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Biomedical subjects

A J Jones

Publications and source records attributed to A J Jones.

At least 19 recordsLinked to original sources
PubMed1991

High resolution analysis of functional determinants on human tissue-type plasminogen activator.

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.

Amino Acid Sequence↗
PubMed1991

Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. Opioids have been shown to inhibit substance P (SP) release from primary afferent neurones (PAN). In addition, opioid receptors have been identified on PAN of the vagus nerves. Sodium cromoglycate (SCG) decreases the excitability of C-fibres in the lung of the dog in vivo. We have utilised a multi-superfusion system to investigate the effect of opioids and SCG on the release of SP from the rat trachea in vitro. 2. Pretreatment of newborn rats with capsaicin (50 mg kg-1 s.c. at day 1 and 2 of life) resulted in a 93.2 +/- 6.3% reduction in tracheal substance P-like immunoreactivity (SP-LI) content when determined by radioimmunoassay in the adult. 3. Exposure to isotonically elevated potassium concentrations (37-90 mM), capsaicin (100 nM-10 microM), and bradykinin (BK; 10nm-1 microM) but not des-Arg9-BK (1 microM) stimulated SP-LI release by a calcium-dependent mechanism. 4. SCG (1 microM and 100 microM) did not affect spontaneous, potassium (60 mM)- or BK (1 microM)-stimulated SP-LI release. 5. Morphine (0.1-100 microM) caused dose-related inhibition of potassium (60 mM)-stimulated SP-LI release with the greatest inhibition of 60.4 +/- 13.7% at 100 microM. The effect of morphine was not mimicked by the kappa-opioid receptor agonist, U50,488H (10 microM) or the delta-opioid receptor agonist, Tyr-(D-Pen)-Gly-Phe-(D-Pen) (DPDPE). 6. The effect of morphine was totally abolished by prior and concomitant exposure to naloxone (100 nM) which had no effect on control release values. 7. We conclude that opioid receptors, predominantly of the MM-opioid receptor subtype, inhibit SP-LI release from PAN in the rat trachea and suggest that centrally inactive MM-opioid receptor agonists may have therapeutic potential in the treatment of asthma.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1991

GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. The role of gamma-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system is well documented. Recently, GABAA and GABAB receptors have been identified in the peripheral nervous system, notably on primary afferent neurones (PAN). We have utilised a multi-superfusion system to investigate the effect of selective GABA receptor agonists and antagonists on the release of substance P (SP) from the rat trachea in vitro. 2. GABA (1-100 microM) did not affect spontaneous release of SP-like immunoreactivity (LI) but caused dose-related inhibition of calcium-dependent potassium (60 mM)-stimulated SP-LI release. The greatest inhibition of 77.7 +/- 18.8% was observed at 100 microM. 3. The inhibitory effect of GABA was mimicked by the GABAB receptor agonist, (+/-)-baclofen (1-100 microM), but not the GABAA receptor agonist, 3-amino-1-propane-sulphonic acid (3-APS, 1-100 microM). Baclofen (100 microM) had no effect on SP-LI release stimulated by capsaicin (1 microM). 4. The inhibitory effect of baclofen (30 microM) was significantly reduced by prior and concomitant exposure to the GABAB receptor antagonist, phacolofen (100 microM) but not the GABAA receptor antagonist, bicuculline (10 microM). Neither antagonist, alone, affected spontaneous or potassium-stimulated SP-LI release. 5. We conclude that activation of pre-synaptic GABAB receptors on the peripheral termini of PANs in the rat trachea inhibits SP-LI release and suggest that GABAB receptor agonists may be of value in the therapeutic treatment of asthma.

Animals↗
PubMed1990

A precise and rapid microtitre plate clot lysis assay: methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis.

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear to influence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and 0.1 s-1, respectively, in excellent agreement with published values. The assay should therefore prove useful in quantitative evaluations of the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗
PubMed1990

Comparison of fine needle aspiration cytology, radioisotopic and ultrasound scanning in the management of thyroid nodules.

In 175 patients presenting with thyroid nodules, the diagnostic value in management of fine needle aspiration and cytology (FNAC), pertechnetate (99mTc) scanning and ultrasound imaging was examined. In 82 patients, the diagnosis was confirmed at operation; in the remaining 93, there was a follow-up period of at least 2 years. Thyroid cancer was found in 13 patients. For FNAC the sensitivity, specificity and positive predictive value for thyroid cancer were 92%, 85% and 41% respectively compared with 82%, 34% and 11% for 99mTc pertechnetate scanning, 75%, 61%, 19% for ultrasound and 73%, 58% and 19% for combined pertechnetate and ultrasound scanning. In 14% of patients, the aspirates were inadequate for cytology at the first examination. FNAC is therefore the preferable initial investigation and usually gives results adequate for a decision on surgical or medical management. With medical management and follow-up, ultrasound is of value in defining the nodule and the appearance of the rest of the gland.

Biopsy, Needle↗
PubMed1990

Age- and position-related heterogeneity of equine tendon extracellular matrix composition.

