Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.     

Activation of caspase-like proteases and induction of apoptosis by isopentenyladenosine in tobacco BY-2 cells

The effects of isopentenyladenosine (iPA) on tobacco (Nicotiana tabacum L.) BY-2 cells were examined. The number of BY-2 cells decreased in a time- and concentration-dependent manner after being exposed to micromolar concentrations of iPA. This decrease was mainly due to a loss of cell viability, since no substantial changes in cell cycle progression were revealed by flow-cytometric analysis. Dying cells exhibited the typical morphological and biochemical hallmarks of apoptosis, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. Caspase-1-like and caspase-3-like proteases also became activated, the former being dominant. Inhibitor-sensitivity studies revealed that although synthetic caspase inhibitors failed to prevent cell death they markedly reduced cell death in tobacco BY-2 cells, Nu-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a specific inhibitor for caspase-1, being the most effective. Our results indicate that caspase-like proteases, and particularly caspase-1-like protease, might be critically implicated in iPA-induced apoptosis of BY-2 cells. Finally, the outcome of inhibiting adenosine kinase by 4-amino-3-iodo-1(beta- D-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine revealed that intracellular phosphorylation of iPA is required for its cytotoxicity to develop.     

Contribution to the coincidence of malignant tumours and some allergic manifestations

Clinical observations during the past decades led us to the early empirical assumptions that adult patients affected by certain types of allergic manifestations may have a lower prevalence of malignant tumors. In order to test those observations as well as some contradictory reports (1-3), we conducted a retrospective study using 32 years of records and statistics of a medium-sized hospital (420 beds) at St. Mary's Hospital, a general, non-chronic, teaching hospital, affiliated with McGill University in Montreal. The study has been realized during two periods: (a) 1965-1989 and (b) 1990-1996.     

Observation of multiple isoforms and specific proteolysis patterns of proliferating cell nuclear antigen in the context of cell cycle compartments and sample preparations

The proliferating cell nuclear antigen (PCNA) is an essential component for eukaryotic chromosomal DNA replication and repair. PCNA forms a homotrimer ring, which may function as a DNA sliding clamp for DNA polymerases and, possibly, a docking station for other replication- and repair-related proteins. Several reports have suggested the existence of different PCNA isoforms. Here we confirm, using high resolution two-dimensional electrophoresis with narrow pH ranges, the existence of three PCNA isoforms in both Chinese hamster and human breast cancer cells. Among the three isoforms, M or main form is the dominant one throughout the cell cycle while the relative amounts of the minor components A (acidic) and B (basic) forms appear to vary during the cell cycle. We also observed that a specific pattern of PCNA proteolysis occurred during isoelectric focusing in spite of high urea (8 M) and detergent (2% 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate), which was largely inhibited by the proteosome inhibitor MG132 or boiling. Interestingly, the proteolysis pattern was mainly observed with samples isolated from cells in S and G2 phases. A similar but much lower level of PCNA proteolysis also occurred in vivo within the nuclei of the cells in S phase. Taken together, our data are consistent with the idea that the existence of the different isoforms and specific proteolysis of PCNA are relevant to its functions in vivo.     

Biologically based risk estimation for radiation-induced CML

Chronic myeloid leukemia (CML) invites biologically based radiation risk modeling because CML is simultaneously well-understood, homogeneous and prevalent. CML is known to be caused by a translocation involving the ABL and BCR genes, almost all CML patients have the BCR-ABL translocation, and CML is prevalent enough that its induction is unequivocally detected among Hiroshima A-bomb survivors. In a previous paper, a linear-quadratic-exponential (LQE) dose- response model was used to estimate the lifetime excess risk of CML in the limit of low doses of γ-rays, R _γ. This estimate assumed that BCR-ABL translocation dose- response curves in stem cells for both neutrons and γ-rays, differ only by a common proportionality constant from dicentric aberration dose-response curves in lymphocytes. In the present paper we challenge this assumption by predicting the BCR-ABL dose response. The predictions are based on the biophysical theory of dual radiation action (TDRA) as it applies to recent BCR-to-ABL distance data in G₀ human lymphocytes; this data shows BCR and ABL geometric distributions that are not uniform and not independent, with close association of the two genes in some cells. The analysis speaks against the previous proportionality assumption. We compute 11 plausible LQE estimates of R _γ, 2 based on the proportionality assumption and 9 based on TDRA predictions. For each estimate of R _γ we also compute an associated estimate of the number of CML target cells, N; the biological basis of the LQE model allows us to form such estimates. Consistency between N and hematological considerations provides a plausibility check of the risk estimates. Within the group of estimates investigated, the most plausible lifetime excess risk estimates tend to lie near R _γ=0.01 Gy^–1, substantially higher than risk estimates based on the proportionality assumption. Received: 10 August 2000 / Accepted: 20 December 2000     

Chronic Exposure of NG108-15 Cells to Amyloid β Peptide (Aβ1–42) Abolishes Calcium Influx via N-Type Calcium Channels

We investigated whether amyloid--peptide (A_1–42) has an effect on the elevations of the intracellular concentration of Ca²⁺ ions ([Ca²⁺]_i) induced by depolarizations of NG108-15 cells and on related Ca²⁺ channels. A_1–42 (10-1000 nM) had no immediate effect on depolarization-induced [Ca²⁺]_i elevations. [Ca²⁺]_i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A_1–42. Nifedipine (1 M) reduced these elevations equally in cells grown in the absence or presence of A_1–42. In contrast, the ability of -conotoxin GVIA to diminish the depolarization-induced [Ca²⁺]_i responses became lost in cells grown in the presence of 100 nM A_1–42. This indicates that the influx of calcium through the N-type Ca²⁺ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A_1–42, presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca²⁺ channels in neurotransmitter release.     

Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

Nucleoli undergo structural and molecular modifications during hibernation

The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.