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Biomedical subjects

J Day

Publications and source records attributed to J Day.

At least 19 recordsLinked to original sources
PubMed1992

The three-dimensional structure of an intact monoclonal antibody for canine lymphoma.

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.

Animals↗
PubMed1992

Crystallization and preliminary X-ray analysis of human endothelin.

Endothelin, a potent regulator of vasoconstriction and hypertension, is a naturally produced peptide of 21 amino acids containing two disulfide bonds. We have crystallized endothelin from humans using the vapor-diffusion technique, characterized the crystals by X-ray diffraction analysis, and have collected the X-ray intensities to a resolution of 1.8 A. The crystals, which demonstrate physical properties similar to most protein crystals and have a comparable solvent content, are hexagonal prisms that frequently grow to lengths of 400 microns and widths of 150 microns. The space group of the crystals is P6(1)22 (or P6(5)22), with a = 27.4, c = 79.6 A. There is one molecule of endothelin in the asymmetric unit of the crystals.

Crystallization↗
PubMed1992

Preliminary crystallographic study of peanut peroxidase.

The cationic isozyme of peroxidase isolated from suspension cultures of peanut cells is a heme-containing and calcium-dependent glycoprotein having four covalently attached oligosaccharide chains. Attempts were made to crystallize the glycoprotein for X-ray diffraction analysis, and these have met with some success. Crystals have now been grown that are suitable for a full three-dimensional structural analysis. The crystals are thin plates and we have shown them to be of the orthorhombic space group P2(1)2(1)2(1) with a = 48.1, b = 97.2, c = 146.2 A. The crystals diffract to beyond 2.8 A resolution, appear to be stable to lengthy X-ray exposure, and contain two molecules of 40,000 daltons each in the asymmetric unit.

Arachis↗
PubMed1992

Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.

Crystallization↗
PubMed1992

Dopaminergic regulation of cortical acetylcholine release.

The extent to which the activity of basal forebrain cholinergic neurons is influenced by dopamine (DA) was investigated using in vivo microdialysis of cortical acetylcholine (ACh). Systemic administration of the DA receptor agonist apomorphine significantly increased dialysate concentrations of ACh. Systemic, but not local, administration of d-amphetamine produced similar effects. Both D1 (SCH 23390) and D2 (haloperidol, raclopride) DA receptor antagonists attenuated the amphetamine-induced increase in cortical ACh release; however, only the D1 antagonist significantly reduced basal output of cortical ACh. These findings suggest that the activity of cortically projecting cholinergic neurons in the nucleus basalis is regulated in an excitatory manner by central dopaminergic neurons and that both D1 and D2 receptors are involved.

Acetylcholine↗
PubMed1992

Regional brain blood flow during prolonged submaximal exercise in ponies.

Experiments were carried out on 8 healthy ponies to examine the effects of prolonged submaximal exercise on regional distribution of brain blood flow. Brain blood flow was ascertained by use of 15-microns-diameter radionuclide-labeled microspheres injected into the left ventricle. The reference blood was withdrawn from the thoracic aorta at a constant rate of 21.0 ml/min. Hemodynamic data were obtained with the ponies at rest (control), and at 5, 15, and 26 minutes of exercise performed at a speed setting of 13 mph on a treadmill with a fixed incline of 7%. Exercise lasted for 30 minutes and was carried out at an ambient temperature of 20 C. Heart rate, mean arterial pressure, and core temperature increased significantly with exercise. With the ponies at rest, a marked heterogeneity of perfusion was observed within the brain; the cerebral, as well as cerebellar gray matter, had greater blood flow than in the respective white matter, and a gradually decreasing gradient of blood flow existed from thalamus-hypothalamus to medulla. This pattern of perfusion heterogeneity was preserved during exercise. Regional brain blood flow at 5 and 15 minutes of exercise remained similar to resting values. However, at 26 minutes of exercise, vasoconstriction resulted in a significant reduction in blood flow to all cerebral and brain-stem regions. In the cerebellum, the gray matter blood flow and vascular resistance remained near control values even at 26 minutes of exercise. Vasoconstriction in various regions of the cerebrum and brainstem at 26 minutes of exertion may have occurred in response to exercise-induced hypocapnia, arterial hypertension, and/or sympathetic neural activation.

Animals↗
PubMed1992

Respiratory muscle perfusion in ponies during prolonged submaximal exercise in thermoneutral environment.

