Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase–cGMP-dependent protein kinase pathway

Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the β-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the γ-globin gene. sGC, an obligate heterodimer of α- and β-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different β-like globin genes showed that, whereas the β-subunit is expressed at similar levels, high-level expression of the α-subunit is preferentially observed in erythroid cells expressing γ-globin but not those expressing β-globin. Also, the levels of expression of the γ-globin gene correlate to those of the α-subunit. sGC activators or cGMP analogs increased expression of the γ-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with β-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the γ-globin gene. Furthermore, increased expression of the γ-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC–PKG pathway constitutes a mechanism that regulates expression of the γ-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the β-globin disorders.

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

Actin Depolymerization Affects Stress-Induced Translational Activity of Potato Tuber Tissue1

Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.

Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas.

Levels of mRNA for the chloroplast-encoded elongation factor Tu (tufA) showed a dramatic daily oscillation in the green alga Chlamydomonas reinhardtii, peaking once each day in the early light period. The oscillation of tufA mRNA levels continued in cells shifted to continuous light or continuous dark for at least 2-3 days. Run-off transcription analyses showed that the rate of tufA transcription also peaked early in the light period and, moreover, that this transcriptional oscillation continued in cells shifted to continuous conditions. The half-life of tufA mRNA was estimated at different times and found to vary considerably during a light-dark cycle but not in cells shifted to continuous light. Light-dark patterns of transcription of several other chloroplast-encoded genes were examined and also found to persist in cells shifted to continuous light or dark. These results indicate that a circadian clock controls the transcription of tufA and other chloroplast-encoded genes.

Suspended matter on the meridional section in the Laptev Sea in August-September 1991

Suspended matter concentration along a meridian section from the Lena River delta to 78°N (~500 km) at ten stations from the surface to the bottom was studied with weight and optical (light attenuation index) techniques. At seven stations average residence time of suspended matter in surface waters was determined by the disequilibrium 234Th method. Average residence time of suspended matter in other depth intervals was calculated by regression dependence between the 234Th/238U ratio and suspended matter concentration. Differential and integral fluxes of suspended matter in the water column were also calculated. Nepheloid matter dominates in suspended matter composition in surface waters. Calculations indicate that, before being buried on the bottom, solid river run-off is resuspended 2.3 times (aver.). Redistribution of nepheloid suspended matter in the near-bottom layer results in formation of a strongly pronounced depocentre - an area of maximal discharge of solid river run-off within the Laptev Sea.

Distribution of grain size and clay minerals in surface sediments of the Kara Sea (Fig 4, 5)

In this paper, we summarize data on terrigenous sediment supply in the Kara Sea and its accumulation and spatial and temporal variability during Holocene times. Sedimentological, organic-geochemical, and micropaleontological proxies determined in surface sediments allow to characterize the modern (riverine) terrigenous sediment input. AMS-14C dated sediment cores from the Ob and Yenisei estuaries and the adjacent inner Kara Sea were investigated to determine the terrigenous sediment fluxes and their relationship to paleoenvironmental changes. The variability of sediment fluxes during Holocene times is related to the post-glacial sea-level rise and changes in river discharge and coastal erosion input. Whereas during the late/middle Holocene most of the terrigenous sediments were deposited in the estuaries and the areas directly off the estuaries, huge amounts of sediments accumulated on the Kara Sea shelf farther north during the early Holocene before about 9 cal kyr BP. The maximum accumulation at that time is related to the lowered sea level, increased coastal erosion, and increased river discharge. Based on sediment thickness charts, echograph profiles and sediment core data, we estimate an average Holocene (0-11 cal kyr BP) annual accumulation of 194,106 t/yr of total sediment for the whole Kara Sea. Based on late Holocene (modern) sediment accumulation in the estuaries, probably 12,106 t/yr of riverine suspended matter (i.e. about 30% of the input) may escape the marginal filter on a geological time scale and is transported onto the open Kara Sea shelf. The high-resolution magnetic susceptibility record of a Yenisei core suggests a short-term variability in Siberian climate and river discharge on a frequency of 300-700 yr. This variability may reflect natural cyclic climate variations to be seen in context with the interannual and interdecadal environmental changes recorded in the High Northern Latitudes over the last decades, such as the NAO/AO pattern. A major decrease in MS values starting near 2.5 cal kyr BP, being more pronounced during the last about 2 cal kyr BP, correlates with a cooling trend over Greenland as indicated in the GISP-2 Ice Core, extended sea-ice cover in the North Atlantic, and advances of glaciers in western Norway. Our still preliminary interpretation of the MS variability has to be proven by further MS records from additional cores as well as other high-resolution multi-proxy Arctic climate records.