The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells. NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner.

Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.     

Photosynthetic action spectra and adaptation to spectral light distribution in a benthic cyanobacterial mat

We studied adaptation to spectral light distribution in undisturbed benthic communities of cyanobacterial mats growing in hypersaline ponds at Guerrero Negro, Baja California, Mexico. Microscale measurements of oxygen photosynthesis and action spectra were performed with microelectrodes; spectral radiance was measured with fiber-optic microprobes. The spatial resolution of all measurements was 0.1 mm, and the spectral resolution was 10 to 15 nm. Light attenuation spectra showed absorption predominantly by chlorophyll a (Chl a) (430 and 670 nm), phycocyanin (620 nm), and carotenoids (440 to 500 nm). Blue light (450 nm) was attenuated 10-fold more strongly than red light (600 nm). The action spectra of the surface film of diatoms accordingly showed activity over the whole spectrum, with maxima for Chl a and carotenoids. The underlying dense Microcoleus population showed almost exclusively activity dependent upon light harvesting by phycobilins at 550 to 660 nm. Maximum activity was at 580 and 650 nm, indicating absorption by phycoerythrin and phycocyanin as well as by allophycocyanin. Very little Chl a-dependent activity could be detected in the cyanobacterial action spectrum, even with additional 600-nm light to excite photosystem II. The depth distribution of photosynthesis showed detectable activity down to a depth of 0.8 to 2.5 mm, where the downwelling radiant flux at 600 nm was reduced to 0.2 to 0.6% of the surface flux.     

Carbon isotopic fractionation in heterotrophic microbial metabolism.

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.     

Maternal mortality and women's condition: “The distress in being a woman”

For millenaria, maternal mortality has been considered as a fatality inherent to women's condition. Thanks to the progress of the obstetric technology, the world M.M.R. fell from 2.000 to 400 per 100.000 births during the past 150 years. At the same time women's condition improved chiefly because of a better level of education. Is there any relationship between them: the decrease of Maternal Mortality and the amelioration of women's general status? Or, in other words, can the decrease of Maternal Mortality Rate be considered as an “indicator” of the situation of women? After a historical review of the importance of the maternal mortality drama as it occured in older times before the “obstetric era”, which began with the improvement of the obstetric technology, this paper will present the actual situation of maternal mortality in relation with the obstetric coverage, the female literacy level and some other socio-economic variables, and in relation with the Gross National Product (G.N.P.) as an indicator of governmental interest in the specific female problems of giving-life.     

Preparing faculty to teach in a problem-based learning curriculum: the Sherbrooke experience.

Over the last 6 years Sherbrooke Medical School has undertaken a major reform of its undergraduate curriculum. A new student-centred, community-oriented curriculum was implemented in September 1987. Problem-based learning (PBL) is now the main educational method. To adequately prepare teachers for the curriculum a series of faculty development programs in pedagogy were offered: first, a 2-day introductory workshop to initiate teachers into educational principles and their application in the new program; second, a 1-year basic training program in medical pedagogy; third, a 1-day workshop on PBL; and fourth, a comprehensive 3-day training program in PBL tutoring. Over 60% of all full-time teachers attended the introductory program and 80% the tutor training program. The 1-year basic training program was completed by 33% of the faculty members. The implementation of these programs, coupled with a high participation rate, resulted in a more student-centred educational philosophy and a greater interest in medical education. This had a significant impact when the new curriculum was instituted. Lessons learned from the experience are discussed.     

Nonhomogeneous labeling of liver mitochondrial acetyl-CoA

The specific activity of carbons 1 and 2 of plasma acetoacetate has been used as a measure of the specific activity of liver mitochondrial acetyl-CoA in tracer studies. To test whether or not acetoacetate actually reflects acetyl-CoA, livers were perfused with a mixture of substrates that are converted to mitochondrial acetyl-CoA: 1 mM lactate, 0.2 mM pyruvate, 0.2 mM acetate, and, where indicated, 0.2 mM octanoate or 0.2 mM alpha-ketoisocaproate. In each experiment, one of these substrates was 13C-labeled. Labeling of mitochondrial acetyl-CoA was assessed by three methods: (i) molar percent enrichment of total tissue acetyl-CoA; (ii) molar percent enrichment of carbons 4 and 5 of tissue citrate, the precursor of which is acetyl-CoA; and (iii) molar percent enrichment of carbons 1 and 2 of perfusate ketone bodies. Nonhomogeneous labeling of liver mitochondrial acetyl-CoA occurred under most conditions, i.e. the enrichments of carbons 4 and 5 of citrate were different from enrichments of carbons 1 and 2 of ketone bodies. Thus, based upon our results obtained in perfused livers, we question the validity of measuring the labeling of carbons 1 and 2 of acetoacetate as a noninvasive probe of liver mitochondrial acetyl-CoA.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.