The digital flexor tendons of the neonate and adult horse have been compared with respect to variation in extracellular matrix composition along their length. Two pepsin-sensitive, acetic acid soluble proteins, molecular weight (Mr) 52 kD (np 52) and Mr 54 kD (np 54), were prominent throughout the length of neonatal tendons. In adult tendon, np 52 and np 54 were less abundant and restricted to the cannon (metacarpal) region. In contrast, a single pepsin- and collagenase-resistant protein of Mr 55 kD (fp 55) was exclusive to the fetlock (metacarpophalangeal joint) region regardless of age, although more distinct in the adult. Pepsin extracted fp 55 precipitated at 2.0 M sodium chloride: 0.5 M acetic acid and was further purified to homogeneity by bacterial collagenase digestion. Analysis of fp 55 amino acid composition revealed the presence of a large proportion of glycine residues (379 of 1001), suggesting a possible homology with the collagen family. These data demonstrate that the composition of equine digital flexor tendons varies with age, is heterogeneous along its length, and suggests that variation in tendon extracellular matrix composition is influenced by functional requirements.

Acetates↗
PubMed1979

Halothane does not inhibit human neutrophil function in vitro.

Various indices of function of neutrophils from normal healthy volunteers have been examined after in vitro exposure to halothane. Random free movement on glass was unaffected, but random migration through millipore filters was slightly increased. There was no significant change in migration in response to casein chemotaxis. Phagocytosis, degranulation and the enhanced non-mitochondrial respiration associated with phagocytosis were unaffected. Electron-microscopic appearance at 30 s after exposure to latex particles was normal in all respects.

Cell Movement↗
PubMed1978

Size and shape of the model lipoprotein complex formed between glucagon and dimyristoylglycerophosphocholine.

Glucagon forms water-soluble lipoprotein particles with dimyristoylglycerophosphocholine at temperatures below the phase-transition temperature of the lipid. The shape and size of this lipoprotein particle were studied by viscometry, sedimentation velocity, sedimentation equilibrium, quasielastic light scattering, and electron microscopy using both negative-staining and freeze-fracture techniques. The lipoprotein particle has an oblate ellipsoid shape with dimensions of 250 X 70 A and an approximate molecular weight of 1.4 X 106. This molecular weight is similar to that found for small unilamellar phospholipid vesicles but is achieved in the presence of glucagon without sonication. The shape of the glucagon lipoprotein particle is similar to that found for the complex formed between some serum apolipoproteins and dimyristoylglycerophosphocholine. From these data, a model for the glucagon-dimyristoylglycerophosphocholine is proposed consisting of a single bilayer of phospholipid with the glucagon incorporated into the bilayer structure in such a manner as not greatly to disturb the average area occupied per phospholipid molecule.

Glucagon↗
PubMed1978

Interaction of the myelin basic protein with the anionic detergent sodium dodecyl sulphate.

The interaction of the myelin basic protein and two peptides derived from it with the anionic detergent SDS (sodium dodecyl sulphate) was studied. At molar ratios of detergent/protein of up to approx. 20:1 the transient increase in turbidity (as measured by increases in A230) is proportional to the ratio. Between ratios of 30:1 and 100:1 the effect of the detergent is constant and maximal. At molar ratios exceeding 100:1 the transient increase in turbidity decreases with increasing amounts of detergent. With increasing ionic strength the rapid development of turbidity is inhibited, whereas the slow decay of turbidity is not affected. Neither of the peptide fragments produced by cleavage of the myelin basic protein at the single tryptophan residue, nor both when mixed, produce measurable turbidity when mixed with SDS. Under similar conditions poly-L-lysine of similar molecular size to the basic protein shows the increase in turbidity but not the decay. The interaction between the protein and SDS is interpreted in molecular terms, which involve the initial ionic interaction of the detergent with protein resulting in aggregation and turbidity in the solution. Within the aggregated complexes molecules rearrange to maximize hydrophobic interactions.

Animals↗
PubMed1978

Pressure enhancement of the depressant effect of halothane on cilial beat.

In order to test the applicability of the critical volume hypothesis of anaesthetic action to the side effects of anaesthesia, we have studied the pressure-anaesthetic interactions on cilial beat. The swimming speed of Tetrahymena pyriformis was determined at one and 137 atmospheres with 0 to 4 per cent halothane. The pressure and anaesthetic effects were synergistic with the halothane dose-response curve being shifted to the left and increased in slope at the high pressure, although high pressure alone had no significant effect. These results are contrary to the predictions of the critical volume hypothesis and appear to demarcate the narcotic effect of anaesthetics from the type of molecular interaction which is concerned in the effect on cilial beat.

Cilia↗
PubMed1977

Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.

Amino Acids↗
PubMed1977

Interaction of glucagon with dimyristoyl glycerophosphocholine.

Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at temperatures below the phase transition leading to a shift in the fluorescence emission maximum of tryptophan from 350 to 338 nm and a 3-fold enhancement of fluorescence intensity as well as a change in the polarization of fluorescence. The circular dichroism properties of the lipid-associated glucagon indicates that it has an increased content of alpha-helix. The phase transition temperature of the lipid as monitored by pyrene excimer fluorescence is not altered by interaction with glucagon although at higher glucagon/lipid ratios a decrease in excimer formation is noted at low temperature. Above the phase transition temperature, the addition of lipid has no effect on the fluorescence emission or circular dichroism of glucagon. Thus this hormone can interact with dimyristoyl glycerophosphocholine and this interaction is stronger below the phase transition temperature than above it.

Amino Acid Sequence↗