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PubMed2002

Collection of genomic DNA by the noninvasive mouthwash method for use in pharmacogenetic studies.

STUDY OBJECTIVE: To determine long-term stability, quantity, and quality of genomic DNA samples collected in buccal cells by the mouthwash method, for use in pharmacogenetic studies. DESIGN: Prospective analysis. SETTING: University pharmacogenomics center in Florida and medical centers in Puerto Rico and the United States participating in a multicenter international trial. SUBJECTS: Ten volunteers at the pharmacogenomics center and 201 participants in the ongoing multicenter clinical trial. INTERVENTION: Stability of genomic DNA was determined by measuring DNA yield from mouthwash samples obtained from six volunteers and stored at room temperature over 90 days and from 201 clinical trial samples that were stored and shipped at room temperature. Whether DNA yield was higher with three 5-ml mouthwash rinses versus one 10-ml rinse was evaluated in four volunteers. Quality of genomic DNA was assessed on 32 randomly selected samples from the six volunteers in the stability study, by determining the success rate of DNA amplification with polymerase chain reaction (PCR) testing and by genotyping. MEASUREMENTS AND MAIN RESULTS: For the stability studies, the quantity of genomic DNA decreased over time with storage at room temperature (overall p < 0.01), with the largest declines occurring at 60 and 90 days. Median DNA recovery at 30 and 90 days was 59% and 28% of that at baseline, respectively. Mean +/- standard deviation, median, and range for recovery of genomic DNA from the 201 samples were 45.2 +/- 55 microg, 25.2 microg, and 1-330 microg, respectively. Median recoveries of DNA from the one-rinse and three-rinse methods were not statistically significantly different (9.1 vs 10.5 microg). All samples were amplified successfully by PCR and genotyped, indicating quality of the DNA samples. CONCLUSION: The mouthwash method for collection of genomic DNA is a simple, inexpensive, and noninvasive method that poses less risk than venipuncture and may be used in a variety of settings. Genomic DNA in mouthwash is stable for prolonged periods at room temperature, and the quantity of DNA recovered from this method is more than sufficient for pharmacogenetic studies. Such an approach should be valuable to pharmacogenetic researchers and others who are conducting genetic research.

DNA↗
PubMed2001

Development of a measure of attitude toward nutrition in patient care.

BACKGROUND: Development of reliable measures of medical student and resident attitudes about nutrition in patient care is needed before the effects of educational interventions or clinical experience can be gauged. This report describes the systematic development of a measure of attitude toward nutrition in patient care. It presents evidence about scale reliability and the absence of response bias that endorses the trustworthiness of data from the measure. METHODS: An eight-step attitude scale development procedure was used to create the Nutrition In Patient care Survey (NIPS). Data from five samples of first- and second-year medical students and first-year medical residents were subjected to factor analysis (PA2, varimax rotation), reliability analyses, and statistical analyses to test for demographic bias in the attitude data. RESULTS: A 45-item attitude measure was developed that contains five subscales derived from the factor analysis: (1) nutrition in routine care (NRC, 8 items); (2) clinical behavior (CB, 20 items); (3) physician-patient relationship (PPR, 8 items); (4) patient behavior/motivation (PBM, 3 items); and (5) physician efficacy (PE, 6 items). Each subscale yields reliable data in terms of internal consistency (alpha coefficients) and stability (test-retest reliability). Medical student and resident demographic variables have negligible influence on attitude scores. DISCUSSION: The NIPS subscales yield reliable data that can be used to assess outcomes in evaluation research on educational or clinical interventions or to predict patient care practices. Systematic attitude scale development increases the likelihood that the resulting measures will produce useful, trustworthy data.

Adult↗
PubMed1988

Stability and self-organization of proteins.

