SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
An optimal, in respect to productivity (activity X stability), enzyme ratio for immobilization of multienzyme systems was calculated by using the kinetic parameters (KM and Vmax), data on the stability and yield of each enzyme during immobilization. The experimental data, obtained during combined immobilization of invertase, mutarotase and glucose oxidase, illustrate the theoretical propositions.
The immune response to viruses partially depends on the biochemical interaction between viral peptides and histocompatibility molecules. In this study, the refolding of recombinant HLA-A*0201 heavy chain and beta 2-microglobulin (beta 2-m) in the presence of peptides from influenza B nucleoprotein (BNP), influenza A matrix protein, and HIV gp120 and their analogues was examined. The plateau value for the amount of refolded complex with three peptides, a 10-mer BNP 85-94 (A86) with alanine substituted for leucine at the P2 anchor residue and two BNP 8-mers, was significantly lower than the native peptide epitope BNP 85-94 or with other peptides tested. To attempt to understand the basis for the lower yield of complex, equilibrium dissociation constants (KdS) for the two 10-mers, BNP 85-94 (A86) and BNP 85-94, were determined from association and dissociation rates and from Scatchard plots, all measured at 10 degrees C. In addition, dissociation rates were measured at 0 degrees, 26 degrees, and 37 degrees C. Although the kinetics were similar at 0 degrees and 10 degrees, at 37 degrees these two complexes had distinct rates of dissociation, resulting in relatively stable or unstable complexes. The behavior of the unstable complexes paralleled the behavior of empty complexes described in vivo; they are unstable at physiologic temperature, produced in low yield, and stabilized by low temperature. Comparison of all of the kinetic data suggests that the equilibrium amounts of the two HLA/peptide complexes (plateau values) result from distinct reaction pathways, i.e., that the molecules that form stable complexes may undergo an additional reaction to those that form unstable complexes.
Progression of benign tumors to malignant cancer is critical since cancerous lesions are capable of metastatic spread and eventually causing death. Inhibitors of the conversion process, therefore, would likely be useful as cancer chemopreventive agents. In this study, we assessed the protective effect of topical application of a polyphenolic fraction isolated from green tea (GTP) against spontaneous as well as benzoyl peroxide (BPO)- and 4-nitroquinoline-N-oxide (4-NQO)-enhanced malignant conversion of chemically induced skin papillomas in SENCAR mice. Papillomas were induced in SENCAR mice by topical application of 7,12-dimethylbenz(a)anthracene as a tumor-initiating agent followed by twice a week application of 12-O-tetradecanoylphorbol-13-acetate as a tumor-promoting agent. Beginning at the 20th week, when papilloma yield was stabilized, enhanced malignant conversion was achieved by twice weekly topical application of either BPO or 4-NQO, whereas spontaneous malignant conversion was associated with topical application of acetone. In these protocols, preapplication of GTP (6 mg/animal) 30 min prior to skin application of acetone, BPO, or 4-NQO resulted in 14, 31, and 29% protection, respectively, in terms of percentage of mice with carcinomas, and 20, 35, and 43% protection in terms of number of carcinomas/mouse. In these experiments, a BPO- and 4-NQO-enhanced rate of malignant conversion was also found to be decreased significantly by the skin application of GTP; however, such effects of GTP were less profound in the cases of spontaneous malignant conversion. The results of this study suggest that, in addition to its chemopreventive effects against tumor initiation and promotion stages of multistage carcinogenesis, green tea also possesses significant protective effects against tumor progression, specifically tumor progression induced by BPO and 4-NQO.
Hexaploid triticale has many advantages over both parental species for both grain and forage production in certain environments. Additional information on environmental stability and heritability would be desirable to develop appropriate selection strategies in the production of superior widely-adapted cultivars. The grain yield of 22 diverse genotypes grown at four ecologically-distinct geographical locations [Quincy, FL, USA (approximate geographical coordinates (AGC) = 30 degreesN 84 degreesW, approximate elevation (AE) = 58 m), Plains, GA, USA (AGC = 32 degreesN 84 degreesW, AE = 76 m), Bozeman, MT USA (AGC = 45 degreesN 111 degreesW, AE = 1458 m), and Aberdeen, ID, USA (AGC = 42 degreesN 112 degreesW, AE = 1360 m)] was measured in two years with winter and spring planting dates only at Bozeman and Aberdeen. Test weight (grain weight in a given volume) was determined for two years at Bozeman and Aberdeen at both planting dates and one year at Quincy. Stability analyses indicated that significant (P < 0.01) variation in means, regression coefficients, and deviation mean squares of the genotypes were present for both characters. Realized heritability (h2) estimates were as follows: grain yield ranged from -0.02 to 0.80 with a mean of 0.57; test weight ranged from 0.63 to 1.05 with a mean of 0.93. The results indicated that substantial genetic variation is present and selection for widely-adapted cultivars would be effective for both characters especially test weight.
