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H T Smith

Publications and source records attributed to H T Smith.

At least 37 records · Page 2Linked to original sources
PubMed1991

Electrolytes in the epidemiology, pathophysiology, and treatment of hypertension.

The data regarding the value of manipulating electrolytes in hypertension are controversial. It appears there are subsets of hypertensive patients who respond with lowering of blood pressure in conjunction with changes in intake of sodium, potassium, and calcium. The information regarding phosphorus and magnesium is less convincing. This paper examines current reports regarding these electrolytes and their role in the pathophysiology and treatment of essential hypertension.

Calcium, Dietary↗
PubMed1989

Immunochemical properties of ubiquitin conjugates in the paired helical filaments of Alzheimer disease.

Immunocytochemical and peptide sequencing studies indicate that the regulatory protein ubiquitin (Ub) is incorporated into the paired helical filaments (PHF) of Alzheimer disease. In this study, we showed that some antibodies raised to PHF recognize epitopes of Ub. Analysis of the Ub sequences recognized by the antibodies raised to PHF, along with the known specificity of several monoclonal antibodies raised to artificial Ub conjugates, indicates the immunochemical representation of Ub residues 34-76 in PHF. The Ub epitopes recognized by antibodies raised to PHF are distinct from those recognized by antibodies raised to artificial Ub conjugates in two respects. First, antibodies that are raised to PHF and that recognize Ub react with PHF equally, whether denatured or not, whereas those raised to artificial Ub conjugates show greater reaction after denaturation. Second, mapping of the epitopes recognized by two monoclonal antibodies to PHF onto Ub indicates a distinction in the Ub residues recognized, compared with monoclonal antibodies raised to artificial Ub conjugates. The proximity of their epitopes to the site of conjugation, as well as their affinity for PHF polypeptides, suggests that the PHF antibodies that recognize Ub may be directed specifically to Ub epitopes defined by the protein conjugated to Ub.

Alzheimer Disease↗
PubMed1989

Ubiquitin: a multifunctional regulatory protein associated with the cytoskeleton.

The discovery that ubiquitin is associated with the neurofibrillary tangles of Alzheimer's disease and inclusion bodies of other neurodegenerative disorders raises the possibility that ubiquitin is playing a role in the pathology. Ubiquitin is a small, highly conserved protein that is found both free and covalently attached to other proteins in all eukaryotic cells. Ubiquitin is conjugated with some proteins that are rapidly degraded and with a select set of regulatory proteins that are relatively stable, implying that ubiquitin is multifunctional. We have produced monoclonal antibodies to ubiquitin and identified the epitopes. Different monoclonal antibodies recognize different ubiquitin-protein conjugates; this could be due to different conformational states of ubiquitin or to steric interference by the conjugated protein. These observations suggest that ubiquitin is structurally and therefore functionally different in different contexts. Ubiquitin is localized in several compartments in cells and this may also affect function. We discovered that it is a normal component of the microtubule network and have identified ubiquitinated proteins in bovine brain that appear associated with microtubules. We discovered that ubiquitin has intrinsic proteolytic activity and proposed that ubiquitin-protein conjugates could function as ad hoc proteases. This proteolytic activity offers a heuristic basis from which to explore ubiquitin's cellular functions. The association of ubiquitin with the cytoskeletal networks suggests that proteolytic processing is a function of these structures. Disruption of these structures in neurodegenerative disorders, improper localization of ubiquitinated components or appearance of abnormal conjugates could lead to altered proteolytic processing pathways and contribute to the pathology of these diseases.

Alzheimer Disease↗
PubMed1988

Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases.

