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At least 109 records · Page 6Linked to original sources
PubMed1999

Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).

Acinetobacter↗
PubMed2000

Poly(allylamine) beads as selective sorbent for preconcentration of formaldehyde and acetaldehyde in high-performance liquid chromatographic analysis.

Formaldehyde and acetaldehyde in water were determined by preconcentration with poly(allylamine) beads, derivatization with 2,4-dinitrophenylhydrazine (DPH) and analysis by HPLC. Poly(allylamine) beads (0.5 g) were used to adsorb formaldehyde and acetaldehyde at 1.2-150 microg l(-1) and 3.5-220 microg l(-1) from water (1 l). The concentration factor is 50 fold. The aldehydes were eluted and derivatized with 2 mM DPH in 0.5 M H2SO4 (10 ml). The time of analysis was 1 h. The detection limits (S/N=3) for formaldehyde and acetaldehyde were 0.6 and 2 microg l(-1), respectively.

Acetaldehyde↗
PubMed2000

Poly(vinyl chloride) ion-selective electrodes for piribedil determination.

Piribedil (PD) ion-selective electrodes have been constructed from poly(vinyl chloride) matrix membrane containing piribedil-tetraphenylborate (PD-TPB) as the electroactive component with dibutylphthalate or dioctylphthalate as the plasticizing solvent mediator. The electrodes displayed a linear response over the concentration range 2.0 x 10(-5) to 10(-2) M PD. The working pH ranges of the electrodes were 3.5-6.4 and 3.0-6.0, and the isothermal coefficients of the cells were 0.00129 and 0.00096 V/degrees C respectively. The electrodes were used for the determination of the diprotonated PD species, the most successful being that based on dioctylphthalate solvent mediator. The electrodes show a linear response over the concentration range of 8.0 x 10(-6) to 10(-2) M PD, with Nernstain slope 30 mV/PD concentration decade when preconditioned by soaking in distilled water for 30 min. The electrodes exhibit good selectivity for the PD with respect to a large number of inorganic cations and organic substances of biological fluids. Piribedil is determined successfully in pure solutions and in tablets or in biological fluids using the standard additions and potentiometric titration methods. The membrane withstood soaking in distilled water for more than 5 months.

Calibration↗
PubMedDate not supplied

[Study of complexes of poly-L-histidine with DNA using hyperchromic spectra].

Complexes of DNA with poly-L-histidine in acidic media were investigated by the method of hyperchromic spectra. Making use of the least squares hyperchromic spectra were partitioned into the components corresponding to the melting of AT and GC base pairs and protonation of cytosine. The protonation of cytosine was found to be proportional to the fraction of broken GC base pairs and was not influenced by the presence of poly-L-histidine. Selectivity of poly-L-histidine towards the base pairs was found to be very weak or absent.

Animals↗
PubMed2004

Pore-bridging poly(dimethylsiloxane) membranes as selective interfaces for vapor-phase chemical sensing.

A new kind of polymer-based sensor is described in which the experimental parameters controlling selectivity and sensitivity are decoupled. The sensor is based on a surface acoustic wave (SAW) gravimetric transducer modified with a high-surface-area, nanoporous alumina coating. A very thin ( approximately 40 nm) poly(dimethylsiloxane) (PDMS) coating resides atop the porous alumina structure. In this configuration, the total surface area of the nanoporous alumina coating controls the sensitivity of the device, while the chemical properties of the PDMS membrane control selectivity. In conventional polymer-based sensors, the polymer plays the dual role of controlling both selectivity (via the chemical composition of the coating) and sensitivity (via the volume of the film). In this paper, we show that PDMS acts as a chemically selective gate that absorbs polar and nonpolar VOCs but does not transport these analytes to the underlying pore volume. In contrast, water vapor is absorbed by the PDMS to a very minor extent but is easily transported through it to the underlying walls of the porous substructure. Specifically, there was little difference in the mass-loading response arising from polar and nonpolar VOCs dosed onto planar and nanoporous SAW devices modified with PDMS. In contrast, SAW devices having nanoporous coatings responded up to 24 times more selectively to water than planar sensors modified identically.

Journal Article↗
PubMed2001

Transcription activation of polyadenylated nuclear rna by rta in human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

