B,Z Conformations and Mechanism of the Z-B-Coil Transitions of the Self-Complementary Deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD Spectroscopy

Abstract

The helical structures of d(C-G-m⁵C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and ¹H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (> 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration).

The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95°C) only the coil form (S) is present. Below 55°C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m⁵C-G-C-G-m⁵C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z ? B ? S. The present data show that even at high salt concentration where only the Z form of d(C-G-m⁵C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

Multiple GTPases participate in the assembly of the large ribosomal subunit in Bacillus subtilis

GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases.     

Changes in dipole membrane potential at the mouse blood–brain barrier enhance the transport of 99mTechnetium Sestamibi more than inhibiting Abcb1, Abcc1, or Abcg2

Cationic ^99mTc-agents like ^99mTc-hexakis-2-methoxyisobutyl isonitrile (^99mTc-MIBI) cannot be used for brain imaging because they do not enter the brain as readily as some uncharged ^99mTc-compounds. The mechanism by which cationic ^99mTc-agents are transported across the blood–brain barrier (BBB) remains unclear. We explored ^99mTc-MIBI transport by in situ mouse brain perfusion to determine the influence of BBB features like the ATP-binding cassette transporters (Abcb1/P-glycoprotein (P-gp), Abcc1/Mrp1, and Abcg2/Bcrp), organic cation transporters (Slc22a1-3/Oct1-3), the transmembrane potential and the dipole membrane potential. P-gp reduced ^99mTc-MIBI transport across the BBB of P-gp-deficient mice 2.2-fold, as confirmed by PSC833 and GF120918 inhibition. Paradoxically verapamil decreased its transport '0.6-fold'. Reducing the BBB dipole membrane potential with tetraphenylborate or phloretin increased ^99mTc-MIBI transport about 12- and 20-fold, respectively. Guanidine, diphenhydramine, and carnitine significantly decreased ^99mTc-MIBI transport, but tetraethylammonium did not. ^99mTc-MIBI transport at the BBB is restricted by P-gp but not by Mrp1 or Bcrp. Some organic cations reduced the influx of ^99mTc-MIBI into the brain independently of Oct1, 2 and 3, but this could be due to their effect on another cation transporter. The membrane dipole potential of the luminal BBB membrane appeared to be the main factor restricting ^99mTc-MIBI permeability.     

Invasive alga <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> makes a strong impact on the Mediterranean sponge <Emphasis Type="Italic">Sarcotragus spinosulus</Emphasis>

Here we present the first observation of the impact of the invasive Caulerpa racemosa var. cylindracea on native photophilic sponge species in the Adriatic Sea, with special focus on Sarcotragus spinosulus. Caulerpa racemosa var. cylindracea is able to completely overgrow the sponge, developing an exceptionally thick canopy with a maximum measured density of 1,887 m of stolons m⁻² and 40,561 fronds m⁻². Necrosis of the sponge surface was significantly correlated with the algal dry biomass, frond number and stolon length. Dense algal canopy, penetration of the algal stolon and rhizoids into the sponge oscula and covering of the ostiae probably diminishes the seawater circulation through the sponge and consequently results in its smothering and even death. We suggest that chemotropism is the reason why C. racemosa penetrates the sponge oscula and establishes such dense canopy on the sponge.     

Macrophage autophagy protects against liver fibrosis in mice

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5^fl/fl LysM-Cre mice, referred to as atg5^−/−) and their wild-type (Atg5^fl/fl, referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5^−/− mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5^−/− mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5^−/− mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5^−/− mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5^−/− macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5^−/− mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.     

The level of pancreatic PLA2 receptor is closely associated with the proliferative state of rat uterine stromal cells

Rat uterine stromal cells (U_III) express pancreatic type PLA₂ (PLA₂-I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA₂-I. There is a dramatic decline in PLA₂-I binding in U_III cells as they progress from a nonconfluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA₂-I changed with the alteration in binding, suggesting that regulation in the PLA₂ binding capacity may have important implications in growth control mechanisms.