DNA damage induces two distinct modes of cell death in ovarian carcinomas.

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cell's ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3sigma deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53- and Chk2-related proteins.

Effects of atrial natriuretic factor on angiotensin-II-and phorbol ester-stimulated protein kinase-C and prostacyclin production in cultured rat aortic smooth muscle cells.

The vasoconstrictor hormone angiotensin-II (AII) raises cytosolic free calcium and stimulates protein kinase-C (PKC) activity in vascular smooth muscle cells (VSMC). Phorbol-12-myristate-13-acetate (PMA) directly activates PKC in these cells. Both of these agonists stimulate prostacyclin production. Several studies have shown that atrial natriuretic factor (ANF) does not interfere with the AII-induced early calcium response. We, therefore, examined the effects of ANF on PKC activity and prostacyclin production in cultured rat aortic VSMC. PKC activity was determined in the membranous and cytosolic fractions after anion exchange chromatography. ANF (10(-7) M) inhibited by 44 +/- 3% and 39 +/- 8% the increase in membranous PKC activity induced by AII and PMA, respectively. ANF (10(-7) M) inhibited PMA-stimulated prostacyclin production, whereas AII-induced prostacyclin production remained unaffected. Thus, our results suggest that some biological effects induced by ANF in VSMC are mediated by an inhibition of membranous PKC activity.

Diagnostic accuracy of Doppler-echocardiography in unselected patients with suspected pulmonary embolism.

This study investigates the diagnostic value of echocardiography in patients with suspected pulmonary embolism. Doppler-echocardiography was performed in fifty consecutive patients, predominantly presenting in the emergency ward, with clinically suspected pulmonary embolism. Patients were classified as having or not pulmonary embolism by a sequential non-invasive strategy including lung scan, D-dimer measurement and lower limb venous compression ultrasonography, pulmonary angiography being performed in case of an inconclusive non-invasive work-up. The prevalence of pulmonary embolism was 36% (18 of 50 patients). Right ventricular dilatation on 2-D echocardiography associated to a tricuspid regurgitation velocity > or =2.7 m/s, corresponding to a pulmonary systolic pressure > or =39 mmHg, were present in 12 of the 18 patients (67%) with and in two of the 32 patients (6.3%) without pulmonary embolism. They were, however, absent in five of the 18 patients (28%), in whom the definite diagnosis of pulmonary embolism was made. The combination of these both echocardiographic criteria yielded a sensitivity of 67% and a specificity of 94%, positive predictive value was 86% and negative predictive value was 83%. The diagnostic performance of these two combined echocardiographic criteria, when present, permitted to reach in patients with a high clinical pre-test probability of pulmonary embolism the post-test probability values above 90%. On the other hand, the absence of these two Doppler-echocardiographic criteria did not allow to exclude pulmonary embolism, except in presence of a low pre-test probability. The findings of our study show that Doppler-echocardiography in patients with high clinical suspicion of pulmonary embolism may represent a potentially useful screening technique for the diagnosis of the disease permitting prompt initiation of treatment. However, the method does not allow to exclude pulmonary embolism in all patients with intermediate or high clinical suspicion of the disease.

Neural stem cells and cell death.

Neural stem cells (NSC) undergo apoptotic cell death as an essential component of neural development. Here, we present the results of our studies on the mechanisms by which NSC undergo cell death in response to neurotoxic insults. As experimental models we used primary culture of adult NSC from the subventricular zone of the rat brain, and the neural stem cell line C17.2 initially derived from developing mouse cerebellum. NSC undergo apoptosis in response to staurosporine (0.25 microM) as well as agents inducing oxidative stress such as 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Exposed cells demonstrate an apoptotic morphology, positive TUNEL staining and phosphatidyl serine exposure as labeled with Annexin V. Using an antibody specific for cytochrome c, we found that cells exposed to staurosporine or DMNQ exhibited diffuse fluorescence throughout the cytosol, implying a release of cytochrome c from the mitochondria. In addition to positive immunoreactivity against the active fragment (p17) of caspase-3, the administration of the pan-caspase inhibitor, zVAD-fmk (40 microM), prevents apoptosis. Both NSC and C17.2 express the Fas receptor, and procaspase-8, but exposure to agonistic Fas mAb (250 ng/ml) fails to induce apoptosis. Pretreatment with cycloheximide or actinomycin D does not influence the cell response to Fas mAb, suggesting that the endogenous inhibitor of caspase-8 FLICE-inhibitory protein (FLIP) is not responsible for the inhibition of the Fas pathway. Thus, it appears that the Fas dependent cell death pathway is not operative in these cells, while the mitochondrial pathway is active and caspase-3 serves as an executioner caspase in the apoptotic machinery. It is known that Fas not only induces apoptosis, but can also deliver growth stimulatory signals through activation of the extracellular-signal regulated kinase (ERK) pathway. The Fas-induced ERK phosphorylation that we detect in C17.2 cells suggests that in NSC Fas may function as a mediator of growth rather than death.

First performance results of PTB's atomic caesium fountain and a study of contributions to its frequency instability.

