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PubMed1981

An improved method for large scale purification of human holo-transcobalamin II.

25 mg of human holo-transcobalamin II with a specific cobalamin-binding capacity of 0.95 mol cobalamin/mol TC II was purified from 122 kg Cohn fraction III with a yield of 73% and a purification factor of 9.34 . 10(5). Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolability attached to 3.3'-diaminodipropylamine-substituted CH-Sepharose, and gel filtration. The high yield of the purification procedure was achieved by improving the stability of apo-transcobalamin II in the eluate of the CM-Sephadex, and by a few other modifications of a former procedure. In the latter, rapid denaturation of apo-transcobalamin II prohibited the use of long term affinity chromatography, which is obligatory for processing large amounts of Crohn fraction. In addition, subfractionation of transcobalamin II into smaller fragments which occurred in SDS-polyacrylamide gel electrophoresis in previous studies, was now reduced, indicating that proteolysis in the CM-Sephadex eluate had been prevented effectively.

Animals↗
PubMed2004

175Yb labeled polyaminophosphonates as potential agents for bone pain palliation.

Ytterbium-175 (T1/2 = 4.2 d, Ebeta(max) = 480 keV) has radionuclidic properties suitable to be used in palliative therapy of bone metastases. 175Yb can be produced in moderate specific activity and good radionuclidic purity by thermal neutron bombardment of natural Yb target. The analysis of the neutron irradiated sample exhibited the presence of 96.2% 175Yb along with 2.1% 169Yb and 1.7% 177Lu at 6 h post-EOB. Four polyaminomethylene phosphonic acid ligands, ethylenediamine tetramethylene phosphonic acid, propylenediamine tetramethylene phosphonic acid, triethylenetetramine hexamethylene phosphonic acid and diethylenetriamine pentamethylene phosphonic acid were synthesized and radiolabeled with 175Yb. Complexation parameters were optimized to achieve maximum yields (92-99%). All complexes were found to retain their stability at room temperature even after 10d of preparation. Biodistribution studies of the complexes carried out in Wistar rats showed significant bone uptake (3-4.4%/g in tibia at 3h post-injection) with rapid clearance from blood and minimum uptake in soft tissues for all the complexes (bone/blood ratio approximately 40-150 and bone/muscles ratio approximately 40-400 at 3 h post-injection). These studies suggest that 175Yb complexes with the phosphonate ligands have potential for use in palliative treatment of painful bone metastases.

Animals↗
PubMed2005

Isolated acetabular revision through the posterior approach: short-term results after revision of a recalled acetabular component.

Isolated acetabular revision is commonly associated with high rates of postoperative dislocation. We hypothesize that the dislocation rate in a series of isolated acetabular revisions for loosening of recalled acetabular components is low when strict intraoperative stability testing is coupled with re-repair of the piriformis tendon. Twenty-six isolated revisions of aseptic, loose recalled acetabular components were performed through the posterior approach by a single surgeon. All piriformis tendon and posterior capsular repairs from the index operation were considered intact at the time of revision. The piriformis tendon was re-repaired in each revision case. The average revision acetabular component was 3 mm larger than the index component. In this select group of isolated acetabular revisions, strict intraoperative stability testing and re-repair of the piriformis tendon yielded zero dislocations.

Acetabulum↗
PubMed2006

Development of a novel bioavailable inhibitor of the calmodulin-regulated protein kinase MLCK: a lead compound that attenuates vascular leak.

Tissue barriers involving epithelial and endothelial cell layers are critical to homeostasis, regulating passage of water, macromolecules, cells and certain classes of small molecules via two distinct cellular mechanisms, transcellular or paracellular. Endothelial or epithelial barrier dysfunction is a key component of pathophysiology in diverse diseases and injuries that have a broad impact on survival and quality of life. However, effective and safe small molecule therapeutics for these disorders are lacking. Success in development would therefore fill a major unmet medical need across multiple disease areas. Myosin light chain kinase (MLCK), a highly specialized calcium/calmodulin (CaM) regulated protein kinase, modulates barrier function through its regulation of intracellular contractile processes. MLCK levels and activity are increased in various animal models of disease and in human clinical disease samples. Our prior work with a genetic knockout (KO) mouse strain for the long form of MLCK, MLCK210, has identified MLCK as a drug discovery target for endothelial and epithelial barrier dysfunction. We describe here the development of a selective, bioavailable, stable inhibitor of MLCK that attenuates barrier dysfunction in mice comparable to that seen with the MLCK KO mice. The inhibitor compound 6 is stable in human microsomal metabolic stability assays and can be synthesized in a high-yielding and facile synthetic process. These results provide a foundation for and demonstrate the feasibility of future medicinal chemistry refinement studies directed toward the development of novel therapies for disorders involving barrier dysfunction.

