Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4⁺ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4⁺ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.     

Enhancement of aromatic amino acid-nucleic acid base stacking interaction by metal coordination to base: fluorescence study on a tryptophan-Pt(II)-guanine ternary complex

In order to investigate the effect of the Pt(II) ion on the stacking interaction between tryptophan and a guanine base, the quenching of Trp fluorescence was monitored for some systems in the absence and presence of the metal ion, and the association constants were obtained by the analysis of Eadie-Hofstee plots. All spectral data suggested that the stacking interaction is enhanced by the Pt(II) coordination to the guanine N7 atom. The result indicates the importance of the metal ion as a bookmark in the specific recognition of a nucleic acid base by an aromatic amino acid residue.     

Concerted actions of DnaA complexes with DNA-unwinding sequences within and flanking replication origin oriC promote DnaB helicase loading

Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA-binding site, which stimulates formation of the Left/Right-DnaA subcomplexes. In addition, a DUE-flanking AT-cluster element (TATTAAAAAGAA) is located just outside of the minimal oriC region. The Left-DnaA subcomplex promotes unwinding of the flanking DUE exposing TT[A/G]T(T) sequences that then bind to the Left-DnaA subcomplex, stabilizing the unwound state required for DnaB helicase loading. However, the role of the Right-DnaA subcomplex is largely unclear. Here, we show that DUE unwinding by both the Left/Right-DnaA subcomplexes, but not the Left-DnaA subcomplex only, was stimulated by a DUE-terminal subregion flanking the AT-cluster. Consistently, we found the Right-DnaA subcomplex–bound single-stranded DUE and AT-cluster regions. In addition, the Left/Right-DnaA subcomplexes bound DnaB helicase independently. For only the Left-DnaA subcomplex, we show the AT-cluster was crucial for DnaB loading. The role of unwound DNA binding of the Right-DnaA subcomplex was further supported by in vivo data. Taken together, we propose a model in which the Right-DnaA subcomplex dynamically interacts with the unwound DUE, assisting in DUE unwinding and efficient loading of DnaB helicases, while in the absence of the Right-DnaA subcomplex, the AT-cluster assists in those processes, supporting robustness of replication initiation.

