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PubMed2004

Bond dissociation energies for radical dimers derived from highly stabilized carbon-centered radicals.

[reaction: see text] The temperature dependence of the dissociation of dimers formed from highly stabilized carbon-centered radicals has been examined. Analysis of the data yields the bond dissociation energy (BDE) for the central head-to-head C-C bond in these compounds. For example, for the dimer derived from 3-phenyl-2-coumaranone, BDE is 23.6 kcal/mol and the C-C bond length 1.596 A, a rather long value for a sigma bond.

Journal Article↗
PubMed1999

The sharkskin instability of polymer melt flows.

Flows of polymeric liquids undergo instabilities whose origins are quite different from those of Newtonian flows, due to their elastic character and the complexity of the fluid/solid boundary condition. This article reviews recent studies of one such instability, the sharkskin phenomenon observed during extrusion of many linear polymers. Key experimental observations are summarized; one important fact that has become clear is the importance of the interaction between the molten polymer and the solid walls of the flow channel, especially near the contact line at the exit of the channel. Recent developments in understanding the relationship between wall slip and disentanglement of wall-adsorbed polymers from the bulk flow are briefly described, and putative heuristic mechanisms relating the instability to slip and contact line motion are presented. Finally, we review mathematical analyses of the stability of viscoelastic shear flows with slip boundary conditions. Some recent analyses yield instability predictions that are consistent with experiments, but further work is required to discriminate between the various mechanisms that have been proposed. (c) 1999 American Institute of Physics.

Journal Article↗
PubMed2005

Mechanistic and kinetic study of the CH3CO + O2 reaction.

Potential-energy surface of the CH3CO + O2 reaction has been calculated by ab initio quantum chemistry methods. The geometries were optimized using the second-order Moller-Plesset theory (MP2) with the 6-311G(d,p) basis set and the coupled-cluster theory with single and double excitations (CCSD) with the correlation consistent polarized valence double zeta (cc-pVDZ) basis set. The relative energies were calculated using the Gaussian-3 second-order Moller-Plesset theory with the CCSD/cc-pVDZ geometries. Multireference self-consistent-field and MP2 methods were also employed using the 6-311G(d,p) and 6-311++G(3df,2p) basis sets. Both addition/elimination and direct abstraction mechanisms have been investigated. It was revealed that acetylperoxy radical [CH3C(O)OO] is the initial adduct and the formation of OH and alpha-lactone [CH2CO2(1A')] is the only energetically accessible decomposition channel. The other channels, e.g., abstraction, HO2 + CH2CO, O + CH3CO2, CO + CH3O2, and CO2 + CH3O, are negligible. Multichannel Rice-Ramsperger-Kassel-Marcus theory and transition state theory (E-resolved) were employed to calculate the overall and individual rate coefficients and the temperature and pressure dependences. Fairly good agreement between theory and experiments has been obtained without any adjustable parameters. It was concluded that at pressures below 3 Torr, OH and CH2CO2(1A') are the major nascent products of the oxidation of acetyl radicals, although CH2CO2(1A') might either undergo unimolecular decomposition to form the final products of CH2O + CO or react with OH and Cl to generate H2O and HCl. The acetylperoxy radicals formed by collisional stabilization are the major products at the elevated pressures. In atmosphere, the yield of acetylperoxy is nearly unity and the contribution of OH is only marginal.

Journal Article↗
PubMed2001

A role for TAF3B2 in the repression of human RNA polymerase III transcription in nonproliferating cells.

RNA polymerase III (Pol III) synthesizes various small RNA species, including the tRNAs and the 5 S ribosomal RNA, which are involved in protein synthesis. Here, we describe the regulation of human Pol III transcription in response to sustained cell cycle arrest. The experimental system used is a cell line in which cell cycle arrest is induced by the regulated expression of the tumor suppressor protein p53. We show that the capacity of cells to carry out Pol III transcription from various promoter types, when tested in vitro, is severely reduced in response to sustained p53-mediated cell cycle arrest. Furthermore, this effect does not appear to be due to direct inhibition by p53. By using complementation assays, we demonstrate that a subcomponent of the Pol III transcription factor IIIB, which contains the proteins TATA-binding protein and TAF3B2, is the target of repression. Moreover, we reveal that TAF3B2 levels are markedly reduced in extracts from cell cycle-arrested cells because of a decrease in TAF3B2 protein stability. These findings provide a novel mechanism of Pol III regulation and yield insights into how cellular biosynthetic capacity and growth status can be coordinated.