We describe a new off-pump coronary bypass technique, the "double suspension-stabilization stitch", performed without the support of mechanical stabilizers. This technique is very cheap and yields good hemodynamic stability, with no epicardial damage.
BACKGROUND: The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC). RESULTS: A single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods. CONCLUSION: The experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.
The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12 mm x 3 mm i.d.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 microg of enzyme dissolved in 800 microl of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4 degrees C. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds' inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous.
Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Zn-pheophytin a. Zn-pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90-300 pmol of Zn-pheophytin a or of about 400-600 pmol of chlorophyll a/4.2 x 10(7) etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-pheophytin a, but was unaffected by higher concentrations of chlorophyll a.
The yield, activity and thermal stability of lipolytic enzymes obtained from the culture fluid filtrates of Rhizopus microsporus, UzLT-1, cultivated at 28, 38 and 48 degrees C (preparations I, II and III, respectively) were investigated. Maximal lipolytic activity was found in Prepartion II, and maximal yield and thermal stability in Preparation III. By disc electrophoresis and DEAE-cellulose chromatography, the presence of three lipolytically active enzymes in the preparations was demonstrated.
The problem of electroviscoelastic Kelvin-Helmholtz waves of Maxwellian fluids under the influence of a vertical periodic electric field is studied in the absence of surface charges. The system is composed of a streaming dielectric fluid sheet of finite thickness embedded between two different streaming semi-infinite dielectric fluids. Due to the streaming flow and the influence of a periodic force, a mathematical simplification is considered. The weak viscoelastic effects are taken into account so that their contributions are demonstrated in the boundary conditions. The approximate equations of motion are solved in the absence of viscoelastic effects. The solutions of the linearized equations of motion and boundary conditions lead to two simultaneous Mathieu equations of damping terms having complex coefficients. Symmetric or antisymmetric deformation that relaxes the coupled Mathieu equations and yields a single Mathieu equation is considered. Stability criteria are discussed and numerical estimation shows that the increase in the sheet thickness plays a destabilizing effect in the presence or in the absence of the field frequency as well as the field intensity. In the absence of the field frequency the velocity ratio between the upper fluid velocity and the sheet velocity has a destabilizing influence, while that between the velocity of the lower fluid and the velocity of the sheet has a stabilizing influence. Moreover, the viscosity ratios have a damping influence while the elasticity ratios have a destabilizing influence. Furthermore, a range of general deformations of the surface deflections is studied. Moreover, the stability behavior for the resonance cases is studied and discussed. The coupled Mathieu equations are analyzed by the multiple scale method. The numerical examination for stability yields some changes in the stability behavior. The fluid sheet thickness plays a stabilizing role in the presence of a constant field while the damping role is observed for the resonance case. Similar results are found for both the stratified velocities and the stratified relaxation times. The dual role of the stratified viscosities is observed in the presence or the absence of the field frequency. Copyright 2000 Academic Press.
A cDNA library was constructed from HL60 human promyelocyte poly(A)+ RNA harvested 3 h after induction of macrophage differentiation with 12-O-tetradecanoyl phorbol-13-acetate in the presence of cycloheximide. We isolated from this library a 1.6-kilobase full-length clone designated b4 whose corresponding mRNA was greatly increased in abundance in cytoplasmic RNA under these conditions. Dideoxy sequencing revealed that this mRNA encoded MONAP (monocyte-derived neutrophil-activating peptide), a 10-kilodalton monokine with neutrophil-specific chemotactic and enzyme-releasing activities. The 3' untranslated region of this mRNA was found to be 1.2 kilobases long and possessed nine copies of the AUUUA sequence known to be associated with regulation of mRNA stability. Actinomycin D chase experiments yielded evidence that cytoplasmic stabilization was one of the means of regulation of MONAP expression. Analysis of cytoplasmic poly(A)- RNA revealed the presence of several discrete truncated species that shared a common 5' end and appeared to be intermediates of degradation. S1 mapping showed that the 3' ends of these molecules were distributed throughout the 3' untranslated region, preferentially in A + U-rich regions, broadly correlating with the distribution of AUUUA sites. Nuclear run-on experiments indicated that transcriptional induction accounted for less than 15% of the accumulation of MONAP mRNA. This mRNA was induced in HL60 cells by treatment with several differentiation-inducing agents: 12-O-tetradecanoyl phorbol-13-myristate alone, sodium butyrate, vitamin D3, and dimethyl sulfoxide. It was also induced in quiescent diploid lung fibroblasts stimulated to divide by serum, and it was constitutively overexpressed by some human tumor lines.