The initiation of bacterial DNA replication requires local duplex unwinding of the chromosomal replication origin oriC, which is regulated by highly ordered initiation complexes. In Escherichia coli, the initiation complex contains oriC, the ATP-bound form of the DnaA initiator protein (ATP–DnaA), and the DNA-bending protein IHF (Fig. 1, A and B), which promotes local unwinding of oriC (1, 2, 3, 4). Upon this oriC unwinding, two hexamers of DnaB helicases are bidirectionally loaded onto the resultant single-stranded (ss) region with the help of the DnaC helicase loader (Fig. 1B), leading to bidirectional chromosomal replication (5, 6, 7, 8). However, the fundamental mechanism underlying oriC-dependent bidirectional DnaB loading remains elusive.Open in a separate windowFigure 1Schematic structures of oriC, DnaA, and the initiation complexes. A, the overall structure of oriC. The minimal oriC region and the AT-cluster region are indicated. The sequence of the AT-cluster−DUE (duplex-unwinding element) region is also shown below. The DUE region (DUE; pale orange bars) contains three 13-mer repeats: L-DUE, M-DUE, and R-DUE. DnaA-binding motifs in M/R-DUE, TT(A/G)T(T), are indicated by red characters. The AT-cluster region (AT cluster; brown bars) is flanked by DUE outside of the minimal oriC. The DnaA-oligomerization region (DOR) consists of three subregions called Left-, Middle-, and Right-DOR. B, model for replication initiation. DnaA is shown as light brown (for domain I–III) and darkbrown (for domain IV) polygons (right panel). ATP–DnaA forms head-to-tail oligomers on the Left- and Right-DORs (left panel). The Middle-DOR (R2 box)-bound DnaA interacts with DnaA bound to the Left/Right-DORs using domain I, but not domain III, stimulating DnaA assembly. IHF, shown as purple hexagons, bends DNA >160° and supports DUE unwinding by the DnaA complexes. M/R-DUE regions are efficiently unwound. Unwound DUE is recruited to the Left-DnaA subcomplex and mainly binds to R1/R5M-bound DnaA molecules. The sites of ssDUE-binding B/H-motifs V211 and R245 of R1/R5M-bound DnaA molecules are indicated (pink). Two DnaB homohexamer helicases (light green) are recruited and loaded onto the ssDUE regions with the help of the DnaC helicase loader (cyan). ss, single stranded.The minimal oriC region consists of the duplex unwinding element (DUE) and the DnaA oligomerization region (DOR), which contains specific arrays of 9-mer DnaA-binding sites (DnaA boxes) with the consensus sequence TTA[T/A]NCACA (Fig. 1A) (3, 4). The DUE underlies the local unwinding and contains 13-mer AT-rich sequence repeats named L-, M-, and R-DUE (9). The M/R-DUE region includes TT[A/G]T(A) sequences with specific affinity for DnaA (10). In addition, a DUE-flanking AT-cluster (TATTAAAAAGAA) region resides just outside of the minimal oriC (Fig. 1A) (11). The DOR is divided into three subregions, the Left-, Middle-, and Right-DORs, where DnaA forms structurally distinct subcomplexes (Fig. 1A) (8, 12, 13, 14, 15, 16, 17). The Left-DOR contains high-affinity DnaA box R1, low-affinity boxes R5M, τ1−2, and I1-2, and an IHF-binding region (17, 18, 19, 20). The τ1 and IHF-binding regions partly overlap (17).In the presence of IHF, ATP–DnaA molecules cooperatively bind to R1, R5M, τ2, and I1-2 boxes in the Left-DOR, generating the Left-DnaA subcomplex (Fig. 1B) (8, 17). Along with IHF causing sharp DNA bending, the Left-DnaA subcomplex plays a leading role in DUE unwinding and subsequent DnaB loading. The Middle-DOR contains moderate-affinity DnaA box R2. Binding of DnaA to this box stimulates DnaA assembly in the Left- and Right-DORs using interaction by DnaA N-terminal domain (Fig. 1B; also see below) (8, 12, 14, 16, 21). The Right-DOR contains five boxes (C3-R4 boxes) and cooperative binding of ATP–DnaA molecules to these generates the Right-DnaA subcomplex (Fig. 1B) (12, 18). This subcomplex is not essential for DUE unwinding and plays a supportive role in DnaB loading (8, 15, 17). The Left-DnaA subcomplex interacts with DnaB helicase, and the Right-DnaA subcomplex has been suggested to play a similar role (Fig. 1B) (8, 13, 16).In the presence of ATP–DnaA, M- and R-DUE adjacent to the Left-DOR are predominant sites for in vitro DUE unwinding: unwinding of L-DUE is less efficient than unwinding of the other two (Fig. 1B) (9, 22, 23). Deletion of L-DUE or the whole DUE inhibits replication of oriC in vitro moderately or completely, respectively (23). A chromosomal oriC Δ(AT-cluster−L-DUE) mutant with an intact DOR, as well as deletion of Right-DOR, exhibits limited inhibition of replication initiation, whereas the synthetic mutant combining the two deletions exhibits severe inhibition of cell growth (24). These studies suggest that AT-cluster−L-DUE regions stimulate replication initiation in a manner concerted with Right-DOR, although the underlying mechanisms remain elusive.DnaA consists of four functional domains (Fig. 1B) (4, 25). Domain I supports weak domain I–domain I interaction and serves as a hub for interaction with various proteins such as DnaB helicase and DiaA, which stimulates ATP–DnaA assembly at oriC (26, 27, 28, 29, 30). Two or three domain I molecules of the oriC–DnaA subcomplex bind a single DnaB hexamer, forming a stable higher-order complex (7). Domain II is a flexible linker (28, 31). Domain III contains AAA+ (ATPase associated with various cellular activities) motifs essential for ATP/ADP binding, ATP hydrolysis, and DnaA–DnaA interactions in addition to specific sites for ssDUE binding and a second, weak interaction with DnaB helicase (1, 4, 8, 10, 19, 25, 32, 33, 34, 35). Domain IV bears a helix-turn-helix motif with specific affinity for the DnaA box (36).As in typical AAA+ proteins, a head-to-tail interaction underlies formation of ATP–DnaA pentamers on the DOR, where the AAA+ arginine-finger motif Arg285 recognizes ATP bound to the adjacent DnaA protomer, promoting cooperative ATP–DnaA binding (Fig. 1B) (19, 32). DnaA ssDUE-binding H/B-motifs (Val211 and Arg245) in domain III sustain stable unwinding by directly binding to the T-rich (upper) strand sequences TT[A/G]T(A) within the unwound M/R-DUE (Fig. 1B) (8, 10). Val211 residue is included in the initiator-specific motif of the AAA+ protein family (10). For DUE unwinding, ssDUE is recruited to the Left-DnaA subcomplex via DNA bending by IHF and directly interacts with H/B-motifs of DnaA assembled on Left-DOR, resulting in stable DUE unwinding competent for DnaB helicase loading; in particular, DnaA protomers bound to R1 and R5M boxes play a crucial role in the interaction with M/R-ssDUE (Fig. 1B) (8, 10, 17). Collectively, these mechanisms are termed ssDUE recruitment (4, 17, 37).Two DnaB helicases are thought to be loaded onto the upper and lower strands of the region including the AT-cluster and DUE, with the aid of interactions with DnaC and DnaA (Fig. 1B) (25, 38, 39). DnaC binding modulates the closed ring structure of DnaB hexamer into an open spiral form for entry of ssDNA (40, 41, 42, 43). Upon ssDUE loading of DnaB, DnaC is released from DnaB in a manner stimulated by interactions with ssDNA and DnaG primase (44, 45). Also, the Left- and Right-DnaA subcomplexes, which are oriented opposite to each other, could regulate bidirectional loading of DnaB helicases onto the ssDUE (Fig. 1B) (7, 8, 35). Similarly, recent works suggest that the origin complex structure is bidirectionally organized in both archaea and eukaryotes (1, 46). In Saccharomyces cerevisiae, two origin recognition complexes containing AAA+ proteins bind to the replication origin region in opposite orientations; this, in turn, results in efficient loading of two replicative helicases, leading to head-to-head interactions in vitro (46). Consistent with this, origin recognition complex dimerization occurs in the origin region during the late M-G1 phase (47). The fundamental mechanism of bidirectional origin complexes might be widely conserved among species.In this study, we analyzed various mutants of oriC and DnaA in reconstituted systems to reveal the regulatory mechanisms underlying DUE unwinding and DnaB loading. The Right-DnaA subcomplex assisted in the unwinding of oriC, dependent upon an interaction with L-DUE, which is important for efficient loading of DnaB helicases. The AT-cluster region adjacent to the DUE promoted loading of DnaB helicase in the absence of the Right-DnaA subcomplex. Consistently, the ssDNA-binding activity of the Right-DnaA subcomplex sustained timely initiation of growing cells. These results indicate that DUE unwinding and efficient loading of DnaB helicases are sustained by concerted actions of the Left- and Right-DnaA subcomplexes. In addition, loading of DnaB helicases are sustained by multiple mechanisms that ensure robust replication initiation, although the complete mechanisms are required for precise timing of initiation during the cell cycle.     