Several degenerative diseases of the nervous system are characterized by the presence of neuronal inclusions. Most of these inclusions are made of abnormal filaments and share epitopes with cytoskeletal proteins. One of these inclusions, the neurofibrillary tangle of Alzheimer disease, has recently been shown to contain ubiquitin, a regulatory protein thought to play a role in the degradation of abnormal proteins. We carried out light and electron microscopic immunocytochemistry with several polyclonal and monoclonal antibodies to investigate the presence of ubiquitin in neuronal inclusions of neurodegenerative diseases. Ubiquitin was present not only in paired helical filaments that form the neurofibrillary tangle of Alzheimer disease, but also in the filamentous components of the inclusion characteristic of Parkinson disease, Pick disease, and progressive supranuclear palsy. In contrast, ubiquitin was not detected in other neuronal inclusions often found in aging and in Alzheimer disease, such as Hirano bodies and granulovacuolar degeneration. Reactivity with monoclonal antibodies suggests differences in the ubiquitin-acceptor proteins present in the inclusions studied. It is concluded that ubiquitin is selectively present in neuronal inclusions of degenerative diseases.

Alzheimer Disease↗
PubMed1988

Ubiquitin is a component of the microtubule network.

Immunofluorescence microscopy was used to study the intracellular localization of ubiquitin. Baby hamster kidney cells (BHK cells) and several other cell lines were probed with a well characterized monoclonal antibody to ubiquitin. The antibody stained a complex cellular structure that we identified as the microtubule network. The anti-ubiquitin antibody bound to the microtubule network at all stages of the cell cycle, and we showed that the apparent association of ubiquitin with the microtubule network is not an artifact of crosslinking of free ubiquitin to the cell structure. Immunoblot procedures demonstrated that tubulin itself was not ubiquitinated. We propose that ubiquitin and/or ubiquitin-protein conjugates are associated with those networks as a new class of microtubule-associated protein. The targeting of ubiquitin to specific sites within the cell by its association with the microtubule network may regulate some of the functions of ubiquitin.

Animals↗
PubMed1987

High-performance liquid chromatographic method for the determination of pindolol in human plasma.

A sensitive, simple and highly reliable high-performance liquid chromatographic method using fluorescence detection is reported for the determination of pindolol in plasma. This method involves a single extraction of pindolol from alkalinized plasma into methyl tert.-butyl ether followed by a back-extraction into dilute hydrochloric acid. Injection of the dilute acid phase directly onto an octyl (LC-8) bonded-phase column provides the final separation, and detection of pindolol is achieved by monitoring the intrinsic fluorescence of pindolol at 315 nm following excitation at 255 nm. The method is sensitive enough to measure with confidence pindolol plasma concentrations of 2 ng/ml using a 2-ml sample. No internal standard is required. This method has been applied to the analysis of 1500 human plasma samples by two different laboratories.

Chromatography, High Pressure Liquid↗
PubMed1987

Multiple-dose pharmacokinetics of clozapine in patients.

After a 2-day buildup, patients were dosed continuously with clozapine solution at three ascending dose levels (37.5, 75, and 150 mg bid for 7 days at each dose level). Following the morning administration on the twenty-third day of dosing a drug holiday was instituted which lasted for a minimum of 48 hr. Serial plasma samples were obtained during each of the periods and during the drug holiday for the calculation of the steady-state parameters AUCSS, CSSmax, and CSSmin at each dose level as well as for the assessment of the terminal elimination rate. Mean parameter values for AUCSS, CSSmax, and CSSmin showed a linearly increasing response with the dose, well described by a straight line passing through the origin. The terminal elimination appeared to follow linear kinetics and had a mean half-life of 15.8 hr (range, 5.8-33 hr).

Adolescent↗
PubMed1987

Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation.

Ubiquitin is a protein of 76 amino acids found in every eukaryotic cell. Although ubiquitin is implicated in ATP-dependent nonlysosomal protein degradation and is also conjugated to specific cellular proteins, the role played by ubiquitin in cellular events has not been defined. We report that purified ubiquitin has intrinsic proteolytic activity and demonstrate that this activity is comparable to that of other well-characterized proteases. Monoclonal antibodies specific to ubiquitin inhibit proteolysis. Ubiquitin has protease activity over a broad pH range with an optimum at pH 8.0. It is stimulated by Ca2+ and is inhibited by high concentrations of phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. Ubiquitin will cleave proteins at a limited number of sites. We propose that the ubiquitination of a protein can convert that protein into an ad hoc specific protease and models are presented as to how this can play a role in regulating a variety of cellular events.