Animals↗
PubMed2005

Carbon dioxide stimulates the production of amylovorin L by Lactobacillus amylovorus DCE 471, while enhanced aeration causes biphasic kinetics of growth and bacteriocin production.

The effects of both oxygen and carbon dioxide on growth of and product formation by Lactobacillus amylovorus DCE 471, a promising new sourdough starter culture, were assessed through controlled, in vitro fermentation experiments, using a temperature of 37 degrees C and a constant pH of 5.4. It was seen that aeration affected both cell growth and amylovorin L production. At aeration rates of 1 l min(-1) and more, the bacterial population was subjected to oxidative stress as reflected by biphasic growth patterns. During the first growth phase, the maximum specific growth rate increased with increasing aeration rates stabilizing at the highest oxygen concentrations. The maximum obtainable cell yields decreased. During the second growth phase, the amylovorin L production was stimulated at the highest aeration. However, amylovorin titers were never higher in the presence of oxygen compared with the anaerobic fermentations. Carbon dioxide did not influence cell growth of L. amylovorus DCE 471. The maximum specific growth rate and the biomass concentrations were merely affected. On the other hand, the maximum soluble bacteriocin titers coincided with the highest carbon dioxide flow rates. These results indicate that mild aeration of type II sourdoughs might enhance both cell yield and amylovorin L production by L. amylovorus DCE 471, thereby contributing to the competitiveness of the strain. Growth in an ecosystem together with yeasts producing carbon dioxide might exert a positive effect on the production of amylovorin L as well.

Bacteriocins↗
PubMed2004

On the origin of colloidal particles in the dispersion polymerization of aniline.

When aniline is oxidized in an aqueous medium in the presence of a steric stabilizer, colloidal polyaniline (PANI) dispersions are obtained. The generally accepted model of the stabilization assumes that the macromolecules of the water-soluble steric stabilizer are adsorbed at the polymer, precipitating during the dispersion polymerization, and provide steric protection against further aggregation. An alternative mechanism of conducting-polymer particle formation is proposed in the present study. We suggest that the steric stabilizer provides a site for adsorption of oligoaniline initiation centers; subsequent polymerization from anchored centers yields particle nuclei that grow to produce colloidal PANI particles. This hypothesis is based on the observation that the colloidal particles are obtained only in the case where the steric stabilizer is introduced in the early stages of polymerization when aniline oligomers are present in the reaction mixture. If the stabilizer had been added during the growth of PANI chains, colloidal dispersions would not have been produced. The process of particle growth is completely analogous to the formation of conducting PANI films on the surface of microparticles and various materials. There, the polymerization of aniline at the surfaces is preferred to the same process proceeding in the bulk of the reaction mixture. While the films grow at the interfaces with the reaction mixture, the dispersion particles similarly emanate from the stabilizer chains. The particle size, the formation of nonspherical morphologies, the importance of the chemical nature of the stabilizer chains, and the general relation between the conducting-polymer film and particle growth are discussed in the light of the proposed model.

Journal Article↗
PubMed2005

An alternative domain near the nucleotide-binding site of Drosophila muscle myosin affects ATPase kinetics.

In Drosophila melanogaster expression of muscle myosin heavy chain isoforms occurs by alternative splicing of transcripts from a single gene. The exon 7 domain is one of four variable regions in the catalytic head and is located near the nucleotide-binding site. To ascribe a functional role to this domain, we created two chimeric myosin isoforms (indirect flight isoform-exon 7a and embryonic-exon 7d) that differ from the native indirect flight muscle and embryonic body-wall muscle isoforms only in the exon 7 region. Germline transformation and subsequent expression of the chimeric myosins in the indirect flight muscle of myosin-null Drosophila allowed us to purify the myosin for in vitro studies and to assess in vivo structure and function of transgenic muscles. Intriguingly, in vitro experiments show the exon 7 domain modulates myosin ATPase activity but has no effect on actin filament velocity, a novel result compared to similar studies with other Drosophila variable exons. Transgenic flies expressing the indirect flight isoform-exon 7a have normal indirect flight muscle structure, and flight and jump ability. However, expression of the embryonic-exon 7d chimeric isoform yields flightless flies that show improvements in both the structural stability of the indirect flight muscle and in locomotor abilities as compared to flies expressing the embryonic isoform. Overall, our results suggest the exon 7 domain participates in the regulation of the attachment of myosin to actin in order to fine-tune the physiological properties of Drosophila myosin isoforms.

Actins↗
PubMed2005

Raltitrexed-Oxaliplatin combination chemotherapy is inactive as second-line treatment for malignant pleural mesothelioma patients.

