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T D Williams

Publications and source records attributed to T D Williams.

At least 55 records · Page 3Linked to original sources
PubMed1999

Solution stability of linear vs. cyclic RGD peptides.

Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2-12 at 50 degrees C. Reversed-phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-first-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.

Chromatography, High Pressure Liquid↗
PubMed1998

Progressive decline in the ability of calmodulin isolated from aged brain to activate the plasma membrane Ca-ATPase.

To identify possible relationships between the loss of calcium homeostasis in brain associated with aging and alterations in the function of key calcium regulatory proteins, we have purified calmodulin (CaM) from the brains of Fischer 344 rats of different ages and have assessed age-related alterations in (i) the secondary and tertiary structure of CaM and (ii) the ability of CaM to activate one of its target proteins, the plasma membrane (PM) Ca-ATPase. There is a progressive, age-dependent reduction in the ability of CaM to activate the PM-Ca-ATPase, which correlates with the oxidative modification of multiple methionines to their corresponding methionine sulfoxides. No other detectable age-related posttranslational modifications occur in the primary sequence of CaM, suggesting that the reduced ability of CaM to activate the PM-Ca-ATPase is the result of methionine oxidation. Corresponding age-related changes in the secondary and tertiary structure of CaM occur, resulting in alterations in the relative mobility of CaM on polyacrylamide gels, differences in the intrinsic fluorescence intensity and solvent accessibility of Tyr99 and Tyr138, and a reduction in the average alpha-helical content of CaM at 20 degreesC. Shifts in the calcium- and CaM-dependent activation of the PM-Ca-ATPase are observed for CaM isolated from senescent brain, which respectively requires larger concentrations of either calcium or CaM to activate the PM-Ca-ATPase. The observation that the oxidative modification of CaM during normal biological aging results in a reduced calcium sensitivity of the PM-Ca-ATPase, a lower affinity between CaM and the PM-Ca-ATPase, and the reduction in the maximal velocity of the PM-Ca-ATPase is consistent with earlier results that indicate the calcium handling capacity of a range of tissues including brain, heart, and erythrocytes isolated from aged animals declines, resulting in both longer calcium transients and elevated basal levels of intracellular calcium. Thus, the oxidative modification of selected methionines in CaM may explain aspects of the loss of calcium homeostasis associated with the aging process.

Aging↗
PubMed1998

Glutathione carbamoylation with S-methyl N,N-diethylthiolcarbamate sulfoxide and sulfone. Mitochondrial low Km aldehyde dehydrogenase inhibition and implications for its alcohol-deterrent action.

S-Methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) and sulfone (DETC-MeSO2) both inhibit rat liver low Km aldehyde dehydrogenase (ALDH2) in vitro and in vivo (Nagendra et al., Biochem Pharmacol 47: 1465-1467, 1994). DETC-MeSO has been shown to be a metabolite of disulfiram, but DETC-MeSO2 has not. Studies were carried out to further investigate the inhibition of ALDH2 by DETC-MeSO and DETC-MeSO2. In an in vitro system containing hydrogen peroxide and horseradish peroxidase, the rate of DETC-MeSO oxidation corresponded to the rate of DETC-MeSO2 formation. Carbamoylation of GSH by both DETC-MeSO and DETC-MeSO2 was observed in a rat liver S9 fraction. Carbamoylation of GSH was not observed in the presence of N-methylmaleimide. In in vitro studies, DETC-MeSO and DETC-MeSO2 were equipotent ALDH2 inhibitors when solubilized mitochondria were used, but DETC-MeSO was approximately four times more potent than DETC-MeSO2 in intact mitochondria. In studies with rats, the dose (i.p. or oral) required to inhibit 50% ALDH2 (ED50) was 3.5 mg/kg for DETC-MeSO and approximately 35 mg/kg for DETC-MeSO2, approximately a 10-fold difference. Furthermore, maximum ALDH2 inhibition occurred 1 hr after DET(-MeSO administration, whereas maximal ALDH2 inhibition occurred 8 hr after DETC-MeSO2 dosing. DETC-MeSO is, therefore, not only a more potent ALDH2 inhibitor than DETC-MeSO2 in vivo, but also in vitro when intact mitochondria are utilized. The in vitro results thus support the in vivo findings. Since oxidation of DETC-MeSO can occur both enzymatically and non-enzymatically, it is possible that DETC-MeSO2 is formed in vivo. DETC-MeSO2, however, is not as effective as DETC-MeSO in inhibiting ALDH2, probably because it has difficulty penetrating the mitochondrial membrane. Thus, even if DETC-MeSO2 is formed in vivo from DETC-MeSO, it is the metabolite DETC-MeSO that is most likely responsible for the inhibition of ALDH2 after disulfiram administration.

