Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Metal-modified nucleobase pairs involving 7,9-dimethylguanine: trans-a2PtII analogues (a = NH3 or CH3NH2) of Watson-Crick GC and homo GG pairs

 The analogy between H-bonded nucleobase pairs and their metalated analogues is extended to the hemiprotonated pair of 7,9-dimethylguanine (7,9-DimeG) and the Watson-Crick and reversed Watson-Crick pair between 7,9-dimethylguaninium (7,9-DimeGH⁺) and 1-methylcytosine (1-MeC). The crystal structure analyses of two model compounds, trans–[Pt(CH₃NH₂)₂(7,9-DimeG-N1)₂](NO₃)₂ (1) and trans–[Pt(NH₃)₂(1-MeC-N3)(7, 9-DimeG-N1)](PF₆)₂· 2.5 H₂O (3a) are reported. Pt binding is through N1 of 7,9-DimeG and N3 of 1-MeC. In solution, 3a exists in a mixture with Watson-Crick and reversed Watson-Crick arrangements of the two bases, depending on solvent, concentration and anions. Received: 16 October 1996 / Accepted: 27 January 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.     

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR)

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR) Immunoglobulin heavy chain (IgH) gene rearrangement analysis was performed on 27 fine needle aspiration (FNA) specimens (13 reactive hyperplasia, 11 B cell non-Hodgkin's lymphoma (B-NHL), one Hodgkin's disease and two suspicious of non-Hodgkin's lymphoma). Satisfactory amplification was achieved in 23/27 cases. A polyclonal pattern was seen in 14 cases (11 reactive hyperplasia, one B-NHL, one suspicious of lymphoma, one Hodgkin's disease). A monoclonal band was seen in nine cases (eight B-NHL, one reactive hyperplasia). Amplification was unsuccessful in four cases. Clonal analysis by PCR-based IgH gene rearrangement analysis can be successfully applied to FNA material and can be useful in diagnosis, but the results must be interpreted in conjunction with morphology and other ancillary information. Analyse de la clonalité en utilisant la PCR sur matériel cytologique L'analyse des réarrangements géniques des chaînes lourdes des immunoglobulines (IgH) a été réalisée sur 27 produits de ponction à l'aiguille fine (13 cas d'hyperplasie réactionnelle, 11 cas de lymphome non hodgkinien de type B (LNH-B), un cas de maladie de Hodgkin et deux cas suspects de lymphome malin non hodgkinien. Une amplification satisfaisante a été obtenue dans 23 cas sur 27. Un aspect polychonal a été observé dans 14 cas (11 hyperplasies réactionnelles, 1 lymphome malin non hodgkinien de type B, 1 cas suspect de lymphome, 1 cas de maladie de Hodgkin). Une bande monoclonale a été observée dans 9 cas (8 LNH-B, 1 hyperplasie réactionnelle). L'amplification n'a pas réussi dans quatre cas. L'analyse de la clonalité par analyse des réarrangements géniques par PCR peut être appliquée avec succès au matériel cytologique qui est obtenu par ponction à l'aiguille fine et elle peut être utile au plan diagnostique, mais les résultats doivent étre interprétés en intégrant les informations morphologiques et les autres données complémentaires. Polymerase Chain Reaction an zytologischem Material Die Analyse schwerer Immunglobulinketten (IgH) wurde an 27 FNP (13 reaktive Hyperplasien, 11 B-NHL, 1 Hodgkin und und zwie verdächtige Fälle) analysiert. Eine ausreichende Amplifikation wurde in 23/27 Fällen erzielt. Ein polyklonales Muster wurde in 14 Fällen beobachtet (11 reaktive Hyperplasien, 1 B-NHL, 1 Hodgkin sowie 1 verdächtiger Fall). Ein monoklonales Band wurde 9 mal gefunden (8 B-NHL, 1 reaktive Hyperplasie). Die Amplifikation war ungenügend in 4 Fällen. Die PCR-Analyse kann für die Diagnostik von Nutzen sein muss jedoch zusammen mit der Morphologie und anderen Informationen interpretiert werden.     

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies.

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.     

HeteroTOCSY-based experiments for measuring heteronuclear relaxation in nucleic acids and proteins

Summary While both ³¹P and ¹¹³Cd are present at locations of interest in many different macromolecular systems, heteronuclear-detected relaxation measurements on these nuclei have been restrained by limitations in either resolution or signal-to-noise ratio. We have developed hetero TOCSY-based methods to overcome both of these problems. Two-dimensional versions of these experiments were utilized to measure ³¹P T₁ and T₂ values in DNA oligonucleotides; the additional resolution offered by a second dimension allowed determination of these values for most of the ³¹P resonances in a DNA dodecamer. The results from the experiments indicated that there was little significant variation in T₁ values for the different phosphates in the DNA dodecamer; however, the T₂ values showed a clear pattern, with lower values in the interior of the sequence than at the ends of the helix. Furthermore, a significant correlation between ³¹P chemical shifts and T₂ values was observed. One-dimensional, frequency-selective versions of these experiments were also developed for use on systems containing a smaller number of heteronuclear spins. These methods were applied to investigate the heteronuclear relaxation properties of ¹¹³Cd in ¹¹³Cd₂LAC9(61), a Cys₆Zn₂ DNA-binding domain. Data from the experiments confirm biochemical evidence that more significant differences occur in the metal-protein interactions between the two metal-binding sites than has been previously identified for proteins containing this motif.     

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Junction formation in aggregated embryonal carcinoma cells

Under the action of supplemental calcium, H6 mouse embryonal carcinoma cell aggregates undergo compaction, a morphological phenomenon similar to mouse embryonic compaction. Formation of various types of cell junctions, especially gap junctions, is associated with compaction of the embryo and we sought to analyze the pattern of junction formation during aggregation and compaction of H6 cells. At 24 hr of aggregation, gap junctions were abundant in both uncompacted and compacted aggregates but quantitative analysis of freeze fracture replicas of these junctions showed a 20-fold increase in the size of the largest gap junctions in compacted aggregates. Such a difference in size could even be detected at 12 hr of aggregation. Tight junctions were not normally formed in 12 hr aggregates but initial stages of tight junction formation could be noticed in 12 hr compacted aggregates. More definitive tight junctions and desmosomes were evident only after 48 hr of aggregation. Thus we have observed that both uncompacted and compacted aggregates can form gap junctions at similar frequencies, suggesting that cell flattening, which contributes to the compacted morphology, is not a requisite for gap junctions. Likewise, generation of the compacted morphology seems to be independent of gap junction formation. This supports the idea that compaction in embryonal carcinoma cells results from calcium-induced cell flattening, probably through the mobilization of cytoskeletal elements. Calcium-dependent features of H6 cell aggregation and compaction enables the independent analysis of separate steps in compaction.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.