The PML and PML/RARα Domains: From Autoimmunity to Molecular Oncology and from Retinoic Acid to Arsenic

Acute promyelocytic leukemia (APL) is specifically associated to a t(15; 17) translocation which fuses a gene encoding a nuclear receptor for retinoic acid, RARα, to a previously unknown gene PML. The PML protein is localized in the nucleus on a specific domain of unknown function (PML nuclear bodies, NB) previously detected with autoimmune sera from patients with primary biliary cirrhosis (PBC). These bodies are nuclear matrix-associated and all of their identified components (PML, Sp100, and NDP52) are sharply upregulated by interferons. We show that autoantibodies against both PML and Sp100 are usually associated in sera with multiple nuclear dot anti-nuclear antibodies and demonstrate that PML is an autoantigen, not only in PBC, but also in other autoimmune diseases. In APL, the PML/RARα fusion interferes with both the retinoic acid (RA) response and PML localization on nuclear bodies, but the respective contribution of each defect to leukemogenesis is unclear. RA induces the terminal differentiation of APL blasts, yielding to complete remissions, and corrects the localization of NB antigens. Arsenic trioxide (As₂O₃) also induces remissions in APL, seemingly through induction of apoptosis. We show that in APL, As₂O₃leads to the rapid reformation of PML bodies. Thus, both agents correct the defect in NB antigen localization, stressing the role of nuclear bodies in the pathogenesis of APL.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

Factors influencing detection of density dependence in British birds

If censuses are taken at less than generation intervals, the number of successive censuses in which a given individual is recorded will depend on longevity. Repeatedly recording the same individuals could produce under-estimates of population variability and influence detection of density dependence. We investigated this possibility in 60 time series of abundances of British birds compiled from the Common Birds Census data and then used simple population models to illustrate the proposed mechanism. Species had average lifespans of 2–10 years and were censused annually. Density dependence was detected (at P<0.05) much more frequently in bird species with long lifespans than in those with short lifespans; 75% of the 12 longest-lived species showed density dependence compared to 46% of all species. Population variability measured in annual censuses (termed annual variability) was lower in bird species with longer lifespans. We used discrete time models based on difference equations to demonstrate how longevity influences population variability and detection of density dependence in series of annual censuses. A model in which only first-year birds experienced density dependence was rejected because annual variability was greater and detection of density dependence was less likely when longevity was greater, the opposite of the observed effects of longevity in birds. A model in which all age classes experienced density dependence gave time series with lower annual variability and in which density dependence was detected more frequently when longevity was greater, which is the pattern observed in British birds. Analysis of data from this model showed that the amount of density dependence actually present caused only small changes in annual variability, whereas detection of density dependence from simulated series was strongly influenced by annual variability. The high annual variability of series from short-lived bird species could mask any density dependence that was present. Correcting for trends lead us to detect density dependence in 75% of the 12 longest lived bird species. There is no reason to believe that this rate is not also representative of short-lived species.     

Glycolytic gene expression in amphibious Acorus calamus L. under natural conditions

Acorus calamus L is an amphibious plant, which is exposed to periods of flooding and consequently hypoxic conditions as a part of its natural life cycle. Previous experiments under laboratory conditions have shown that the plant can survive for two months in the complete absence of oxygen, and that during this period the expression of genes encoding the glycolytic enzymes fructose-1,6-bisphosphate aldolase (ALD), pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is induced in leaves and rhizomes (Bucher and Kuhlemeier, 1993). Here we studied the expression of ALD and ADH through two years in the natural habitat of A. calamus. Under natural conditions roots and rhizomes were always submerged but newly grown leaves emerged in spring; in autumn the leaves senesced and the whole plant was submerged again. High Ald and Adh mRNA levels in leaf and rhizome were found only in winter when the leaves were entirely submerged. Upon leaf emergence in spring the mRNA levels rapidly declined. Under controlled experimental conditions expression of Ald and Adh was not induced by low temperature. The combination of laboratory and field experiments supports the hypothesis that oxygen deprivation rather than low temperature is a major regulator of glycolytic gene expression in A. calamus. The possible role of other environmental factors is also discussed.Abbreviations ADH alcohol dehydrogenase - Adh gene encoding ADH - ALD cytoplasmic fructose-1,6-bisphosphate aldolase - Ald gene encoding ALD - PDC pyruvate decarboxylase - Pdc gene encoding PDC     