Aldehyde Dehydrogenase↗
PubMed1998

Loss of conformational stability in calmodulin upon methionine oxidation.

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.

Amino Acid Sequence↗
PubMed1998

Fatty acid binding protein, a major protein in the flight muscle of migrating western sandpipers.

Migratory flight in birds is fueled primarily by fatty acid oxidation imposing a requirement for high rates of fatty acid: (a) transport; (b) uptake; and (c) delivery to intracellular sites of beta-oxidation. Muscle fatty acid binding protein (M-FABP) is a cytosolic protein involved in the intracellular transport of fatty acids. Its expression appears to be correlated with muscle fatty acid oxidation capacity. The M-FABP was isolated for the first time from a long distance migrant bird using: (i) size exclusion; (ii) anion exchange; and (iii) hydroxyapatite chromatography. M-FABP has a molecular weight of approximately 14,000 Da and an isoelectric point of pH 4.8. A partial amino acid sequence of the protein demonstrated homology to M-FABPs from other species (80% identical to human heart FABP). It was estimated that M-FABP comprises approximately 14 and 21% of total cytosolic protein of the pectoralis and heart, respectively; the highest values yet reported from any vertebrate muscle. The abundance of M-FABP in these tissues suggests that the protein may play a key role in fatty acid supply during endurance flight. Thus, it is proposed that a seasonal increase in M-FABP expression could be a component of physiological preparation for migration.

Amino Acid Sequence↗
PubMed1998

Solubility enhancement of phenol and phenol derivatives in perfluorooctyl bromide.

Perfluorinated solvents are gaining popularity as pulmonary ventilation fluids, but they suffer from poor solvent quality in concurrent drug delivery applications. The present study examines the use of a hydrophobic solubilizing agent capable of interacting with model drug solutes by hydrogen bonding with the purpose of enhancing solubility in perfluorooctyl bromide (PFOB). A series of solubilizing agents containing a ketone carbonyl to act as a hydrogen bond acceptor and a perfluoroalkyl chain to maintain the solubility of the putative complex in PFOB are investigated. The solubility of phenol in PFOB is enhanced to the greatest extent by 1-(4-perfluorobutyl phenyl)-1-hexanone (III) where the ketone carbonyl is protected from the electron withdrawing effects of the perfluorobutyl chain by a phenyl ring. Experiments with solubilizers lacking the ketone group suggest that pi-pi bond interactions of III with phenol do not significantly enhance solubility. For a series of phenol derivatives, a rank-order correlation exists between the magnitude of solubility enhancement by III, as reflected by the calculated association constants, and the Hammett sigma parameter of the phenols. Because the O-methyl-substituted phenols do not have the ability to hydrogen bond, their solubility is not enhanced by the presence of III. The results of the present study indicate that solubility of model drug hydrogen bond donating compounds can be enhanced in PFOB by the presence of fluorocarbon-soluble hydrogen bond acceptors.

Chromatography, High Pressure Liquid↗
PubMed1998

Enzymatic reduction of 3-nitrotyrosine generates superoxide.

