The accuracy of electronic working length determination.

AIM: To determine in vivo the accuracy of two impedance quotient apex locators under clinical conditions. METHODOLOGY: Electronic working length determination was carried out before extraction in 79 human teeth with 93 root canals. In 51 root canals, the determination was performed using the apex locator Justy II(R) (Hager & Werken, Duisburg, Germany); in 42 canals, the apex locator Endy 5000(R) (Loser, Leverkusen, Germany) was used. A root canal instrument was fixed at working length with composite material prior to extraction followed by the exposure of a radiograph. After histological preparation of the apical region, the teeth were examined under a light microscope. The distance of the file tips to the target intervals 'minor foramen - major foramen' and 'apical canal constriction' was determined. These values were compared with the calculated working lengths, determined by radiographic assessment. The data were statistically analysed by a paired t-test. RESULTS: For both apex locators and both target intervals, no significant differences between the electronic and radiographical assessments were recorded. The probability of determining the area between minor and major foramen was 82.4% for Justy II and 81% for Endy 5000. However, accurate determination of the apical constriction was only successful in 51% (Justy II) and 64.3% (Endy 5000) of canals. Variation of the inaccurate measurements was higher for Endy 5000 than for Justy II. CONCLUSIONS: Under clinical conditions, it is possible to determine the region between the minor and major apical foramen with electronic length measuring devices (ELD). However, use of these devices does not result in precise determination of the apical constriction.

The H1 receptor agonist 2-(3-chlorophenyl)histamine activates Gi proteins in HL-60 cells through a mechanism that is independent of known histamine receptor subtypes.

In dibutyryl-cAMP-differentiated HL-60 cells, histamine H1 and formyl peptide receptors mediate increases in the cytosolic Ca2+ concentration ([Ca2+]i) via pertussis toxin-sensitive G proteins of the Gi family. We compared the effects of 2-(3-chlorophenyl)-histamine (CPH) [2-[2-(3-chlorophenyl)-1H-imidazol-4-yl] ethanamine], one of the most potent and selective H1 receptor agonists presently available, with those of histamine and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in these cells. CPH increased [Ca2+]i through Ca2+ mobilization and Ca2+ influx. Unlike histamine-induced rises in [Ca2+]i, those induced by CPH were not desensitized in a homologous manner, and there was no cross-desensitization between CPH and histamine. Like fMLP, CPH activated phospholipases C and D, tyrosine phosphorylation, superoxide anion formation, and azurophilic granule release. The effects of CPH on [Ca2+]i, phospholipase D, and superoxide anion formation were inhibited by pertussis toxin. CPH and fMLP stimulated high affinity GTP hydrolysis by Gi proteins in HL-60 membranes. They also enhanced binding of guanosine-5'-O-(3-thio)triphosphate and GTP azidoanilide to, and cholera toxin-catalyzed ADP-ribosylation of, Gi protein alpha subunits. Histamine receptor antagonists did not inhibit the stimulatory effects of CPH, and CPH did not reduce fMLP binding in HL-60 membranes. Our data suggest that CPH activates Gi proteins in HL-60 cells through a receptor agonist-like mechanism that is, however, independent of known histamine receptor subtypes and formyl peptide receptors. CPH may be an agonist at an as yet unknown histamine receptor subtype or, by analogy with other cationic-amphiphilic substances, may activate G proteins directly. Future studies will have to take into consideration the fact that CPH, in addition to activating H1 receptors, may show other, most unexpected, stimulatory effects on G protein-mediated signal transduction processes.

Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex.

A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.

Degradation of inositol 1,3,4,5-tetrakisphosphates by porcine brain cytosol yields inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate.

Inositol 1,3,4,5-tetrakisphosphates (Ins(1,3,4,5)P4), 32P-labelled in positions 4 and 5 were prepared enzymatically, using [4-32P]-phosphatidylinositol 4-phosphate (PtdInsP) and [5-32P]phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates, respectively. Degradation studies of Ins(1,3,4,5)P4, using an enriched phosphatase preparation from porcine brain cytosol, led to the formation of two inositol trisphosphate isomers which were identified as inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). This novel degradation pathway of Ins(1,3,4,5)P4 to Ins(1,4,5)P3 provides an additional source for the generation of Ins(1,4,5)P3, involving a 3-phosphatase.

In vitro synthesis of 32P-labelled phosphatidylinositol 4,5-bisphosphate and its hydrolysis by smooth muscle membrane-bound phospholipase C.

For studies of phospholipase C (PLC) activity in cell-free systems, 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2) was prepared enzymatically by phosphorylating phosphatidylinositol 4-phosphate (PIP) in the presence of [gamma-32P]ATP using a PIP kinase partially purified from bovine retinae. PLC activity was determined by incubating membranes of DDT1 MF-2 cells with 32P-PIP2 and measuring remaining non-hydrolyzed substrate as well as accumulation of the hydrolysis product, inositol trisphosphate (IP3). Guanine nucleotides stimulated PIP2 hydrolysis and IP3 release. Additional increase in IP3 accumulation was observed with adrenaline plus guanine nucleotides.