X-ray crystallography and NMR provide insight into the three-dimensional structure of proteins at high resolution yielding a relatively small number of stable "topologies". Their free energies of stabilization are minute compared to the total molecular energy. Molecular adaptation to extremes of physical conditions at the protein level refers to marginal alterations of the intramolecular interactions; clearcut predictions with respect to the correlation of protein structure and protein stability are at present not possible. The acquisition of the native structure of proteins is determined by the kinetically accessible minimum of potential energy. The overall mechanism may be described as a hierarchical condensation with elements of secondary/super-secondary structure and subdomains/domains as intermediate stages. In the case of oligomeric and multimeric proteins, folding and association of subunits follow a consecutive uni-bimolecular mechanism, the kinetics of which can be simulated with high precision. The "code of protein folding" is still unknown.

Drug Stability↗
PubMed2006

Structural and functional characterization of peptide-beta2m fused HLA-A2/MART1(27-35) complexes.

The uses of soluble HLA class I/peptide complexes to monitor antigen reactive T cells are often hampered by their low-yield and high-cost production. As an alternative strategy, the peptide-beta(2)m fused, 2-component (2C) HLA class I/peptide complex has been developed, but its application is limited due to the lack of the comparison of its structural and functional characteristics with those of its conventional 3-component (3C) counterpart. In this study, we have demonstrated that the 2C and 3C HLA-A2/MART1(27-35) complexes have a similar chromatographical profile and comparable stability, but the former has 2.5 times higher yield and significantly higher binding ability with HLA-A2/MART1(27-35) complex-specific receptors than the latter. Furthermore, the 2C complex has a comparable ability to stimulate specific CTL proliferation, but appears to be more effective in eliciting the cytotoxicity of antigen-specific CTL, as compared to its 3C counterpart.

Antigens, Neoplasm↗
PubMed1996

Stable formulations of recombinant human growth hormone and interferon-gamma for microencapsulation in biodegradable microspheres.

PURPOSE: The successful development of controlled release formulations for proteins requires that the protein not be denatured during the manufacturing process. The major objective was to develop formulations that stabilize two recombinant human proteins, human growth hormone (rhGH) and interferon-gamma (rhIFN-gamma), at high protein concentrations (> 100 mg/mL) in organic solvents commonly used for microencapsulation, methylene chloride and ethyl acetate. METHODS: Several excipients were screened to obtain the maximum solubility of each protein. These formulations (aqueous, lyophilized, milled, spray dried, or isoelectric precipitate) were then rapidly screened by emulsification in the organic solvent followed by recovery into excess buffer. Additional screening was performed with solid protein that was suspended in the organic solvent and then recovered with excess buffer. The recovery of native protein was determined by native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The selected formulations were encapsulated in polylactic-coglycolic acid (PLGA) microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water (S/O/W) methods. The initial protein released from the microspheres incubated at physiological conditions was analyzed by SEC-HPLC, CD, and biological assays. RESULTS: The stability of a given formulation in the rapid screening method correlated well with stability during encapsulation in PLGA microspheres. Formulations of rhGH containing Tween 20 or 80 resulted in lower recovery of native protein, while trehalose and mannitol formulations (phosphate buffer, pH 8.0) yielded complete recovery of native rhGH. Other additives such as carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, respectively, and then encapsulated in PLGA microspheres. The protein was released from these microspheres in its native state. Lyophilized formulations of rhGH yielded analogous results indicating the ability of trehalose and mannitol to stabilize the protein. Small solid particles of rhGH generated by spray drying (both air and freeze-drying) formulations containing Tween 20 or PEG were stable in ethyl acetate, but not methylene chloride. Similar results were also obtained with rhIFN-gamma (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and trehalose were observed to stabilize the protein during exposure to the organic solvents resulting in the release of native rhIFN-gamma from PLGA microspheres. CONCLUSIONS: The rapid screening method allowed the development of stable concentrated protein solutions or solid protein formulations that could be successfully encapsulated in PLGA microspheres. The excipients observed to stabilize these proteins function by preferential hydration of the protein, and in the dry state (e.g., trehalose) may stabilize the protein via water substitution yielding a protective coating around the protein surface. Studies of other proteins should provide further insight into this mechanism of protein stabilization during encapsulation.

Chemical Precipitation↗
PubMed2002

Study of the complexation behavior of tenoxicam with cyclodextrins in solution: improved solubility and percutaneous permeability.

Complexation of tenoxicam (TEN) with gamma-, HPgamma-, beta,-, HPbeta-, and Mbeta-cyclodextrin (CD) in aqueous solution at pH 7.4 has been investigated using phase solubility diagrams. TEN formed soluble complexes with 1:1 stoichiometry with all the CDs studied, although the inclusion stability constants (K1:1) obtained had low values. The presence of propylene glycol (PG) in the dissolution medium decreased the stability constants and led to a higher fraction of free drug by competitive displacement and by an increase in the lipophility of the media. Among the CDs tested, MbetaCD was chosen for further studies since TEN-MbetaCD complexes yielded the best results: good solubility and the highest stability constant. The effect of MbetaCD and PG on the TENpartitioning coefficient was also studied in skin-buffer systems. Although each substance reduced the partitioning value, the combination of PG and MbetaCD increased this parameter. The noticeable increase in solubility of the drug found in the presence of MbetaCD allowed the formulation of carbopol gels with higher doses of TEN and a reduced amount of cosolvent. The presence of MbetaCD improved the percutaneous penetration of TEN through abdominal rat skin by increasing the solubility of the drug in the vehicle and by affecting the partitioning behavior of TEN in the skin. In addition, TEN retention in the skin was found to be related to the flux values attained with the corresponding gels.

Animals↗
PubMed1998

Effects of copper on the structure and function of factor VIII subunits: evidence for an auxiliary role for copper ions in cofactor activity.

Factor VIII, a divalent metal ion-dependent heterodimer, contains a single copper atom, but the role of this metal in the structure and function of the cofactor is unclear. Earlier results showed that the dissociated heavy and light chains of factor VIII could be recombined in the presence of Ca(II) or Mn(II) but not Cu(II) to yield functional protein [Fay, P. J. (1988) Arch. Biochem. Biophys. 262, 525-531]. Inclusion of Cu(I) or Cu(II) inhibited the Mn(II)- or Ca(II)-dependent reconstitution of factor VIII with an IC50 approximately 10 micro M. The heavy chain was the susceptible subunit with inhibition by copper ion resulting from its reduced affinity for light chain. On the other hand, Mn(II)-dependent factor VIII reconstitutions performed with Cu(II) light chain and native heavy chain occurred at an accelerated rate (approximately 10-fold) and yielded an enhanced activity ( approximately 50%), likely reflecting an increased specific activity of the heterodimer. Cu ions enhanced the activity of EDTA-treated factor VIII in the presence of Ca(II) but not in its absence, suggesting that EDTA-treated factor VIII is not equivalent to separated subunits and that copper ions are auxiliary to ions that mediate reconstitution. Conformational analyses showed that the ellipticities and extrinsic fluorescence of both subunits were differentially affected by Cu(II) and Mn(II). These structural effects were fully reversed by EDTA. The metal ions had little if any effect on the conformation of intact factor VIII or the A1/A3-C1-C2 dimer. Mn(II) and Cu(II) stabilized the factor VIII light chain, and the latter stabilized the A1 subunit derived from the heavy chain, yielding similar thermal denaturation profiles that were distinct from that observed for the Ca(II)-stabilized subunits. Thus both subunits of factor VIII bind copper ions, and the effects of this binding differ from the interactions observed with Ca(II) or Mn(II). These data support a model where copper in factor VIII likely functions to increase specific activity of the heterodimer rather than directly mediating the intersubunit interaction.

Calcium↗
PubMed1997

Identification of a novel allele at the human NAT1 acetyltransferase locus.

