Biomedical subjects

R J Harris

Publications and source records attributed to R J Harris.

At least 109 records · Page 6Linked to original sources

PubMed1990

Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.

This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.

Amino Acid Sequence↗

PubMed1990

Characterization of a soluble form of human CD4. Peptide analyses confirm the expected amino acid sequence, identify glycosylation sites and demonstrate the presence of three disulfide bonds.

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen-presenting cells bearing the class II major histocompatibility (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells. This 368-amino-acid glycoprotein contains two potential sites of N-linked glycosylation (Asn-271 and Asn-300) and six cysteine residues. Amino-terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P.J. et al. (1985) Cell 42, 93-104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed-phase HPLC after digestion of S-carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin-digested rCD4 confirmed that the carboxy-terminus of the protein was Pro-368. Enzymatic digestion of non-reduced rCD4 generated disulfide-bonded fragments that demonstrated the presence of disulfide bonds between Cys-16 and Cys-84, Cys-130 and Cys-159, and between Cys-303 and Cys-345. The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N-glycosidase F demonstrated that both potential N-glycosylation sites are utilized. The tryptic map of rCD4 treated with endo-beta-N-acetylglucosamine H demonstrated that only complex-type oligosaccharides are attached to Asn-271, while Asn-300 has high-mannose or hybrid structures attached in addition to complex-type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn-300 or Asn-271 while no galactosamine was observed. This suggests that rCD4 contains no O-linked oligosaccharides.

Amino Acid Sequence↗

PubMed1990

Event order versus syntactic structure in recall of adverbial complex sentences.

Two experiments using recall of sentences examined two contrasting principles of clause ordering in a type of complex sentences: the main-clause-first (syntactic) principle and the event-order (or temporal-causal order) principle. In Experiment 1, these two principles were studied in complex sentences with a main clause and a subordinate adverbial clause-e.g., "When she heard the thunder, she stopped playing Frisbee." In sentences of this type the subordinate clause typically describes a temporally or causally antecedent event, while the main clause describes a subsequent event in the temporal-causal sequence. These two principles make opposite predictions on what is the psychologically simpler or preferred order of the two clauses in this type of complex sentence. Results of Experiment 1 showed an overall preference in memory for main-clause-first order. In Experiment 2, complex sentences with a main clause and a subordinate clause not temporally or causally related were also used in a sentence recall task. Similar results were obtained. The implication of these findings for the determinants of linguistic structures was discussed.

Adult↗

PubMed1990

A double-blind study of hyposensitization with an alginate-conjugated extract of Dermatophagoides pteronyssinus (Conjuvac) in patients with perennial rhinitis. II. Immunological aspects.

In a 2-year double-blind placebo controlled study an immunological evaluation was carried out on 33 patients (15 males, 18 females, mean age 29.2 years) with mite-induced perennial rhinitis who were submitted to specific immunotherapy (IT) with an alginate-conjugated extract of D. pteronyssinus. The behaviour of IgE, IgG, IgG1 and IgG4 antibodies specific to D. pteronyssinus and its major allergen Der p1 was characterized by assessment of their changes in serum, and changes in IgG in nasal secretions during the treatment. The placebo-treated patients did not show any significant variation in the levels of specific antibodies, while in the actively treated patients we found: a statistically significant decrease (P less than 0.005) of specific IgE, a statistically significant increase of specific IgG (P less than 0.005), IgG1 (P less than 0.005) and IgG4 (P less than 0.005) in serum and a statistically significant increase (P less than 0.001) of specific IgG in nasal secretions. The IgG response showed an early relative predominance of the IgG1 subclass and a late absolute predominance of IgG4 subclass, that confirmed the model of IgG4 restriction in prolonged allergen stimulation. No correlation was found between immunological and clinical data.

Adjuvants, Immunologic↗

PubMed1989

Purification of recombinant human tissue factor.

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.

Amino Acid Sequence↗

PubMed1989

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping.

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.

Amino Acid Sequence↗

PubMed1989

An experimental study of the effect of nimodipine in primate subarachnoid haemorrhage.

Acute subarachnoid haemorrhage was produced in baboons by a transorbital vessel avulsion technique. Half the animals were pretreated with an intravenous infusion of the calcium antagonistic nimodipine, in a dosage comparable with clinical levels. The severity of the haemorrhage, as measured by changes in intracranial pressure, cerebral perfusion pressure, cerebral blood flow and reactivity, and evoked potentials, was not greater in the group receiving nimodipine. Changes in extracellular K+ and pH were much less marked in animals receiving nimodipine. It is suggested that nimodipine (a) has a protective effect at a cellular level against the ionic changes of ischaemia, (b) does not alter the mechanical severity of subarachnoid haemorrhage.

Acute Disease↗

PubMed1989

Defective fibrinolysis in Behçet's syndrome: significance and possible mechanisms.

Reduced fibrinolytic activity has frequently been reported in Behcet's syndrome, but both the underlying mechanism and its relation to the development of thrombosis are unclear. The fibrinolytic activity was studied in seven patients with Behçet's syndrome and 12 patients with idiopathic oral ulceration. The patients with Behçet's disease had significantly reduced resting tissue plasminogen activator(t-PA) both by euglobulin fibrin plate lysis and enzyme linked immunosorbent assay (ELISA), but the tissue plasminogen activator inhibitor(t-PAI) was not significantly different from the control value. After stimulation by venous occlusion the euglobulin fibrin plate lysis results showed three good responders and four poor responders, but neither t-PA antigen nor t-PAI concentrations showed any clear difference between the two groups. Interestingly, three out of the four patients with a history of thrombosis were good responders. The group with oral ulceration showed no significant difference from the controls for any of these measurements. These findings suggest that the reduced resting fibrinolytic activity is usually due to decreased production of t-PA, but that the defective response to stimulation may be multifactorial in origin. The fibrinolytic abnormalities appear unrelated to the development of thrombosis.

Adult↗

PubMed1988

Lyme disease involving the temporomandibular joint.

Lyme disease is an increasingly common problem that has spread rapidly across the United States. A case of the disease and its correlation to TMJ pain and dysfunction in a 35-year-old female is described. The patient was diagnosed and cured of her symptoms with antibiotics, possibly sparing her from surgery.

Adult↗

PubMed1988

Brain tissue concentrations of ATP, phosphocreatine, lactate, and tissue pH in relation to reduced cerebral blood flow following experimental acute middle cerebral artery occlusion.

Local CBF (LCBF) was compared with the corresponding local tissue concentration of ATP, phosphocreatine (PCr), and lactate in anaesthetized baboons subjected to focal ischaemia produced by middle cerebral artery occlusion (MCAO). LCBF hydrogen electrodes were implanted in cortical regions where MCAO had been previously shown to produce severe and penumbral ischaemia and in posterior regions where blood flow is not altered. Metabolites were assayed in small tissue samples collected either by cryoprobe biopsy in the regions where LCBFs were measured (series 1) or by sampling appropriate regions of the rapidly frozen brain (series 2). Subsequent topographical study of brain tissue pH with umbelliferone was performed in this latter series. The results from these two series are compared and discussed in terms of the more appropriate way to perform simultaneous electrode measurements and analysis of tissue samples for studying focal ischaemia in the primate brain. They confirm that the concentrations of ATP and PCr decrease, and that lactate level increases, with decreasing blood flow. These metabolites tended to change more rapidly below a blood flow threshold, rather than showing a steady decrease as the blood flow was reduced, although the variability of the data precluded us from establishing this with confidence. Topographical study of tissue pH often showed sharp boundaries between zones of very low pH and regions with normal pH.

Adenosine Triphosphate↗

PubMed1988

Abnormal fibrinolytic activity in systemic lupus erythematosus and possible mechanisms.

Plasma fibrinolytic activity was measured in 34 patients with systemic lupus erythematosus (SLE) and 12 normal subjects. Patients with SLE showed a significantly reduced resting level of plasma tissue plasminogen activator (t-PA) compared to normal. The reduction in t-PA was demonstrated both by a functional assay (fibrin-plate lysis, FP) and an immunochemical assay (ELISA). Measurement of the fibrinolytic response following venous occlusion allowed division of the patients into two groups. In the first (24 patients), there was a normal increase in t-PA response, demonstrated both by the functional and immunochemical assays. In the second (10 patients), there was a significantly reduced plasma t-PA response measured by FP. Seven of the patients in this second group, which included four patients with histological evidence of vasculitis, showed a similar failure of t-PA response (ELISA) after venous occlusion. These results suggest that their impaired fibrinolytic response may be related to defective t-PA release secondary to endothelial cell damage. The remaining three patients in the latter group had a normal t-PA (ELISA) response despite a reduced FP response, suggesting the presence of an inhibitor.

