Evidence for Different MCM Subcomplexes with Differential Binding to Chromatin inXenopus

MCM proteins are molecular components of the DNA replication licensing system inXenopus.These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes. We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-boundXenopusMCM proteins during the cell cycle. In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones. The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control. Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits. Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3. Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes.     

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of ¹H, ¹³C or ¹⁵N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 °C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.     

Compensatory changes in Photosystem II electron turnover rates protect photosynthesis from photoinhibition

Exposure of algae or higher plants to bright light can result in a photoinhibitory reduction in the number of functional PS II reaction centers (n) and a consequential decrease in the maximum quantum yield of photosynthesis. However, we found that light-saturated photosynthetic rates (P_max) in natural phytoplankton assemblages sampled from the south Pacific ocean were not reduced despite photoinhibitory decreases in n of up to 52%. This striking insensitivity of P_max to photoinhibition resulted from reciprocal increases in electron turnover ( )through the remaining functional PS II centers. Similar insensitivity of P_max was also observed in low light adapted cultures of Thalassiosira weissflogii (a marine diatom), but not in high light adapted cells where P_max decreased in proportion to n. This differential sensitivity to decreases in n occurred because was close to the maximum achievable rate in the high light adapted cells, whereas was initially low in the low light adapted cells and could thus increase in response to decreases in n. Our results indicate that decreases in plant productivity are not necessarily commensurate with photoinhibition, but rather will only occur if decreases in n are sufficient to maximize or incident irradiance becomes subsaturating.     

Low activity of amine-oxidases and accumulation of conjugated polyamines in disfavour of organogenic programs in Chrysanthemum leaf disc explants

Foliar discs (8 mm diameter) from expanding leaves of the middle part of vegetative shoots of Chrysanthemum morifolium Ramat raised in vitro were induced to form directly on specific media in vitro either roots or vegetative buds, or callus. The budding programme, on its specific medium, was deviated to callus formation by the addition of 2 mM β-OH-E (β-OH-ethyldrazine, an inhibitor of diamine oxidase). Conversely vegetative buds instead of callus were formed on the callus medium in the presence of 2 mM DFMO (difluoromethylornithine, an inhibitor of ornithine decarboxylase). Callus formation was characterized by high accumulation of free and particularly conjugated polyamines (PA), very low or undetectable activities of diamine- and polyamine oxidases, and transglutaminase. DFMO-deviation of callus initiation in favour of bud formation lowered the accumulation of PA and increased the activity of amine-oxidases. The high catabolism of PA in the organogenic (rooting, budding) programs was questioned as to its role in developmental processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

EXPERIMENTAL EVIDENCE FOR HOST RACES IN MISTLETOE (PHORADENDRON TOMENTOSUM)

In order to test the hypothesis that mistletoes (Phoradendron tomentosum) are differentially adapted for the host species that they occur on, mistletoe seeds from the three most common hosts in central Texas (hackberry, Celtis occidentalis, elm, Ulmus crassifolia, and mesquite, Prosopsis glandulosa) were planted onto different individuals of each of the three hosts. Germination of seeds and formation of haustorial disks by seedlings were followed in the subsequent 17 months. Germination of seeds was very high for all nine combinations of maternal (source) and seed (experimental) hosts (range 82%–98%). There were no significant differences in seed germination between the two groups when source and experimental hosts were the same species and when they were different species. In contrast, development of haustorial disks when source and experimental hosts were the same species was significantly greater than when experimental host and source host species were different. The data suggest that populations of mistletoes are genetically differentiated such that early seedling development is greatest when there is correspondence between maternal and seedling host species.     

Cell death and lumen formation in spheroids of MCF‐7 cells

3D (three‐dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F‐actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF‐7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.