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Biomedical subjects

A Dong

Publications and source records attributed to A Dong.

At least 55 records · Page 3Linked to original sources
PubMed1994

Characterization of the guanidine hydrochloride-denatured state of iso-1-cytochrome c by infrared spectroscopy.

Infrared spectroscopy has been used to monitor residual ordered structure in the denatured state of wild-type and two mutants of iso-1-cytochrome c. The technique used involves a careful digital subtraction procedure that removes spectral contributions from buffer, water vapor, and the denaturant guanidine hydrochloride. Reliable and reproducible spectra can be produced using these methods. The data for iso-1-cytochrome c show upon denaturation a shift of the structure-sensitive amide I infrared band away from the spectral region associated with random structure. Second-derivative resolution enhancement of the amide I absorption band uncovers several bands which can be associated with various residual ordered structures in the denatured state. Gradual changes in the amide I band after denaturation are also observed as the guanidine hydrochloride concentration is increased. Two single-site mutants of iso-1-cytochrome c, which have been shown to have more compact denatured states than the wild-type protein, exhibited denatured-state infrared spectra with significant differences from the wild-type protein spectra. These observations provide new insight into the characteristics of protein denatured states.

Amides↗
PubMed1994

Effects of calcium, magnesium, and phosphorylcholine on secondary structures of human C-reactive protein and serum amyloid P component observed by infrared spectroscopy.

The secondary structures of human C-reactive protein (CRP) and serum amyloid P component (SAP) in D2O-based solutions in the presence or absence of calcium, magnesium, and phosphorylcholine have been investigated using Fourier transform infrared spectroscopy. Quantitative analysis provided estimations of about 50% beta-sheet, 12% alpha-helix, 24% beta-turn, and 14% unordered structure for CRP and about 54% beta-sheet, 12% alpha-helix, 25% beta-turn, and 9% unordered structure for SAP. With both proteins significant calcium-dependent changes were observed in conformation-sensitive amide I regions assigned to each type of structure. The CRP spectrum was also affected by magnesium, but the changes differed from those induced by calcium. The SAP spectrum was not affected by magnesium. Phosphorylcholine in the presence of calcium also affected the spectrum of CRP but not the spectrum of SAP. Our present study provides the first direct comparison of the secondary structures of the pentraxins human CRP and SAP and hamster female protein (Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S., and Coe, J. E. (1992) Biochemistry 32, 9364-9370). These findings suggest that the three pentraxins have similar secondary structure compositions and calcium-dependent conformational changes, but differ significantly in their responses to phosphorylcholine and magnesium. Such properties are expected to be relevant to the incompletely understood roles of these highly conserved proteins including binding to nuclear proteins, complement activation, and association with amyloids.

C-Reactive Protein↗
PubMed1993

Destabilizing effects of replacing a surface lysine of cytochrome c with aromatic amino acids: implications for the denatured state.

A series of mutations at the highly solvent-exposed lysine 73 of iso-1-cytochrome c have been prepared by site-directed mutagenesis. These mutations were designed to probe denatured-state effects on the unfolding equilibrium of this protein. The hydrophilic amino acid Lys was replaced with the hydrophobic amino acids Met, Tyr, Phe, and Trp. The idea was to induce stabilizing hydrophobic interactions in the unfolded state, while having little effect on the folded-state energy due to the high solvent exposure of this site. Fourier transform infrared spectral analyses indicate that none of these mutations significantly affect the native fold of the protein. The stability of each protein to guanidine hydrochloride denaturation was monitored at 25 degrees C by circular dichroism spectroscopy. All four hydrophobic mutants decreased the value of delta Go uH2O, the free energy of unfolding of the protein in the absence of denaturant, by 1.0-1.5 kcal/mol. The delta Go uH2O values for these proteins correlate linearly (correlation coefficient of 0.98) with the hydrophobicity of the amino acid at position 73 of the sequence. These data are consistent with the idea that the position-73 mutants are more buried in the denatured state than in the native state, suggestive of a compact denatured state where such interactions would be possible.

Base Sequence↗
PubMed1993

Probing heart cytochrome c oxidase structure and function by infrared spectroscopy.

IR spectra directly probe specific vibrators in bovine heart cytochrome c oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to Fe2+ a3 as two conformers each in isolated immobile environments sensitive to Fea and/or CuA oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN- that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fea, CuA, and CuB with associated changes in protein conformation of likely significance in O2 reduction and H(+)-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of alpha-helix (approximately 60%) and subtle changes with redox level and exposure to anesthetics. N2O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.