Cell Cycle↗
PubMedDate not supplied

Influences of process parameters on preparation of microparticle used as a carrier system for omega - 3 unsaturated fatty acid ethyl esters used in supplementary nutrition.

Microparticles were prepared by complex coacervation to encapsulate eicosapentaenoic acid ethyl ester (EPA-EE) for incorporation into foods as a nutrition supplement. Gelatin and acacia were used in the coacervation process. With an increasing oil/polymer ratio, both yield and encapsulation rate decreased; with an increasing homogenization time, the yield remained constant while the encapsulation rate slightly increased. Several particle hardening techniques were examined and their influence on particle structure, yield and encapsulation rate were examined. Ethanol hardening was compared to cross-linking with dehydroascrobic acid with respect to both yield and encapsulation rate. The particle diameters for both formulations were similar (ethanol: 38.4 +/- 4.1 microm; cross-linking: 41.8 +/- 3.0 microm). Spray-drying of the coacervates led to the smallest particles (5.2 +/- 1.1 microm), lowest yield and encapsulation rate. All microencapsulation products were assayed for their storage stability over 4 weeks with respect to the oxidation of the encapsulated omega - 3 unsaturated fatty acid ester inside the particles. Hardening with ethanol showed the lowest amount of peroxides: particle wall cross-linking by dehydroascorbic acid and spray-drying were observed to be less protective. All microparticles were characterized for their internal structure with confocal laser scanning microscopy (CLSM) after fluorescence labelling of the polymers, in order to localize the oil phase and visualize the distribution of the polymers in the coacervates. With increasing homogenization time, the internal structure changed stepwise from a capsule structure (core/wall) towards a matrix structure. For all experiments, a homogeneous distribution for both polymers, gelatin and acacia was observed inside the particle wall. No influence of the different particle hardening procedures on the polymer distribution was found.

Dehydroascorbic Acid↗
PubMed1996

A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities.

A sulfated polysaccharide named calcium spirulan (Ca-SP) has been isolated from a sea alga, Spirulina platensis, as an antiviral component. The anti-human immunodeficiency virus type 1 (HIV-1) and anti-herpes simplex virus type 1 (HSV-1) activities of Ca-SP were compared with those of dextran sulfate (DS) as a representative sulfated polysaccharide. Anti-HIV-1 activities of these agents were measured by three different assays: viability of acutely infected CD4-positive cells, or a cytopathology assay; determination of HIV-1 p24 antigen released into culture supernatants; and inhibition of HIV-induced syncytium formation. Anti-HSV-1 activity was assessed by plaque yield reduction. In addition, their effects on the blood coagulation processes and stability in the blood were evaluated. These data indicate that Ca-SP is a potent antiviral agent against both HIV-1 and HSV-1. Furthermore, Ca-SP is quite promising as an anti-HIV agent because even at low concentrations of Ca-SP an enhancement of virus-induced syncytium formation was not observed, as was observed in DS-treated cultures, Ca-SP had very low anticoagulant activity, and showed a much longer half-life in the blood of mice when compared with that of DS. Thus, Ca-SP can be a candidate agent for an anti-HIV therapeutic drug that might overcome the disadvantages observed in many sulfated polysaccharides. When the role of chelation of calcium ion with sulfate groups was examined by removing calcium or its replacement by sodium, the presence of calcium ion in the molecule was shown to be essential for the dose-dependent inhibition of cytopathic effect and syncytium formation induced by HIV-1.

Animals↗
PubMed1992

Both mutations in G6PD A- are necessary to produce the G6PD deficient phenotype.