Semliki forest virus capsid protein inhibits the initiation of translation by upregulating the double-stranded RNA-activated protein kinase (PKR)

We investigated the possible translational role which elevated concentrations of highly purified Semliki Forest virus (SFV) capsid (C)-protein molecules may play in a cell-free translation system. Here we decomonstrate that in the absence of double-stranded RNA high concentrations of C protein triggered the phosphorylation of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. Activated PKR in turn phosphorylated its natural substrate, the subunit of eukaryotic initiation factor 2 (eIF-2), thereby inhibiting initiation of host cell translation. These findings were further strengthened by experiments showing that during natural infection with SFV the maximum phosphorylation of PKR coincided with the maximum synthesis of C protein 4–9 hours post infection. Thus, our results demonstrate that high concentrations of C-protein molecules may act in a hitherto novel mechanism on PKR to inhibit host cell protein synthesis during viral infection.     

Different properties of the inward rectifying potassium conductance of aleurone protoplasts from dormant and non-dormant barley grains

Isogenic dormant and non-dormant barley grains provide a useful system to study the molecular mechanisms of grain dormancy and the role of plant hormones in this process. As ion fluxes are associated with dormancy-related plant hormone responses, we compared the properties of the inward rectifying potassium conductance in aleurone protoplasts isolated from dormant and non-dormant Triumph grains and in germinating Himalaya grains. Maximal conductance, voltage dependency of steady-state activation, activation and deactivation kinetics were studied in the whole-cell patch-clamp configuration. Activation and deactivation time courses were single exponential. No differences in the above described properties were found between the protoplasts isolated from non-dormant Triumph and Himalaya grains. However, the maximal conductance (corrected for cell size) in protoplasts from dormant Triumph grains was much smaller (65%), and activation time constants were much larger as compared to protoplasts from non-dormant grains. No differences were found in the deactivation kinetics in the three different types of protoplasts. The half-maximal activation potential was slightly more negative in protoplasts from dormant grains than from non-dormant grains.     

Crystal and molecular structure of polymeric [MnCl2(bpy)]n

A crystal and molecular structure determination of MnCl₂(bpy) showed that it exists as polymeric octahedral [MnCl₂(bpy)]_n. [MnCl₂(bpy)_n crystallizes in the monoclinic space group I2/a with A = 7.007(1), B = 9.200(1), C = 16.495(1) Å, β = 91.313(5)° and Z = 4. On the basis of 979 unique observed reflections with I 2.5σ(I) the structure was refined to R = 0.032.     

MONITORING ULTRAVIOLET-B-INDUCED DNA DAMAGE IN INDIVIDUAL DIATOM CELLS BY IMMUNOFLUORESCENT THYMINE DIMER DETECTION1

We developed a method to investigate the effect of ultraviolet-B radiation (UVBR) on the formation of thy-mine dimers in microalgal DNA that can be used for both laboratory and in situ research. Antibody labeling of dimers was followed by a secondary antibody (fluorescein isothiocyanate) staining to allow visualization of DNA damage with flow cytometry or fluorescence microscopy. Thymine dimer-specific fluorescence in nuclear DNA of the marine diatom Cyclotella sp. was linearly related to the UVBR dose. Simultaneous measurements of cellular DNA content showed that the vulnerability of G2 cells to DNA damage did not differ significantly from the vulnerability of G1 cells. The formation and removal of thymine dimers in Cyclotella sp. cells was monitored for 3 consecutive days at two realistic UVBR irradiance levels. Thy-mine dimers were removed within 24 h when exposed to a saturating photosynthetically active radiation intensity following the UVBR treatment. This new method allows the study of UVBR-induced DNA damage on a cell-to-cell basis. It is also feasible for field studies because cells remain intact and can be recognized readily after antibody treatment.