Plasma membrane rafts complete cholesterol synthesis by participating in retrograde movement of precursor sterols

Mammalian cells synthesize significant amounts of precursor sterols, in addition to cholesterol, at the endoplasmic reticulum (ER). The newly synthesized sterols rapidly move to the plasma membrane (PM). The mechanism by which precursor sterols move back to the ER for their enzymatic processing to cholesterol is essentially unknown. Here we performed pulse-chase experiments and showed that the C29/C30 sterols rapidly move from the PM to the ER and are converted to cholesterol. The retrograde precursor sterol transport is largely independent of the Niemann-Pick type C proteins, which play important roles in late endosomal cholesterol transport. In contrast, disrupting lipid rafts significantly retards the conversion of C29/C30 and C28 sterols to cholesterol, causing the accumulation of precursor sterols at the PM. Our results reveal a previously undisclosed function of the PM lipid rafts: they bring cholesterol biosynthesis to completion by participating in the retrograde movement of precursor sterols back to the ER.     

Genetic variations in rice in vitro cultures at the <Emphasis Type="Italic">EPSPs–RPS20</Emphasis> region

In vitro cultures of plant cells have often been utilized to generate genetic variations, which are designated somaclonal variations. Little is known about the major genetic alterations in the cultured cells and the nature of these genetic changes. Here, we examined different lines of rice Oc cells that have been cultured for more than 20 years on agar media or in liquid media. We surveyed 35 clones obtained from PCR amplification of the 3-kb EPSPs–RPS20 region. The sequence divergence among the Oc cells was even greater than that between Japonica and Indica rice cultivars. The divergent sequences appeared to be maintained as multiple copies in a single cell. Surprisingly, the nucleotide substitutions in the Oc cells were characterized by an extremely high frequency of transition mutations of A/T-to-G/C, a feature which is similar to that of the mutations caused by chemical mutagens such as 5-bromouracil and 2-aminopurine. Although no replacements in the exons of this region were observed among the AA-genome Oryza species, our results revealed that the nucleotide substitutions of the cultured cell lines occurred more frequently at replacement sites in the exons than at synonymous sites. These distinct mutation biases found in rice in vitro cultures might contribute importantly to somaclonal variations. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users. Y. Noro and T. Takano-Shimizu equally contributed to this study.     

Bioreductive deposition of platinum nanoparticles on the bacterium Shewanella algae

An environmentally friendly method using the metal ion-reducing bacterium Shewanella algae was proposed to deposit platinum nanoparticles. Resting cells of S. algae were able to reduce aqueous PtCl(6)(2-) ions into elemental platinum at room temperature and neutral pH within 60min when lactate was provided as the electron donor. Biogenic platinum nanoparticles of about 5nm were located in the periplasm--a preferable, cell surface location for easy recovery of biogenic nanoparticles.     

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA^Tyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA^Tyr(GΨA). Structural information for TyrRS–tRNA^Tyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNA^Tyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA^Tyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA^Tyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA^Tyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.     

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.     

Regulation of ion transport in eel intestine by the homologous guanylin family of peptides

Since the gene expression of guanylin peptides and their receptors, guanylyl cyclase Cs, is enhanced in the intestine of seawater (SW)-adapted eels compared with fresh water (FW)-adapted fish, the guanylin family may play an important role in SW adaptation in eels. The present study analyzed the effect of three homologous guanylin peptides, guanylin, uroguanylin and renoguanylin, on ion movement through the eel intestine, and examined the target of guanylin action using Ussing chambers. The middle and posterior parts of the intestine, where water and ion absorption occurs actively in SW eels, exhibited serosa-negative transepithelial potential, while the anterior intestine was serosa-positive. Mucosal application of each guanylin in the middle or posterior intestine reduced the short-circuit current (Isc) dose dependently and reversed it at high doses, and reduced electric tissue resistance. The effects were greater in the middle intestine than in the posterior intestine. All three guanylins showed similar potency in the middle segment, but guanylin was more potent in the posterior segment. 8-bromo cGMP mimicked the effect of guanylins. The intestinal response to guanylin was smaller in FW eels. The mucosal presence of NPPB utilized as a CFTR blocker, but not of other inhibitors of the channels/transporters localized on the luminal surface in SW fish intestine, inhibited the guanylin-induced decrease in Isc. In eels, therefore, the guanylin family may be involved in osmoregulation by the intestine by binding to the receptors and activating CFTR-like channels on the mucosal side through cGMP production, perhaps resulting in Cl(-) and HCO3(-) secretion into the lumen.     

Notch2 is required for formation of the placental circulatory system, but not for cell-type specification in the developing mouse placenta

We have previously reported that a mutation in the ankyrin repeats of mouse Notch2 results in embryonic lethality by embryonic day 11.5 (E11.5), showing developmental retardation at E10.5. This indicated that Notch2 plays an essential role in postimplantation development in mice. Here, we demonstrate that whole embryo culture can circumvent developmental retardation of Notch2 mutant embryos for up to 1 day, suggesting that the lethality was primarily caused by extraembryonic defects. Histological examinations revealed delayed entry of maternal blood into the mutant placenta and poor blood sinus formation at later stages. Notch2-expressing cells appeared around maternal blood sinuses. Specification of trophoblast subtypes appeared not to be drastically disturbed and expression of presumptive downstream genes of Notch2 signaling was not altered by the Notch2 mutation. Thus, in the developing mouse placenta, Notch2 is unlikely to be involved in cell fate decisions, but rather participates in formation of maternal blood sinuses. In aggregation chimeras with wild-type tetraploid embryos, the mutant embryos developed normally until E12.5, but died before E13.5. The chimeric placentas showed a restored maternal blood sinus formation when compared with the mutant placentas, but not at the level of wild-type diploid placentas. Therefore, it was concluded that the mutant suffers from defects in maternal blood sinus formation. Thus, Notch2 is not cell autonomously required for the early cell fate determination of subtypes of trophoblast cells, but plays an indispensable role in the formation of maternal blood sinuses in the developing mouse placenta.