Animals↗
PubMed1992

Secondary structure of the pentraxin female protein in water determined by infrared spectroscopy: effects of calcium and phosphorylcholine.

The secondary structure of hamster female protein in aqueous solutions in the presence or absence of calcium and phosphorylcholine has been investigated using Fourier transform infrared spectroscopy. Our present studies provide the first evaluation of the secondary structure of FP and its calcium- and phosphorylcholine-dependent conformational changes. Quantitative analysis indicated that FP is composed of 50% beta-sheet, 11% alpha-helix, 29% beta-turn, and 10% random structures. Calcium- and phosphorylcholine-dependent infrared spectral changes were observed in regions assigned to beta-sheet, alpha-helix, turn, and random structures. The infrared-based secondary structure compositions were used as constraints to compute theoretical locations for the different secondary structures along the amino acid sequence of the FP protein. Two putative calcium-binding sites were proposed for FP (residues 93-109 and 150-168) as well as other members of the pentraxin family on the basis of the theoretical secondary structure predictions and the similarity in sequence between the pentraxins and EF-hand calcium-binding proteins. The changes in protein conformation detected upon binding of calcium and phosphorylcholine provide a mechanism for the effects of these ligands on physiologically important properties of the protein, e.g., activation of complement and association with amyloids.

Alpha-Globulins↗
PubMed1992

Computer-assisted neurosurgery: simulation and automation.

Instrumentation has been developed to automate, accurately guide, and integrate microsurgery laser resection equipment that is used in stereotactic brain surgery. The instrument is a computer-controlled laser-guiding system. The improvement in the accuracy comes from relying on a certain well-referenced three-dimensional surgical orientation, and computer-preplanned tumor contours, rather than only the surgeons hand-eye orientation in the operating room.

Automation↗
PubMed1991

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.

Acetylglucosamine↗
PubMed1990

Protein secondary structures in water from second-derivative amide I infrared spectra.

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.

Alcohol Dehydrogenase↗
PubMed1989

Distal His----Arg mutation in bovine myoglobin results in a ligand binding site similar to the abnormal beta site of hemoglobin Zurich (beta 63 His----Arg).

Carbon monoxide binding to a myoglobin mutant with distal arginine in place of histidine has been examined. The mutant is derived from a cDNA clone for Mb mRNA from fetal bovine skeletal muscle. The mutation only slightly perturbs visible/Soret spectra whereas the infrared spectrum of liganded CO is greatly modified to become nearly identical to Hb Zurich beta-subunit spectrum. The mutant IR spectra differ substantially from spectra of wild-type MbCO and normal HbCO beta-subunit. For both the Mb and the Hb the distal His----Arg mutation increases the affinity for CO and reduces the number of observed conformers. These results demonstrate that this mutation greatly reduces the differences between Mb and Hb in the structure and properties of its ligand binding sites.

Animals↗
PubMed1989

Comparison of the secondary structures of human class I and class II major histocompatibility complex antigens by Fourier transform infrared and circular dichroism spectroscopy.

We have examined the secondary structures of human class I and class II histocompatibility antigens in solution by Fourier transform infrared spectroscopy and circular dichroism in order to compare the relative amounts of alpha-helix, beta-sheet, and other structures, which are crucial elements in the comparison of the protein structures. Quantitation of infrared spectra of papain-solubilized HLA-A2, HLA-B7, and DR1 in phosphate buffer gave alpha-helix contents of 17%, 8%, and 10% and beta-sheet contents of 41%, 48%, and 53%, respectively. By circular dichroism, papain-solubilized HLA-A2, HLA-B7, and DR1 were also found to have comparable alpha-helix contents (e.g., 8%, 20%, and 17%, respectively). Circular dichroism analysis for beta-sheet gave 29% for papain-solubilized HLA-B7 and 42% for papain-solubilized DR1. The value for papain-solubilized HLA-A2 (74%) was anomalous. It is proposed that Trp-107 of HLA-A2, missing in both HLA-B7 and DR1, may be responsible for much of the anomaly. Due to the uncertainties inherent in quantitation of the amounts of secondary structures by both spectral methods, the differences in the contents of alpha-helix and beta-sheet in the three proteins are not considered significant. However, differences in the nature of the beta-sheet structures are suggested by infrared spectroscopy. These results provide physical evidence for an overall structure of class II antigens modeled on that of class I antigens.

Buffers↗
PubMed1988

Infrared spectroscopy of a single cell--the human erythrocyte.

