Human–vegetation interactions during the Holocene in North America

Vegetation History and Archaeobotany - Between the initial colonization of North America and the European settlement period, Indigenous American land use practices shaped North American landscapes...     

Polar body formation in Spisula oocytes: function of the peripheral aster

Activated Spisula oocytes proceed through meiotic stages rapidly and in near synchrony, providing an excellent system for analyzing polar body formation. Our previous studies suggested that cortical spreading of the metaphase peripheral aster determines spatial features of the cortical F-actin ring that is generated prior to extrusion of the polar body. We tested this hypothesis by experimentally altering the number and cortical contact patterns of peripheral asters. Such alteration was achieved by (a) lovastatin-induced arrest at metaphase I, with and without hexylene glycol modification, followed by washout; and (b) cytochalasin-D inhibition of extrusion of the first polar body, with washout before extrusion of the second polar body. Both methods induced simultaneous formation of two or more cortically spreading asters, correlated with subsequent formation of double, or even triple, overlapping F-actin rings during anaphase. Regardless of pattern, ring F-actin was deposited near regions of greatest astral microtubule density, indicating that microtubules provided a positive stimulus to which the cortex responded indiscriminately. These results strongly support the proposed causal relationship between peripheral aster spreading and biogenesis of the F-actin ring involved in polar body formation.     

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell''s stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.     

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine‐subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R‐like enzyme, which possesses both processive exo‐ and endonuclease activities, whereas the latter is a distributive RNase D‐like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs—hDIS3 and hDIS3L—with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.     

Formation and function of the polar body contractile ring in Spisula

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility-previously assumed-was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.     

Synthesis, crystal structure, magnetic properties and EPR studies of Cu/Hg bimetallic thiocyanato-bridged coordination polymer

The new Cu/Hg bimetallic thiocyanato-bridged coordination polymer, [Cu(2-bzpy)Hg(SCN)₄]_n, has been synthesized and characterized by elemental analysis, magnetic measurements, EPR, and X-ray crystallography. X-ray diffraction analysis reveals that the title complex structure creates 3D network.