Electronic phase transition and an anomalous ordered phase in Ba2Ti13O22 with 3d1 ions on a triangle-based lattice.

We found that Ba(2)Ti(13)O(22) with Ti(3+) (3d(1)) ions on a triangle-based lattice exhibits a phase transition at T(c)~200 K, below which the increase of electrical resistivity and decrease of magnetic susceptibility were observed. Transmission electron microscopy and optical reflectivity measurements indicate that the low-temperature phase of the present compound shares characteristics in common with a charge-density-wave state with remnant carriers, although a commensurate wave vector of the modulation and a linear temperature dependence of the magnetic susceptibility below T(c) suggest an exotic ordered state.

Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.

Predicting habitual water intake from lifestyle questions.

OBJECTIVE: Previous studies have used selective recall and descriptive dietary record methods, requiring considerable effort for assessing food and water intake. This study created a simplified lifestyle questionnaire to predict habitual water intake (SQW), accurately and quickly assessing the habitual water intake. We also evaluated the validity using descriptive dietary records as a cross-sectional study. SUBJECTS AND METHODS: First, we used crowdsourcing and machine learning to collect data, predict water intake records, and create questionnaires. We collected 305 lifestyle-related questions as predictor variables and selective recall methods for assessing water intake as an outcome variable. Random forests were used for the machine learning models because of their interpretability and accurate estimation. Random forest and single regression correlation analysis were augmented by the synthetic minority oversampling that trained the model. We separated the data by sex and evaluated our model using unseen hold-out testing data, predicting the individual and overall habitual water intake from various sources, including non-alcoholic beverages, alcohol, and food. RESULTS: We found a 0.60 Spearman's correlation coefficient for total water intake between the predicted and the selective recall method values, reflecting the target value to be achieved. This question set was then used for feasibility tests. The descriptive dietary record method helped to obtain a ground-truth value. We categorized the data by gender, season, and source: non-alcoholic beverages, alcohol, food, and total water intake, and the correlation was confirmed. Consequently, our results showed a Pearson's correlation coefficient of 0.50 for total water intake between the predicted and the selective recall method values. CONCLUSIONS: We hypothesize that dissemination of SQW can lead to better health management by easily determining the habitual water intake.

A SOS3 homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na+ exclusion trait.

Genes that enable crops to limit Na(+) accumulation in shoot tissues represent potential sources of salinity tolerance for breeding. In barley, the HvNax4 locus lowered shoot Na(+) content by between 12% and 59% (g(-1) DW), or not at all, depending on the growth conditions in hydroponics and a range of soil types, indicating a strong influence of environment on expression. HvNax4 was fine-mapped on the long arm of barley chromosome 1H. Corresponding intervals of ∼200 kb, containing a total of 34 predicted genes, were defined in the sequenced rice and Brachypodium genomes. HvCBL4, a close barley homologue of the SOS3 salinity tolerance gene of Arabidopsis, co-segregated with HvNax4. No difference in HvCBL4 mRNA expression was detected between the mapping parents. However, genomic and cDNA sequences of the HvCBL4 alleles were obtained, revealing a single Ala111Thr amino acid substitution difference in the encoded proteins. The known crystal structure of SOS3 was used as a template to obtain molecular models of the barley proteins, resulting in structures very similar to that of SOS3. The position in SOS3 corresponding to the barley substitution does not participate directly in Ca(2+) binding, post-translational modifications or interaction with the SOS2 signalling partner. However, Thr111 but not Ala111 forms a predicted hydrogen bond with a neighbouring α-helix, which has potential implications for the overall structure and function of the barley protein. HvCBL4 therefore represents a candidate for HvNax4 that warrants further investigation.

Expression and function of a nonglycosylated major histocompatibility class I antigen.

