori strains. To confirm their presence, structural studies and additional serological experiments were undertaken on H. pylori strains suspected of carrying type 1 chain epitopes. These investigations revealed that the O-chain region of H. pylori strain UA948 carried both Lea (type 1) and Lex (type 2) blood group determinants. The O-chain from H. pylori UA955 LPS expressed the terminal Lewis disaccharide (type 1 chain) and Lex and Ley antigens (type 2). The O-chain of H. pylori J223 LPS carried the type 1 chain precursor Lec, the H-1 epitope (Led, type 1 chain) and an elongated nonfucosylated type 2 N-acetyllactosamine chain (i antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a single H. pylori strain may carry O-chains with type 1 and 2 Lewis blood groups simultaneously. That monoclonal antibodies putatively specific for the Leb determinant can detect glycan substructures (Le disaccharide, Lec, and Led) of Leb indicates their nonspecificity. The expression of both type 1 and 2 Lewis antigens by H. pylori LPSs mimics the cell surface glycomolecules present in both the gastric superficial (which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type 2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS blood group antigen may have a dissimilar role in H. pylori adaptation.

Mechanisms of docosahexaenoic acid accretion in the fetal brain.

Docosahexaenoic acid (DHA, 22:6 n-3) is the major polyunsaturated fatty acid (PUFA) in the adult mammalian brain. DHA is an essential fatty acid (FA) since it, or its short chain precursor, alpha-linolenic acid (LnA, 18:3 n-3), have to be obtained in the diet. Moreover, dietary n-3 FA deficiency is associated with biochemical changes in the brain and with disturbances in vision and other neurological parameters. Under normal nutritional conditions, fetal brain DHA accumulation is substantial, with a "DHA accretion spurt" being demonstrated in the last period of gestation. This accumulation is supported by the maternal supply of DHA or LnA, but selectivity of DHA accumulation is probably a placental function whose mechanism is lately being clarified. The fetal gastrointestinal (G-I) tract may be instrumental in supplying DHA to the fetal brain under certain conditions, such as following intra-amniotic administration of ethyl-docosahexaenoate (Et-DHA). In this pathway, DHA is supplied independently of the maternal metabolism, and the fetal liver is apparently involved. The fetal G-I tract may be advantageous for DHA supply in cases of maternal-placental insufficiency resulting in intrauterine growth retardation. The fetal brain itself is capable of metabolizing LnA to DHA, without the participation of the fetal liver, thus contributing to the accumulation of its own DHA during one of the most crucial periods of its development.

Strong Precursor-Pore Interactions Constrain Models for Mitochondrial Protein Import

Mitochondrial precursor proteins are imported from the cytosol into the matrix compartment through a proteinaceous translocation pore. Import is driven by mitochondrial Hsp70 (mHsp70), a matrix-localized ATPase. There are currently two postulated mechanisms for this function of mHsp70: 1) The “Brownian ratchet” model proposes that the precursor chain diffuses within the pore, and that binding of mHsp70 to the lumenal portion of the chain biases this diffusion. 2) The “power stroke” model proposes that mHsp70 undergoes a conformational change that actively pulls the precursor chain through the pore. Here we formulate these two models quantitatively, and compare their performance in light of recent experimental evidence that precursor chains interact strongly with the walls of the translocation pore. Under these conditions the simulated Brownian ratchet is inefficient, whereas the power stroke mechanism seems to be a plausible description of the import process.

Studies on calix(aza)crowns, II. Synthesis of novel proximal doubly bridged calix[4]arenes by intramolecular ring closure of syn 1,3-and 1,2-ω-chloroalkylamides

Inherently chiral calix[4]arenes ( 9,10) with carboxamide bridges spanning the proximal positions on the lower rim and 18 achiral counterparts have been synthesized by double intramolecular cyclization of 7b,8b 1,3-and 17 1,2-bis-chloroalkylamides. The conformational analysis of the 1.3- and 1,2-disubstituted calixarene intermediates 5–8 and 15–17 proved an equilibrium of two distorted cone conformations. The success of ring closure was strictly dependent on the chain length of the open chain precursors.

A new synthetic route to (3 R,4 S)-3-hydroxy-4-phenylazetidin-2-one as a taxol side chain precursor

A new synthetic route to (3 R,4 S)-3-hydroxy-4-phenylazetidin-2-one, an important precursor for the paclitaxel side chain, has been developed using intramolecular cyclization of N-( p-methoxyphenyl) (2 S,3 R)-2-acetoxy-3-bromo-3-phenylpropionamide which can be easily obtained by catalytic asymmetric dihydroxylation of N-( p-methoxyphenyl)- trans-cinnamide, followed by bromoacetylation.

The H4P Heavy Chain of Inter-α-inhibitor Family Largely Differs in the Structure and Synthesis of Its Prolin-Rich Region from Rat to Human

The family of plasma proteins collectively referred to as Inter-α-Inhibitor (IαI) family is comprised of a set of multi-polypeptide molecules and a single-chain molecule designated IαIH4P. Although the 4 heavy chain precursors H1P to H4P that lead to these molecules are evolutionarily related, only H4P harbours a Pro-rich region (PRR) in its C-terminal third. A comparison of hepatic H4P cDNAs in human and rat has now unraveled an extensive variability of this PRR. Within the rat PRR, 6 repeats of a Gly-X-Pro motif participate in a collagen-like pattern that is absent in human. Within the human PRR, a domain that is absent in rat can be transcribed or deleted by alternative splicing which results in two variant forms of human H4P. In rat liver, the single mRNA is up-regulated by an acute, systemic inflammation whereas neither mRNA is up-regulated in human liver. Finally the shortest human mRNA is also transcribed in peripheral blood mononuclear cells where it is down-regulated by bacterial lipopolysaccharides. Therefore, in contrast to what is seen for theITIH1to-3genes, the rat and humanITIH4gene transcriptions and products thereof present marked differences, which suggests species-specific functions for IαIH4P.

Effects of nitro substituents on the properties of a ferroelectric liquid crystalline side chain polysiloxane

The syntheses of chiral liquid crystalline side chain polysiloxanes with lateral nitro substituents in the mesogenic core are described. The influence of the substituents and substituent positions on phase behaviour and electro-optical properties are investigated and compared. The lateral nitro groups strongly affect the phase behaviour of the side chain precursors as well as the liquid crystalline polymers. Properties in smectic A and C* phases are discussed with respect to substituent position. One side chain precursor exhibits very large spontaneous polarization of ∼700 nC cm–2 .

STRUCTURE AND MECHANICAL PROPERTIES OF BIMODAL PDMS NETWORKS IN THE SWOLLEN STATE

We have investigated mechanical properties of bimodal networks, which have a two-peak distribution of the length of the network chains between crosslinks, in equilibrium swollen state. The bimodal networks were prepared by end-linking mixtures of short and long poly (dimethylsiloxane) (PDMS) chains with tetra-functional crosslinker. The ratio of molecular mass of the short and long PDMS chains was ca. 11. The stress-strain relationships have been investigated as a function of molar fraction of short chains. The networks with more than 98mol% short chains are brittle and they have high elastic moduli. When the molar fractions of short chains fall below 95mol%, the networks become markedly extensible. The stress-strain relations of unimodal networks and bimodal network with small fraction of short chains obey the prediction of the classical theory of rubber elasticity, while those of other bimodal networks deviate from the theoretical prediction. The dependence of network structure on the composition of precursor chains has been estimated from the analysis of the stress-strain behavior. The structure model proposed in small angle X-ray scattering (SAXS) study on bimodal networks has been re-examined on the basis of the results of mechanical experiments in this study.