Adult↗

PubMed1987

pH, K+, and PO2 of the extracellular space during ischaemia of primate cerebral cortex.

pH and K+ from the extracellular space, PO2, and CBF have been measured in the same region during progressive ischaemia of primate cerebral cortex. As blood flow was reduced, the other changes had the following sequence. PO2 fell rapidly to 30% of control levels at regional CBF (rCBF) of 30 ml 100 g-1 min-1. As CBF was further reduced, PO2 continued to fall. pH remained stable until around 20 ml 100 g-1 min-1, below which pH fell rapidly, with an exponential increase in H+ concentration. K+ showed the well-known relationship to CBF, remaining normal until around 10 ml 100 g-1 min-1, below which K+ rose rapidly. pHe and log K+ were lin-early related and confirmed that pH fell by 0.3 U before K+ rose significantly, and fell by 0.6 U before the massive rise in K+. The mechanisms involved in this sequence of events and the role of pH changes in the development of the so-called "ischaemic penumbra" are discussed.

Animals↗

PubMed1986

Recent trends in the relative economic status of older adults.

Contrary to theoretical expectations and previous empirical research based on modernization and aging theory, this study demonstrates that there has been no decline in the relative income of the older population from 1945 to 1980. Furthermore, recent increases in the relative earnings of older workers between 1967 and 1977 are documented, independent of structural changes in educational and occupational levels. Rather than representing a reversal of previous trends, conceptual and measurement issues appear to account for the differences in findings. Although each older cohort does experience a decline in relative earnings between 1967 and 1977, newer cohorts entering old age have higher relative earnings than the older cohorts whom they replace, accounting for aggregate improvements in relative earnings.

Aged↗

PubMed1984

Extracellular pH, potassium, and calcium activities in progressive ischaemia of rat cortex.

We measured the relationships between changes in extracellular pH (pHe), potassium (Ke), and calcium (Cae) activities and DC potential (DCe) in progressive ischaemia of rat cerebral cortex. pHe and Ke, or Cae and Ke, were measured at the same point simultaneously, using triple-barrelled, double-ion-sensitive microelectrodes. Ischaemia was produced using bilateral carotid artery occlusion and hypotension in rats under 50% N2O-0.4% halothane anaesthesia. Unilateral carotid artery occlusion did not affect blood flow, but bilateral occlusion reduced flow to approximately 40% of normal. Autoregulation of blood pressure (BP) changes was lost after bilateral occlusion, and so progressive hypotension produced a linear decrease in flow. pHe began to decrease at high levels of flow (30-35 ml 100 g-1 min-1) and showed stepwise acidotic shifts with reductions in BP. Ke was affected at flows of approximately 15 ml 100 g-1 min-1, during which time it was critically dependent on BP. When Ke reached 6 mM, it increased rapidly to 40 mM and was associated with a negative shift in DCe. When Ke reached approximately 10 mM, Cae decreased rapidly to approximately 0.1 mM. pHe had reached 6.87 when Ke increased rapidly and showed a transient alkalotic shift of approximately 0.14 units at that time. Possible mechanisms for the sequence of ion changes described are discussed.

Animals↗

PubMed1984

Cerebral extracellular calcium activity in severe hypoglycemia: relation to extracellular potassium and energy state.