DNA-binding antibiotics such as intercalators, narrow groove binders, and other substances modify duplex DNA, making it an altered substrate for DNA helicases. The intercalators daunorubicin, actinomycin D, echinomycin, and elsamicin, the narrow groove binders distamycin and mithramycin, and the plant toxin teniposide, each representing a different chemical class, block SV40 large T antigen DNA helicase action with IC50 values ranging from 4 x 10(-8) to 2 x 10(-6) M. A partially purified human HeLa cell DNA helicase is also potently blocked by daunorubicin, distamycin, and teniposide. Because eukaryotic cells contain helicases of varying abundance, specificity, and type, this site of action for DNA-binding antibiotics may help explain antibiotic potency and specificity for DNA or RNA inhibition. The antihelicase effect of the antibiotic-double-stranded DNA complex may be central to the anticancer activities of these substances. An additional interesting correlation is the antihelicase action of DNA-intercalating antibiotics and their DNA-binding preference for G-C base pair sites. The G-C base pair binding preference of the intercalating antibiotics may result from evolutionary selection because of the higher G-C binding stability, compared with A-T binding stability. The combination of the higher base pair stability at G-C regions and increased duplex DNA stability induced by intercalating antibiotic yields a total additive stability of the intercalator-G-C base pair complex that resists helicase action.
BACKGROUND: Prostate cancer gene 3 (PCA3) encodes a prostate-specific mRNA that has shown promise as a prostate cancer diagnostic tool. This report describes the characterization of a prototype quantitative PCA3-based test for whole urine. METHODS: Whole-urine specimens were collected after digital rectal examination from 3 groups: men scheduled for prostate biopsy (n = 70), healthy men (<45 years of age with no known prostate cancer risk factors; n = 52), and men who had undergone radical prostatectomy (n = 21). PCA3 and prostate-specific antigen (PSA) mRNAs were isolated, amplified, and quantified by use of Gen-Probe DTS400 Systems. Prostate biopsy results were correlated with the PCA3/PSA mRNA ratio, and PSA mRNA concentrations were used to normalize PCA3 signals and confirm the yield of prostate-specific RNA. Assay precision, specimen stability, and mRNA yield were also evaluated. RESULTS: The specimen informative rate (fraction of specimens yielding sufficient RNA for analysis) was 98.2%. In this clinical research study, ROC curve analysis of prebiopsy specimens yielded an area under the curve of 0.746; sensitivity was 69% and specificity 79%. Serum PSA assay specificity was 28% for this same group. PCA3 and PSA mRNAs were undetectable in postprostatectomy specimens except for one man with recurrent prostate cancer. Assay interrun CVs were < or =12%. Both mRNAs were stable in processed urine up to 5 days at 4 degrees C and after 5 freeze-thaw cycles. CONCLUSION: The APTIMA PCA3 assay combines simple specimen processing with precise assays and existing instruments and could add specificity to the current algorithm for prostate cancer diagnosis.
In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive Phi angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.
