Foreword: from the TRANSCRIPTOME conferences to the SYSTEMOSCOPE international consortium

This thematic issue issue of the Comptes rendus Biologies contains review articles, original papers and conference reports presented at the first two TRANSCRIPTOME conferences From Functional Genomics to Systems Biology and IMAGE Consortium Invitational workshops (Paris, November 2000 and Seattle, March 2002), and discussed during the inaugural meetings of the SYSTEMOSCOPE International Consortium (Paris, June 2003). We describe the founding principles, missions, working plan and policy for partnership and industrial development of SYSTEMOSCOPE to promote the study of the complexity of biological systems by integrating scientific, medical, ethical and economic issues in implementation of interdisciplinary projects for human health.     

<Emphasis Type="Italic">Tropicibacter phthalicus</Emphasis> sp. nov., A Phthalate-Degrading Bacterium from Seawater

An aerobic, Gram-negative bacterial strain, designated KU27E1^T, which degrades phthalate and dimethylphthalate, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile rods with polar flagella. Strain KU27E1^T grew at 15–30°C, pH 6.0–8.0, in the presence of 1.0–2.0% (w/v) NaCl. The 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria, and was most closely related to Tropicibacter naphthalenivorans (96.8%). The predominant respiratory lipoquinone was ubiquinone-10, and the major cellular fatty acid was C_18:1ω7c (88.5%). The G+C content of genomic DNA was 58.7 mol%. Based on the physiological, chemotaxonomic, and phylogenetic data, strain KU27E1^T is suggested to represent a novel species of the genus Tropicibacter, for which the name Tropicibacter phthalicus sp. nov. is proposed. The type strain of Tropicibacter phthalicus is designated as KU27E1^T (=JCM 17793^T = KCTC 23703^T).     

Maricurvus nonylphenolicus gen. nov., sp. nov., a nonylphenol-degrading bacterium isolated from seawater

A novel aerobic, Gram-negative bacterial strain, designated KU41E(T), which degrades p-n-nonylphenol, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile, curved rods with a single polar flagellum. Strain KU41E(T) grew at 20-35 °C, pH 7.0-8.0, in the presence of 1.0-4.0% NaCl. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were summed feature 3 (C(15:0) iso 2OH and/or C(16:1) ω7c, 28.4%), C(18:1) ω7c (19.8%), and C(16:0) (17.0%). The DNA G + C content was 48.6 mol%. The 16S rRNA gene sequence analysis indicated that strain KU41E(T) is affiliated with the order Alteromonadales within the class Gammaproteobacteria and is most closely related to Pseudoteredinibacter isoporae SW-11(T) (93.6% similarity) and Teredinibacter turnerae T7902(T) (91.9% similarity). On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41E(T) is suggested to represent a novel species of a new genus, for which the name Maricurvus nonylphenolicus gen. nov., sp. nov. is proposed. The type strain of M. nonylphenolicus is KU41E(T) (=JCM 17778(T)).     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Functional reconstitution and characterization of the Arabidopsis Mg2+ transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg²⁺) plays critical role in many physiological processes. The mechanism of Mg²⁺ transport has been well documented in bacteria; however, less is known about Mg²⁺ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg²⁺ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg²⁺ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg²⁺ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg²⁺ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg²⁺ uptake. The AtMRS2-10-mediated Mg²⁺ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co²⁺ and Ni²⁺; however, it was not inhibited by Ca²⁺, Fe²⁺, or Fe³⁺. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al³⁺. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships.     

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.     

Gene pathway analysis of the mechanism by which the Rho-associated kinase inhibitor Y-27632 inhibits apoptosis in isolated thawed human embryonic stem cells

Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1β, TGF-β, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1β, TGF-β, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-β receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.     

Activation of macrophage-stimulating protein by human airway trypsin-like protease

Macrophage-stimulating protein (MSP) circulates as a proform protein and requires proteolytic processing for activation. Respiratory ciliated cells express the MSP receptor, recepteur d'origine nantais (RON), at the apical surface, which reportedly has an important role in ciliary function. Like RON, human airway trypsin-like protease (HAT) is also expressed at the apical surface of ciliated cells. Here we show that HAT cleaves proMSP at the physiological activation site, Arg483-Val484. MSP processed by HAT could induce chemotactic responses and morphological changes of peritoneal macrophages. In human respiratory epithelial cells, knock down of HAT expression reduced proMSP processing and RON autophosphorylation. We suggest that HAT is important for MSP-RON signaling in the respiratory tract.     

Altered expression of diacylglycerol kinase isozymes in regenerating liver

The liver possesses the capacity to restore its function and mass after injury. Liver regeneration is controlled through complicated mechanisms, in which the phosphoinositide (PI) cycle is shown to be activated in hepatocytes. Using a rat partial hepatectomy (PH) model, the authors investigated the expression of the diacylglycerol kinase (DGK) family, a key enzyme in the PI cycle, which metabolizes a lipid second-messenger diacylglycerol (DG). RT-PCR analysis shows that DGKζ and DGKα are the major isozymes in the liver. Results showed that in the process of regeneration, the DGKζ protein, which is detected in the nucleus of a small population of hepatocytes in normal liver, is significantly increased in almost all hepatocytes. However, the mRNA levels remain largely unchanged. Double labeling with bromodeoxyuridine (BrdU), an S phase marker, reveals that DGKζ is expressed independently of DNA synthesis or cell proliferation. However, DGKα protein localizes to the cytoplasm in normal and regenerating livers, but immunoblot analysis reveals that the expected (80 kDa) and the lower (70 kDa) bands are detected in normal liver, whereas at day 10 after PH, the expected band is solely recognized, showing a different processing pattern of DGKα in liver regeneration. These results suggest that DGKζ and DGKα are involved, respectively, in the nucleus and the cytoplasm of hepatocytes in regenerating liver.     

Planar cell polarity protein localization in the secretory ameloblasts of rat incisors

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.