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Biomedical subjects

J Sutherland

Publications and source records attributed to J Sutherland.

At least 55 records · Page 3Linked to original sources
PubMed1995

Large amplitude twisting motions of an interdomain hinge: a disulfide trapping study of the galactose-glucose binding protein.

The galactose--glucose binding protein possesses two structural domains bordering a ligand binding cleft, with three polypeptide strands serving as a flexible hinge connecting the two domains. The hinge is known to bend, enabling the cleft to open by an angle of at least 18 degrees. Here the twisting motions of the hinge were examined by placing pairs of engineered cysteines on the perimeter of the cleft to generate six stable di-cysteine proteins. Each cysteine pair introduced reactive sulfhydryls into both rims of the cleft, one in the N-terminal domain and the other in the C-terminal domain. Collisions between sulfhydryls in different domains were trapped by disulfide formation, yielding sensitive detection of large amplitude domain rotations. When the cleft was occupied by the ligand D-glucose, counterclockwise hinge twist rotations were detected with amplitudes up to 36 degrees, and frequencies ranging from 10(1) to 10(3) collisions s-1. Removal of ligand from the cleft increased the range of twist angles 3-fold and the frequency of motions up to 10(2)-fold. Thus, in this representative hinged cleft protein, large amplitude hinge twist motions occur on biologically relevant timescales. The functional implications of such motions are discussed.

Binding Sites↗
PubMed1995

Genetic construction and characterization of the diphtheria toxin-related interleukin 15 fusion protein DAB389 sIL-15.

A gene fusion encoding DAB389 sIL-15 was constructed in which the catalytic and transmembrane domains of native diphtheria toxin (DAB389) are genetically linked to the N-terminus of simian interleukin 15 (sIL-15). It was demonstrated that the cytotoxic action of DAB389 sIL-15 is mediated through the IL-15 receptor. Since toxicity may be blocked with chloroquine, it was concluded that following binding to the IL-15 receptor, the fusion toxin is internalized by receptor-mediated endocytosis and must pass through an acidic compartment in order to facilitate the delivery of the catalytic domain to the cytosol of target cells. As a non-toxic control, the ADP-ribosyltransferase defective mutant DA(E149S)B389 sIL-15 was constructed. It was demonstrated that both sIL-15 and DA(E149S)B389 sIL-15 stimulate protein and DNA synthesis in IL-15 receptor-positive CTLL-2 cells in vitro.

Amino Acid Sequence↗
PubMed1995

A prospective study of rectal methohexital: efficacy and side effects in 648 cases.

Rectal methohexital has been used for nearly 30 yr in pediatric anesthesia. Despite this long and increasingly varied use, no large prospective series has been published detailing safety and efficacy. This study prospectively evaluated the efficacy, safety, and side effects of this medication in a series of 648 cases. On 553 of 648 occasions (85%), the child fell asleep after a single 30-mg/kg dose of 10% methohexital. Sleep was less likely in patients with myelomeningocele or who were receiving oral phenobarbital or phenytoin. When sleep occurred, the average time to onset of sleep was 6 min. Most patients who remained awake 15 min after drug were sedated. Defecation (10%) and hiccups (13%) were common but benign side effects. Partial airway obstruction and/or desaturation to Spo2 < or = 93% occurred in 26 patients (4%), but was resolved with blow-by oxygen and/or jaw-thrust in all but two cases. These two patients (0.3% of total) required aggressive airway intervention by the supervising anesthesiologist. Apnea did not occur in any patient. Methohexital has a high efficacy rate for sleep (85%) or sedation (96%), and has a relatively rapid onset. Significant respiratory side effects occur infrequently, but can be life threatening if not properly managed.

Administration, Rectal↗
PubMed1995

Human trks: molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins.

Using molecular cloning techniques, human homologs of the known members of the trk family of neurotrophin receptors have been cloned and sequenced. Overall, there is a high degree of similarity between the human sequences and those from other mammals; however, there are differences in splicing patterns. There are two spliced forms of the extracellular domain of trkC in the human, a finding that has not been described in other species. In contrast, fewer spliced forms were detected of the intracellular domains of human trkB and trkC than has been described in other mammals. Northern analysis and in situ hybridization experiments indicate that the human trks are expressed in a similar pattern to that described in other mammals. Expression of the trk extracellular domains as fusion proteins with IgG heavy chain yields soluble molecules that mimic intact trks in their binding specificity and affinity. These soluble chimeras block the biological activity of their cognate neurotrophin(s) in vitro.

Amino Acid Sequence↗
PubMed1994

Composition of the coagulant polysaccharide fraction from Strychnos potatorum seeds.

The composition of the coagulant polysaccharide fraction from Strychnos potatorum seeds is described. This fraction comprises a 1:1.7 mixture of a galactomannan and a galactan. The structure of these polysaccharides is also discussed. In addition, the coagulant properties of the polysaccharide fractions of two other Strychnos species, innocua and nux-vomica, have been assayed.