Antibodies, Monoclonal↗
PubMed1986

Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched-chain glycoprotein.

Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino termini, one of which corresponds precisely to the amino terminus of ubiquitin. This observation extends the province of this conserved polypeptide to the cell surface and leads to a proposed model of the receptor complex as a core polypeptide modified by glycosylation and ubiquitination. Independent antibodies to ubiquitin serve to identify additional cell surface species, an indication that ubiquitination of cell surface proteins may be more general. It is proposed that functional binding of lymphocytes to lymph node high endothelial venules might involve the ubiquitinated region of the receptor; if true, cell surface ubiquitin could play a more general role in cell-cell interaction and adhesion.

Amino Acid Sequence↗
PubMed1986

Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species.

The lymphocyte cell surface receptor for the high endothelial venules (HEV's) of peripheral lymph nodes is specifically recognized by the monoclonal antibody MEL-14. Three independent complementary DNA (cDNA) clones, each of which encodes the protein ubiquitin, were detected by virtue of the expression of the MEL-14 antigenic determinant on cDNA-beta-galactosidase bacterial fusion proteins. The antigenic determinant defined by MEL-14 resides in the carboxyl terminal 13-amino-acid proteolytic peptide of ubiquitin, but is undetected in intact undenatured ubiquitin and other cellular ubiquitinated proteins. Antisera and monoclonal antibodies to ubiquitin determinants bind to the surface of both HEV-receptor positive and negative cell lines. The MEL-14-identified cDNA clones hydridize to RNA transcripts that encode tandemly repeated ubiquitins. Sequence analysis of these polyubiquitin cDNA's does not identify a leader sequence for export to the cell surface. The expression of the MEL-14 epitope of ubiquitin depends upon its local environment. The steady-state levels of expression of the ubiquitin messenger RNA's do not correlate with either the tissue derivation of the RNA or the expression of the lymphocyte HEV receptor. Regulation of the expression of the HEV receptor is not likely to reflect the transcriptional control of ubiquitin genes, but rather to reflect control of the expression of the HEV core polypeptide or its level or form of ubiquitination.

Amino Acid Sequence↗
PubMed1985

Catabolism of the anion transport protein in human erythrocytes.

We identified the catabolic products of protein 3 in human erythrocytes. Protein 3, the major protein of the erythrocyte membrane, functions in anion transport and reacts covalently with tritiated 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid ([3H]DIDS), a very selective inhibitor of anion transport. In this study, [3H]DIDS was used to label protein 3 in the membranes of normal cells and those from a donor heterozygous for a variant of protein 3, defined by its elongated amino-terminal end. Both types of cells contained [3H]DIDS-labeled peptides other than protein 3. A protein fragment of 60K molecular weight was found in normal cells, whereas both 60K and 63K fragments were identified in cells from the heterozygote. These peptides are identical with those generated by treatment of intact erythrocytes with Pronase or chymotrypsin. A polyclonal rabbit antibody specific for the purified 60K fragment of protein 3 was used to detect this protein and its products in the erythrocyte membrane. Autoradiographs of membrane peptides that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and allowed to react with the monospecific antibody showed, in addition to protein 3, a 60K fragment and fragments in the 40K region and in the 20-30K region. Cells containing the protein 3 variant yielded two fragments showing a 3K difference in molecular weight in all three regions, demonstrating that degradation of protein 3 is identical in normal erythrocytes and those heterozygous for the variant. This observation also confirms the common derivation of the fragments from protein 3.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Semi-automated high-performance liquid chromatographic method for the determination of cyclosporine in plasma and blood using column switching.

