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Biomedical subjects

R B Harris

Publications and source records attributed to R B Harris.

At least 145 records · Page 8Linked to original sources
PubMed1985

Level of satiety: GABA and pentose shunt activities in three brain sites associated with feeding.

The hypothesis addressed was that metabolic activity within specific brain areas may be altered to depict peripheral metabolic status. Sixty-three female Sprague-Dawley rats (225 g) received 150, 100, or 50% of normal intake by gastric intubation for 7 days. The incentive for spontaneous feeding would be inhibited in 150% fed rats (anoretic), stimulated in 50% fed rats (hungry), and maintained in 100% fed rats (control). Glucose flux through the gamma-aminobutyric acid shunt of the ventrolateral hypothalamus was 32% lower in hungry rats and 35% higher in anoretic rats relative to control values. Glucose flux through the pentose shunt of the ventromedial hypothalamus was 111% lower in hungry rats and 152% higher in anoretic rats relative to control values. Pentose shunt activity in the area postrema nucleus of the solitary tract (AP NTS) was 116% lower in hungry rats and 60% higher in anoretic rats relative to control values; however, hungry and anoretic rats had AP NTS pentose shunt activities that were not different from control values but were different from each other. The data demonstrate that within selective brain sites, specific pathways for glucose oxidation are affected by energy intake and may be used by the rat to assess and respond to changes in peripheral energy status.

Animals↗
PubMed1984

Atrial tissue contains a metallo dipeptidyl carboxyhydrolase not present in ventricular tissue: partial purification and characterization.

A new membrane-bound dipeptidyl carboxyhydrolase has been identified in bovine atrial tissue, and has been partially purified after extraction with Triton X-100. This enzyme, found in quantities of 0.01-0.03 units/g tissue assayed with Bz-Gly-His-Leu, is potentially capable of hydrolyzing atriopeptin II to atriopeptin I. The enzyme is located in the microsomal fraction and in sucrose density fractions enriched for atrial granules. The enzyme is completely inhibited by reagents for heavy metals such as EDTA, o-phenanthroline, dithiothreitol, and mercaptoethanol. The latter two compounds are also disulfide reagents. The atrial enzyme is also inhibited by D-2-methyl-3-mercaptopropanoyl-L-Pro(Captopril), 3-mercaptopropanoyl-L-Pro, 2-D-methylsuccinyl-L-Pro, and bradykinin potentiating factor, all inhibitors of the angiotensin I-converting enzyme. However, the atrial enzyme differs from the converting enzyme in a number of kinetic and molecular properties. Its activity increases with ionic strength, but the atrial enzyme does not have a chloride dependence for Bz-Gly-His-Leu hydrolysis; the pH optimum, 7.3, is slightly lower, and it is 5500 times less sensitive to the very potent converting enzyme inhibitor, D-Cys-L-Pro. The strokes radius of the atrial enzyme is 5.00 nm as compared to 4.10 nm, and its molecular weight is 240,000 compared to 145,000. Ventricular tissue, which does not contain the atrial peptides, does not contain the dipeptidyl carboxyhydrolase enzyme.

Animals↗
PubMed1984

Recovery of body weight from below "set point" in mature female rats.

In this study we determined the time course of changes in body composition and of serum concentrations of some hormones in mature rats returning to "set point" following weight loss. Groups of six female rats were fed ad libitum for 10 days. Carcass composition of one group was determined. A control group continued to eat ad libitum. The others were restricted to 5 g per rat per day for 22 days. Carcass composition of groups of rats was determined after 0, 2, 6, 13 and 20 days of refeeding. Of 60 g of body weight lost during restriction, 21 g were fat and 12 g were protein. The fat was replaced by the sixth day of refeeding. Protein and body weight were recovered by the thirteenth day. Calculated efficiency of energy retention was increased until body fat was replete. This was not due to a decrease in metabolism of brown fat, measured in vitro. Serum thyroxine (T4), L-3,3',5'-triiodothyronine (rT3), insulin, corticosterone and free fatty acids did not change with body composition. T3 (L-3,3',5-triiodothyronine) decreased in restricted rats and returned to control levels when body weight, protein and food intake had been restored. These results do not support the concept that body weight is controlled by regulating body fat content.