The major histocompatibility class I antigens, expressed in most somatic cells, have carbohydrate moieties. We constructed mutant mouse MHC class I genes in which codons for the N-linked glycosylation sites were replaced by those of other amino acids. L cell transformants expressing the nonglycosylated class I antigens allowed us to investigate biological roles of carbohydrates with the highest specificity possible. The nonglycosylated antigen was unchanged in its overall serological specificities, and was recognized by alloreactive cytotoxic T cells. Further, the antigen was capable of mediating cytotoxic activity of vesicular stomatitis virus-specific T cells. These studies indicate that carbohydrates are not essential for immunological function of the MHC class I antigens. Cell surface expression of the nonglycosylated antigen was markedly reduced as compared with the native antigen, which was not attributable to accelerated degradation or rapid shedding. We conclude that the primary role of carbohydrates of the class I antigens is to facilitate the intracellular transport of the nascent proteins to the plasma membrane. The possible involvement of carbohydrate-receptor interactions in this process is discussed.

Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis.

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.

Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes.

The cytotoxicity of reactive oxygen intermediates (ROIs) has been implicated in the destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Thioredoxin (TRX), a redox (reduction/oxidation)-active protein, has recently been shown to protect cells from oxidative stress and apoptosis. To elucidate the roles of oxidative stress in the development of autoimmune diabetes in vivo, we produced nonobese diabetic transgenic mice that overexpress TRX in their pancreatic beta cells. In these transgenic mice, the incidence of diabetes was markedly reduced, whereas the development of insulitis was not prevented. Moreover, induction of diabetes by streptozotocin, an ROI-generating agent, was also attenuated by TRX overexpression in beta cells. This is the first direct demonstration that an antioxidative and antiapoptotic protein protects beta cells in vivo against both autoimmune and drug-induced diabetes. Our results strongly suggest that oxidative stress plays an essential role in the destruction of beta cells by infiltrating inflammatory cells in IDDM.

Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor-deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock.

Formation of a three-dimensional network of V trimers in A2V13O22 (A=Ba, Sr).

We found that in A2V13O22 (A=Ba, Sr), which contains a trilayer slab of VO in the sodium-chloride structure with periodically missing ions, the trimerization of V ions occurs at 290 K (A=Ba) and 380 K (A=Sr). V trimers form a three-dimensional network, but some V ions remain untrimerized in these compounds. The suppression of magnetic susceptibility with trimerization and the existence of a Curie tail at low temperatures, together with the result of NMR measurement, indicate that the V trimers are spin singlet, whereas the untrimerized V ions have a magnetic moment; i.e., there is a spontaneous separation between nonmagnetic and magnetic ions in the crystal.

Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2.

The role of Lefty2 in left-right patterning was investigated by analysis of mutant mice that lack asymmetric expression of lefty2. These animals exhibited various situs defects including left isomerism. The asymmetric expression of nodal was prolonged and the expression of Pitx2 was upregulated in the mutant embryos. The absence of Lefty2 conferred on Nodal the ability to diffuse over a long distance. Thus, Nodal-responsive genes, including Pitx2, that are normally expressed on the left side were expressed bilaterally in the mutant embryos, even though nodal expression was confined to the left side. These results suggest that Nodal is a long-range signaling molecule but that its range of action is normally limited by the feedback inhibitor Lefty2.

Physical analysis of the complex rye (Secale cereale L.) Alt4 aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco.

Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library's suitability for investigating Al tolerance genes. The library provides 6 x genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus.

Suppression of transcription factor PDX-1/IPF1/STF-1/IDX-1 causes no decrease in insulin mRNA in MIN6 cells.

The insulin gene transcription factor PDX-1/IPF1/STF-1/ IDX-1 plays a key role in directing beta cell-specific gene expressions. Recently, impairment of PDX-1 expression or activity has been observed in beta cell-derived HIT cells cultured under high glucose concentrations, and this has been suggested as a possible cause of the decrease in insulin gene transcription. To investigate the pathophysiological significance of PDX-1 as a determinant of the rate of insulin gene transcription, we suppressed its expression in beta cell-derived MIN6 cells using an antisense oligodeoxynucleotide (ODN) and searched for possible changes in the beta cell-specific gene expression. Treatment of MIN6 cells with an 18-mer phosphorothioate ODN complementary to a sequence starting at the translation initiation codon of PDX-1 caused a potent, concentration-dependent reduction in PDX-1 expression; addition of 2 microM antisense ODN could reduce PDX-1 expression to 14+/-4% of the control. There was also a decrease in its DNA binding to the insulin gene A element. Despite such suppression of PDX-1, Northern blot analysis revealed no decrease in the amount of insulin mRNA in the MIN6 cells. Similarly, no changes were detected in the transcription of the glucokinase or islet amyloid polypeptide gene, for which PDX-1 was shown to function as a transcription factor. Thus, our findings dispute the physiological significance of PDX-1 in determining the rate of insulin gene transcription. This means that other components constituting the transcription-controlling machinery need to be evaluated in order to understand the molecular basis of impaired insulin biosynthesis such as that observed due to glucose toxicity.

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.

Interferons increase transcription of a major histocompatibility class I gene via a 5' interferon consensus sequence.

Interferons (IFNs) augment expression of major histocompatibility class I genes in many cells. To study the effect of IFNs on transcription of class I genes, we prepared and tested activity of chloramphenicol acetyltransferase (CAT) hybrid genes in which the cat gene is under the control of the 5' flanking region of the murine H-2Ld gene. NIH 3T3 cells transiently transfected with a cat construct having the sequence from position -210 to -134 showed a four- to fivefold increase in CAT activity when treated with IFN-alpha/beta. This sequence contains the IFN consensus sequence (ICS) shared among IFN-inducible genes, as well as the class I regulatory element (CRE) that controls up and down regulation of class I gene expression. To determine the precise sequence requirement for the IFN action, the ICS and CRE were independently placed upstream of the class I or a heterologous simian virus 40 promoter, and CAT activity was tested. The ICS, but not the CRE, enhanced activity of both promoters by about twofold upon exposure to IFN-alpha/beta, although greater responses were noted when the ICS and CRE were combined. These results demonstrate that the ICS alone is capable of enhancing promoter activity in response to IFN-alpha/beta treatment and that the CRE exerts a synergistic effect. Further, we show that the ICS functions as an inducible enhancer since it acts regardless of its orientation and distance in the simian virus 40 promoter.

Binding of multiple nuclear factors to the 5' upstream regulatory element of the murine major histocompatibility class I gene.

Transcription of mouse major histocompatibility complex class I genes is controlled by the conserved class I regulatory element (CRE) in the 5' flanking region. The CRE, approximately 40 base pairs long, acts as a negative control element in undifferentiated F9 embryonal carcinoma cells which do not express the major histocompatibility complex genes. The same element, however, acts as a positive control element in cells expressing the genes at high levels. To investigate the molecular basis of the regulatory role of the CRE, we studied the binding of nuclear proteins to the CRE of the H-2Ld gene by gel mobility shift and methylation interference experiments. Nuclear extracts from L fibroblasts and LH8 T lymphocytes revealed three distinct factors that bind discrete sequences within the CRE. The three sequences correspond to the inverted and direct repeats within the CRE. In contrast, F9 extracts exhibited factor binding to only two of the three sequences and lack a major factor detected in the above two cell types. Protein-binding sites within each of the three sequences were identified by methylation interference experiments. These data were in full agreement with results obtained by a competition assay performed with a series of mutant oligonucleotides containing a few nucleotide substitutions in each of the three regions. The results illustrate complex DNA-protein interactions in which several independent proteins bind to overlapping sequences in the CRE in a cell type-specific fashion.

Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas.

Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development.

Loss of hippocampal CA3 pyramidal neurons in mice lacking STAM1.