At the Physikalisch-Technische Bundesanstalt (PTB), an atomic caesium fountain was constructed. Ramsey fringes with a full width at half maximum (FWHM) of 0.86 Hz were obtained by launching the atoms to a height of 83 cm above the cooling region (40 cm above the microwave cavity center). A first measurement of the homogeneity of the magnetic flux density yields 0.33 nT (rms), only 0.16% of the mean value of 0.205 muT used in normal operation. The inherent elementary noise contributions of the fountain and of a thermal beam atomic clock are compared in some detail.

Biosynthesis of cytochalasans. XI. New results on the incorporation of phenylalanine into cytochalasin D by Zygosporium masonii [1].

Incorporation of L-[2-2H]phenyl-[2-2H]alanine and L-phenyl-[2-13C, 15N]alanine into cytochalasin D by Zygosporium masonii involved the complete loss of both the alpha-2H- and the alpha-15N-atom. Incorporation of a mixture of L-phenyl-[15N]alanine and L-[U-14C]phenylalanine into cytochalasin D and protein amino acids (phenylalanine, leucine, isoleucine) was accompanied by a substantial loss of 15N with respect to 14C. These effects are attributed to rapid exchange reactions taking place while L-phenylalanine is part of the intracellular pool of amino acids. In addition, the medium- and concentration-dependent incorporation of the carbon skeleton of exogeneous D-phenylalanine into cytochalasin D is reported. In a peptone-based complex medium, D-phenyl-alanine is poorly incorporated. Throughout the whole concentration range (0-250 mg/l), the incorporation rates are less than 10% of those of L-phenylalanine. In a minimal medium containing NH4NO3 as nitrogen source however, D-phenylalanine is preferred over the natural enantiomer by a factor of 1.28 up to 6.78, depending on the concentrations of exogeneous D- and L-phenylalanine. These effects are attributed to the medium-dependent activities of different amino acid transport systems responsible for the uptake of D- and L-phenylalanine in Z. masonii.

[Recent progress of natural product chemistry and potential application in fertility control].

PIP: The present work deals with gossypol, a male contraceptive derived from cottonseed oil, and antifertility agent zoapatanol which has been isolated from a Mexican plant. It also discusses 2 natural products of microbial origin. They exhibit remarkable biological activities. The 1st group, the trichothecene family (verrucarins, roridins, baccharins) show antibiotic, antifungal, antiviral, insecticidal, and cytostatic (antileukemic) activity. The 2nd class of compounds are the cytochalasans (e.g., cytochalasans A, B, and D, chaetoglobosins). They exhibit cytoplasmic cleavage in mammalian cell cultures and cell movement. Some cytochalasans are characterized by other interesting biological activities. Tests for antifertility have not been carried out yet. Biogenesis and approaches to the total synthesis of both classes of natural products are outlined. (author's modified)^ieng

Myocardial fatty acid oxidation during ischemia and reperfusion.

Inhibition of fatty acid oxidation is an early event in myocardial ischemia that most likely contributes to tissue injury by the accumulation of potentially toxic intermediates such as acylCoA and acylcarnitine. After reperfusion both myocardial oxygen consumption and fatty acid oxidation may rapidly recover to preischemic levels, even when contractile function remains depressed. The mechanisms underlying the apparent dissociation between contractile function and oxidative metabolism early during reperfusion are still controversial. In isolated rat hearts subjected to 60 min of no-flow ischemia myocardial oxygen consumption and oxidation of palmitate were lowered during reperfusion by 3 mM of NiCl2 and by 6 microM of ruthenium red. The results provide indirect evidence for the hypothesis that intracellular calcium transport may be involved in the mechanisms responsible for the high oxidative metabolic rate early after reperfusion.

High performance liquid chromatographic assay of disaccharides and oligosaccharides produced by the digestion of glycosaminoglycans with chondroitin sulphate lyases.

In high performance liquid chromatographic procedures hitherto described, SiO2, NH2 and RP columns have been used for the analysis of disaccharides produced by the digestion of glycosaminoglycans with the chondroitin sulphate lyases AC and ABC. The use of a potent anion exchanger offers the following advantages over these columns: superior separation characteristics for non-sulphated disaccharides, and improved column performance, coupled with more stable analytical conditions. Elution with dilute saline solutions permits separation of the two non-sulphated disaccharides from chondroitin and hyaluronate. The sequential application of chondroitinase AC and ABC permits the determination of hyaluronate, the chondroitin sulphate isomers and the dermatan sulphate isomers by high performance liquid chromatographic separation of the products of enzymatic hydrolysis. In a previously described method, hyaluronate lyase was used for the determination of hyaluronate. It has been found, however, that omission of the hyaluronate lyase step results in superior accuracy in the high performance liquid chromatographic separation of the non-sulphated disaccharides. The enzymatic analysis of human articular cartilage glycosaminoglycans has repeatedly yielded a fraction which is not digestable by chondroitinase AC, but is completely digestable by chondroitinase ABC. More extensive characterization has disclosed that this fraction differs structurally from chondroitin sulphate. Enzymatic characterization indicates that it should presumably be assigned to dermatan sulphate.

