Collective oscillations in developing cells: insights from simple systems

From hormonal secretion to gene expression, multicellular dynamics are rich in oscillatory regulation. When organized in space and time, periodic cell-cell signaling can give rise to long-range coordination of gene expression and cell movement in tissues. Lack of synchrony of the oscillations on the other hand can serve as a source of initial divergence of cell fate in stem cells. How properties of individual cells can account for collective rhythmic behaviors at the tissue level remains elusive in most cases. Recently, studies in chemical reactions, synthetic gene circuits, yeast and social amoeba Dictyostelium have greatly enhanced our view of collective oscillations in cell populations. From these relatively simple systems, a unified view of how excitable and oscillatory regulations could be tuned and coupled to give rise to tissue-level oscillations is emerging. The review focuses on recent progress in cyclic adenosine monophosphate oscillations in Dictyostelium and highlights similarities and differences with other systems. We will see that the autonomy of single-cell level oscillations and different ways in which cells are coupled influence how group-level information can be encoded in collective oscillations.     

Effects of rosuvastatin on electronegative LDL as characterized by capillary isotachophoresis: the ROSARY Study

Electronegative LDL, a charge-modified LDL (cm-LDL) subfraction that is more negatively charged than normal LDL, has been shown to be inflammatory. We previously showed that pravastatin and simvastatin reduced the electronegative LDL subfraction, fast-migrating LDL (fLDL), as analyzed by capillary isotachophoresis (cITP). The present study examined the effects of rosuvastatin on the more electronegative LDL subfraction, very-fast-migrating LDL (vfLDL), and small, dense charge-modified LDL (sd-cm-LDL) subfractions. Patients with hypercholesterolemia or those who were being treated with statins (n = 81) were treated with or switched to 2.5 mg/d rosuvastatin for 3 months. Rosuvastatin treatment effectively reduced cITP cm-LDL subfractions of LDL (vfLDL and fLDL) or sdLDL (sd-vfLDL and sd-fLDL), which were closely related to each other but were different from the normal subfraction of LDL [slow-migrating LDL (sLDL)] or sdLDL (sd-sLDL) in their relation to the levels of remnant-like particle cholesterol (RLP-C), apolipoprotein (apo) C-II, and apoE. The percent changes in cm-LDL or sd-cm-LDL caused by rosuvastatin were correlated with those in the particle concentrations of LDL or sdLDL measured as LDL-apoB or sdLDL-apoB and the levels of HDL-C, RLP-C, apoC-II, and apoE. In conclusion, rosuvastatin effectively reduced both the vfLDL subfraction and sd-cm-LDL subfractions as analyzed by cITP.     

Stochastic thermodynamic limit on E. coli adaptation by information geometric approach

Biological systems process information under noisy environment. Sensory adaptation model of E. coli is suitable for investigation because of its simplicity. To understand the adaptation processing quantitatively, stochastic thermodynamic approach has been attempted. Information processing can be assumed as state transition of a system that consists of signal transduction molecules using thermodynamic approach, and efficiency can be measured as thermodynamic cost. Recently, using information geometry and stochastic thermodynamics, a relationship between speed of the transition and the thermodynamic cost has been investigated for a chemical reaction model. Here, we introduce this approach to sensory adaptation model of E. coli, and examined a relationship between adaptation speed and the thermodynamic cost, and efficiency of the adaptation speed. For increasing external noise level in stimulation, the efficiency decreased, but the efficiency was highly robust to external stimulation strength. Moreover, we demonstrated that there is the best noise to achieve the adaptation in the aspect of thermodynamic efficiency. Our quantification method provides a framework to understand the adaptation speed and the thermodynamic cost for various biological systems.     

Identification,cDNA cloning and possible roles of seed-specific rice asparaginyl endopeptidase,REP-2

We previously showed that two major cysteine endopeptidases, REP-1 and REP-2, were present in germinated rice ( Oryza sativa L.) seeds, and that REP-1 was the enzyme that digests seed storage proteins. The present study shows that REP-2 is an asparaginyl endopeptidase that acts as an activator of REP-1, and we separated it into two forms, REP-2alpha (39 kDa) and REP-2beta (40 kDa), using ion-exchange chromatography and gel filtration chromatography. Although analysis of the amino terminals revealed that 10 amino acids of both forms were identical, their isoelectric points were different. SDS-PAGE/immunoblot analysis using an antiserum raised against legumain, an asparaginyl endopeptidase from jack bean, indicated that both forms were present in maturing and germinating rice seeds, and that their amounts transiently decreased in dry seeds. Northern blot analysis indicated that REP-2 mRNA was expressed in both maturing and germinating seeds. In germinating seeds, the mRNA was detected in aleurone layers but not in shoot and root tissues. Incubation of the de-embryonated seeds in 10(-6) M gibberellic acid induced the production of large amounts of REP-1, whereas REP-2beta levels declined rapidly. Southern blot analysis showed that there is one gene for REP-2 in the genome, indicating that both REP-2 enzymes are generated from a single gene. The structure of the gene was similar to that of beta-VPE and gamma-VPE isolated from Arabidopsis thaliana.     

Corynebacterium diphtheriae: identification and characterization of a channel-forming protein in the cell wall

The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8_r(−) Tox⁻ (= ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.     

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.     

Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”