Spin-trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) was used to demonstrate that 3-nitrotyrosine (nitrotyrosine) promotes the formation of substantial amounts of reactive oxygen species (O2.- and *OH), when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor. Spin adduct formation is strongly inhibited by the presence of superoxide dismutase (SOD); spin adduct formation requires aerobic conditions. Nitration of leucine enkephalin, a tyrosine-containing pentapeptide, results in a similar generation of O2*- and *OH species. Both nitrotyrosine and nitrated leucine enkephalin stimulate acetylated ferricytochrome c reduction in the presence of NAD(H)-cytochrome c reductase with typical Michaelis-Menten kinetics and Km's of 104 +/- 14 and 0.78 +/- 0.11 microM, respectively. No stimulation of acetylated ferricytochrome c reduction is observed in the presence of SOD. Catalase and the metal chelators DTPA and deferoxamine mesylate do not influence observed stimulation of acetylated ferricytochrome c reduction by nitrotyrosine. Nitration of two tyrosines (of four) within the sequence of the 6.5-kDa globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2*- generation implying steric restrictions for BPTI-reductase interactions. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferricytochrome c. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine may be enzymatically reduced to the corresponding nitro anion radical (ArNO2*-) which is then oxidized by molecular oxygen to yield O2*- and regenerate ArNO2. Thus, once formed in vivo, nitrotyrosine may act to promote oxidative stress by means of repetitive redox cycling.

Chromatography, High Pressure Liquid↗
PubMed1998

Low-frequency arterial pressure fluctuations do not reflect sympathetic outflow: gender and age differences.

Low-frequency arterial pressure oscillations (Mayer waves) have been proposed as an index of vascular sympathetic outflow. However, cross-sectional differences in these pressure oscillations may not reflect different levels of sympathetic nervous outflow in humans. Three groups of healthy subjects with characteristically different sympathetic nervous outflow were studied: young females (n = 10, 18-28 yr), young males (n = 11, 18-29 yr), and older males (n = 13, 60-72 yr). Average R-R interval, arterial pressures, and systolic pressure variability at the Mayer wave frequency (0.05-0.15 Hz) did not differ among the three groups. Diastolic pressure Mayer wave variability was similar in young females vs. young males (39 +/- 10 vs. 34 +/- 5 mmHg2) and lower in older males vs. young males (14 +/- 2 mmHg2; P < 0.05). In contrast, muscle sympathetic activity was lowest in young females (892 +/- 249 total activity/min) and highest in older males (3,616 +/- 528 total activity/min; both P < 0.05 vs. young males: 2,505 +/- 285 total activity/min). Across the three groups, arterial pressure Mayer wave variability did not correlate with any index of sympathetic activity. Our results demonstrate that arterial pressure Mayer wave amplitude is not a surrogate measure of vascular sympathetic outflow.

Adolescent↗
PubMed1997

Identification of oxidation-sensitive peptides within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+-ATPase.

We have examined the oxidative sensitivity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) membranes, exposing isolated SR membranes to the thermolabile water soluble free radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Incubation with up to 702 microM AAPH-derived radicals results in a concentration- and time-dependent inhibition of calcium-dependent ATPase activity correlating with the loss of monomeric Ca2+-ATPase polypeptides, and the concomitant appearance of higher molecular weight species. However, no oxidant-induced protein fragmentation is detected. The observed formation of oxidant-induced bityrosine accounts for the intermolecular Ca2+-ATPase cross-links, as well as intramolecular cross-links. The oxidation of sulfhydryl groups to disulfides as another possible source of intermolecular cross-links has been ruled out after examination of SDS -PAGE performed under both reducing and non-reducing conditions. Exposure of the SR membranes to AAPH-derived radical species results in a small degree of lipid peroxidation that is not correlated with enzyme inactivation, suggesting that modification of membrane-spanning peptides is not related to enzyme inactivation. Six cytoplasmic peptides have been identified that are modified by exposure to AAPH or, alternatively, to hydrogen peroxide, suggesting that these regions of the Ca2+-ATPase are generally sensitive to oxidants. These oxidized peptides were identified after separation by reversed-phase HPLC followed by N-terminal sequencing and amino acid analysis as corresponding to the following sequences of the Ca2+-ATPase: (i) Glu121 to Lys128, (ii) His190 to Lys218, (iii) Asn330 to Lys352, (iv) Gly432 to Lys436, (v) Glu551 to Arg604, and (vi) Glu657 to Arg671. The Glu551 to Arg604 peptide, located within the nucleotide binding domain, was found to participate in the formation of intermolecular bityrosine cross-links with the identical Glu551 to Arg604 peptide from a neighboring Ca2+-ATPase polypeptide chain.