Carbohydrate Conformation↗
PubMed1992

Human monoclonal antibodies specific for blood group antigens demonstrate multispecific properties characteristic of natural autoantibodies.

A panel of 72 human monoclonal antibodies with specificities for blood group antigens, A, Rh D, Rh C, Rh c, Rh E, Rh e, Rh G, Jka and Jkb, has been established from the peripheral blood of deliberately immunized donors. Previous work has established that the antibodies are highly specific for their respective blood group antigens, and a number of them are in routine clinical use as blood grouping reagents. This panel was screened for reactivity against six unrelated foreign and autoantigens by ELISA, for rheumatoid factor activity by ELISA and agglutination techniques, and for reactivity with a number of different tissues by immunofluorescence. Binding of the monoclonal antibodies to unrelated exo- and autoantigens was commonly seen amongst the antibodies of the IgM class, and to a lesser degree amongst the IgG class, with reaction patterns similar to those given by natural autoantibodies. Only five of the IgM antibodies failed to demonstrate any unexpected cross-reactivities and these included 1/13 anti-D, 2/7 anti-E, 1/13 anti-c and 1/2 anti-A. We propose that rather than natural autoantibodies representing a distinct population of immunoglobulins, multispecificity (polyspecificity, or polyreactivity) may be a feature of antibodies produced in response to exogenous antigens. The implications of this for the study of autoantibodies are discussed.

Antibodies, Monoclonal↗
PubMed1991

Paradoxical relationship between substrates and agonist-induced contractions of opossum esophageal body and sphincter in vitro.

Possible differences in the abilities of esophageal body and lower esophageal sphincter muscles to utilize substrates to support agonist-induced contractions were studied. Strips of longitudinal, circular, and lower esophageal sphincter muscle from the opossum esophagus were first contracted to approximately 70% of the maximal contraction elicited by acetylcholine, histamine, or substance P. The tissues were then exhausted by exposure to 5 x 10(-4) M carbachol and a 15% O2-5% CO2-80% N2 gas mixture for 90 min. They were next reequilibrated with one of a number of alternative substrates and 95% O2-5% CO2 for 3 hr. Responses to the initial agonist doses were again noted and compared to controls. The alternative substrates were: 2-deoxyglucose, glucose, fructose 1-6 diphosphate, pyruvate, lactate, acetate, butyrate, caprylate, histidine, leucine, aspartate, alanine, succinate, acetoacetone, and beta-hydroxybutyrate. The results obtained show qualitative differences in the ability of the three muscle types to use these substrates. More importantly, however, the ability of any one substrate to support contractions was a function of the agonist used to stimulate the muscle. The evidence suggests, therefore, that not all pharmacologic receptors have equal access to intracellular energy sources.

Acetylcholine↗
PubMed1991

Human monoclonal antibodies against blood group antigens preferentially express a VH4-21 variable region gene-associated epitope.

An anti-idiotypic antibody has been raised which recognizes human immunoglobulins with cold agglutinin activity of anti-I/i specificity. The pattern of reactivity of the antibody indicates that the structural basis for the epitope is located in the VH4-21 gene segment of the VHIV family, which is preferentially utilized by these cold reactive antibodies. Using this antibody, epitope expression was investigated in a panel of 72 human monoclonal allo-antibodies specific for human blood group antigens, as compared with a control panel of 39 randomly selected human monoclonal IgM antibodies of unknown specificities. The anti-blood group panel included 44 IgM and 28 IgG monoclonal antibodies against a variety of blood group antigens including the A antigen, Rh C, c, D, E, e, G antigens, and the Kidd antigens Jka and Jkb. The epitope was expressed by 64% (28/44) of the IgM anti-blood group antibodies and by 21% (6/28) of the IgG antibodies, but by only 7.7% (3/39) of the control IgM antibodies. These data indicate that the human alloimmune response to blood group antigens is biased in the use of VH gene families, with a preference for the VH4-21 gene segment of the VHIV family, or closely related gene segments. The fact that this mirrors the findings for the autoimmune cold agglutinins suggests a link in immunoglobulin gene usage between antibodies against structurally diverse antigens on the red cell surface.

Antibodies, Monoclonal↗
PubMed1991

Human monoclonal antibodies to human blood group antigens Kidd Jka and Jkb.