The high prevalence of glucose 6-phosphate dehydrogenase (G6PD) deficiency in African populations is due almost entirely to the enzyme variant A-, which differs from the wild-type G6PD B by two amino acid replacements, 68 Val-->Met and 126 Asn-->Asp. The non-deficient polymorphic variant G6PD A contains only the mutation 126 Asn-->Asp. The frequencies of the G6PD A and of the G6PD A- genes in parts of Africa are both about 0.2. The 68 Val-->Met mutation has not been found in a B background. This could be because the 68 Val-->Met mutation happened to arise in an A gene in the first instance, or because the 68 Val-->Met mutation alone is not sufficient to cause G6PD deficiency. We have approached this question by producing G6PD B, A, A-, and G6PD 68 Val-->Met in a bacterial expression system and analysing their biochemical properties. With each single mutation we found a slight decrease in both the specific activity and the yield of enzyme when compared to G6PD B. When both mutations were introduced together, there was a roughly additive effect on specific activity, but a much more drastic effect on enzyme yield (4% of normal). This synergistic effect was also demonstrated on thermal stability, especially at low NADP concentrations. Comparable results were produced when the replacement 119 Gln-->Glu was studied instead of 126 Asn-->Asp. We infer that the coexistence of the two mutations is responsible for enzyme deficiency in G6PD A- because they act synergistically in causing instability of the enzyme.

Alleles↗
PubMed2006

Synthesis of fluorescent cyclic cytosine nucleosides and their fluorescent properties upon incorporation into oligonucleotides.

We report here the synthesis and spectroscopic characterization of a new fluorescent pyrimidopyrimidoindole nucleoside derivative (dC(PPI)) modified at the cytosine base. The photophysical properties of dC(PPI) were examined by fluorescent spectroscopy and quantum chemical calculations. It was found that dC(PPI)-labeled oligonucleotides gave almost the same thermal stability as that of the corresponding unmodified sequences. In addition, the quantum yield of dC(PPI) in the double-stranded state was significantly higher than that in the single-stranded state.

DNA Probes↗
PubMed1995

Engineered turns of a recombinant antibody improve its in vivo folding.

Using recombinant antibodies functionally expressed by secretion to the periplasm in Escherichia coli as a model system, we identified mutations located in turns of the protein which reduce the formation of aggregates during in vivo folding or which influence cell stability during expression. Unexpectedly, the two effects are based on different mutations and could be separated, but both mutations act synergistically in vivo. Neither mutation increases the thermodynamic stability in vitro. However, the in vivo folding mutation correlates with the yield of oxidative folding in vitro, which is limited by the side reaction of aggregation. The in vivo folding data also correlate with the rate and activation entropy of thermally induced aggregation. This analysis shows that it is possible to engineer improved frameworks for semi-synthetic antibody libraries which may be important in maintaining library diversity. Moreover, limitations in recombinant protein expression can be overcome by single amino acid substitutions.

Blotting, Western↗
PubMed2001

Hexa-histidine tag as a novel alternative for one-step direct labelling of a single-chain Fv antibody fragment with 99m Tc.

From genetic material of hybridoma cells, we have generated a recombinant single-chain antibody fragment (scFv antibody) specific to carcinoembryonic antigen (CEA), which can substitute an intact murine monoclonal immunoglobulin G1 (IgG1) antibody, also developed by our group, and used in clinical practice for many years. In this paper, we examine a novel one-step method for direct 99mTc labelling of a recombinant anti-CEA scFv fragment through a C-terminal peptide tag containing a six-histidine sequence. This C-terminal peptide tag does not affect antigen binding, and was employed as a strategy for the one-step method of direct 99mTc labelling of a recombinant antibody fragment, based on the criteria of Zamora and Rhodes (Zamora PO, Rhodes BA. Imidazoles as well as thiolates in proteins bind technetium-99m. Bioconj Chem 1992; 3: 493-498). This is a novel technique for the rapid labelling of molecules, suitable for in vivo trials. The method yields >95% labelling efficiency without major effects on biological or in vitro stability.

Animals↗
PubMed2003

Ossiculoplasty with total hydroxylapatite prostheses anatomical and functional outcomes.