Amidines↗
PubMed1997

Albinism: an update and review of the literature.

BACKGROUND: Albinism can be a diagnostic challenge to the optometrist, with ocular albinism the entity most likely to be overlooked or misdiagnosed. Albinism should be suspect in a child with nystagmus. METHODS: Albinism is best diagnosed by electron microscopy of skin or hair bulbs. Measuring the flash visually evoked response (VER) is the best way to confirm the abnormal decussation of the optic nerve fibers. Transillumination of the iris can be performed to see if it lacks pigment on its posterior surface. Optometrists should also be alert for nystagmus, strabismus, lack of stereopsis, and poor fusional capacity. Most people with albinism have photophobia and reduced acuity, and many have defective hearing. RESULTS: This review describes the various kinds of albinism and summarizes associated ocular manifestations with pertinent forms of the disorder. CONCLUSIONS: Optometrists are responsible for detection of albinism and provision of optical aids and related advice to afflicted patients. These patients also need appropriate counselling and genetic studies of family members.

Albinism, Oculocutaneous↗
PubMed1996

Oxidative modification of a carboxyl-terminal vicinal methionine in calmodulin by hydrogen peroxide inhibits calmodulin-dependent activation of the plasma membrane Ca-ATPase.

In order to investigate the possibility that calmodulin (CaM) may be a principal target of reactive oxygen species (ROS) produced under conditions of oxidative stress, we have examined wheat germ CaM for the presence of highly reactive sites that correlate with the loss of function. Using reversed-phase HPLC and FAB mass spectrometry after proteolytic digestion, we have identified the sites of modification by hydrogen peroxide. We find that one of the vicinal methionines (i.e., Met146 or Met147) near the C-terminus of CaM is selectively oxidized. The ability of CaM to bind and to activate the plasma membrane (PM)-Ca-ATPase from erythrocytes was measured. There is a 30-fold decrease in the calcium affinity of oxidatively modified CaM. While there is a little change in the binding constant between the carboxyl-terminal domain of calcium-saturated CaM and a peptide homologous to the autoinhibitory sequence of the PM-Ca-ATPase, we find that there is a 9-fold reduction in the affinity of the amino-terminal domain of CaM with respect to the ability to bind target peptides. The extent of oxidative modification to one of the vicinal methionines near the carboxyl-terminal domain correlates with the loss of CaM-dependent activation of the PM-Ca-ATPase. The presence of oxidatively modified CaM prevents native CaM from activating the PM-Ca-ATPase, indicating that the oxidatively modified CaM binds to the autoinhibitory sequence on the Ca-ATPase in an altered nonproductive conformation. We suggest that the functional sensitivity of CaM to the oxidation of one of the C-terminal vicinal methionines permits CAM to serve a regulatory role in modulating cellular metabolism under conditions of oxidative stress. The predominant oxidation of a methionine near the carboxyl terminal of CaM is rationalized in terms of the enhanced solvent accessibility of these vicinal methionines.

Amino Acid Sequence↗
PubMed1996

Gas-phase reactions of glycine, alanine, valine and their N-methyl derivatives with the nitrosonium ion, NO+.