Three IgM human monoclonal antibodies to Jkb, and one IgM human monoclonal antibody to Jka were produced from the lymphocytes of two immunized donors. Two of the anti-Jkb monoclonal antibodies (MS-7 and MS-9) are of the IgM (kappa) isotype and one (MS-8) is an IgM(lambda). The anti-Jka monoclonal antibody (MS-15) is of the IgM(kappa) isotype. They are all specific for their respective antigens, and give positive agglutinations in saline by the immediate spin technique, even against Jk(a+b+) cells. The heterohybridomas have been shown to be suitable for bulk culture and produce levels of antibody that reach 18 micrograms/ml in the spent culture supernatant. They offer considerable advantages over currently available reagents in terms of stability, simplicity and speed of use, and will provide a reliable and unlimited supply of what are at the moment rare and unsatisfactory antibodies.

Antibodies, Monoclonal↗
PubMed1991

The influence of epidural pethidine on shivering during lower segment caesarean section under epidural anaesthesia.

A prospective study of 94 patients undergoing elective lower segment caesarean section under epidural anaesthesia was performed in order to determine the incidence of shivering and the influence of epidural pethidine. Epidural anaesthesia was established with bupivacaine 0.5% with adrenaline, with or without additional lignocaine 2% with adrenaline, to total 20-25 ml. With the injection of epidural local anaesthesia an extra 5 ml of solution was administered into the epidural space--pethidine 25 mg preservative-free, in normal saline, or normal saline alone. Patient, administering anaesthetist and observer were blinded to the nature of the test substance. Shivering was assessed by an independent observer and subsequently rated by the patient. Other side-effects also recorded were nausea, vomiting, pruritus and drowsiness. The incidence of shivering was 36% in the control (saline) group and 11% in the pethidine group. The difference was highly significant (P less than 0.01). There was no significant difference in the incidence of maternal nausea, vomiting, drowsiness or pruritus, or neonatal Apgar scores. Cord blood samples were assayed for pethidine, revealing low or absent pethidine concentrations.

Adult↗
PubMed1990

Identification of taurocholate binding sites in ileal plasma membrane.

Intestinal absorption of bile salts occurs by passive processes throughout the length of the small intestine, whereas active carrier-mediated uptake is localized to the ileum. Although previous studies have extensively characterized brush-border transport of bile acids, their extrusion across the basolateral membrane is less well understood. Because previous reports had failed to show specific bile acid binding sites except with the use of photolabeled bile salt derivatives, we sought to identify and characterize the binding parameters of the physiological bile salt taurocholate in ileal and jejunal plasma membrane subfractions. Brush-border membrane (BBM) and basolateral membrane (BLM) fractions were rapidly and simultaneously isolated from the small intestinal mucosa. BBM fractions were isolated with enrichments of 50- to 54-fold for leucine aminopeptidase, whereas the basolateral membrane enrichment of Na(+)-K(+)-ATPase, its specific marker enzyme, was 22- to 25-fold. Contamination from intracellular organelles was minimal. Binding of [14C]taurocholate was demonstrated in both jejunal as well as ileal plasma membrane fractions. However, only ileal binding demonstrated saturation, reversibility, and susceptibility to proteolytic enzymes. [14C]taurocholate binding to BBM fractions also showed competition with bile acids but was not altered by pH or alkylating agents. In contrast, binding of taurocholate to the basolateral membrane showed optimal pH between 6.5 and 7.5 and was inhibited by thiol and alkylating agents. Kinetic analysis of specific ileal BBM and BLM binding showed the parameters for BBM as 288 +/- 70 microM and 2.4 +/- 0.6 nmol/mg protein and for BLM as 6.6 +/- 0.7 microM and 0.56 +/- 0.01 nmol/mg protein for dissociation constant and maximum binding capacity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Human monoclonal antibodies to C, c, E, e and G antigens of the Rh system.

A panel of heterohybridomas secreting human IgG and IgM monoclonal antibodies to the C, c, G, E, and e antigens of the Rh system has been established. Both classes of antibody have been shown to react with red blood cells carrying their respective antigens; those of the IgM class being able to directly agglutinate unmodified red blood cells in saline. The heterohybridomas have been shown to be suitable for bulk culture and produce levels of antibody in the range of 15-73 micrograms/ml in the spent culture supernatant.

Antibodies, Monoclonal↗
PubMed1990

The influence of weight-bearing exercise on articular cartilage of meniscectomized joints. An experimental study in sheep.