Adipose Tissue, Brown↗
PubMed1984

tert.-Butyl aminocarbonate (tert.-butyloxycarbonyloxyamine)--a new acylating reagent for amines.

tert.-Butyl amino carbonate (1, tert.-butyloxycarbonyloxyamine) was prepared by reaction of hydroxylamine with di-tert.-butyl dicarbonate (2). 1 was used to acylate different amino acids or amines in water or in aqueous dioxane. 1 was also prepared in situ and used to acylate amino acids directly. 1 reacted 1.5-2.5 times faster than 2 with all amines studied either in water or 50% (v/v) dioxane. Remarkably, 1 retained its ability to acylate amines even in acid solution; the rate of acylation of L-Phe at pH 6.5 (15 M-1 X min-1) was about 20% of the rate at pH 10 (72 M-1 X min-1). 2 was not an acylating reagent below pH 7.0 and as expected, the rate at pH 8.3 (4 M-1 X min-1) was about 10% of that at pH 9.3 (39 M-1 X min-1). Pure BOC-amino acids were obtained in high yield and could be quantitatively deprotected by standard procedures.

Acylation↗
PubMed1984

The relationship between energy expenditure and environmental temperature in congenitally obese and non-obese Zucker rats.

The energy expenditure of normal and congenitally obese adult female Zucker rats has been measured by continuous indirect calorimetry for periods of 3-10 days at ambient temperatures varied from 30 to 5 degrees C. Rectal temperatures were also recorded. Exposure to cold caused no ill-effects in normal or obese rats. The rectal temperatures of obese rats were about 1 degree C lower than those of normal rats. The rectal temperatures of normal rats did not change measurably with ambient temperature; in obese rats rectal temperature rose slightly as ambient temperature fell. In normal and obese rats, energy expenditure showed a smooth, steeply sloping, negative relationship to ambient temperature. Energy expenditure per rat was higher in obese than in normal rats at all temperatures. The two slightly curvilinear regressions were nearly 'parallel', with a separation of about 40 kJ/day per rat at the mid-point. This study therefore does not confirm suggestions that obese Zucker rats suffer from a defect in the level of energy expenditure, or in their capacity to increase it when exposed to cold. It is suggested that in both normal and obese rats the level of energy expenditure was determined by thermoregulatory control. The greater heat production of obese rats may have been a response to their lower core temperature. A steady state in which greater heat production is associated with lower core temperature implies lower insulation between body core and surface. This could be due to greater blood flow.

Animals↗
PubMed1984

Specific depletion of body fat in parabiotic partners of tube-fed obese rats.

One member in each of 15 parabiosed pairs of rats was fed twice its normal food intake as four tube-fed meals per day. Seven other pairs ate ad libitum. Partners of overfed rats ate approximately 90% of the intake of individual members of ad libitum pairs. After 46 days of overfeeding, blood samples were taken and the rats were killed for carcass analysis. Tube-fed parabiotic rats had gained a considerable amount of fat and some protein. Their partners had a normal lean body mass but very little fat. Serum corticosterone, reverse triiodothyronine, free fatty acids, and beta-hydroxybutyrate were the same in all parabionts. Serum triiodothyronine and insulin were increased and growth hormone was decreased in obese rats. Serum thyroxine and triiodothyronine were increased and glucose was decreased in their parabiotic partners. The results are discussed as evidence for a humoral factor that crossed the parabiotic union and acted as a "lipid-depleting" agent in the partners of overfed rats.

3-Hydroxybutyric Acid↗
PubMed1984

The vasculitis of atrophie blanche (livedoid vasculitis) and abdominal aortic pathology.

Seven of forty-two patients with livedoid vasculitis whose cases were reviewed at the Mayo Clinic were found to have abdominal aortic disease, in the form of aortic calcification and atherosclerosis or aneurysm. None of these patients had any associated systemic mesenchymal disease (lupus erythematosus, periarteritis nodosa, rheumatoid arthritis, Sjögren's syndrome, or carcinomatous diseases). Three patients received graft replacements for aneurysm or vascular occlusive disease. Removal of the aneurysm in one case was associated with resolution of the livedoid vasculitis. Abdominal aortic changes should be considered in patients with diagnosed livedoid vasculitis.

Aged↗
PubMed1983

Dipeptide-hydroxamates are good inhibitors of the angiotensin I-converting enzyme.

The inhibition constants (Ki) and modes of inhibition have been determined for a series of dipeptide-hydroxamate compounds with bovine lung parenchyma angiotensin I-converting enzyme (peptidyldipeptide carboxy-hydrolase, E.C. 3.4. 15.1). The hydroxamido function was borne by aspartic, glutamic, or aminoadipic acid and extended by 2, 3 or 4 bond lengths from the proline amide bond. L-glu(NHOH)-L-pro (Ki = 3.4 microM) and D,L-aminoadipicyl (NHOH)-L-pro (Ki = 1.2 microM) were the best competitive inhibitors of the hydrolysis of benzoyl-gly-his-gly but were not effective as affinity ligands for purification of the enzyme.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1983

Polyacrylamide gels which contain a novel mixed disulfide compound can be used to detect enzymes that catalyze thiol-producing reactions.

The synthesis of N-[5-(hydroxyethyl)dithio-2-nitrobenzoylaminoethyl] acrylamide (I) is described. If the disulfide bond in this compound is reduced with thiol reagents, an intense yellow color develops (epsilon 412 V 13,600 at pH 7.4) due to essentially the same chromophore as 5-thio-2-nitrobenzoic acid, the reduced form of 5,5'-dithiobis(2-nitrobenzoic acid)(Ellman's reagent). Polyacrylamide gels were prepared that were crosslinked with N,N'-methylenebisacrylamide and which contained I as an integral part of the polymerized acrylamide chain. Acetylcholinesterase (from electric eel and human brain tissue slices) and alkaline phosphatase (from Escherichia coli and calf intestine) were subjected to electrophoresis and then the gels were immersed in an appropriate thiol-substrate buffer (acetylthiocholine and cysteamine-S-phosphate, respectively). A yellow band developed rapidly in the acrylamide gel at the site of enzyme activity. Electrophoresis on the mixed disulfide-polyacrylamide gel proved to be a rapid and sensitive technique to detect very small amounts of enzyme (approximately 0.02 fmol acetylcholinesterase) and should have wide application for detecting other enzymes that hydrolyze thiol substrates.

Acetylcholinesterase↗
PubMed1983

Glutamic acid is an active site residue of angiotensin I-converting enzyme. Use of the Lossen rearrangement for identification of dicarboxylic acid residues.

A set of chemical reactions was used to show that one glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme is esterified with the alkylating agent p-[N,N-bis(chloroethyl)amino] phenylbutyryl-L-Pro (chlorambucyl-L-Pro), an affinity label for this enzyme (Harris, R. B., and Wilson, I. B. (1982) J. Biol. Chem. 257, 811-815). The same procedure was used to confirm that a glutamic acid residue of carboxypeptidase A alpha is esterified by reaction with bromoacetyl-N-methyl-L-phenylalanine (Haas, G. M., and Neurath, H. (1971) Biochemistry 10, 3535-3546). In the procedure described in this paper, the esterified residue at the active site is converted to the hydroxamic acid by reaction with hydroxylamine and the hydroxamic acid is subject to the Lossen rearrangement. If a glutamic acid residue was esterified, 1 eq of 2,4-diaminobutyric acid will be formed. Aspartyl esters will give 2,3-diaminopropionic acid. The diamino acids can be quantitatively measured using the short column of an amino acid analyzer if the amount of lysine and histidine is largely decreased by modification with suitable side chain protecting groups. With carboxypeptidase A, the reactions were done on the whole undigested enzyme. With the converting enzyme, we first cleaved the esterified enzyme with cyanogen bromide. Twenty-nine cleavage peptides were separated on high performance liquid chromatography and one of these contained all of the bound radioactive inhibitor. This active site peptide was then subjected to the derivatization and Lossen procedures, and 1 eq of 2,4-diaminobutyric acid was obtained.

Amino Acids↗
PubMed1982

Irreversible inhibition of bovine lung angiotensin I-converting enzyme with p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and chlorambucyl L-proline and with evidence that an active site carboxyl group is labeled.

Bovine lung angiotensin I-converting enzyme is rapidly and irreversibly inactivated by p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and by the chlorambucil derivative of L-proline (chlorambucyl-proline). Chlorambucil is a nitrogen mustard alkylating agent that is used as an antineoplastic drug. At any one concentration, the inactivation is pseudo-first order with time. Inhibition by both substances is active site directed as suggested by the formation of a reversible enzyme-inhibitor complex prior to the alkylation reaction and by the fact that L-Phe-L-Pro, a reversible inhibitor which is competitive with substrate, is also competitive with both irreversible inhibitors in protecting the enzyme against inactivation. The second order rate constant for inactivation increases in the pH range 5-8 and reaches a value of 3.5 X 10(3) M-1 . min-1 for chlorambucil and 4.8 X 10(2) M-1 . min-1 for chlorambucyl-proline. Chlorambucyl [U-14C]L-proline reacts 1:1 with the converting enzyme and the uptake of radioactivity paralleled the loss of enzyme activity with and without protection by Phe-Pro. Once bound, the radioactive chlorambucyl proline was released (as the dihydroxy derivative) by hydroxide ion with a second order rate constant of 2.2 M-1 . min-1 at 25 degrees C. The radioactive label is also removed by hydroxylamine at pH 10. The lability of the irreversibly bound inhibitor in alkali and in hydroxylamine indicates that an ester bond is formed by the alkylation of an aspartic acid or glutamic acid side chain.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1982

Physicochemical characteristics of homogeneous bovine lung angiotensin I-converting enzyme. Comparison with human serum enzyme.

Angiotensin I-converting enzyme was purified to electrophoretic homogeneity (12 units/mg) from bovine lung tissue and from human serum using an affinity gel described previously (Harris et al., (1981) Anal. Biochem. 111, 227-234). The isoelectric point (4.5), molecular weight (145 000), S20,W (8.1), amino acid composition and carbohydrate content of the lung enzyme are all similar to the values obtained for the human serum enzyme. The NH2-terminus of the lung enzyme (Ala) is different from that of the serum enzyme (Tyr) but the COOH-terminal sequences are identical (-Leu-Ser-OH). Pure bovine lung enzyme was reduced and carboxyamidomethylated with iodo (14C1) acetamide to the extent predicted by the number of cysteine residues. Since no radioactivity was incorporated into denatured enzyme that was not reduced, all of the cysteine residues must be in the form of disulfide bonds. Reverse-phase HPLC was used to separate peptides obtained from the lung enzyme after degradation with either trypsin or cyanogen bromide. The number of peptides resolved (42 after trypsin, 31 after cyanogen bromide), were only 20% fewer than the number predicted from the amino acid analysis and therefore the possibility that the converting enzyme (a single polypeptide chain) might be a fused dimer is excluded.

Amino Acid Sequence↗
PubMed1981

Partial purification of biologically active, low molecular weight, human antihemophilic factor free of Von Willebrand factor. I. Partial characterization and evidence for disulfide bond(s) susceptible to limited reduction.

Partially purified (approx. 5000-fold), low molecular weight human antihemophilic factor, free of detectable Von Willebrand factor (ristocetin cofactor activity or Von Willebrand antigen), was prepared from fresh citrated plasma by limited reduction with 1 mM dithiothreitol and chromatography on Sepharose CL-4B, Sephadex G-100, and polyelectrolyte E-5. The ratio of antihemophilic factor activity to Von Willebrand factor activity or antigen was greater than 27 000 : 1. The antihemophilic factor activity could be neutralized with homologous antibody and could be further increased with thrombin. The Mr (approx. 116 000) was determined by calibrated gel permeation chromatography, electrophoresis in 5% polyacrylamide gels with sodium dodecyl sulfate and by electrophoresis in large-pore acrylamide gels without it. Since the low Mr antihemophilic factor could be prepared by treating fresh rather than fresh-frozen plasma with dithiothreitol, it was concluded that partial reduction of the antihemophilic factor with this reagent helped to maintain the antihemophilic factor in a low Mr form. When iodo[l-14C]acetamide was used to alkylate the reduced plasma proteins prior to purification, the molecular weight of the purified antihemophilic factor remained low despite numerous purification steps. By this means, one of four radioactive proteins (Mr 116 000) in the final preparation was bound specifically to homologous antihemophilic factor antibody and attributed to 14C-labeled antihemophilic factor. While the data suggest that antihemophilic factor in fresh plasma contains one or more dithiothreitol-sensitive intramolecular disulfide bonds, the possibility of disulfide linkages with other proteins(s) cannot be excluded.

Chemical Phenomena↗