Humans possess two N-acetyltransferase isozymes (NAT1 and NAT2). We cloned and sequenced a novel NAT1 allele (Genbank HSU 80835) that contained nucleotide substitutions at -344 (C-->T), -40 (A-->T), 445 [G-->A(Val-->Ile)], 459 [G-->A(silent)], 640 [T-->G(Ser-->Ala)], a 9 base pair deletion between nucleotides 1065 and 1090, and 1095 (C-->A). The novel NAT1 allele which we have designated NAT1*17 is similar to NAT1*11 except for a G445A substitution (Val149-->Ile) in the NAT1 coding region. The G445A (Val149-->Ile) substitution yielded no significant changes in levels of immunoreactivity, as detected by Western blot, nor in intrinsic stability of the recombinant N-acetyltransferase protein. However, the G445A (Val149-->Ile) substitution yielded expression of recombinant NAT1 protein that catalyzed the N-acetylation of aromatic amines and the O- and N,O-acetylation of their N-hydroxylated metabolites at rates up to 2-fold higher than wild-type recombinant human NAT1.

Acetylation↗
PubMed2004

Stability and repair of DNA in hyperthermophilic Archaea.

Evolutionary and physiological considerations argue that study of hyperthermophilic archaea should reveal new molecular aspects of DNA stabilization and repair. So far, these unusual prokaryotes have yielded a number of genes and enzymatic activities consistent with known mechanisms of excision repair, photo-reversal, and trans-lesion synthesis. However, other DNA enzymes of hyperthermophilic archaea show novel biochemical properties which may be related to DNA stability or repair at extremely high temperature but which remain difficult to evaluate rigorously in vivo. Perhaps the most striking feature of the hyperthermophilic archaea is that all of them whose genomes have been sequenced lack key genes of both the nucleotide excision repair and DNA mismatch repair pathways, which are otherwise highly conserved in biology. Although the growth properties of these micro-organisms hinder experimentation, there is evidence that some systems of excision repair and mutation avoidance operate in Sulfolobus spp. It will therefore be of strategic significance in the next few years to formulate and test hypotheses in Sulfolobus spp. and other hyperthermophilic archaea regarding mechanisms and gene products involved in the repair of UV photoproducts and DNA mismatches.

Archaea↗
PubMed1997

Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding.

A growing body of evidence indicates that the most common cystic fibrosis-causing mutation, DeltaF508, alters the ability of the cystic fibrosis transmembrane conductance regulator (CFTR) protein to fold and transit to the plasma membrane. Here we present evidence that the DeltaF508 mutation affects a step on the folding pathway prior to formation of the ATP binding site in the nucleotide binding domain (NBD). Notably, stabilization of the native state with 4 mM ATP does not alter the temperature-dependent folding yield of the mutant DeltaF508 NBD1 in vitro. In contrast, glycerol, which promotes DeltaF508-CFTR maturation in vivo, increases the folding yield of NBD1DeltaF and reduces the off pathway rate in vitro, although it does not significantly alter the free energy of stability. Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of DeltaF508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain. These results demonstrate that DeltaF508 is a kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native state ligands to promote folding. The implications for protein folding in general are that the primary sequence may not necessarily simply define the most stable native structure, but rather a stable structure that is kinetically accessible.

Adenosine Triphosphate↗
PubMed1995

Tubercidin stabilizes microtubules against vinblastine-induced depolymerization, a taxol-like effect.

A sensitive assay for the detection of microtubule-stabilizing agents [1] was used to screen an extensive collection of cyanobacterial and microalgal extracts. The hydrophilic extract of the cyanobacterium, Plectonema radiosum (UH isolate IC-70-1), exhibited microtubule-stabilizing activity. Bioassay-directed purification of the active compound yielded tubercidin (7-deazaadenosine), a potent cytotoxic nucleoside analog. Further studies revealed that tubercidin protected a population of cellular microtubules against vinblastine-induced depolymerization, a microtubule-stabilizing, taxol-like effect. The microtubule-stabilizing effect of tubercidin is dose dependent and limited by the cytotoxicity of the agent. Tubercidin represents another natural product that interacts with microtubules and is one of the few to cause microtubule stabilization.