Unilateral medial meniscectomy was undertaken in 29 purebred adult merino sheep. Arthrotomy (without meniscectomy) was conducted in 11 animals, and six were used as nonoperated controls. All animals were followed with either three- or six-month active or passive postoperative management. Active animals traversed a total of 360 km after three months and 1040 km after six months. The passive group was housed in pens allowing limited weight-bearing exercise. After death, morphologic changes were recorded and cartilages from the medial and lateral joint regions were examined for water, collagen, and proteoglycan (as hexuronate) content. Proteoglycan aggregation and extractability under nondissociative (0.4 molar GuHCl) and dissociative (4.0 molar GuHCl) conditions were also determined. The collagen content of the medial cartilage of the passively maintained meniscectomized animals was reduced, relative to external controls. Although proteoglycan content was elevated in medial cartilage of the passive group three months postoperatively, these levels returned to the control range after six months. However, low-salt (0.4 molar GuHCl) extractability of proteoglycans still remained high. All cartilages of control and meniscectomized joints showed an elevation of proteoglycan content in the active groups three months postoperatively. The cartilages in the medial region of meniscectomized animals showed the largest increase, but these levels declined after six months. Proteoglycan aggregation and water content were still elevated relative to controls six months postoperatively. The collagen levels in the three-month or six-month actively maintained meniscectomized group were not distinguishable from control values. Morphologically, joints of the passively and actively maintained animals showed focal surface fibrillation and erosions. However, in the active group, osteophytes were common and well developed six months postoperatively. These studies indicate that, while weight-bearing exercise after meniscectomy appears to be beneficial to the quality of the cartilaginous matrix, it is also accompanied by osteophytosis and cartilage hyperplasia.

Animals↗
PubMed1989

Evidence for an immune-mediated mechanism in food-induced migraine from a study on activated T-cells, IgG4 subclass, anti-IgG antibodies and circulating immune complexes.

Various immunological studies have revealed controversial outcomes on pathogenetic mechanisms of food-induced migraine. In order to better define the immune status of this disease we studied 21 patients for circulating immune complexes (CIC). Six out of them were also studied for lymphocyte subpopulations, IgG4 and anti-IgG antibodies, before (TO), 4 hours after (T4), and 72 hours after (T72) a specific challenge with 250 ml of milk. The ClqSp assay was used to determine CIC. Lymphocyte subpopulations were defined by the following monoclonal antibodies (Mab): OKT3, OKT4, OKT8, 4F2, H366, TAC, 5E9, L.243 and DA6.231. IgG4 subclass was assessed by using a mouse specific Mab. Anti-IgG antibodies were determined by using HPLC. The result showed an increased incidence of CIC (28.6%) when compared to the control group (10%). Total T-cells (OKT3+) showed a marked increase at T4 (p less than 0.01) and a subsequent decrease at T72 (p less than 0.02). Interestingly, T-activated cells (4F2+ and TAC+) showed a parallel trend at T4 (respectively p less than 0.02 and less than 0.01) and a subsequent decrease at T72 only for the Tac+ cells (p less than 0.05). Also K and NK cells (H366+) showed an early increase at T4 (p less than 0.05). No significant variation was observed in the study of the expression of DR antigens (L.243 and DA6.231) at the three times. IgG4 and anti-IgG antibodies values showed no variation in their time-course.+

Adolescent↗
PubMed1989

Molecular and phenotypic analysis of patients with deletions within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene.

Eighty unrelated individuals with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) were found to have deletions in the major deletion-rich region of the DMD locus. This region includes the last five exons detected by cDNA5b-7, all exons detected by cDNA8, and the first two exons detected by cDNA9. These 80 individuals account for approximately 75% of 109 deletions of the gene, detected among 181 patients analyzed with the entire dystrophin cDNA. Endpoints for many of these deletions were further characterized using two genomic probes, p20 (DXS269; Wapenaar et al.) and GMGX11 (DXS239; present paper). Clinical findings are presented for all 80 patients allowing a correlation of phenotypic severity with the genotype. Thirty-eight independent patients were old enough to be classified as DMD, BMD, or intermediate phenotype and had deletions of exons with sequenced intron/exon boundaries. Of these, eight BMD patients and one intermediate patient had gene deletions predicted to leave the reading frame intact, while 21 DMD patients, 7 intermediate patients, and 1 BMD patient had gene deletions predicted to disrupt the reading frame. Thus, with two exceptions, frameshift deletions of the gene resulted in more severe phenotype than did in-frame deletions. This is in agreement with recent findings by Baumbach et al. and Koenig et al. but is in contrast to findings, by Malhotra et al., at the 5' end of the gene.

Chromosome Aberrations↗
PubMed1988

Human ribosomal RNA genes: orientation of the tandem array and conservation of the 5' end.

The multiple copies of the human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters that map to the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. Concerted evolution of the gene family is thought to be mediated by interchromosomal recombination between rDNA repeat units, but such events would also result in conservation of the sequences distal to the rDNA on these five pairs of chromosomes. To test this possibility, a DNA fragment spanning the junction between rDNA and distal flanking sequence has been cloned and characterized. Restriction maps, sequence data, and gene mapping studies demonstrate that (i) the rRNA genes are transcribed in a telomere-to-centromere direction, (ii) the 5' end of the cluster and the adjacent non-rDNA sequences are conserved on the five pairs of chromosomes, and (iii) the 5' end of the cluster is positioned about 3.7 kb upstream from the transcription initiation site of the first repeat unit. The data support a model of concerted evolution by interchromosomal recombination.

Biological Evolution↗