OBJECTIVE: To evaluate the results of partial and total ossicular reconstruction prostheses entirely made of hydroxylapatite. STUDY DESIGN: A retrospective review of cases followed-up between 1997 and 2000. SETTING: Tertiary referral center. PATIENTS: One hundred adult patients (60 men and 40 women) were studied. The mean age was 55 years (range, 25 to 65 years). Fifty patients had a previous tympanoplasty. The series included 45 cholesteatomas (45%), 37 chronic otitis media without cholesteatoma (37%), 13 retraction pockets (13%), and 5 posttraumatic ossicular displacements (5%). INTERVENTION: A one-stage ossiculoplasty with a partial ossicular reconstruction prosthesis (n = 65) or a total ossicular reconstruction prosthesis (n = 35) was performed. This was associated with a simple mastoidectomy in 70 cases and a radical mastoidectomy in 10 cases. MAIN OUTCOME MEASURES: All patients were followed-up at 1 year, and 50 were reexamined 2 years after surgery. Clinical and audiometric data were collected in a database at each visit. RESULTS: A prosthetic extrusion was noted in two cases (2%). A prosthetic displacement occurred in 10 cases (10%). A revision surgery was carried out in 10 cases because of an ossiculoplasty failure or a disease recurrence. At 1 year, the residual air-bone gap was lower in cases with a partial ossicular reconstruction prosthesis than those with a total ossicular reconstruction prosthesis (16 +/- 9.3 versus 23 +/- 13.7 dB, p < 0.05). The gain in mean air conduction threshold was 21 +/- 12.8 dB in cases of partial ossicular reconstruction prosthesis and 19 +/- 11.3 dB in cases of total ossicular reconstruction prosthesis (not significant). Revision surgery, canal wall-down mastoidectomy, or cholesteatoma yielded poorer functional results. CONCLUSION: Partial ossicular reconstruction prosthesis and total ossicular reconstruction prosthesis entirely made of dense hydroxylapatite yielded high rates of air-bone gap reduction and high anatomic stability.

Adult↗
PubMed2006

A responder analysis of memantine treatment in patients with Alzheimer disease maintained on donepezil.

OBJECTIVE: The objective of this study was to examine the clinical utility of memantine for moderate-to-severe Alzheimer disease (AD) using responder analyses. METHOD: Data from a previously published 24-week, randomized, double-blind, placebo-controlled trial of 10 mg memantine twice a day in patients with moderate-to-severe AD (N = 404) on stable donepezil therapy were evaluated using three sets of responder criteria. Response rates were calculated and analyzed for the intention-to-treat population using a generalized estimating equations model. The following outcomes were examined separately and in combination: the Alzheimer's Disease Cooperative Study-Activities of Daily Living 19-Item Inventory (ADCS-ADL19), Severe Impairment Battery (SIB), Clinician's Interview-Based Impression of Change Plus Caregiver Input (CIBIC-Plus), and Neuropsychiatric Inventory (NPI). RESULTS: When treatment response required cognitive improvement relative to baseline, memantine yielded higher response rates than placebo. When treatment response was defined as stabilization of individual outcomes, memantine resulted in significantly higher response rates than placebo for all outcomes, with number needed to treat (NNT) ranging from 8-10. More conservative definitions of response that required simultaneous stabilization on multiple outcome measures again favored memantine treatment for six of 10 combinatorial definitions. CONCLUSIONS: These responder analyses may assist clinicians in evaluating the impact of memantine in a relevant clinical scenario, i.e., in patients with AD previously stabilized on a cholinesterase inhibitor. The current results indicate that in this setting, memantine produces both improvement and stabilization of symptoms, across multiple outcomes, and thus provides a clinically important treatment benefit for patients with moderate-to-severe AD.

Activities of Daily Living↗
PubMedDate not supplied

Assessing attachment security with the Attachment Q Sort: meta-analytic evidence for the validity of the observer AQS.

The reliability and validity of the Attachment Q Sort (AQS; Waters & Deane, 1985) was tested in a series of meta-analyses on 139 studies with 13,835 children. The observer AQS security score showed convergent validity with Strange Situation procedure (SSP) security (r = .31) and excellent predictive validity with sensitivity measures (r = .39). Its association with temperament was weaker (r = .16), which supports the discriminant validity of the observer AQS. Studies on the stability of the observer AQS are still relatively scarce but they have yielded promising results (mean r = .28; k = 4, n = 162). It is concluded that the observer AQS, but not the self-reported AQS, is a valid measure of attachment.

Child↗
PubMed1999

Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications.

Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.

Biocompatible Materials↗
PubMed1973

Size and shape in biology.