Interaction of oxindanac and frusemide in man.

Oxindanac, a moderately active cyclo-oxygenase inhibitor in vitro, is a new antiinflammatory agent under clinical investigation. Its effects on frusemide-induced natriuresis have now been studied. Eight male volunteers receiving frusemide 40 mg b.d. were also given either oxindanac 300 mg b.d. or placebo in two consecutive periods separated by a treatment-free period, according to a randomized cross-over study design. Urinary prostaglandin excretion (PGF2 alpha) fell by 75% after 3 days on oxindanac. Frusemide-induced renin activity reached 66% of the control value in the presence of oxindanac. However, the natriuresis induced by frusemide did not differ significantly whether oxindanac or placebo was administered, despite the inhibitory action of the former on prostaglandin synthesis in vivo.

Substrate competition in postischemic myocardium. Effect of substrate availability during reperfusion on metabolic and contractile recovery in isolated rat hearts.

Normal myocardium can derive energy for contraction and relaxation from oxidative metabolism of a variety of substrates. This investigation examined the influence of substrate availability early during reperfusion on the substrate pattern of oxidative metabolism and recovery of contractile function. For this purpose, isovolumically beating isolated rat hearts, perfused retrogradely with erythrocyte-supplemented buffer containing 0.4 mmol/L palmitate and 11 mmol/L glucose, were subjected to 40 minutes of no-flow ischemia. Hearts were reperfused with medium containing selected concentrations of palmitate and glucose. The substrate pattern for oxidative metabolism was determined on the basis of myocardial release of 14CO2 after equilibration of the hearts during the initial 15 minutes of reperfusion with either [1-14C]palmitate or [U-14C]glucose. In continuously perfused control hearts, glucose oxidation was largely inhibited by palmitate. During postischemic reperfusion, oxidation of glucose was increased by 59% (P < .05) and 467% (P <.01) in hearts reperfused after the ischemic period with 11 mmol/L glucose plus 0.4 or 1.2 mmol/L palmitate, respectively. Oxidation of palmitate was concomitantly reduced during reperfusion at low (0.4 mmol/L) but not at high (1.2 mmol/L) palmitate concentration. Compared with hearts reperfused with medium containing 0.4 mmol/L palmitate as sole substrate, hearts reperfused with medium containing 11 mmol/L glucose with 0.4 mmol/L palmitate exhibited lower left ventricular diastolic pressure (69 +/- 5 versus 90 +/- 3 mm Hg [mean +/- SEM], P < .05), less release of creatine kinase (31 +/- 5 versus 59 +/- 7 U/g wet wt, P < .05), and better recovery of left ventricular pressure development (26 +/- 9 versus 6 +/- 4 mm Hg, P < .05). Omission of palmitate or increasing the palmitate concentration to 1.2 mmol/L did not significantly alter postischemic myocardial contracture and enzyme release. The findings support the view that glucose oxidation early during reperfusion may be crucial for functional recovery. The results further indicate that interaction of substrates of oxidative metabolism is altered in severely injured postischemic myocardium. Inhibition of glucose oxidation by fatty acids was partially reversed during reperfusion.

Effect of magnesium administered during postischemic reperfusion on myocardial oxidative metabolism in isolated rat hearts.

To determine the effect of magnesium on myocardial function and oxidative metabolism after reperfusion, isolated rat hearts perfused retrogradely with erythrocyte-enriched medium (0.4 mM palmitate bound to 0.4 mM albumin, 11 mM glucose) were subjected to 60 minutes of no-flow ischemia followed by 60 minutes of reperfusion. Untreated postischemic hearts exhibited after 15 minutes of reperfusion recovery of myocardial oxygen consumption to 65% of the preischemic value despite persistent depression of left ventricular isovolumic pressure development to 21%. Magnesium (15 mM) administered during the initial 30 minutes of reperfusion reduced myocardial oxygen consumption of reperfuse myocardium by 35%. Oxidation of [1-14C]palmitate was slightly more reduced (-55%) than oxidation of [U-14C]glucose (-42%). Magnesium did not influence ultimate recovery of contractile function and cumulative myocardial release of creatine kinase. Thus, 15 mM magnesium administered during reperfusion elicited a reduction of oxidative metabolism. However, magnesium did not modify myocardial injury.

Absolute frequency measurement of the 435.5-nm (171)Yb+-clock transition with a Kerr-lens mode-locked femtosecond laser.

We have measured the frequency of the 6s(2)S(1/2)(2)-5d D(3/2)(2) electric-quadrupole transition of (171)(Yb) (+) with a relative uncertainty of 1x10(-14) , nu(Yb)=688358 979309312Hz +/-6Hz . We used a femtosecond frequency comb generator to phase-coherently link the optical frequency derived from a single trapped ion to a cesium-fountain-controlled hydrogen maser. This measurement is one of the most accurate measurements of optical frequencies ever reported, and it represents a contribution to the development of optical clocks based on a (171)Yb(+)-ion standard.