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C J Kirk

Publications and source records attributed to C J Kirk.

At least 37 records · Page 2Linked to original sources
PubMed1997

Interpretation of the Ws statistic: application to an integrated trauma system.

BACKGROUND: Comparison of trauma survival rates between institutions and reference data bases is hampered by different injury severity mixes. To overcome this, a standardized comparison using a stratified W statistic (Ws) has been proposed. Ws enables comparisons but does not represent actual survival rates at an institution. METHOD, DISCUSSION, AND RESULTS: This discussion paper uses, as an example, data from 632 patients with blunt injuries treated by an integrated trauma system comprising a helicopter emergency medical service and the Royal London Hospital. Using the United Kingdom Major Trauma Outcome Study as a reference data set, Ws is calculated and its usefulness discussed. The problem of overprediction of survival is discussed. A modification of the Ws method is used to allow for this and to estimate the actual survival rate. CONCLUSION: Ws is useful for comparison between centers. Trauma and Injury Severity Score methodology overpredicts survival in certain probability of survival intervals. Adjustment can be made to estimate the actual survival rate. When applied to an integrated trauma system, such adjustments demonstrate overall improved survival, most noticeable in a more severely injured subgroup.

Diagnosis-Related Groups↗
PubMed1997

Early activation defects in T lymphocytes from aged mice.

Aging affects both calcium signals and protein kinase cascades in mouse T lymphocytes. The decline in calcium signal development largely represents differences between naive and memory T cells; the latter are resistant to increases in calcium concentration, and are more common in aged mice. Aging leads to declines in phosphorylation of a wide range of substrates in T cells stimulated by either anti-CD3 antibodies or by substances, such as phorbol myristate acetate (PMA) or ionomycin, that act at intracellular sites, but some phosphoproteins respond only in old T cells, and others respond regardless of age. Tyrosine phosphorylation of the CD3 zeta chain declines with age, both in resting T cells and after activation, but the proportion of Zap-70 that is bound to CD3 zeta increases in T cells from old mice. Zap-70 function and phosphorylation of CD3 zeta-associated Zap-70 change only slightly after stimulation of T cells by anti-CD3 and anti-CD4, and are at similar levels in activated old and young T cells. Nonetheless, induction of Raf-1, MEK, and ERK kinase activity declines with age in CD4 T cells. The effect of aging on T-cell activation is not simply an overall decline in signal intensity, but a set of qualitative changes that differ among subsets and depend at least partly on the nature of the stimulus.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Thyroid-stimulating hormone rapidly stimulates inositol polyphosphate formation in FRTL-5 thyrocytes without activating phosphoinositidase C.

The thyroid-stimulating hormone (TSH) receptor is widely regarded as one of a limited number of G-protein-coupled receptors that activate both adenylate cyclase and phosphoinositidase C (PIC) via G-proteins, but the existing experimental evidence for TSH-stimulated PtdIns(4,5)P2 hydrolysis remains inconclusive. We have compared the effects of TSH and of ATP (acting via P2-purinergic receptors) on the inositol lipids and polyphosphates of [2-3H]inositol-labelled FRTL-5 rat thyroid cells. ATP initiated a rapid decrease in 3H-labelled PtdIns4P and PtdIns(4,5)P2, whereas TSH did not. Stimulation with ATP and, less consistently, with noradrenaline (acting via alpha-adrenergic receptors) provoked rapid formation of Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(1,4)P2, confirming activation of PtdIns(4,5)P2 hydrolysis. No concentration of TSH provoked detectable accumulation of Ins(1,4,5)P3 or Ins(1,4)P2 during the first few minutes of stimulation. However, an InsP3 [with the chromatographic properties of Ins(1,3,4)P3] and two InsP4 isomers [neither of which was Ins(1,3,4,5)P4] accumulated quickly in TSH-stimulated cells. ATP immediately provoked a large increase in intracellular calcium concentration ([Ca2+]i) in Indo 1-AM-loaded cells. TSH provoked a small and delayed [Ca2+]i elevation in only some experiments. We therefore confirm that activation of P2-purinergic receptors and alpha 1-adrenergic receptors provokes PIC activation, an accumulation of Ins(1,4,5)P3 and its metabolites and rapid [Ca2+]i mobilization in FRTL-5 cells. By contrast, TSH provokes no rapid PIC-catalysed PtdIns(4,5)P2 hydrolysis or immediate [Ca2+]i mobilization. These results fail to support the widespread view that the TSH receptor of FRTL-5 cells signals, in part, through PIC activation. Our results suggest that TSH activates another, still undefined, mechanism that causes accumulation of an InsP3 and two isomers of InsP4.

Adenosine Triphosphate↗
PubMed1996

Children falling from a height in London.

