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Biomedical subjects

W R Harris

Publications and source records attributed to W R Harris.

At least 19 recordsLinked to original sources
PubMed1992

Equilibrium model for speciation of aluminum in serum.

An equilibrium model for the speciation of Al3+ in normal serum has been developed in which 81% of the aluminum is bound to transferrin. The remaining 19% exists primarily as Al(PO4)(OH)-, with minor amounts of citrate and hydroxide species. The model meets several key criteria. The concentration of free aluminum is below the upper limit set by the solubility product of aluminum phosphate. The fraction of low-molecular-mass aluminum species agrees with experimental studies on the fraction of ultrafiltrable aluminum in both normal and uremic serum. Lastly, the model accounts for the relative effectiveness of EDTA, citrate, and desferrioxamine B for binding aluminum in serum by including competition from zinc, magnesium, and calcium for these ligands. Uncertainties in the available thermodynamic database are discussed.

Aluminum↗
PubMed1991

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.

Animals↗
PubMed1991

Fractures of the spine in diffuse idiopathic skeletal hyperostosis.

Fractures of the spine in diffuse idiopathic skeletal hyperostosis (DISH) have rarely been reported. Only four cases could be found in the world literature. Eight new cases with nine fractures are reported in this study. The critical features are the frequent delays in diagnosis (three of eight patients) and the high rate of immediate and delayed neurologic deficit (seven of eight patients). Two fracture patterns occurred in this group. The first type occurred through the midportion of an ankylosed segment of the spine and involved the vertebral body (five fractures). The second type occurred at the top or bottom of a fused segment (four fractures). The latter were disk disruptions or odontoid fractures. This is a marked difference from spinal fractures in ankylosing spondylitis, in which the majority are transdiskal fractures. The difference can be explained on the basis of the different pathology of these two disease processes. Careful evaluation of patients with DISH who sustain trauma is critical. Treatment of this rare injury should be early stabilization of the spine to avoid complications of nonunion, deformity, neurologic injury, and death.

Aged↗
PubMed1990

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain.

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.

Amino Acid Sequence↗
PubMed1990

Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences.

A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).

Amino Acid Sequence↗
PubMed1990

Structural analysis of bovine pancreatic thread protein.

Pancreatic thread protein (PTP) forms double helical threads in the neutral pH range after purification, undergoing freely reversible, pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Gross et al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing greater than 99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH2-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca2(+)-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.

Amino Acid Sequence↗
PubMed1990

Kinetic analysis of the separate phosphorylation events in the phosphorylase kinase reaction.

Glycogen phosphorylase, a dimer of identical subunits, is activated by phosphorylase kinase-catalyzed phosphorylation of one serine residue in each subunit. In this paper, the effect of the phosphorylation of one subunit on the phosphorylation of the other subunit was examined. The three forms of phosphorylase, phosphorylase b (nonphosphorylated), phosphorylase ab (one subunit phosphorylated), and phosphorylase a (both subunits phosphorylated), were separated by anion-exchange high-performance liquid chromatography (HPLC). Purified phosphorylase ab was found to be stable under the conditions of the phosphorylase kinase assay. Initial rate kinetics showed that phosphorylase kinase had a lower KM for phosphorylase ab (3.9 +/- 0.24 microM) than for phosphorylase b (14.9 +/- 2.6 microM). Using the HPLC separation as a simultaneous assay for the three forms of phosphorylase during the phosphorylase kinase reaction, it was found that the pseudo-first-order rate constant for the second phosphorylation step (k2) was 3.7 times greater than that for the first step (k1). The activator AMP reduced the ratio k2/k1 from 3.7 without AMP to 1.4. When the monomeric gamma delta complex of phosphorylase kinase subunits was used as the enzyme, the ratio k2/k1 was 2.1, compared to 3.7 with the multimeric holophosphorylase kinase. One explanation for these data is that phosphorylation of one subunit of phosphorylase b causes conformational changes that make the other subunit a better substrate for the kinase. In this context, the effect of AMP is to reduce the conformational differences between phosphorylases b and ab, and the gamma delta complex is less sensitive to the conformational differences between the two forms of phosphorylase.

Animals↗
PubMed1990

Site-specific rate constants for iron removal from diferric transferrin by nitrilotris(methylenephosphonic acid) and pyrophosphate.

The kinetics of iron removal from human serum diferric transferrin by nitrilotris(methylenephosphonic acid) (NTP) and pyrophosphate (PPi) have been studied in 0.1 M, pH 7.4, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonate buffer at 25 degrees C using urea/polyacrylamide gel electrophoresis. The four microscopic rate constants required for a complete description of iron removal from the two transferrin metal-binding sites have been measured at 100 mM concentrations of NTP and PPi. There is very good agreement between the rate constants determined by electrophoresis in this study and the corresponding rate constants determined spectrophotometrically for monoferric transferrins that have been labeled at the empty binding site with substitutionally inert Co(III). The results validate the use of cobalt-labeled transferrins as models for kinetic studies on iron removal from diferric transferrin.

Cobalt↗
PubMed1990

Site selectivity in the binding of inorganic anions to serum transferrin.

Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding.

Anions↗
PubMed1990

Differences in heart rate and blood pressure in children with conduct disorder, major depression, and separation anxiety.

