Thein vitro effect of theophylline on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown in the catfish,Clarias batrachus

The effects of theophylline (a phosphodiesterase inhibitor) and cAMP on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown was investigatedin vitro in catfish (Clarias batrachus) oocytes. Folliculated oocytes incubated with 17α, 20ß-dihydroxy-4-pregnen-3-one at the concentration of 1 μg/ml induced 93.2 ± 2.23% germinal vesicle breakdown. When the oocytes were prestimulated with 17α,20ß-dihydroxy-4-pregnen-3-one for 6 h and then treated with different concentrations of theophylline, there was a significant drop in the frequency of germinal vesicle breakdown at the concentrations 2.0, 1.5 and 1.0 mM. However, theophylline was found to be incapable of inhibiting germinal vesicle breakdown at its lowest concentration (0.5 inM). In the time course study, significant inhibition of germinal vesicle breakdown was recorded when 1 mM theophylline was added up to 30 h of 17α,20ß-dihydroxy-4-pregnen-3-one Stimulation but the inhibitory effect of theophylline gradually (time dependent manner) declined if the stimulatory time of 17α,20ß-dihydroxy-4-pregnen-3-one was increased. A similar inhibition of germinal vesicle breakdown was also recorded with various concentrations of cAMP. Except 0.5 mM, all the higher concentrations of cAMP significantly inhibited 17α,20ß-dihydroxy-4-pregnen-3-one induced germinal vesicle breakdown.     

Free and total thyroid hormones in humans at extreme altitude

Alterations in circulatory levels of total T₄ (TT₄), total T₃ (TT₃), free T₄ (FT₄), free T₃ (FT₃), thyrotropin (TSH) and T₃ uptake (T₃U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized lowlanders, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT₃ and FT₄ occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T₃U was found to be higher at high altitudes. A decline in TSH and hormone binding proteins and an increase in the free moiety of the hormones is indicative of a subtle degree of tissue hyperthyroidism which may be playing an important role in combating the extreme cold and hypoxic environment of high altitudes.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS-0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow-fiber or packed-bed-type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated alpha-phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore-conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N-acetylneuraminic acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less detectable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS-0 cells had terminal N-glycolylneuraminic acid but no detectable N-acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N-acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore-conjugated carbohydrates. (c) 1995 John Wiley & Sons, Inc.     

Neurotoxic Effect of Intranigral Injection of 1-Methyl-4-Phenylpyridinium on GABA-Containing Neurons and Its Relation to Circling Behavior

Abstract: The ionic species 1-methyl-4-phenylpyridinium (MPP⁺) seems to be the metabolite responsible for the damage to dopaminergic neurons occurring after administration of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In the present study we show that the unilateral stereotaxic microinjection of MPP⁺ into the substantia nigra pars reticulata in rats produces immediately intense and long-lasting (up to 96 h) contralateral turning behavior in a dose-dependent manner. This behavioral effect was correlated with a dose- and time-dependent decrease (up to 90%) of glutamate decarboxylase activity and with a notable loss of neurons in the injected nigra reticulata. GABA levels in the injected nigra were also decreased, whereas the dopamine concentration in the ipsilateral striatum was not affected at 24 h, when maximal behavioral effects were observed. The circling behavior was prevented by the dopamine carrier blocker nomifensine only during the first 2 h, whereas the dopamine receptor antagonist haloperidol was ineffective. The results indicate that MPP⁺ is toxic for inhibitory GABAergic neurons in the nigra pars reticulata and, furthermore, suggest that disruption of the function of these GABAergic neurons may be involved in the abnormal motor behavior produced by the injection of MPP⁺ in the substantia nigra.     

Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

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.