OBJECTIVES: To determine the frequency and geographical distribution of children falling from a height in London and to suggest possible causes and preventative measures. METHODS: All relevant cases attended by the Helicopter Emergency Medical Service (HEMS) in a three and a half year period were reviewed and the locations related to the boroughs. The rates, per 1,000 resident children, were compared with socioeconomic indices for the boroughs concerned. In addition, a survey was undertaken of window fittings and maintenance in high rise flats close to one particular incident. RESULTS: A total of 90 incidents were attended involving 91 patients (64 male, 27 female) of whom five died. HEMS attends approximately one third of incidents involving serious trauma. In the study period the maximum frequency was 0.2 fallers per 1,000 resident children, occurring in three boroughs. In three boroughs there were no fallers. There was no overlap in socioeconomic indices between the five boroughs with the highest fall rates and the five with the lowest. The building survey found a high incidence of faulty window catches, a slow response rate for repair, and a lack of safety advice for residents. CONCLUSIONS: The frequency of falling is related to urban deprivation, poor maintenance, and lack of safety information. A combination of regulation and targeted education could substantially decrease deaths and injuries in children from this cause.

Accidental Falls↗
PubMed1995

Regulation of neurite outgrowth from differentiated human neuroepithelial cells: a comparison of the activities of prothrombin and thrombin.

The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP6 dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P3 generation and intracellular Ca2+ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P3 were detected after stimulation with prothrombin, and no rapid synchronized release of Ca2+ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca2+ concentration, similar to those produced by low concentrations of thrombin, after approximately 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P2 and Ca2+ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca2+ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205-->A). Prothrombin-mediated intracellular Ca2+ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.

Blotting, Western↗
PubMed1995

Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells.

1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.

Animals↗
PubMed1995

The disability status of injured patients measured by the functional independence measure (FIM) and their use of rehabilitation services.

The type and severity of disability following major trauma was evaluated using the Functional Independence Measurement (FIM) in 93 patients brought to the Royal London Hospital (RLH) by helicopter. The range of values for FIM is from 18 (dependent) to 126 (fully independent) in the six sections of self-care, sphincter control, mobility, locomotion, communication and social cognition. The sections are divided into 18 separate items and graded 1-7. Forty-eight patients were discharged directly to home with a median FIM score of 124; 11 were transferred to another acute hospital with a median FIM of 63 and seven went to rehabilitation unit with a median FIM of 58. At six months, 79 per cent of the patients reported no disability and 89 per cent of the original 93 patients were at home with a median FIM of 126. The mean amount of rehabilitation provided at the RLH for all patients was 11 h 20 min with a mean in-patient length of stay of 14 days. The actual and optimal amount of therapy for rehabilitation worked out at less than 1 h per day in the acute hospital. FIM is a useful, practical and simple methodology for recording disability in the acute hospital. It provides a measure for assessing the original disability, its progress and residual limitations. Nurses, doctors and therapists can use it for establishing care plans and goals as well as deciding the transfer of the patient to the most appropriate place for future care.

Activities of Daily Living↗
PubMed1995

Purification and expansion of human Schwann cells in vitro.

The ability to culture cells from the human nervous system provides new insight into the pathophysiology of neurological diseases and could be crucial to the development of gene replacement therapies and neural transplantation. We report that the proliferation of human Schwann cells isolated from paediatric and adult nerves is sustained in vitro by recombinant glial growth factor. Agents that increase intracellular cyclic cAMP were also mitogenic towards Schwann cells but suppress growth of contaminating fibroblasts. As the lifespan of highly enriched cultures can be extended for up to twelve population doublings, large numbers of cells can be generated from nerve biopsies.

Adult↗
PubMed1995

Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation.

Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Characterisation of a novel Ca2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca2+ mobilization.

Bis-phenol, a phenolic antioxidant, is an inhibitor of sarcoplasmic reticulum (SR), endoplasmic reticulum (ER) and plasma membrane Ca2+ ATPases. The concentration of bis-phenol giving half-maximal inhibition of the SR Ca(2+)-ATPase is 2 microM. On binding to the SR Ca(2+)-ATPase it shifts the E2 to E1 transition towards the E2 state and slows the transition between E2 to E1. Bis-phenol completely inhibits Ca(2+)-dependent ATP hydrolysis and Ca2+ uptake by rat cerebellar microsomes at a concentration of 30 microM. The plasma membrane Ca(2+)-ATPase is also completely inhibited at similar concentrations, however, the Na+/K(+)-ATPase is only marginally affected. Other inhibitors of the ER Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibit Ca2+ uptake by approximately 75%. Bis-phenol therefore inhibits all types of ER Ca(2+)-ATPases present in cerebellum. This inhibitor is also able to mobilize Ca2+ from intracellular Ca2+ stores, including those sensitive to InsP3, in intact HL-60 cells.

Adenosine Triphosphate↗
PubMed1994

The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells.

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form.

Adenosine Triphosphate↗
PubMed1994

Inhibition of porcine brain inositol 1,3,4-trisphosphate kinase by inositol polyphosphates, other polyol phosphates, polyanions and polycations.

We have partially purified an enzyme activity that phosphorylates inositol 1,3,4-trisphosphate from porcine brain, rat liver and bovine testis by FPLC chromatography on Q-Sepharose anion-exchange resin and Heparin-agarose. The products of this reaction were inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate. The same enzyme appears to be responsible for both 6-kinase and 5-kinase activities against inositol 1,3,4-trisphosphate (the 6-kinase: 5-kinase activity ratio is approximately 4 to 1), has a pH optimum of approximately 6.8 and requires Mg2+ for activity. The Km values of the enzyme for inositol 1,3,4-trisphosphate and ATP were approximately 0.5 microM and approximately 100 microM, respectively. Inositol 3,4,5,6-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate are all competitive inhibitors with K(i) values of 0.4 microM, 3 microM and 5 microM, respectively, well within their likely intracellular concentration ranges: they inhibited 6-kinase and 5-kinase activities equally. 2,3-Bisphosphoglycerate and spermine were also competitive inhibitors, with K(i) values of 0.8 mM an 12 mM, respectively. Dextran sulphate was a non-competitive inhibitor with a Ki of approximately 15 microM, and poly-L-lysine (IC50 approximately 200 microM), polyvinylsulphate (IC50 approximately 250 microM) and heparin (IC50 approximately 2 mg/ml) also inhibited. Inhibition by these compounds suggests that inositol 3,4,5,6-tetrakisphosphate (and to a lesser extent inositol 1,3,4,5-tetrakisphosphate and other naturally occurring intracellular ions) may restrict the synthesis of inositol 1,3,4,6-tetrakisphosphate and hence regulate the rate of inositol penta- and hexakisphosphate synthesis from receptor-generated inositol phosphates.

Animals↗
PubMed1994

Pharmacological characterization of linear analogues of vasopressin generated by the systematic substitution of positions 1 and 6 by L-amino acids.

Eighteen linear analogues of [Arg8]vasopressin (AVP) were synthesized by systematically substituting the cysteine residues at positions 1 and 6 with a range of L-amino acids. Screening by competition ligand binding revealed that the combinations of amino acid residues tolerated at these positions was very restricted with respect to retention of vasopressin receptor (VPR) binding. Consequently, only three of the eighteen analogues investigated, [Pro1,Met6]AVP, [Gly1,Met6]AVP and [Phe1,Lys6]AVP, bound to the V1a receptor. Furthermore, these three peptides were all selective for the V1a receptor rather than the V1b, V2 and vasotocin receptors. In addition, although very homologous to the natural agonist, these analogues were in fact antagonists at V1a receptors. These data provide insights into the biophysical requirements at positions 1 and 6 of linear ligands for binding to V1a receptors and furthermore, supply clues to the nature of the receptor:ligand interaction.

Amino Acid Sequence↗
PubMed1994

Bovine testis and human erythrocytes contain different subtypes of membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphomonoesterases.

1. We have purified membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatases from bovine testis and human erythrocytes by chromatography on several media, including a novel 2,3-bisphosphoglycerate affinity column. 2. The enzymes have apparent molecular masses of 42 kDa (testis) and 70 kDa (erythrocyte), as determined by SDS/PAGE, and affinities for Ins(1,4,5)P3 of 14 microM and 22 microM respectively. 3. The two enzymes hydrolyse both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and are therefore type I Ins(1,4,5)P3 5-phosphatases [nomenclature of Hansen, Johanson, Williamson and Williamson (1987) J. Biol. Chem. 262, 17319-17326]. 4. On chromatofocusing, the partially purified testicular enzyme migrates as two peaks of activity, with pI values of about 5.8 and 5.5. The erythrocyte enzyme exhibits only the latter peak. 5. The testis 5-phosphatase is labile at 37 degrees C, but its activity can be maintained in the presence of 50 mM phorbol dibutyrate (PdBu). After PdBu treatment, a third form of the enzyme, with pI about 6.2, appears on chromatofocusing, but without change in its Km or Vmax. 6. Consideration of the properties of these enzymes and of the 5-phosphatases from other tissues suggests that type I Ins(1,4,5)P3 5-phosphatases are of two well-defined subtypes. We propose that these be termed type Ia [typified by the testis enzyme: approximately 40 kDa, higher affinity for Ins(1,4,5)P3] and Type Ib [typified by the erythrocyte enzyme: approximately 70 kDa, lower affinity for Ins(1,4,5)P3].

Animals↗