A sensitive, semi-automated high-performance liquid chromatographic method utilizing column switching is described for the determination of cyclosporine in plasma and blood. This method involves a short and improved manual protein precipitation of the sample followed by an automated clean-up of the supernatant. After automatic loading of the clean supernatant onto an LC-8 column for initial separation, the segment containing cyclosporine is loaded (automatically) onto an LC-18 column for final separation and quantitation. Cyclosporine is detected by its ultraviolet absorption at 202 nm. The rate of analysis was four samples per h running 24 h per day (ca. 100 samples per day). The method is sensitive enough to measure with confidence cyclosporine concentrations of 8 microgram/l in plasma and 20 microgram/l in blood with a linear response up to 2500 microgram/l using only 0.5 ml of sample. No internal standard is required. The method was applied continuously (24 h per day) to approximately 1000 samples without deterioration in method parameters.

Chromatography, High Pressure Liquid↗
PubMed1983

Enzymatic trimethylation of lysine-72 in cytochrome c.

The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowering the pI values of the protein than the chemical modification. Furthermore, the lowering of the pI value of cytochrome c by enzymatic methylation is highly dependent on the urea concentration. The presence of urea reduces the effect of methylation on the protein molecule and the difference in pI values virtually disappears with the increasing concentration of urea (6 M), which essentially disrupts the protein tertiary structure.

Animals↗
PubMed1982

The reaction of the trifluoromethylphenylcarbamylated lysine-13 derivative of horse cytochrome c with cytochrome oxidase.

The kinetics of oxidation of horse cytochrome c and the trifluoromethylphenylcarbamylated lysine-13 derivative by cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) were compared using both spectrophotometric and polarographic methods under different experimental conditions. The rate constants measured spectrophotometrically in 0.025 M tris-cacodylate buffers were similar with the two cytochrome at pH 7.8, but those with the derivative were slightly higher at pH 6. Rates measured with polarographic assays in these buffers were the same with the horse and the derivative cytochromes c at pH 6, but at pH 7.8 the rates with the derivative were less at cytochrome c concentrations between 0.05 and 0.5 micro M and were greater at higher concentrations. The pH optima in the polarographic assays of the derivative and the native pigments were different in 0.025 M Tris-cacodylate buffers; in spectrophotometric assays at pH 7.8 the trifluoromethylphenylcarbamylated lysine-13 cytochrome c showed a greater sensitivity to changes in ionic strength than did the native cytochrome. The variations in apparent Km and V values calculated from spectrophotometric and polarographic assays with the two cytochromes cannot be explained as due to changes in binding of cytochrome c to cytochrome oxidase. The large excess of O2 uptake seen in polarographic assays with horse cytochrome c over that expected from spectrophotometric measurements was not apparent with the trifluoromethylphenylcarbamylated lysine-13 derivative. Thus, the derivative seems to have decreased ability to form the combination of cytochrome c with the oxidase giving high turnover rates.

Animals↗
PubMed1981

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase.

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.

Animals↗
PubMed1980

Involvement of lysines-72 and -79 in the alkaline isomerization of horse heart ferricytochrome c.

Spectrophotometric titrations of five singly modified horse heart ferricytochromes c, specifically (trifluoromethyl)phenylcarbamylated (CF3PhNHCO-) or trifluoroacetylated (CF3CO-) at lysines-13, -72, and -79, were carried out. The CF3PhNHCO-Lys-13, Lys-79, and CF3CO-Lys-79 derivatives all underwent alkaline isomerization with loss of the 695-nm band to low-spin species with an apparent pK of about 8.9, as did the unmodified cytochrome. However, modification of lysine-72 appeared to alter the reaction pathway since the CF3PhNHCO-Lys-72 derivative isomerized to a high-spin form with an apparent pK of 9.3, while the CF3CO-Lys-72 derivative isomerized to a low-spin species with an apparent pK of 9.6, indicating that lysine-72 may be the normal sixth iron ligand in the native protein alkaline isomer. These results, together with those of other workers, suggest a model for the alkaline transition in which replacement of the methionine iron ligand is dependent on a number of factors, including the local availability and relative affinities of possible ligands for the heme iron and the effects of ionic and hydrophobic interactions on the tertiary structure of the molecule.

Animals↗