Methods for obtaining the infrared spectrum of a single erythrocyte by infrared microscopy have been developed. The spectrum contains the amide I, II, and III bands characteristic of protein secondary structure near 1650, 1550, and 1300 cm-1, respectively. Bound carbon monoxide exhibits a readily measured band at 1951 cm-1 for 12C16O and 1907 cm-1 for 13C16O. Both amide and CO bands are similar to those found for purified hemoglobin A. Spectra can be obtained in H2O or D2O media under physiologically relevant conditions. Single cell infrared spectroscopy (SCIR) permits the qualitative and quantitative determination of differences among individual red cells. These results suggest many potential applications for SCIR for the measurements of properties of individual cells at the molecular level under physiologically relevant conditions.

Carbon Monoxide↗
PubMed1987

The anesthetic nitrous oxide affects dioxygen utilization by bovine heart and bean seed mitochondrial particles.

Nitrous oxide affects dioxygen utilization by both bean seed and bovine heart submitochondrial particles when either succinate or reduced cytochrome c are used as substrates. Bovine heart particles exhibit reversible, dose-dependent partial inhibition of respiratory activity when exposed to N2O. Bean seed particle respiration is stimulated by low levels of N2O, but higher concentrations are inhibitory. These findings can be explained in terms of one locus of anesthetic action: cytochrome c oxidase, the terminal component of the mitochondrial respiratory chain. Alterations in respiration rates are expected to make important contributions to anesthesia in animals and to control of germination in plants.

Animals↗
PubMed1986

Limitations of urinary mutagen assays for monitoring occupational exposure to antineoplastic drugs.

The sensitivity of the Salmonella reversion test of Ames as a screen for accidental absorption of 17 antineoplastic agents by drug handlers was evaluated. Dilutions of each drug were added to agar inoculated with each of two Salmonella typhimurium strains (TA98 and TA100); control plates contained no test drug. Colonies were counted after incubation at 36 degrees C for 48 hours. The drugs were tested in the presence of a liver preparation to provide metabolic activation of mutagenicity. Urine samples collected from patients after doses of three mutagenic drugs were extracted and tested with the Ames test. For 11 of the 17 drug solutions, no mutagenic activity was seen, but many of these 11 were toxic to the organisms. The most highly mutagenic drugs were doxorubicin and cisplatin, with mechlorethamine, carmustine, dacarbazine, and cyclophosphamide exhibiting less mutagenic activity. Urine from patients treated with doxorubicin or cyclophosphamide showed mutagenicity, but the results suggested that the quantity of these drugs that would have to be absorbed to produce a definite reaction in urine is unlikely to be achieved by drug handlers who use standard precautions. Because of its lack of sensitivity and the potential effects of environmental and dietary factors on the results, this bacterial mutagenicity test should not be used routinely for detection of accidental absorption of antineoplastic drugs.

Antineoplastic Agents↗
PubMed1982

Serum vitamin B12 and blood cell values in vegetarians.

Serum vitamin B12 and complete blood count values were determined for 83 volunteer subjects from an American vegetarian society conference (USA). Among subjects who did not supplement their diets with vitamin B12 or multiple vitamin tablets, 92% of the vegans (total vegetarians), 64% of the lactovegetarians, 47% of the lacto-ovovegetarians and 20% of the semivegetarians had serum vitamin B12 levels less than 200 pg/ml (normal = 200-900 pg/ml). However, their complete blood count values did not deviate greatly from those found for nonvegetarians, even though some had been vegans or lactovegetarians for over 10 years. Macrocytosis among the vegetarians was minimal; none had mean corpuscular volume greater than 103 fl.

Adolescent↗
PubMed1976

Feasibility of routine testing for hepatitis B surface antigen in hospital employees and restriction of carriers.

In 1972-73, 48 hospital staff members were tested selectively for hepatitis B surface antigen (HBsAg); 4 (8.3%) were found to be HBsAg-positive. In 1974-75, 1415 staff members were tested routinely before employment and at periodic health examination; 25 (1.8%) were found to be HBsAg-positive. Of the HBsAg-positive staff members 55.2% were Asians, this proportion being significantly (P less than 0.05) greater than that of any other ethnic group, and 31.0% were southern Europeans. Nurses and laboratory technologists were the largest professional groups among the HBsAg-positive staff, each accounting for 20.7%. Our results indicate that it is impractical to carry out routine testing of hospital staff for HBsAg. Selective testing and restriction from work in their units is proposed for staff of the renal and peritoneal dialysis units, the emergency department and the intravenous team and dietary staff who handle food directly.

Adult↗