The changes in extracellular Ca2+ (Cae) and K+ (Ke) activities were studied in the rat brain during insulin-induced hypoglycemia. At about the time of onset of isoelectric EEG in severe insulin-induced hypoglycemia (300-g male Wistar rats under 70% N2O anaesthesia), there was an increase in Ke which, at approximately 13 mM, was associated with a fall in Cae. Ke peaked at 48 +/- 12 mM, and Cae at 0.18 +/- 0.28 mM. This ion change began to normalise, but before recovery was complete a second ion change, of magnitude similar to that of the first, occurred from which the cells did not recover. The Cae recovered to only 66% of normal in the time available before the second depolarisation. Measurements on brains frozen at different stages during the sequence of ion changes revealed that ATP and phosphocreatine (PCr) concentrations and energy charge (EC) were not reduced before the first depolarisation. During the first depolarisation there was a 72% decrease in PCr and a 37% fall in ATP level, leading to a 23% drop in EC. These levels decreased further by the 10th minute of isoelectricity , but only the fall in ATP concentration was significant. The results indicate that the first ion change was a spreading depression and that cellular energy state was not the only factor in determining the response of tissue in the early stages of the comatose state.

Animals↗

PubMed1984

Influence of severe hypoglycemia on brain extracellular calcium and potassium activities, energy, and phospholipid metabolism.

In the cerebral cortices of rats, during insulin-induced hypoglycemia, changes in the concentrations of labile phosphate compounds [ATP, ADP, AMP, and phosphocreatine (PCr)] and glycolytic metabolites (lactate, pyruvate, and glucose) as well as phospholipids and free fatty acids (FFAs) were studied in relation to extracellular potassium and calcium activities. Changes in extracellular calcium and potassium activities occurred at approximately the onset of isoelectricity . The extracellular calcium activity dropped from 1.17 +/- 0.14 mM to 0.18 +/- 0.28 mM and the potassium activity rose from 3.4 +/- 0.94 mM to 48 +/- 12 mM (means +/- SD). Minutes prior to this ionic change the levels of ATP, PCr, and phospholipids were unchanged while the levels of FFAs remained unchanged or slightly elevated. Following the first ionic change the steady-state levels of ATP decreased by 40%, from 2.42 to 1.56 mumol/g. PCr levels decreased by 75%, from 4.58 to 1.26 mumol/g. Simultaneously, the levels of FFAs increased from 338 to 642 nmol/g, arachidonic acid displaying the largest relative increase, 33 to 130 nmol/g. The first ionic change was followed by a short period of normalization of ionic concentrations followed by a sustained ionic change. This was accompanied by a small additional decrease in ATP (to 1.26 mumol/g). The FFA levels increased to 704 nmol/g. There was a highly significant negative correlation between the levels of FFAs and the energy charge of the tissue. The formation of FFAs was accompanied by a decrease in the phospholipid pool. The largest relative decrease was observed in the inositol phosphoglycerides, followed by serine and ethanolamine phosphoglycerides.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1983

Nutritional survey of Bangladeshi children aged under 5 years in the London borough of Tower Hamlets.

The main deficiencies in the diet of Bangladeshi infants and children are vitamin D, iron, and later, calories. Protein intake is a little low in some, but in general is satisfactory. Calcium and vitamin C intakes are adequate. The proportions of food are often inappropriate, mainly because of a high carbohydrate intake. Weaning tends to be late, with predominant milk drinking, even into the second year of life. Convenience baby foods, containing mainly carbohydrate are the major components of the diet until well into the second and third years of life, without the conversion to family food seen in white children. Vitamin D is given to fewer than half the children. It may be given to fewer still if the observation that bottles were often unopened is true for many families.

Ascorbic Acid↗

PubMed1982

Calcium ions and calcium antagonists in ischaemia.

The effects of a calcium antagonist, nimodipine, were tested on the response of the cerebral circulation to arterial pCO2 and blood pressure changes. The effects of reduced blood flow upon oedema formation and extracellular ion homeostasis under nimodipine preloading were studied. Both open and closed skull primate models were used, with alpha-chloralose anaesthesia. Nimodipine infusion increased basal blood flow in the open skull, but not the closed skull animals. Autoregulation to increased blood pressure was little affected. Responses to arterial pCO2 changes and autoregulation to reduced blood pressure were severely impaired. Residual blood flow after middle cerebral artery occlusion was significantly higher with nimodipine than in controls. The threshold levels of blood flow for the development of cortical oedema and for disturbance of ion homeostasis were, however, increased, suggesting that nimodipine interferes with cellular energy metabolism and increases the susceptibility of tissue to ischaemic damage.

Animals↗