The gas-phase reactions of the nitrosonium ion, NO+ with the amino acids glycine, alanine and valine and their N-methyl derivatives were investigated under chemical ionization mass spectrometric (CIMS) conditions. Two products were observed in all cases: the formation of the iminium ion and the formation of an [M-H]+ ion. The latter product is consistent with a reaction channel involving hydride abstraction by NO+, and was confirmed by (i) examining the Ar+CI mass spectra of the same amino acids under similar source conditions and (ii) examining the unimolecular fragmentation reactions of the [M + H]+ ions of the N-nitroso-N-methyl derivatives of each of the amino acids in a tandem mass spectrometer. Further insights into the reaction of glycine with NO+ were obtained by performing ab initio calculations (at the MP2/6-31G* parallel HF/6-31G* level). These results indicate that four reactions are thermodynamically viable for glycine: (i) hydride abstraction; (ii) iminium ion formation (with concomitant loss of HONO and CO); (iii) diazonium ion formation; and (iv) diazonium ion formation followed by loss of N2. Possible reasons why reactions (iii) and (iv) are not observed are discussed, and comparisons with solution reactivity and the gas-phase reactivity of NO+ are also made.

Alanine↗
PubMed1996

Oxygen-centered spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2H-imidazole 1-oxides.

The hydroxyl and some alkoxyl spin adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) are difficult to assign due to the remarkable similarity of their EPR spectra. The utility of resolving superhyperfine (SHF) structure followed by computer simulations has been demonstrated to assist in the assignment of EPR spectra with close values of hyperfine splitting constants, e.g., DMPO/ .OH and DMPO/.OR. Here, .OR is the alkoxyl radical derived from thermal decomposition of 2,2' -azobis (2-amidinopropane) hydrochloride (AAPH). In addition, two other spin traps, derivatives of 2H-imidazole 1-oxide, namely, 2,2,4-trimethyl-2H-imidazole 1-oxide (TMIO) and 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO), have been used in a model study to develop a procedure for distinguishing between oxygen-centered spin adducts. These results are compared with those for DMPO. TMIO and DMPIO spin traps provide more distinguishable individual spectra with .OH and AAPH-derived .OR radicals than the DMPO spin trap. The formation of DMPO/.OR(AAPH) and DMPIO/ .OR(AAPH) spin adducts was confirmed by mass spectrometry. The comparison of spin trapping by DMPO and 2H-imidazole 1-oxides using typical biological sources of other oxygen-centered radicals reveals application limits of these spin traps. For example, 2H-imidazole 1-oxides do not form superoxide spin adducts in the xanthine/xanthine oxidase system. Also, for the first time, experimental evidence is presented for SHF structure in spectra of TMIO and DMPIO spin adducts with .OH/.OD and .CH3/ .CD3 radical species.

Animals↗
PubMed1996

Decreased plasma membrane calcium transport activity in aging brain.

We have assessed the functional properties of both calmodulin (CaM) and the plasma membrane Ca(2+)-ATPase in brains of young, middle aged, and old Fisher 344 rats. Under optimal conditions of saturating Ca2+ and ATP, the CaM-activated Ca(2+)-ATPase activity was decreased with increasing age, particularly when CaM isolated from the brains of aged rats was used to stimulate the enzyme. In the case of CaM, structural modifications within the primary sequence of the protein from aged brains were identified. We found that during normal biological aging approximately 6 methionine residues were modified to their corresonding sulfoxide per CaM, and no other amino acids were modified. Some aspects of the age-related decline in the effectiveness of CaM as an activator of Ca(2+)-ATPase could be simulated using a range of reactive oxygen species (including hydrogen peroxide and oxoperoxynitrite) and, in the latter case, the extent of oxidative modification of specific methionine residues was directly related to their surface accessibility. The pattern of oxidative modification of the methionines in the aged CaM was less straightforward, though both in vitro oxidation of CaM and aging within the brain markedly decreased the functional properties of this important Ca(2+)-regulating protein.

Aging↗