Animals↗
PubMed2006

Screening for ligands using a generic and high-throughput light-scattering-based assay.

Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.

Binding Sites↗
PubMed2001

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins.

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.

ATP-Binding Cassette Transporters↗
PubMed1995

Studies of the reaction of acetaldehyde with deoxynucleosides.

The reaction of acetaldehyde with deoxynucleosides was studied in buffered solutions at room temperature (22-24 degrees C) and neutral pH. Reaction products were obtained with all deoxynucleosides with the exception of thymidine, as shown by reversed-phase HPLC analysis. The order of reactivity was dGuo > dAdo > dCyd, for which three, two and one reaction products, respectively, were obtained. We report here data on the kinetics of the reactions, the stability of the adducts at physiological pH, product yields, UV-spectroscopic data at different pH values, and describe the synthesis, isolation and structural characterization by FAB/MS and NMR of the stable adducts of acetaldehyde with dGuo. Furthermore, the formation of adducts with dGuo by the cooperative reaction of Aa with ethanol was studied.

Acetaldehyde↗
PubMed2006

7-Dialkylamino-1-alkylquinolinium salts: highly versatile and stable fluorescent probes.

7-Dialkylamino- and 7-alkylsulfenyl-1-alkylquinolinium salts have been synthesized using a novel synthetic approach. The key intermediate, 7-fluoro-1-methylquinolinium iodide, was shown to possess high reactivity toward nitrogen and sulfur nucleophiles, and the kinetics of this nucleophilic aromatic substitution reaction was investigated. A wide variety of compounds were synthesized and characterized spectroscopically. High fluorescence quantum yields were observed, and this was attributed to the rigid molecular architecture. The thermal and photochemical stability of a number of compounds was investigated, and it was demonstrated that 7-dialkylamino-1-methylquinolinium salts have superior stability compared to a number of hemicyanine dyes and rigid charge-transfer probes. Based on the high quantum yields, the large Stokes shifts, and in particular, the high thermal and photochemical stability, it is concluded that 7-dialkylamino-1-methylquinolinium salts are excellent color-shifting, mobility-sensitive fluorescent probes for polymer characterization and other demanding applications.

Fluorescent Dyes↗
PubMedDate not supplied

Cysteine analogs of recombinant barley ribosome inactivating protein form antibody conjugates with enhanced stability and potency in vitro.

Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.

Animals↗
PubMed2004

Rice bran stabilization and rice bran oil extraction using ohmic heating.

Ohmic heating has been shown to increase the extraction yields of sucrose from sugar beets, apple juice from apples, beet dye from beet root, and soymilk from soybeans. Rice bran is a byproduct of the rice milling process that has economic potential by virtue of highly nutritious rice bran oil contained within the bran. In this study, ohmic heating was used to stabilize rice bran and to improve rice bran oil extraction yield as compared to microwave heating and a control (no heating). Results showed that ohmic heating is an effective method for rice bran stabilization with moisture addition. Free fatty acid concentration increased more slowly than the control for raw bran samples subjected to ohmic heating with no corresponding temperature rise, indicating that electricity has a non-thermal effect on lipase activity. Ohmic heating increased the total percent of lipids extracted from rice bran to a maximum of 92%, while 53% of total lipids were extracted from the control samples. Lowering the frequency of alternating current significantly increased the amount of oil extracted, probably due to electroporation. Ohmic heating was successfully applied to rice bran despite its high oil content. This could have important implications for the enhanced extraction of non-polar constituents.

Electric Impedance↗
PubMed2001

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus.

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an alpha-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.

Carbohydrate Conformation↗