The condensation reaction between 2-pyridinecarboxaldehyde and diethylenetriamine, 3-[(2-aminoethyl)amino]propylamine, and 3,3'-iminobis(propylamine) in a 2:1 molar ratio yields ligands that may be isolated exclusively in the dissymmetric (cyclic) isomeric forms L(A), L(B)/L(B*), and L(C). The template effect of a metal center (Fe(II), Ni(II), and Zn(II)) results in the ring opening of L(C) including one hexahydropyrimidine ring and one (long) propylene bridge. The resulting symmetric bis-Schiff base isomeric form L(C') is stabilized through pentacoordination, yielding [Fe(II)L(C')(NCS)](NCS) (3), [Ni(II)L(C')(NCS)](NCS) (6), and [Zn(II)L(C')(NCS)](NCS) (9). The same metal centers are too bulky to exert a template effect on L(A) including one imidazolidine ring and one (short) ethylene bridge. L(A) acts as a tetradentate ligand yielding [Fe(II)L(A)(NCS)2] (1), [Ni(II)L(A)(NCS)2] (4), and [Zn(II)L(A)(NCS)2] (7). The template effect of the metal center is selective toward the ligand L(B)/L(B*) including a hexahydropyrimidine (imidazolidine) ring and the shorter ethylene (longer propylene) bridge. The Fe(II) cation is small enough to exert a template effect, resulting in the ring opening of L(B)/L(B*). The resulting bis-Schiff base L(B') is stabilized through pentacoordination, yielding [Fe(II)L(B')(NCS)](NCS) (2). Ni(II) is too bulky to promote the ring opening of L(B)/L(B*): L(B) acts as a tetradentate ligand, yielding [Ni(II)L(B)(NCS)2] (5) (the L(B*) isomer is totally converted to L(B)). The coordinative requirements and stereochemical preference of the bulkier Zn(II) cation allow neither the ring opening of L(B)/L(B*) nor the tetracoordination of L(B) or L(B*) but stabilize the novel tetradentate dissymmetric form L(B degrees) in [Zn(II)L(B degrees)(NCS)2].H2O (8) (L(B degrees) results from MeOH addition across the imine bond of L(B)). Density functional theory calculations performed for Ni(II) and Zn(II) complexes of the L(B)/L(B*)/L(B degrees) set of ligands allowed one to compare the relative stabilities of all possible isomers, showing that the most stable ones correspond to those experimentally obtained: isomerization, or methanol addition across the imine bond, of the tetradentate ligand depends on the relative stabilities of all possible isomeric complexes.
Bacterial production of a plasmid-encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA- LexA+ and RecA+ LexA1(Ind-) backgrounds. In a LexA1(Ind-) context, we have observed lesser toxicity and higher productivity than in the wild-type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression. Also, a negative effect of the incubation temperature on the growth of producing cells was also detected. By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein. Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition. Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth.
Inclusion complexes of phenytoin (DPH) with 6-O-alpha-D-glucosyl (G1)- and 6-O-alpha-D-maltosyl (G2)-beta-cyclodextrins (beta-CyDs) were prepared in a molecular mixing ratio of 1:1. The advantages of these preparations in terms of dissolution characteristics and the oral absorbency of DPH were evaluated in comparison with the known solid dispersions of polyvinylpyrrolidone K-30 and sodium deoxycholate (DC-Na). The results of a phase-solubility study indicated that G1- and G2-beta-CyDs provided higher solubility for DPH than 2-hydroxypropyl (HP)-beta-CyD. Irrespective of inclusion ability, the DPH/beta-CyD complexes allowed faster dissolution rates than those of the known dispersions in JP 1st and 2nd mediums. The dissolution behavior of the DPH/DC-Na dispersion was considerably different between the 1st and 2nd mediums. The complexation by the sugar-modified derivatives yielded a higher stability of dissolved DPH in the JP 2nd medium than that yielded by K-30 or DC-Na. The safe estimation of carriers themselves indicated that G1- and G2-bet-CyDs did not damage the small intestine, while 10 mM DC-Na showed some damage. Compared with the DPH/K-30 dispersion, the preparations with the sugar-modified beta-CyDs were more effective in enhancing the absorbability of DPH after oral administration. These results clearly suggest that complexation with G1- and G2-beta-CyDs are useful forms for the oral delivery of DPH. The advantage of these complexes is that they produce an increased level of DPH available for gastrointestinal absorption. Additionally, G2-beta-CyD is recommended as a safe and potent additive for DPH.
The E/K coil, a heterodimeric coiled-coil, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and as an expression and affinity purification tag. In this design, heterodimer formation is specified through the placement of charged residues at the e and g positions of the heptad repeat such that the E coil contains all glutamic acid residues at these positions, and the K coil contains all lysine residues at these positions. The affinity and stability of the E/K coil have been modified to allow a greater range of conditions for association and dissociation. Increasing the hydrophobicity of the coiled-coil core, by substituting isoleucine for valine, gave increases in stability of 2.81 and 3.73 kcal/mol (0.47 kcal/mol/substitution). Increasing the alpha-helical propensity of residues outside the core, by substituting alanine for serine, yielded increases in stability of 2.68 and 3.28 kcal/mol (0.41 and 0.45 kcal/mol/substitution). These sequence changes yielded a series of heterodimeric coiled-coils whose stabilities varied from 6.8 to 11.2 kcal/mol, greatly expanding their scope for use in protein engineering and biomedical applications.