Heart rate and blood pressure of children and adolescents admitted to a psychiatric hospital were compared among those diagnosed conduct disorder, major depressive disorder, and separation anxiety disorder. Subjects with conduct disorder had a lower heart rate compared to subjects without a conduct disorder diagnosis; and subjects with separation anxiety disorder had higher heart rate and systolic blood pressure compared to subjects without an anxiety disorder diagnosis. Subjects with major depressive disorder had higher systolic blood pressure than subjects with conduct disorder but no difference in heart rate. The findings are consistent with conduct disorder being associated with decreased noradrenergic function and anxiety/depressive disorder being associated with increased noradrenergic function.

Adolescent↗
PubMed1990

Chondrosarcoma complicating total hip arthroplasty in Maffucci's syndrome.

A 70-year-old woman who was followed for 60 years for Maffucci's syndrome required a total hip arthroplasty (THA) of the right hip for degenerative arthritis. This did not relieve pain. Three years later, chondrosarcoma of the femur developed around the femoral component and was fatal. The roentgenographic changes after the THA were incorrectly interpreted as being caused by infection.

Aged↗
PubMed1989

Human epidermal growth factor (EGF) receptor sequence recognized by EGF competitive monoclonal antibodies. Evidence for the localization of the EGF-binding site.

Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.

Amino Acid Sequence↗
PubMed1989

Attention deficit disorder symptoms and urine catecholamines.

The symptoms of hyperactivity, impulsivity, and concentration deficits associated with attention deficit disorder (ADD) may be related, in part, to alterations in dopaminergic and noradrenergic functioning. In this study we correlate the above symptoms with 24-hour urinary catecholamines and their metabolites in emotionally disturbed boys divided into two groups based on their plasma dopamine-beta-hydroxylase (DBH) activities and also divided into the following diagnostic groups: conduct disorder, undersocialized; conduct disorder, socialized; and subjects without conduct disorder. Boys in the low DBH group showed significant correlations between the ADD symptoms and the biochemical measures. The low DBH group may be more genetically homogeneous with regard to catecholamine function, making relationships between catecholamine function and behavior more visible. The group of boys with conduct disorder, socialized had higher 24-hour urinary norepinephrine and vanillylmandelic acid output. The relationship between monoamines and their metabolites appeared to differ among diagnostic groups.

Attention Deficit Disorder with Hyperactivity↗
PubMed1989

Calculations of the distribution of zinc in a computer model of human serum.

During the last decade, investigators employing a wide variety of separation techniques have reported that serum albumin, rather than serum transferrin, is the major carrier protein for labile zinc. However, in contrast to these studies, a simple comparison of the zinc binding constants for albumin and transferrin suggests a significant role for transferrin with regard to serum zinc transport. In the present study multicomponent equilibrium calculations that incorporate recently determined binding constants for zinc-transferrin as well as equilibrium constants for the binding of bicarbonate, sulfate and phosphate to apotransferrin have been used to model the distribution of labile zinc in normal human serum. Anion binding has a major impact on the predicted zinc distribution, lowering the percentage of zinc bound to transferrin from 56% to 11%. The model gives approximately 1% zinc as low-molecular-weight complexes. The improved model results are in good agreement with biological studies on the distribution of zinc among serum proteins.

Bicarbonates↗
PubMed1988

Equilibrium studies on the binding of cadmium(II) to human serum transferrin.

The binding of cadmium(II) to human serum transferrin in 0.01 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid with 5 mM bicarbonate at 25 degrees C has been evaluated by difference ultraviolet spectroscopy. Equilibrium constants were determined by competition versus three different low molecular weight chelating agents: nitrilotriacetic acid, ethylenediamine-N,N'-diacetic acid, and triethylenetetramine. Conditional equilibrium constants for the sequential binding of two cadmium ions to transferrin under the stated experimental conditions are log K1 = 5.95 +/- 0.10 and log K2 = 4.86 +/- 0.13. A linear free energy relationship for the complexation of cadmium and zinc has been prepared by using equilibrium data on 243 complexes of these metal ions with low molecular weight ligands. The transferrin binding constants for cadmium and zinc are in good agreement with this linear free energy relationship. This indicates that the larger size of the cadmium(II) ion does not significantly hinder its binding to the protein.

Cadmium↗
PubMed1988

The bicarbonate-dependence of zinc(II)-transferrin binding.

The binding of zinc(II) to human serum transferrin has been studied as a function of the solution concentration of sodium bicarbonate in 100 mM, pH 7.4 hepes buffer at 25 degrees C. The apparent molar absorptivity of the zinc-transferrin complex has been determined from the initial slopes of titration curves of delta epsilon versus the ratio of [Zn]/[Tf]. This absorptivity represents the difference between the positive absorbance of the ternary Zn-HCO3-Tf species in the sample cuvette and the negative absorbance of binary HCO3-Tf species in the reference cuvette. Higher concentrations of bicarbonate increase the degree of saturation of apo-Tf with bicarbonate and thus increase the apparent absorptivity of the zinc-Tf complex. Titrations of apo- and monoferric transferrins with bicarbonate indicate that there is little, if any, difference in the bicarbonate binding constants of the two specific transferrin binding sites. An equilibrium constant of log K = 2.49 has been used to calculate the degree of saturation of the C-terminal binding site with bicarbonate. The zinc-binding affinity of this site depends linearly on this degree of saturation. The scatter in the zinc-binding constants of the weaker N-terminal site precludes a similar analysis of the bicarbonate-dependence of binding at this site. The results strongly support the previous proposal that binding of the synergistic bicarbonate anion is responsible for the uv absorption observed upon addition of bicarbonate to apoTf.

Bicarbonates↗