Human herpesvirus 8 (HHV-8) (also known as Kaposi's sarcoma-associated herpesvirus) encodes a novel noncoding polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) during the early phase of lytic replication. PAN RNA is the most abundant transcript of HHV-8, comprising 80% of total poly(A)-selected transcripts in HHV-8-infected cells during lytic replication. We directly measured the abundance of PAN RNA by visualizing 1.1- to 1.2- kb PAN RNA in an ethidium bromide-stained gel from poly(A)-selected RNA. We further pursued the mechanisms by which PAN RNA expression is induced to such high levels. rta, an immediate-early gene of HHV-8, is a transactivator that is sufficient and necessary to activate lytic gene expression in latently infected cells. Ectopic expression of Rta was previously shown to induce PAN RNA expression from the endogenous viral genome and activate the PAN promoter in a reporter system. Here, we have identified the Rta-responsive element (RRE) in the PAN promoter. Deletion analysis revealed that the RRE is present in a region between nucleotides -69 and -38 of the PAN promoter. A promoter construct containing the 69 nucleotides upstream of the transcription start site of the PAN promoter was activated by Rta in the absence or presence of the HHV-8 genome. Rta activated the PAN promoter up to 7,000-fold in 293T cells and 2,000-fold in B cells. Electrophoretic mobility shift assays demonstrated that Rta formed a highly stable complex with the RRE of the PAN promoter. Our study suggests that Rta can induce PAN RNA expression by direct binding of Rta to the RRE of the PAN promoter. This study has highlighted an important mechanism controlling PAN RNA expression and also provides a model system for investigating how Rta transactivates gene expression during lytic replication.

Animals↗
PubMedDate not supplied

Characterization of the expression of cellular retrovirus genes and oncogenes in feline cells.

Expression of endogenous retrovirus genes and two different cellular oncogenes (c-onc genes) was examined at the transcriptional level in a variety of normal and lymphoma/leukemia tissues of the domestic cat. The two oncogenes, c-myb(related to avian myeloblastosis virus) and c-myc(related to avian myelocytomatosis virus) were selected for their association with the induction of hematopoietic malignancies, when present in the transforming retroviruses. Tissue-specific expression of endogenous feline leukemia virus (FeLV)-related genes was detected in cellular subpopulations of the cat placenta by in situ method of hybridization. Gel blotting analysis of placental poly(A)-selected RNA revealed that the FeLV-related RNA species were primarily subgenomic, representing the env gene region of the endogenous provirus elements. Like the endogenous retrovirus genes, c-myb and c-myc loci of the cat genomic DNA were also transcribed at differential levels in normal tissues of the cat. Dot-blot hybridization analysis showed that the expression of these two oncogenes was linked to growth and development as it varied with the gestational age of the fetus and from fetal to adult tissues. Among the major hematopoietic organs, spleen and bone marrow contained both c-myb and c-myc transcripts, while thymus preferentially expressed the c-myb gene. In contrast to the low level of c-myc expression in fetal thymus tissues, enhanced c-myc expression was detected in all five thymomas tested and also in several other neoplasms including two granulocytic leukemias. The feline c-myb gene was not very active in granulocytic leukemias or in three of the five thymomas. RNA gel blotting analysis of poly(A)-selected RNA of a thymoma and its lymph node metastasis showed identical pattern of c-myc transcripts.

Animals↗
PubMed2003

Selective phenolic acylation of 10-hydroxycamptothecin using poly (ethylene glycol) carboxylic acid.

Selective acylation of the phenolic hydroxyl group of 10-hydroxycamptothecin has been accomplished using phenyl dichlorophosphate. Additional modification of the 10-OH as an ether permits a 20-acyl derivative to be synthesized. This result along with data from a 6-hydroxyquinoline model strongly suggests that powerful intermolecular hydrogen bonding exists in the parent molecule.

Acylation↗
PubMed2006

Bundling and diameter selectivity in HiPco SWNTs poly(p-phenylene vinylene-co-2,5-dioctyloxy-m-phenylene vinylene) composites.

Temperature-dependent (TD) Raman measurements at laser excitation 514.5 nm were performed at different concentrations. The spectral profile of the radial breathing modes were investigated up to a polymer concentration of 1 g/L and were found to be dominated by approximately 1.2-1.4 nm diameter tubes at room temperature. Upon heating above the glass transition of the polymer (60 degrees C) the smaller tubes around approximately 0.9 nm increased significantly in relative intensity. This suggests that below the glass transition of the polymer (60 degrees C) RBMs within the composite are damped and spectral changes cannot be interpreted as diameter selective solubilization. The observed RBM damping at room temperature only occurred up to a concentration of approximately 1.2 x 10(-4) g/L and below this no damping was observed. Photoluminescence intensity (PL) measurements were taken for a range of PmPV concentrations, in which HiPco single walled carbon nanotubes (SWNTs) at 100%, 10%, 1%, 0.1%, 0.01%, and 0% mass fractions were added. Fitting of the concentration dependence to a dynamic absorption/desorption model indicates that the polymer interacts with nanotube bundles until a critical concentration of approximately 1.2 x 10(-4) g/L is reached, below which the nanotubes are isolated. The polymer and or solvent has a significant effect on the debundling and aggregation within these systems. Aggregation and/or interaction with the polymer at higher concentrations can effect the RBM profile in the composite at ambient temperatures, providing an incomplete representation of the selection of diameters present within composites at a particular wavelength.