Distribution of blood flow among various respiratory muscles was examined in 8 healthy ponies during submaximal exercise lasting 30 minutes, using radionuclide labeled 15-microns diameter microspheres injected into the left ventricle. From the resting values (40 +/- 2 beats/min; 37.3 +/- 0.2 C), heart rate and pulmonary arterial blood temperature increased significantly at 5 (152 +/- 8 beats/min; 38.6 +/- 0.2 C), 15 (169 +/- 6 beats/min; 39.8 +/- 0.2 C), and 26 (186 +/- 8 beats/min; 40.8 +/- 0.2 C) minutes of exertion, and the ponies sweated profusely. Mean aortic pressure also increased progressively as exercise duration increased. Blood flow increased significantly with exercise in all respiratory muscles. Among inspiratory muscles, perfusion was greatest in the diaphragm and ventral serratus, compared with external intercostal, dorsal serratus, and scalenus muscles. Among expiratory muscles, blood flow in the internal abdominal oblique muscle was greatest, followed by that in internal intercostal and transverse thoracic muscles, in which the flow values remained similar. The remaining 3 abdominal muscles had similar blood flow, but these values were less than that in the internal intercostal, transverse thoracic, and internal abdominal oblique muscles. Blood flow values for all inspiratory and expiratory muscles remained similar for the 5 and 15 minutes of exertion. However, at 26 minutes, blood flow had increased further in the diaphragm, external intercostal, internal intercostal, transverse thoracic, and the external abdominal oblique muscle as vascular resistance decreased. On the basis of our findings, all respiratory muscles were activated during submaximal exercise and their perfusion had marked heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Bronchial circulation during prolonged exercise in ponies.

Tracheal, bronchial, and renal flow were studied in 8 healthy ponies at rest and during exercise performed on a treadmill at a speed setting of 20.8 km/h and 7% grade (incline) for 30 minutes. Blood flow was determined with 15-microns-diameter radionuclide-labeled microspheres that were injected into the left ventricle when the ponies were at rest, and at 5, 15, and 26 minutes of exertion. Heart rate and mean aortic pressure increased from resting values (40 +/- 2 beats/min and 124 +/- 3 mm of Hg, respectively) to 152 +/- 8 beats/min and 133 +/- 4 mm of Hg at 5 minutes of exercise, to 169 +/- 6 beats/min and 143 +/- 5 mm of Hg at 15 minutes of exercise, and to 186 +/- 8 beats/min, and 150 +/- 5 mm of Hg at 26 minutes of exercise. Tracheal blood flow at rest and during exercise remained significantly (P less than 0.05) less than bronchial blood flow. Tracheal blood flow increased only slightly with exercise. Vasodilation caused bronchial blood flow to increase throughout exercise. Pulmonary arterial blood temperature of ponies also increased significantly (P less than 0.05) with exercise and a significant (P less than 0.005) correlation was found between bronchial blood flow and pulmonary arterial blood temperature during exertion. At 5 minutes of exercise, renal blood flow was unchanged from the resting value; however, renal vasoconstriction was observed at 15 and 26 minutes of exercise. We concluded that bronchial circulation of ponies increased with exercise in close association with a rise in pulmonary arterial blood temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.

Animals↗
PubMed1991

Characterization of two crystal forms of cytochrome c from Valida membranaefaciens.

Two different crystal forms of cytochrome c from the yeast Valida membranaefaciens have been grown by vapor diffusion from polyethylene glycol 4000. Both have been characterized by X-ray diffraction. The first crystal form was grown at 277 K but proved unstable and dissolved when the temperature was increased above 288 K. This crystal was of triclinic space group P1 and had unit-cell dimensions of a = 68.4, b = 35.7, c = 33.24 A, alpha = 116.9, beta = 114.9, gamma = 75.0 degrees. Following dissolution of the triclinic crystals, the second crystal form also grew at 277 K but remained stable even at room temperature. It was of orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 27.95, b = 63.68, and c = 64.28 A. Although rather small in size, both crystal forms diffracted X-rays well and the resolution of the patterns extended beyond 3 A.

Crystallization↗
PubMed1991

Characterization of crystals of an intact monoclonal antibody for canine lymphoma.

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.

Animals↗
PubMed1991

Preliminary investigation of crystals of the neutral lipase from Pseudomonas fluorescens.