Inference of S-system models of genetic networks using a cooperative coevolutionary algorithm

MOTIVATION: To resolve the high-dimensionality of the genetic network inference problem in the S-system model, a problem decomposition strategy has been proposed. While this strategy certainly shows promise, it cannot provide a model readily applicable to the computational simulation of the genetic network when the given time-series data contain measurement noise. This is a significant limitation of the problem decomposition, given that our analysis and understanding of the genetic network depend on the computational simulation. RESULTS: We propose a new method for inferring S-system models of large-scale genetic networks. The proposed method is based on the problem decomposition strategy and a cooperative coevolutionary algorithm. As the subproblems divided by the problem decomposition strategy are solved simultaneously using the cooperative coevolutionary algorithm, the proposed method can be used to infer any S-system model ready for computational simulation. To verify the effectiveness of the proposed method, we apply it to two artificial genetic network inference problems. Finally, the proposed method is used to analyze the actual DNA microarray data.     

Further studies on the relationship between tyrosine supply and catecholamine production in cultured adrenal chromaffin cells.

To elucidate a possible role of tyrosine supply as a factor modulating catecholamine biosynthesis in the adrenergic cell, the transport of [14C]tyrosine into cultured bovine adrenal chromaffin cells was first examined, and the relationship between [14C]tyrosine transport and [14C]catecholamine formation was then investigated. Under the conditions which were routinely employed to determine the rate of catecholamine biosynthesis, tyrosine was taken up into the cells in a manner independent of extracellular Na+ and Ca2+, and this uptake was also insensitive to ouabain and various metabolic inhibitors. The stimulation of these cells with high K+ and other secretagogues caused no significant alteration in the uptake. While, tyrosine transport was markedly inhibited by tyrosine analogues and other L-aromatic amino acids, and this inhibition was accompanied by the reduction of [14C]catecholamine formation. In contrast, tyrosine transport was markedly enhanced by flavone, and this enhancement was also accompanied by the augmentation of catecholamine production under the same experimental conditions. These results seem to indicate that the transport of tyrosine into the cells may be closely related to catecholamine formation within the cells, thus providing an evidence for a possible role of tyrosine supply as one of the factors affecting catecholamine production in the adrenal chromaffin cell.     

A Japanese child with geleophysic dysplasia caused by a novel mutation of FBN1

Geleophysic dysplasia (GD) is a rare disorder characterized by severe short stature, short hands and feet, limited joint mobility, skin thickening, characteristic facial features (e.g., a “happy” face), and cardiac valvular disorders that often result in an early death. The genes ADAMTSL2 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif-like 2) and FBN1 (fibrillin 1) were recently identified as causative genes for GD. Here, we describe a 10-year-old Japanese female with GD who was born to non-consanguineous parents. At the age of 11 months, she was referred to our hospital because of very short stature for her age (− 4.4 standard deviations of the age-matched value) and a “happy” face with full cheeks, a shortened nose, hypertelorism, and a long and flat philtrum, characteristic of GD. Her hands and feet were small, her skin was thickened, and her joint mobility was generally limited. She had cardiac valvular disorders and history of recurrent respiratory failure. Mutation analysis revealed no abnormalities in ADAMTSL2. However, analysis of FBN1 revealed a novel heterozygous mutation (c.5161T > T/G) in exon 41, which encodes transforming growth factor-β-binding protein-like domain 5 (TB5). GD is an extremely rare disorder and, to our knowledge, only one case of GD with an FBN1 mutation has been reported in Japan. Similar to the previously reported cases of GD, the mutation in the current patient was located in the TB5 domain, which suggests that abnormalities in this domain of FBN1 are responsible for GD.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells.