Resonance Raman characterization of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

 Di-heme Pseudomonas stutzeri cytochrome c ₄ has been characterized by electronic absorption and resonance Raman spectroscopies in the ferric and ferrous forms at pH 7.5 and at room temperature. The data indicate that the two hemes are inequivalent. It is proposed that the N-terminal contains a more relaxed heme as a consequence of the relative orientation of the methionine and histidine ligands with respect to the N-Fe-N directions of the heme plane. This causes a weakening of the Fe-S bond with concomitant partial dissociation of the methionine and the formation of an Fe-aquo bond. Heme group relaxation is further accompanied by less distortion of the heme group than that associated with cytochrome c, expansion of the "core" and a negative shift of the redox potential. Received: 17 December 1996 / Accepted: 6 March 1997     

An evaluation of ‘rapid review’ as a method of quality control of cervical smears using the AxioHOME microscope

An evaluation of ‘rapid review’ as a method of quality control of cervical smears using the AxioHOME miscroscope One method of quality control which has recently been recommended by professional bodies in the UK is the ‘rapid review’ method. This involves the microscopic 30 s review of all negative cervical smears with the intention of flagging potential missed abnormalities. Although it has been suggested that rapid review is better than 10% random rescreening of negative smears, the efficiency and efficacy of this method of quality control have not been thoroughly evaluated. We have used the AxioHOME system, which can record the area of a slide covered and the screening time, to investigate slide coverage during rapid review quality control, as performed by 15 cytoscreeners and MLSOs reviewing a test set of 22 slides each. The test set comprised 18 negative slides, three positive slides, and one unsatisfactory slide. We have recorded two distinct methods of rapid review in use amongst cytotechnologists, the step method and the whole slide method. The data show that rapid review takes longer on average than the recommended 30 s, the mean screening times being 76 s and 82 s for the step and whole slide methods, respectively. Abnormal smears were missed on three of 15 occasions by the step method (sensitivity 80%, positive predictive value 85%), and on seven of 30 occasions by the whole slide method (sensitivity 76.6%, positive predictive value 45%). However, the 95% confidence intervals were wide (57.7–90.7% for the step method, and 51.9–95.7% for the whole slide method). Analysis of scanning tracks and screening rates shows significant flaws in the methodology of rapid review. Abnormal cells were not identified, although dyskaryotic cells were included in the scanning track on nine occasions, seven using the whole slide method and two using the step method. On one occasion (using the step method) abnormal cells were not identified because they were not included in the scanning track. Further research is in progress to determine optimal methods of rapid review, and whether the rapid review technique is as effective as automated screening systems for quality assurance in cytology. Evaluation de la technique de ‘Relecture Rapide’ comme méthode de contrôle de qualité des frottis cervico-utérins, à l'aide du microscope AxioHOME Une des méthodes de contrôle de qualité récemment recommandée par le corps professionnel du Royaume Uni est la méthode dite de ‘relecture rapide’. Cette méthode consiste en une deuxième lecture d'une durée de trente secondes de tous les frottis cervicaux négatifs et dont l'objectif est de détecter les anomalies ayant pu échapper au premier examen. Bien qu'il ait été suggéré que cette méthode de relecture rapide soit meilleure que la relecture de 10% des frottis négatifs tirés au sort, le rendement et l'efficacité de cette méthode de contrôle de qualité n'ont pas été évalués complètement. Nous avons utilisé le système AxioHOME capable d'enregistrer la plage de la lame qui a été explorée ainsi que le temps de lecture afin d'étudier la surface explorée au cours de cette relecture rapide telle qu'elle a pu être pratiquée par quinze cytotechniciens et MLSOs, chacun ayant relu une série test de vingt deux lames. Cette série test comprenait dix huit lames négatives, trois lames positives et un frottis non satisfaisant. Nous avons noté que les cytotechniciens utilisaient deux méthodes de relecture rapide différentes, la méthode ‘pas à pas’ et la méthode ‘globale’. Les données montrent que la relecture rapide prend, en moyenne, un temps supérieur aux trente secondes recommandées, la moyenne des temps de lecture étant de 76 secondes et de 82 secondes respectivement pour la méthode ‘pas à pas’ et la méthode ‘globale’. Les anomalies n'ont pas été détectées dans trois cas sur quinze par la méthode ‘pas à pas’ (sensibilité 80%, valeur prédictive positive 85%), et dans 7 cas sur 30 par la méthode globale (sensibilité 76,6%, valeur prédictive positive 45%). Toutefois, l'intervalle de confiance à 95% est important (57,7%-90,7% pour la méthode ‘pas à pas’ et 51,9%-95,7% pour la méthode globale). L'analyse des surfaces balayées et des taux de détection montre des points faibles significatifs de cette méthodologie de relecture rapide. Dans 9 cas, les anomalies n'ont pas été identifiées alors que des cellules dyskaryotiques étaient présentes dans les plages balayées au cours de la relecture (sept utilisant la méthode globale et deux utilisant la méthode ‘pas à pas’). Dans un cas (avec la méthode ‘pas à pas’) les cellules anormales n'ont pas été identifiées parce qu'elles étaient absentes des plages de relecture. Des études sont en cours afin de déterminer quelles sont les méthodes optimales de relecture rapide et si ces techniques de relecture rapide sont aussi efficaces que les systèmes de lecture automatisée pour l'assurance de qualité en cytologie cervico-utérine. ‘Rapid Review’ als Methode der Qualitätskontrolle gynäkologischer Abstriche, Überprüfung mit dem AxioHOME-Mikroskop Die empfohlene ‘Rapid Review’ Kontrolle aller negativen Abstriche in nur 30 Sekunden anstelle des Nachscreenens von 10% der Präparate ist in ihrer Zuverlässigkeit bislang nicht überprüft worden. Mit Hilfe des AxioHOME-Mikroskops ist es möglich sowohl die ausgewertete Fläche, als auch die erforderliche Zeit zu erfassen. 15 Auswerter prüften mit der Methode jeweils einen Testsatz von 22 Präparaten. Er enhielt 18 negative, 3 positive und 1 nichtauswertbaren Abstrich. Getestet wurden zwei verschiedene Vorgehensweisen: die schrittweise und die das ganze Präparat erfassende. Beide erfordern mehr Zeit als 30 Sekunden; der mittlere Zeitaufwand betrug für die Schrittmethode 76 und für die Ganzheitsmethode 82 Sekunden. Die Schrittmethode verfehlte 3/15 Anomalien (Sensitivität 80%, positiver prädiktiver Wert 85%), die Ganzheitsmethode 7/30 (Sensitivität 76,6%, positiver prädiktiver Wert 45%). Der 95% Konfi denzbereich reichte für die Schrittmethode von 57,5–90,7% und für die Ganzheitsmethode von 51,9–95,7%). Die Analyse deckt wesentliche Schwachstellen des Rapid Review-Verfahrens auf. In 9 Fällen lagen nicht erkannte Zellatypien in den kontroll ierten Bahnen (7 bei des Schrittmethode, 2 bei der Ganzheitsmethode). Einmal lagen sie bei der Schrittmethode ausserhalb der geprüften Bahnen. Weitere Studien werden prüfen ab das Rapid Review-Verfahren automatisierten Systemen vergleichbar ist.     

Multilocus DNA fingerprints in the plant Cynoglossum officinale L. and their use in the estimation of selfing

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale . Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four-base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding 'between-gel' comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing.     

Interleukin-6 involvement in mesothelioma pathobiology: inhibition by interferon α immunotherapy

A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon (IFN) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P<0.001), as did treatment with recombinant human (rhu) IFN (P<0.001). Neither anti-IL-6 antibody nor rhuIFN had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFN treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFN and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.     

Model of synchronized population bursts in electrically coupled interneurons containing active dendritic conductances

We constructed a computer model of 128 interneurons, each with multiple dendritic branches and an axonal segment. The model neurons were interconnected by gap junctions between dendritic compartments, as are known to occur in rat and guinea-pig hilar interneurons. The model contained no excitatory synapses. In the presence of low-frequency spontaneous action potentials, the model generated synchronized population bursts, when gap junction resistance was 50 M and there were at least two gap junctions per neuron on average. Population bursts occurred only when the dendrites of model neurons were electrically excitable. Consistent with experiment, somatic hyperpolarization during the population burst uncovered partial spikes. In the model, partial spikes originated in electrically active dendrites driven by coupled dendrites. This model may account for population bursts in hilar interneurons that occur in 4-aminopyridine (4AP) together with blockers of GABA_A and excitatory amino acid (EAA) receptors.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

Intrinsic factor secretion from isolated human gastric mucosal cells

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[⁵⁷Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H⁺ production as measured by amino[¹⁴C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H₂ receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10⁶ cells per h under basal conditions and 14.3 fmol in response to histamine.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .