Relationship between ornithine decarboxylase activity and hexamethylene bisacetamide-induced differentiation of murine erythroleukemia cells

A transitory increase in ornithine decarboxylase (ODC) activity is shown not to be a prerequisite for the differentiation induced by hexamethylene bisacetamide (HMBA) in murine erythroleukemic (MEL) cells. On the contrary, conditions are described, where inhibition of the ODC activity with alpha-difluoromethyl ornithine (DFMO) stimulated the induced differentiation. Polyamine analysis demonstrated that a reduction in intracellular putrescine and spermidine occurred in MEL cells before commitment to erythrodifferentiation. The presence of DFMO increased the rapidity and the amplitude of these changes. No effect of dexamethasone on these changes in ODC activity or intracellular polyamines was observed.     

Cancer risk from chronic exposures to chemicals and radiation: a comparison of the toxicological reference value with the radiation detriment

This article aims at comparing reference methods for the assessment of cancer risk from exposure to genotoxic carcinogen chemical substances and to ionizing radiation. For chemicals, cancer potency is expressed as a toxicological reference value (TRV) based on the most sensitive type of cancer generally observed in animal experiments of oral or inhalation exposure. A dose–response curve is established by modelling experimental data adjusted to apply to human exposure. This leads to a point of departure from which the TRV is derived as the slope of a linear extrapolation to zero dose. Human lifetime cancer risk can then be assessed as the product of dose by TRV and it is generally considered to be tolerable in a 10^–6–10^–4 range for the public in a normal situation. Radiation exposure is assessed as an effective dose corresponding to a weighted average of energy deposition in body organs. Cancer risk models were derived from the epidemiological follow-up of atomic bombing survivors. Considering a linear-no-threshold dose-risk relationship and average baseline risks, lifetime nominal risk coefficients were established for 13 types of cancers. Those are adjusted according to the severity of each cancer type and combined into an overall indicator denominated radiation detriment. Exposure to radiation is subject to dose limits proscribing unacceptable health detriment. The differences between chemical and radiological cancer risk assessments are discussed and concern data sources, extrapolation to low doses, definition of dose, considered health effects and level of conservatism. These differences should not be an insuperable impediment to the comparison of TRVs with radiation risk, thus opportunities exist to bring closer the two types of risk assessment.

     

Seasonal variations of P compounds and their concentrations in two coastal lagoons (Herault,France)

This article concerns seasonal variations in the phosphate concentrations in two coastal lagoons near Montpellier (Mediterranean coast, France). The o-P concentration in the overlying water is highest during summer. The role of the sediment, particularly that of the different P fractions in the sediment, is discussed. Significant variations, especially in the FeOOH ≈ P fraction, occur. For both Tot-P_sed and the Fe00H≈P fraction a gradient from surface to bottom is observed, as well as a distinct decrease in the FeOOH≈P fraction in the surface sediments during summer and autumn. Variations in the FeOOH≈P fraction appear to be compensated by variations in the CaC0₃≈P fraction. These variations appear to be determined by the ferric hydroxide concentration. This compound represents only a small part (maximally 15%) of the total iron in the sediments and is related to the dissolved oxygen content of the immediately overlying water. Besides the fractions o-P, Fe(OOH)≈P, a large part of the CaC0₃≈P fraction is potentially bioavailable. A large proportion of the Tot-P_sed is therefore bioavailable.[/p]     

Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase.

The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane.     

Evidence for new C-terminally truncated variants of α- and β-tubulins

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the –EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same –EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with –EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.     

Phosphate in the sediments of the Gulf of Lions (NW Mediterranean Sea), relationship with input by the river Rhone

We investigated P-input by the Rhone river into the Mediterranean Sea taking into account P trapped in the surface sediment of the Gulf of Lions. Total phosphate concentration was determined every cm in the upper 10 cm-layer of sediments sampled at 11 stations in the Gulf of Lions during two cruises (March 1998 and January 1999). Two low downward gradients, one East–West and another North–South, with distance to the Rhone river mouth were found. Except at one station, total phosphate concentration in surface sediments was found to be constant with depth down to 10 cm. Values for individual stations ranged between 400 and 700 g g^–1 with an average value of 547 g g^–1 (st. dev. = 63 g g^–1) for the whole gulf. The low variability in total-P concentration in sediments is in contrast to the large variability in suspended matter load of the river Rhone and suggests the dominance of authigenic P removal mechanisms in P burial. The total P-pool in the upper 10 cm-layer of the sediments in the gulf was estimated at 562 kt, with about 80% trapped into the shelf and 20% into the slope. Annual P-deposition was estimated as 7.2–12.4 kt y^–1, from the P-pool in the sediment and the sedimentation rates. This is equivalent to a previous estimation of the river Rhone input, estimated to be about 6.5–12.2 kt y^–1. As the Rhone is the major river flowing into the Mediterranean Sea, total P in surface sediments of the Gulf of Lions should be taken into account in P-budgets at the scale of the Mediterranean Sea.     

The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.     

Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.     

Evidence of the cost of the production of microcystins by Microcystis aeruginosa under differing light and nitrate environmental conditions

The cyanobacterium Microcystis aeruginosa is known to proliferate in freshwater ecosystems and to produce microcystins. It is now well established that much of the variability of bloom toxicity is due to differences in the relative proportions of microcystin-producing and non-microcystin-producing cells in cyanobacterial populations. In an attempt to elucidate changes in their relative proportions during cyanobacterial blooms, we compared the fitness of the microcystin-producing M. aeruginosa PCC 7806 strain (WT) to that of its non-microcystin-producing mutant (MT). We investigated the effects of two light intensities and of limiting and non-limiting nitrate concentrations on the growth of these strains in monoculture and co-culture experiments. We also monitored various physiological parameters, and microcystin production by the WT strain. In monoculture experiments, no significant difference was found between the growth rates or physiological characteristics of the two strains during the exponential growth phase. In contrast, the MT strain was found to dominate the WT strain in co-culture experiments under favorable growth conditions. Moreover, we also found an increase in the growth rate of the MT strain and in the cellular MC content of the WT strain. Our findings suggest that differences in the fitness of these two strains under optimum growth conditions were attributable to the cost to microcystin-producing cells of producing microcystins, and to the putative existence of cooperation processes involving direct interactions between these strains.     

Dual role of CD38 in microglial activation and activation-induced cell death

Microglia, the resident immune cells of the CNS, are normally quiescent but become activated after infection or injury. Their properties then change, and they promote both repair and damage processes. The extent of microglial activation is regulated, in part, by activation-induced cell death (AICD). Although many apoptotic aspects of the microglial AICD mechanism have been elucidated, little is known about the connection between the activation step and the death process. Using mouse primary microglial cultures, we show that the ectoenzyme CD38, via its calcium-mobilizing metabolite cyclic-ADP-ribose (cADPR), helps promote microglial activation and AICD induced by LPS plus IFN-gamma (LPS/IFN-gamma), suggesting that CD38 links the two processes. Accordingly, CD38 expression and activity, as well as the intracellular calcium concentration ([Ca2+]i) in the primary microglia were increased by LPS/IFN-gamma treatment. Moreover, CD38 deficiency or treatment with cADPR antagonists conferred partial resistance to LPS/IFN-gamma-induced AICD and also reduced [Ca2+]i. Microglial activation, indicated by induced expression of NO synthase-2 mRNA and production of NO, secretion and mRNA expression of TNF-alpha and IL-12 p40, and expression of IL-6 mRNA, was attenuated by CD38 deficiency or cADPR-antagonist treatment. The observed effects of CD38 on microglial activation are probably mediated via a cADPR-dependent increase in [Ca2+]i and the effect on AICD by regulation of NO production. Our results thus suggest that CD38 significantly affects regulation of the amount and function of activated microglia, with important consequences for injury and repair processes in the brain.