The neutral lipase from the bacteria Pseudomonas fluorescens, marketed under the trade name LpL-200S, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8.5. The crystals are monoclinic prisms and are of space group C2 with a = 91.00 A, b = 47.17 A, c = 35.21 A and beta = 121.43 degrees. There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1.6 A resolution and the crystals are extremely robust in terms of X-ray exposure.

Crystallization↗
PubMed1991

Self testing for diabetes mellitus.

OBJECTIVE: To develop a simple, economically viable, and effective means of population screening for diabetes mellitus. DESIGN: A postal request system for self testing for glycosuria with foil wrapped dipsticks. Preprandial and postprandial tests were compared with a single postprandial test. The subjects were instructed how to test, and a result card was supplied on which to record and return the result. All those recording a positive test result and 50 people recording a negative result were invited for an oral glucose tolerance test. SETTING: General practice in east Suffolk, list size 11534. PATIENTS: All subjects aged 45-70 years registered with the practice were identified by Suffolk Family Health Services Authority (n = 3057). The 73 subjects known to have diabetes from the practice's register were excluded, leaving 2984 subjects, 2363 (79.2%) of whom responded. 1167 subjects completed the single test and 1196 the two tests. MAIN OUTCOME MEASURES: Response rate and number of patients with glycosuria. Sensitivity, specificity, and positive predictive value of a single postprandial test and preprandial and postprandial tests. Number of new cases of diabetes identified and cost of screening. RESULTS: Of the patients completing the single postprandial test, 29 had a positive result, an oral glucose tolerance test showed that eight (28%) had diabetes, six (21%) impaired glucose tolerance, and 14 (48%) normal glucose tolerance. 44 of the group who tested before and after eating had a positive result; nine (20%) had diabetes, five (11%) impaired tolerance, and 26 (11%) normal tolerance. Screening cost 59p per subject and 81 pounds per case detected. Of the 17 people with previously undiagnosed diabetes, eight were asymptomatic and 11 had not visited their general practitioner in the past three months. CONCLUSIONS: A postal request system for self testing for postprandial glycosuria in people aged 45-70 is a simple and effective method of population screening for diabetes mellitus.

Aged↗
PubMed1991

Cholinergic activity in the rat hippocampus, cortex and striatum correlates with locomotor activity: an in vivo microdialysis study.

The possible relationship between behavioral arousal and acetylcholine release in the striatum, hippocampus and frontal cortex was investigated in rats. In vivo microdialysate concentrations of acetylcholine and choline from these brain structures, and photocell beam interruptions (as a measure of behavioral arousal), were measured simultaneously under three conditions: after injections of 1) vehicle or 2) scopolamine (0.4 mg/kg), and 3) before and after the beginning of the rats' night cycle. Dialysate concentrations of ACh in all 3 brain structures and locomotor activity were increased after scopolamine and the onset of the lights out condition. Vehicle injections transiently increased ACh in the hippocampus and cortex and caused short-lasting increases in locomotor activity. Under all conditions, the release of ACh from each of the 3 brain structures correlated with the level of locomotor activity.

Acetylcholine↗
PubMed1991

Synergy between preactivated photofrin-II and tamoxifen in killing retrofibroma, pseudomyxoma and breast cancer cells.

Exposure of photoactive compounds to light prior to their use in biological systems (preactivation) results in the generation of tumour cell specific metastable cytotoxic species that are no longer dependent on the light energy. Thus, preactivation renders the photoactive compounds suitable for systemic use. We have examined the in vitro effect of preactivated photofrin-II and tamoxifen in retroperitoneal fibroma, pseudomyxoma and male breast carcinoma cell lines. These cells were found to be non-responsive to tamoxifen and were negative for oestrogen receptors. Incubation of these cells with 0.5 microgram/ml preactivated photofrin-II and tamoxifen (less than 10(-6) mol/l) resulted in a significantly enhanced (P less than 0.001) inhibition of DNA synthesis compared with either agent alone. This synergistic effect between tamoxifen and preactivated photofrin-II was determined by multiple drug effect analysis. Treatment of cells with preactivated photofrin-II did not cause the increased expression of oestrogen receptors. These observations suggest that a combination of antihormonal drugs with preactivated compounds may be of clinical value.

Breast Neoplasms↗
PubMed1990

Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci.

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.

Amino Acid Sequence↗