STAM1, a member of the STAM (signal transducing adapter molecule) family, has a unique structure containing a Src homology 3 domain and ITAM (immunoreceptor tyrosine-based activation motif). STAM1 was previously shown to be associated with the Jak2 and Jak3 tyrosine kinases and to be involved in the regulation of intracellular signal transduction mediated by interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Here we generated mice lacking STAM1 by using homologous recombination with embryonic stem cells. STAM1(-/-) mice were morphologically indistinguishable from their littermates at birth. However, growth retardation in the third week after birth was observed for the STAM1(-/-) mice. Unexpectedly, despite the absence of STAM1, hematopoietic cells, including T- and B-lymphocyte and other hematopoietic cell populations, developed normally and responded well to several cytokines, including IL-2 and GM-CSF. However, histological analyses revealed the disappearance of hippocampal CA3 pyramidal neurons in STAM1(-/-) mice. Furthermore, we observed that primary hippocampal neurons derived from STAM1(-/-) mice are vulnerable to cell death induced by excitotoxic amino acids or an NO donor. These data suggest that STAM1 is dispensable for cytokine-mediated signaling in lymphocytes but may be involved in the survival of hippocampal CA3 pyramidal neurons.

Tissue-specific autoregulation of the stat3 gene and its role in interleukin-6-induced survival signals in T cells.

Signal transducer and activator of transcription 3 (STAT3) mediates signals of various growth factors and cytokines, including interleukin-6 (IL-6). In certain IL-6-responsive cell lines, the stat3 gene is autoregulated by STAT3 through a composite IL-6 response element in its promoter that contains a STAT3-binding element (SBE) and a cyclic AMP-responsive element. To reveal the nature and roles of the stat3 autoregulation in vivo, we generated mice that harbor a mutation in the SBE (stat3(mSBE)). The intact SBE was crucial for IL-6-induced stat3 gene activation in the spleen, especially in the red pulp region, the kidney, and both mature and immature T lymphocytes. The SBE was not required, however, for IL-6-induced stat3 gene activation in hepatocytes. T lymphocytes from the stat3(mSBE/mSBE) mice were more susceptible to apoptosis despite the presence of IL-6 than those from wild-type mice. Consistent with this, IL-6-dependent activation of the Pim-1 and junB genes, direct target genes for STAT3, was attenuated in T lymphocytes of the stat3(mSBE/mSBE) mice. Thus, the tissue-specific autoregulation of the stat3 gene operates in vivo and plays a role in IL-6-induced antiapoptotic signaling in T cells.

Gene transfer into muscle by electroporation in vivo.

Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. Applications of this method have been limited by the relatively low expression levels of the transferred gene. We investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA expressing interleukin-5 (IL-5) as the vector. The tibialis anterior muscles of mice were injected with the plasmid DNA, and then a pair of electrode needles were inserted into the DNA injection site to deliver electric pulses. Five days later, the serum IL-5 levels were assayed. Mice that did not receive electroporation had serum levels of 0.2 ng/ml. Electroporation enhanced the levels to over 20 ng/ml. Histochemical analysis of muscles injected with a lacZ expression plasmid showed that in vivo electroporation increased both the number of muscle fibers taking up plasmid DNA and the copy number of plasmids introduced into the cells. These results demonstrate that gene transfer into muscle by electroporation in vivo is more efficient than simple intramuscular DNA injection.

Method for determining a coupling function in coupled oscillators with application to Belousov-Zhabotinsky oscillators.

A coupling function that describes the interaction between self-sustained oscillators in a phase equation is derived and applied experimentally to Belousov-Zhabotinsky (BZ) oscillators. It is demonstrated that the synchronous behavior of coupled BZ reactors is explained extremely well in terms of the coupling function thus obtained. This method does not require comprehensive knowledge of either the oscillation mechanism or the interaction among the oscillators, both of these being often difficult to elucidate in an actual system. These facts enable us to accurately analyze the weakly coupled entrainment phenomenon through the direct measurement of the coupling function.