Arguments based on elastic stability and flexure, as opposed to the more conventional ones based on yield strength, require that living organisms adopt forms whereby lengths increase as the (2/3) power of diameter. The somatic dimensions of several species of animals and of a wide variety of trees fit this rule well. It is a simple matter to show that energy metabolism during maximal sustained work depends on body cross-sectional area, not total body surface area as proposed by Rubner (1) and many after him. This result and the result requiring animal proportions to change with size amount to a derivation of Kleiber's law, a statement only empirical until now, correlating the metabolically related variables with body weight raised to the (3/4) power. In the present model, biological frequencies are predicted to go inversely as body weight to the (1/4) power, and total body surface areas should correlate with body weight to the (5/8) power. All predictions of the proposed model are tested by comparison with existing data, and the fit is considered satisfactory. In The Fire of Life, Kleiber (5) wrote "When the concepts concerned with the relation of body size and metabolic rate are clarified, . . . then compartive physiology of metabolism will be of great help in solving one of the most intricate and interesting problems in biology, namely the regulation of the rate of cell metabolism." Although Hill (23) realized that "the essential point about a large animal is that its structure should be capable of bearing its own weight and this leaves less play for other factors," he was forced to use an oversimplified "geometric similarity" hypothesis in his important work on animal locomotion and muscular dynamics. It is my hope that the model proposed here promises useful answers in comparisons of living things on both the microscopic and the gross scale, as part of the growing science of form, which asks precisely how organisms are diverse and yet again how they are alike.

Anatomy, Comparative↗
PubMed2006

Humanization of the bispecific epidermal growth factor receptor x CD3 diabody and its efficacy as a potential clinical reagent.

PURPOSE: Bispecific antibodies (BsAb) have been exploited as both cancer immunodiagnostics and cancer therapeutics and show promise in clinical trials of cancer imaging and therapy. For development of BsAbs as clinical reagents, we have focused on construction of small recombinant BsAbs, called bispecific diabodies. Here, we constructed and characterized a humanized bispecific diabody. EXPERIMENTAL DESIGN: We have reported significant antitumor activity of an anti-epidermal growth factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3) in in vitro cytotoxicity assays and in vivo. We humanized the Ex3 diabody (hEx3) by grafting the complementarity-determining region and compared its biological properties with those of Ex3. We also tested its physiologic stability and ability to alter survival in xenografted mice. RESULTS: The final yield of hEx3 was 10 times that of Ex3, and refolded hEx3 and Ex3 showed identical binding profiles in EGFR-positive cell lines and EGFR-transfected Chinese hamster ovary cells. hEx3 showed dose-dependent cytotoxicity to EGFR-positive cell lines, which could be specifically inhibited by parental monoclonal antibody IgGs against EGFR or CD3 antigens. The heterodimeric structure was retained in PBS for 6 months, and growth inhibition was maintained after incubation under physiologic conditions. Coadministration of hEx3 with T-LAK cells and interleukin-2 prolonged the survival of nude mice with human colon carcinoma. CONCLUSIONS: The humanized diabody hEx3 is an attractive molecule for cancer therapy and may provide important insights into the development of EGFR-based cancer-targeting reagents.

Animals↗
PubMed1989

Immunogold-silver cytochemistry using a capillary action staining system.

An improved method for immunogold-silver staining is described. Rat monoclonal antibodies to T-lymphocyte surface membrane antigens were employed to demonstrate T-cells and their subsets in frozen sections of mouse spleen and in smears of peripheral blood mononuclear cells. A capillary action staining system was used to perform all stages of the procedure, substantially reducing the technical effort involved, saving reagents, and improving reproducibility and control of batch staining. The use of a light-stable silver enhancement reagent simplified the development of the reaction product. Technical modifications were incorporated to obtain good preservation of tissue morphology when employing this reagent on frozen sections and to stabilize the reaction product. The improved immunostaining procedure was rapid, sensitive, and yielded better staining quality, with excellent contrast and minimal nonspecific precipitation of silver.

Animals↗
PubMed2005

A theoretical study of hypothalamo-pituitary-adrenocortical axis dynamics.

Does the hypothalamo-pituitary-adrenocortical (HPA) axis itself generate oscillations? The affirmative answer to this question is commonly assumed, because a regular daily rhythm of its hormones is observed. We offer another plausible explanation of the origin of this pattern: HPA just responds to an external pacemaker. A qualitative mathematical model is constructed wherein all the terms in the equations are physicochemically interpretable. Linear stability analysis shows that this system does not generate oscillations. Computer simulation yields oscillations that are the system's response to an external pulsing activator, implying that the observed pattern does not have to be an intrinsic property of this system.

Adrenal Cortex Hormones↗