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Biomedical subjects

D E Macfarlane

Publications and source records attributed to D E Macfarlane.

At least 55 records · Page 3Linked to original sources
PubMed1985

Flavobacterium odoratum ventriculitis treated with intraventricular cefotaxime.

A 6-week-old infant admitted to the University Hospital of the West Indies with hydrocephalus later developed ventriculitis. A heavy growth of Flavobacterium odoratum susceptible to gentamicin and cefotaxime was recovered from the ventricular fluid. Since intraventricular therapy was envisaged, a Pudenz reservoir was installed and ventricular fluid aspirated every 24 h to monitor treatment. Initial therapy consisted of intravenous cefotaxime, 50 mg/kg q.i.d. for 4 days. No significant reduction in the number of organisms in the ventricular fluid was achieved with this regimen. Intravenous therapy was therefore discontinued. On day 5 intraventricular therapy began with 5 mg cefotaxime 24 h for 6 days, followed by 1 mg/24 h for 4 days. Daily monitoring of intraventricular fluid indicated a high degree of antibacterial activity with rapid elimination of bacteria. Ventricular fluid remained sterile 10 days after therapy stopped. The Pudenz reservoir was removed, a ventriculoperitoneal shunt installed, and the patient discharged from hospital 4 days later without noticeable sequelae.

Bacterial Infections↗
PubMed1985

Cerebrospinal fluid C-reactive protein in the laboratory diagnosis of bacterial meningitis.

Samples of cerebrospinal fluid from 112 cases of suspected meningitis were tested for the presence of C-reactive protein (CRP), using a qualitative and quantitative slide test. Bacterial meningitis was confirmed in 34 patients, based on CSF and blood culture results, and/or elevated CSF white blood cell (WBC) count and typical biochemical profile. There were 8 patients with early onset, and 3 who had received prior antimicrobial therapy among the 5 neonates, 23 children, and 6 adults with bacterial meningitis. Organisms recovered from CSF, and/or blood, included Haemophilus influenzae 14, Streptococcus pneumoniae 9, Streptococcus group B-5, Staphylococcus aureus 2, E. coli 2 and Klebsiella pneumoniae 1. Slide test was positive for CRP in 33 cases, giving a sensitivity of 97% which compared favourably with elevated CSF protein 33%, decreased CFS glucose 64.7% CSF glucose/blood glucose less than 1/2, 85%, raised CSF WBC 38.2%, raised CSF PMN 61.7%, CSF culture positive 88.2%, and CSF gram-positive 82.5%. Slide test was positive for CRP in 1 of 78 CSF samples negative for bacterial meningitis, giving a specificity of 98%. It was concluded that testing of CSF for CRP is a simple, rapid and accurate method for the laboratory diagnosis of bacterial meningitis, which is particularly appropriate for areas lacking adequate laboratory facilities.

Adult↗
PubMed1984

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets.

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Acute graft-versus-host disease resulting from normal donor blood transfusions.

2 fatal cases of graft-versus-host disease (GvHD) occurred following blood product transfusions given to patients receiving standard chemotherapy for Hodgkin's disease. GvHD was established by HLA typing, clinical course, and compatible skin biopsy. 23 cases of GvHD following transfusion of blood products from normal donors are also reviewed. It should be suspected when fever or rash appear 1-2 weeks after transfusion of unirradiated blood products into a compromised host or when pancytopenia following chemotherapy is prolonged or unexpectedly severe. Prevention of GvHD by irradiation of granulocytes, platelets and packed red blood cells given to immunosuppressed patients is recommended to prevent this often fatal disease.

Adult↗
PubMed1983

Use of benzyldimethyl-n-hexadecylammonium chloride ("16-BAC"), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells.

A discontinuous polyacrylamide gel system operating at pH 4.0-1.5 which resolves proteins bearing base labile groups extracted from intact cells is described. It uses potassium phosphate buffer in the running and stacking gel and glycine as the trailing ion component. Proteins are solubilized with urea and benzyldimethyl-n-hexadecylammonium chloride, a cationic detergent. The utility of the system is illustrated by fluorographs of the pattern of protein methylation in blood platelets and the HL60 promyelocyte cell line.

Blood Platelets↗
PubMed1983

2-Methylthioadenosine[beta-32P]diphosphate. An agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets.

2-Methylthio-ADP and its radioactive analogue [beta-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin E1. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [beta-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was approximately 3.5 X 10(6) M-1 S-1, and the first order dissociation rate was 0.024 s-1, both measured at 23 degrees C. The dissociation equilibrium constant (approximately 15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin E1-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Ki = 3.5 microM), ATP (7 microM), 2-azido-ADP (0.12 microM), inosine diphosphate (IDP, 150 microM), guanosine diphosphate (GDP, 350 microM), and AMP (800 microM). Binding of 2-methylthio-ADP was also blocked by the non-cell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 32 degrees -40 degrees C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was approximately 15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket.

Adenosine Diphosphate↗
PubMed1983

Clonidine and para-aminoclonidine, partial agonists for the alpha2-adrenergic receptor on intact human blood platelets.

Human blood platelets display alpha2-adrenergic receptors, which promote platelet aggregation and inhibit the adenylate cyclase. We investigated the effects of the antihypertensive agent clonidine and its analogue para-aminoclonidine on this receptor in the intact human platelet to determine their pharmacological effects and their ability to bind to the receptor by radioligand displacement. Both agents potentiated platelet aggregation induced by a submaximal concentration of ADP. Epinephrine-induced aggregation, on the other hand, was antagonized by clonidine and para-aminoclonidine in a dose-dependent fashion. Both agents inhibited the accumulation of cyclic AMP in platelets exposed to prostaglandin E1 and a phosphodiesterase inhibitor, but to a lesser extent than the inhibition caused by epinephrine. Both antagonized this excess inhibitory action of epinephrine. Clonidine and para-aminoclonidine blocked the binding of [3H]yohimbine (a selective alpha 2-adrenergic antagonist) to intact platelets with half-maximal effects at 0.3 and 0.7 microM, respectively. No evidence for the existence of a second class of binding sites with high affinity for clonidine was seen in intact platelets, either by this technique or by direct binding of [3H]clonidine. It is concluded that these two agents are partial agonists for the alpha 2-adrenergic receptors on blood platelets and that this receptor exists predominantly in the low-affinity state in the intact cell.

Adrenergic alpha-Agonists↗
PubMed1982

Prostacyclin biosynthesis in vascular endothelium is not inhibited by cyclic AMP. Studies with 3-isobutyl-1-methylxanthine and forskolin.

We have previously reported (Proc. Natl. Acad. Sci. 79, 495-499, 1982) that the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), stimulates cyclic AMP accumulation and inhibits prostacyclin (PGI2) production in primary monolayer cultures of human umbilical vein endothelium. The present study was carried out to determine whether these effects are causally related. Incubation of endothelial monolayers with the diterpene, forskolin, increased the intracellular concentration of cyclic AMP by 10-fold. Despite this marked increase in cyclic AMP, neither baseline production of PGI2 nor release in response to stimulation by thrombin or the divalent cation ionophore, A23187, was affected. Both forskolin and isoproterenol were found to potentiate the effect of IBMX on cyclic AMP accumulation without causing further inhibition of PGI2 biosynthesis. Inhibition of cyclic nucleotide phosphodiesterase activity with 2,6-bis-(diethanolamino)-4-piperidinopyrimido-[5,4-d]pyrimidine increased cyclic AMP levels to the same extent as IBMX; however, this agent had no effect on PGI2 biosynthesis. These findings demonstrate that increases in the intracellular concentration of cyclic AMP have no short-term effects on PGI2 biosynthesis in vascular endothelium and suggest that inhibition of PGI2 production by IBMX is the result of some other, cyclic AMP-independent action of the drug.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Parallel observation of the occupancy of the alpha 2-adrenergic receptor in intact platelets and its ability to inhibit the adenylate cyclase.