Journal Article↗
PubMed2000

Selective enzymatic degradations of poly(L-lactide) and poly(epsilon-caprolactone) blend films.

Solution cast films were prepared from poly(L-lactide) (PLLA) and poly(epsilon-caprolactone) (PCL) as well as from three blends, namely B75, B50, and B25 with PLLA/PCL proportions of 75/25, 50/50, and 25/75, respectively. The enzymatic degradation of square samples (10 x 10 x 0.2 mm) cut from the films was investigated at 37 degrees C in a pH = 8.6 Tris buffer containing proteinase K or in a pH = 7.0 phosphate buffer containing Pseudomonas lipase. It was confirmed that proteinase K can degrade amorphous domains of PLLA, but cannot degrade crystalline PLLA or PCL. In contrast, Pseudomonas lipase can degrade both amorphous and crystalline PCL but cannot degrade PLLA. The two faces of solution cast films showed different morphologies due to the solvent evaporation process. The lower face appeared more crystalline than the upper face because of the plasticizing effect of solvent entrapped inside which allowed crystallization to proceed. Therefore, the lower face was more resistant to enzymatic attack by proteinase K in the cases of PLLA and the blends. The two polymers in the blends exhibited well separated crystalline domains. PCL seemed to constitute the continuous phase of the blends with formation of large size spherulites when the PCL content was over 50%. The selective degradation of PCL or PLLA components revealed the inner morphology of the blends where microspherelike or islandlike patterns were observed.

Calorimetry, Differential Scanning↗
PubMed1993

Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA).

The tissue response and in vivo molecular stability of injection-molded polyhydroxyacids--polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA, 5-22% VA content)--were studied. Polymers were implanted subcutaneously in mice and extirpated at 1, 3, and 6 months in order to study tissue response and polymer degradation. All polymers were well tolerated by the tissue. No acute inflammation, abscess formation, or tissue necrosis was observed in tissues adjacent to the implanted materials. Furthermore, no tissue reactivity or cellular mobilization was evident remote from the implant site. Mononuclear macrophages, proliferating fibroblasts, and mature vascularized fibrous capsules were typical of the tissue response. Degradation of the polymers was accompanied by an increase in collagen deposition. For the polylactide series, the inflammatory response after 1 month of implantation was less for materials containing the D-unit in the polymer chain, whereas in the case of the polyhydroxybutyrate/valerates, the number of inflammatory cells increased with increasing content of the valerate unit in the polymer chain. Between 1-3 months, there was slightly more tissue response to the PHB and PHB/VA polymers than to PLA. This response is attributed to the presence of leachable impurities and a low molecular weight soluble component in the polyhydroxybutyrate/valerates. At 6 months, the extent of tissue reaction was similar for both types of polymers. All polylactides degraded significantly (56-99%) by 6 months. For a poly(L-lactide) series, degradation rate in vivo decreased with increasing initial molecular weight of the injection-molded polymer. Several samples showed pronounced bimodal molecular weight distributions (MWD), which may be due to differences in degradation rate, resulting from variability in distribution of crystalline and amorphous regions within the samples. This may also be the result of two different mechanisms, i.e., nonenzymatic and enzymatic, which are involved in the degradation process, the latter being more extensive at the later stage of partially hydrolyzed polymer. The PHB and PHB/VA polymers degraded less (15-43%) than the polylactides following 6 months of implantation. Generally, the polymer with higher valerate content (19%, 22%) degraded most. The decrease in molecular weight was accompanied by a narrowing of the MWD for PHB and copolymers; there was no evidence of a bimodal MWD, possibly indicating that the critical molecular weight that would permit enzyme/polymer interaction had not been reached. Weight loss during implantation ranged from 0-50% for the polylactides, whereas for the PHB polymers weight loss ranged from 0-1.6%.

Absorption↗
PubMedDate not supplied

Selective synthesis of heterobifunctional poly(ethylene glycol) derivatives containing both mercapto and acetal terminals.

A novel synthetic route to heterobifunctional poly(ethylene glycol) (PEG) derivatives containing both mercapto and acetal terminal groups was established in this study using anionic ring opening polymerization of ethylene oxide (EO) using potassium 3, 3-diethoxypropanolate (PDP) as the initiator, followed by the successive conversion of the end-alkoxide group to a methanesulfonic group, and then to an ethyldithiocarbonate moiety. Molecular functionalities of the acetal and the mercapto terminal groups of the heterotelechelic PEG (acetal-PEG-SH) thus prepared were confirmed to 1.00 and 0.85, respectively, indicating that the reaction proceeds almost quantitatively. The obtained acetal-PEG-SH products, including 2-pyridyldithio derivatives, have a promising utility for bioconjugation in the fields of medicine and biology.

Acetals↗
PubMed2006

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells.

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2',5'-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.

2',5'-Oligoadenylate Synthetase↗
PubMed2006

A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel.

The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.

Alanine↗