Within a single-institution phase II trial, we investigated the antitumor activity of the Raltitrexed-Oxaliplatin combination as second-line therapy for malignant pleural mesothelioma (MPM). Fourteen patients were enrolled and all were assessable for response. The trial was then closed because chemotherapy, though well tolerated, yielded no objective responses. The best response observed was disease stabilization in 4 patients only (28.57%), while the other 10 patients (71.42%) progressed despite treatment. Median time to progression (TTP) was 8 weeks (average: 9.85, range: 7-20), while median overall survival was just 14 weeks (average: 21.69, range: 9-66+).

Adult↗
PubMed2004

Characterization of the temperature- and pressure-induced inverse and reentrant transition of the minimum elastin-like polypeptide GVG(VPGVG) by DSC, PPC, CD, and FT-IR spectroscopy.

We investigated the temperature- and pressure-dependent structure and phase behavior of a solvated oligopeptide, GVG(VPGVG), which serves as a minimalistic elastin-like model system, over a large region of the thermodynamic phase field, ranging from 2 to 120 degrees C and from ambient pressure up to approximately 10 kbar, applying various spectroscopic (CD, FT-IR) and thermodynamic (DSC, PPC) measurements. We find that this octapeptide behaves as a two-state system which undergoes the well-known inverse-temperature folding transition occurring at T approximately 36 degrees C, and, in addition, a slow trend reversal at higher temperatures, finally leading to a reentrant unfolding close to the boiling point of water. Furthermore, the pressure-dependence of the folding/unfolding transition was studied to yield a more complete picture of the p, T-stability diagram of the system. A molecular-level picture of these processes, in particular on the role of water for the folding and unfolding events of the peptide, presented with the help of molecular-dynamics simulations, is presented in a companion article in this issue.

Biomimetic Materials↗
PubMed2002

Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties.

A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library. The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction. Surprisingly, these V(H) dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies. The very close similarity of these molecules to human V(H)s makes them potentially very useful as therapeutic dAbs.

Amino Acid Sequence↗
PubMed2001

Structure of epitopes recognized by the antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptors: extrapolation to the structure of acetylcholine-binding domain.

Using the alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor (nAChR) and Ala-substituted peptide analogues, amino acid residues critical for specific monoclonal antibody (mAb) binding were identified. By means of 2D nuclear magnetic resonance (2D-NMR) analysis followed by molecular modeling, it was found that mAb binding resulted in stabilization of the free alpha3(181-192) peptide flexible conformation yielding an extended structure with residues 6-11 of the peptide being in direct contact with the Ab. Since the Ab binds the native AChR as well, it is suggested that the corresponding fragment of AChR alpha3 subunit is exposed to solution and also appears in extended conformation.

Alanine↗
PubMed2003

Synthesis and angiogenetic activity in the chick chorioallantoic membrane model of thymosin beta-15.

Thymosin beta-15 (Tbeta15), a 44 amino acid peptide (MW = 5173) localized in human prostate and breast cancer tissues was successfully synthesized in multigram quantities using Fmoc solid-phase peptide synthesis. The synthesized product was shown to have the right structure by ESI and MALDI mass spectral techniques and amino acid analysis. Relatively high yield was achieved, which might be due to enhanced acid stability of the p-cyanotrityl resin used. The effect of the synthesized Tbeta15 on the angiogenesis process was investigated using the chick chorioallantoic membrane (CAM) in vivo model. At concentrations above 1 microg/10 microl per disc, Tbeta15 exhibited a positive effect on angiogenesis, comparable to the effect of the intense angiogenetic factor phorbol 12-myristate 13-acetate at a standard concentration of 0.1 microg/10 microl per disc. The results of this study contribute to the further elucidation of the biological regulatory role of thymosin peptides and provide helpful information in the investigation of their possible therapeutic potential.

Allantoin↗
PubMed2002

Characterization of a Vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate.

Enterovirus 71 (EV71) is a neurotrophic virus that causes seasonal morbidity and mortality in children throughout the world with increasing frequency in recent years. Because of the lack of an effective antiviral agent, primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. Poliovirus vaccine technology, both live attenuated and inactivated, killed virus vaccines, can be adopted for use with EV71 because of their relatedness. In this study, we have characterized a laboratory-adapted EV71 strain, YN3-4a, which exhibits different characteristics from those of its parent isolate, neu, in having a rapid growth rate in Vero cells, a larger plaque size, and a lower LD(50) in newborn mice. The YN3-4a can be produced at a high viral titer of up to 10(10) tissue culture infective dose (TCID(50)) when grown in Vero cells, an approved substrate for virus vaccine production. Mouse antiserum raised against YN3-4a can neutralize a broad range of strains of EV71 isolated at different times from a variety of geographic regions. On passage in Vero cells, YN3-4a remained genetically and phenotypically stable. Many of the above-described features, such as high viral yield, strong immunogenicity, broad-based antigenic coverage, and passage stability, are desirable features in a prototype virus for the development of an inactivated viral vaccine.

Animals↗
PubMed1975

Linkage and assembly of polymeric IgA immunoglobulins.

The intersubunit linkage of polymeric IgA immunoglobulins was determined from studies of the products of reductive and cyanogen bromide cleavage. Under conditions of limited dithioerythritol reduction tetramer IgA molecules were cleaved to yield two monomers and a J chain containing dimer. The stability of the dimer and the conservation of the J chain disulfides indicated that the J chain joins two monomer subunits. Evidence confirming the J chain dimer clasp was obtained from the depolymerization of tetramer and dimer IgA by cyanogen bromide treatment. The cleavage studies also showed that (a) the S-S bonds directly joining the other subunits are located at the same penultimate alpha chain half-cystines that constitute the site of J chain attachment and (b) during limited reduction the monomer-monomer bonds undergo interchange to release subunits without a concomitant generation of alpha chain thiols. These linkage data provide strong support for the assembly of IgA and IgM polymers by sequential disulfide exchanges beginning with the formation of a J chain containing dimer.

Chemical Phenomena↗
PubMed2006

The binding of DYNLL2 to myosin Va requires alternatively spliced exon B and stabilizes a portion of the myosin's coiled-coil domain.

The myosin Va light chain DYNLL2 has been proposed to function as an adaptor to link the myosin to certain cargo. Here, we mapped the binding site for DYNLL2 within the myosin Va heavy chain. Copurification and pull-down experiments showed that the heavy chain contains a single DYNLL2 binding site and that this site resides within a discontinuity in the myosin's central coiled-coil domain. Importantly, exon B, an alternatively spliced, three-amino acid exon, is a part of this binding site, and we show in the context of full-length myosin Va that this exon is required for DYNLL2-myosin Va interaction. We investigated the effect of DYNLL2 binding on the structure of a myosin Va heavy chain fragment that contains the DYNLL2 binding site and flanking sequence, only parts of which are strongly predicted to form a coiled coil. Circular dichroism measurements revealed a DYNLL2-induced change in the secondary structure of this dimeric myosin fragment that is consistent with an increase in alpha-helical coiled-coil content. Moreover, the binding of DYNLL2 considerably stabilizes this heavy chain fragment against thermal denaturation. Analytical ultracentrifugation yielded an apparent association constant of approximately 3 x 10(6) M(-1) for the interaction of DYNLL2 with the dimeric myosin fragment. Together, these data show that alternative splicing of the myosin Va heavy chain controls DYNLL2-myosin Va interaction and that DYNLL2 binding alters the structure of a portion of the myosin's coiled-coil domain. These results suggest that exon B could have a significant impact on the conformation and regulatory folding of native myosin Va, as well as on its interaction with certain cargos.

Alternative Splicing↗
PubMedDate not supplied

Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor.

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment.

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 degrees C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.

Bioreactors↗
PubMed1998

Halide Effects in the Synthesis of Mixed Uranium(IV) Aryloxide-Halide Compounds.

Several uranium(IV) aryloxide and mixed aryloxide-halide compounds of the formula UX(4)(-)(z)()(OAr)(z)() (X = Cl, Br, I; OAr = 2,6-di-tert-butylphenoxide; z = 2, 3, 4) have been prepared by the reaction of KOAr with the appropriate uranium tetrahalide in tetrahydrofuran (THF). The complex UI(4)(CH(3)CN)(4) reacts with 2 equiv of KOAr in THF to yield the compound I(2)U(OAr)(2) (1). The utility of this reaction is limited by the instability of UI(4)(CH(3)CN)(4) in THF, however, and isolated yields of the THF adduct of 1 do not exceed 50%. In contrast, UBr(4)(CH(3)CN)(4) is stable in THF solution and reacts with 2 equiv of KOAr to yield the dibromide, Br(2)U(OAr)(2)(THF) (4.THF), in 77% yield. Under identical reaction conditions, UCl(4) reacts to yield the complex [K(THF)(4)][UCl(3)(OAr)(2)] (5) in 68% yield. The uranium center in compound 5 is coordinated by two trans aryloxide ligands and three chloride ligands in a trigonal bipyramidal arrangement. Two chloride ligands of each unit are also coordinated to a potassium ion, forming an infinite chain in the lattice. Compounds 1 and 5 will further react with 1 equiv of KOAr to yield the compounds XU(OAr)(3) (2, X = I; 6, X = Cl). Both UI(4)(CH(3)CN)(4) and UCl(4) will react with a 4.2 equiv of KOAr to yield the previously characterized compound U(OAr)(4) (3). The stability of the mixed aryloxide-halide uranium complexes toward ligand redistribution reactions has been investigated.

Journal Article↗