The binding of the selective alpha2-adrenergic receptor antagonist [methyl-3H]yohimbine to intact human blood platelets was studied in parallel with an assessment of the ability of epinephrine to inhibit their accumulation of cyclic AMP in response to prostaglandin E1 and a phosphodiesterase inhibitor. Specifically bound [3H]yohimbine dissociated in a monoexponential fashion, and equilibrium binding showed 200-400 sites/platelet with a dissociation constant of 2-7 nM. The concentration of epinephrine which inhibited cyclic AMP accumulation was one-quarter of that required for an equal degree of inhibition of [3H]yohimbine binding. Conversely, the concentration of yohimbine required to inhibit the action of epinephrine on the adenylate cyclase was 10 times higher than that required for an equal degree of saturation of the alpha2-adrenoreceptor. The ability of epinephrine and the partial agonist p-aminoclonidine to compete with [3H]yohimbine for binding was not influenced by agonist-occupation of the ADP receptor, which also inhibits the adenylate cyclase. These results are not compatible with the concept that each adenylate cyclase unit is coupled to an alpha 2-receptor, nor do they indicate that the agonist-occupied alpha 2-receptor forms a complex with the enzyme which persists for the duration of the inhibitory effect. However, it is suggested that these results are compatible with a persistent inhibition of the cyclase being induced by a brief interaction between the agonist-occupied receptor and the adenylate cyclase.

Adenine↗
PubMed1981

Combined selective descriptive medium for the culture of Neisseria gonorrhoeae.

A selective identification medium for Neisseria gonorrhoeae was devised by incorporating antibiotics and a mixture of sugars into a yeast extract agar. The results were compared with those using a standard combined selective/non-selective blood agar medium. Recovery rates of N gonorrhoeae from specimens from 208 patients cultured on both media were comparable, but the selective descriptive medium identified 68 cultures of N gonorrhoeae correctly, 64 of them after 24 hours' incubation.

Bacteriological Techniques↗
PubMed1981

Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets.

The influence of extracellular ADP on cyclic AMP accumulation within intact human platelets was studied. ADP inhibited the increase in cyclic AMP which occurs when platelets are exposed to prostaglandin E1 or I2. The degree of inhibition varied in the range 70-95% , and was half maximal at ADP concentrations of between 0.3 and 2 microM. Other naturally occurring diphosphates, i.e. GDP, IDP and UDP, were at least 100 fold less effective than ADP, and UDP at 1mM partially reversed the effect of ADP. The effect by ADP was completely reversed by ATP, but only attenuated to a minor degree of 10 mM EDTA. Increasing concentrations of ADP caused a progressive degree of inhibition of cyclic AMP accumulation, and the kinetics of this inhibition were compatible with a simple saturable process with no cooperativity. ADP added 10 seconds after prostaglandin E1 blocked cyclic AMP accumulation within 1-2 seconds, and addition of ATP after ADP and prostaglandin I2 relieved the inhibition due to ADP within 2-3 seconds. The action of ADP was blocked by sulphydryl reagents including N-substituted maleimides, cytochalasin A, NBD chloride and p-mercuribenzene sulphonate. The data were considered to be consistent with mediation of the ADP effect through a sulphydryl-bearing specific extracellular receptor coupled to the adenylate cyclase.

Adenosine Diphosphate↗
PubMed1980

Improved media for the culture of Neisseria gonorrhoeae.

The ANM medium of Hafiz and McEntegart (1976) was found to be deficient in ability to support the growth of various strains of N. gonorrhoeae. Strains that did grow required a large starting inoculum which invariably suffered a substantial drop in the number of viable organisms during the first 2 h. Investigation of the various deficiencies led to the development of liquid (G77L) and solid (G77S)media which were shown to have several advantages over existing media for the cultivation of N. gonorrhoeae. These media, which are simple to prepare, do not contain blood or serum and yet gave excellent growth. The inclusion of antibiotics, glucose and phenol red allowed the selective isolation and partial identification of N. gonorrhoeae from routine specimens. These media also gave good results in fermentation tests for the identification of neisseriae.

Animals↗