Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

The affinity of hemoglobin for oxygen affects ventilatory responses in mutant mice with Presbyterian hemoglobinopathy

The purpose of this study was to test whether chronically enhanced O2 delivery to tissues, without arterial hyperoxia, can change acute ventilatory responses to hypercapnia and hypoxia. The effects of decreased hemoglobin (Hb)-O2 affinity on ventilatory responses during hypercapnia (0, 5, 7, and 9% CO2 in O2) and hypoxia (10 and 15% O2 in N2) were assessed in mutant mice expressing Hb Presbyterian (mutation in the beta-globin gene, beta108 Asn --> Lys). O2 consumption during normoxia, measured via open-circuit methods, was significantly higher in the mutant mice than in wild-type mice. Respiratory measurements were conducted with a whole body, unrestrained, single-chamber plethysmograph under conscious conditions. During hypercapnia, there was no difference between the slopes of the hypercapnic ventilatory responses, whereas minute ventilation at the same levels of arterial PCO2 was lower in the Presbyterian mice than in the wild-type mice. During both hypoxic exposures, ventilatory responses were blunted in the mutant mice compared with responses in the wild-type mice. The effects of brief hyperoxia exposure (100% O2) after 10% hypoxia on ventilation were examined in anesthetized, spontaneously breathing mice with a double-chamber plethysmograph. No significant difference was found in ventilatory responses to brief hypoxia between both groups of mice, indicating possible involvement of central mechanisms in blunted ventilatory responses to hypoxia in Presbyterian mice. We conclude that chronically enhanced O2 delivery to peripheral tissues can reduce ventilation during acute hypercapnic and hypoxic exposures.     

Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease

Cerebral amyloid angiopathy (CAA) due to beta-amyloid (Abeta) is one of the specific pathological features of familial Alzheimer's disease. Abeta mainly consisting of 40- and 42-mer peptides (Abeta40 and Abeta42) exhibits neurotoxicity and aggregative abilities. All of the variants of Abeta40 and Abeta42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the Abeta40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type Abeta40. Similar tendency was observed for Abeta42 mutants at positions 22 and 23 whose neurotoxicity was 50-200 times stronger than that of the corresponding Abeta40 mutants, suggesting that these Abeta42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-Abeta42 and D23N-Abeta42 was similar to that of wild-type Abeta42, E22Q-Abeta42 and E22K-Abeta42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of Abeta mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that beta-sheet content of the Abeta mutants correlates with their aggregation. However, beta-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue beta-turn significantly enhanced the aggregative ability.     

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Negative regulation of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1

SHPS-1 is a receptor-type glycoprotein that binds and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and thereby negatively modulates intracellular signaling initiated by various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems through its association with CD47 (integrin-associated protein) on adjacent cells. Furthermore, recent studies with fibroblasts derived from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region suggested that the intact protein contributes to cytoskeletal function. Mice homozygous for this SHPS-1 mutation have now been shown to manifest thrombocytopenia. These animals did not exhibit a defect in megakaryocytopoiesis or in platelet production. However, platelets were cleared from the bloodstream more rapidly in the mutant mice than in wild-type animals. Furthermore, peritoneal macrophages from the mutant mice phagocytosed red blood cells more effectively than did those from wild-type mice; in addition, they exhibited an increase both in the rate of cell spreading and in the formation of filopodia-like structures at the cell periphery. These results indicate that SHPS-1 both contributes to the survival of circulating platelets and down-regulates the macrophage phagocytic response.     

Identification of the Anti-proliferative protein Tob as a MAPK substrate

Mitogen-activated protein kinases (MAPKs) regulate a wide variety of cellular functions by phosphorylating their specific substrates. Here we have identified Tob as a novel substrate of MAPK. Tob, a member of the Tob and B-cell translocation gene anti-proliferative protein family, is shown to negatively regulate the proliferation of osteoblasts and T cells. In this study, our two-hybrid screening has identified Tob as an ERK2-interacting protein. Biochemical analyses have then shown that ERK MAPK (ERK2) and JNK/SAPK (JNK2) bind to and phosphorylate Tob in vitro. ERK catalyzes the phosphorylation more efficiently than JNK. When the ERK pathway is activated in cells, phosphorylation of Tob is induced. An ERK-binding or -docking site locates in the N-terminal portion of Tob, and phosphorylation sites reside in the C-terminal stretch region. The docking is crucial for efficient phosphorylation. Mutant forms of Tob, in which serines are replaced by glutamic acids to mimic phosphorylation, show a much reduced ability to inhibit the cell cycle progression to S phase from G(0)/G(1) phase, as compared with wild-type Tob, indicating that ERK phosphorylation negatively regulates the anti-proliferative function of Tob.     

An attempt to promote neo-vascularization by employing a newly synthesized inhibitor of protein tyrosine phosphatase

Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of alpha,alpha-difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF-induced angiogenesis. DFPMPA-3, the most effective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its effectiveness was proven through neo-vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy.     

Diverse variation of reproductive barriers in three intraspecific rice crosses

Reproductive barriers are thought to play an important role in the processes of speciation and differentiation. Asian rice cultivars, Oryza sativa, can be classified into two main types, Japonica and Indica, on the basis of several characteristics. The fertility of Japonica-Indica hybrids differs from one cross to another. Many genes involved in reproductive barriers (hybrid sterility, hybrid weakness, and gametophytic competition genes) have been reported in different Japonica-Indica crosses. To clarify the state of Japonica-Indica differentiation, all reproductive barriers causing deviation from Mendelian segregation ratios in F(2) populations were mapped and compared among three different Japonica-Indica crosses: Nipponbare/Kasalath (NK), Fl1084/Dao Ren Qiao (FD), and Fl1007/Kinandang puti (FK). Mapping of reproductive barriers was performed by regression analysis of allele frequencies of DNA markers covering the entire genome. Allele frequencies were explained by 33 reproductive barriers (15 gametophytic and 18 zygotic) in NK, 32 barriers (15 gametophytic and 17 zygotic) in FD, and 37 barriers (19 gametophytic and 18 zygotic) in FK. The number of reproductive barriers in the three crosses was similar; however, most of the barriers were mapped at different loci. Therefore, these reproductive barriers formed after Japonica-Indica differentiation. Considering the high genetic similarity within Japonica and Indica cultivars, the differences in the reproductive barriers of each cross were unexpectedly numerous. The reproductive barriers of Japonica-Indica hybrids likely evolved more rapidly than other genetic elements. One possible force responsible for such rapid evolution of the barriers may have been the domestication of rice.     

Real-time kinetic analyses of the interaction of ricin toxin A-chain with ribosomes prove a conformational change involved in complex formation

Ricin toxin A-chain (RTA), a ribosome-inactivating protein from seeds of the castor bean plant (Ricinus communis), inactivates eukaryotic ribosomes by hydrolyzing the N-glycosidic bond of a single adenosine residue in a highly conserved loop of 28S rRNA, but does not act on prokaryotic ribosomes. We investigated the interaction of rat liver 80S ribosomes with RTA using an optical biosensor based on surface plasmon resonance (BIAcore instrument), which allows real-time recording of the interaction. RTA was coupled to the dextran gel matrix on the sensor chip surface through a single thiol group that is not involved in the enzymatic action. The interaction of rat ribosomes with RTA, which was greatly affected by the Mg(2+) concentration and ionic strength, was usually measured at 5 mM Mg(2+), 50 mM KCl, and pH 7.5. The modes of interaction of intact and RTA-depurinated rat liver ribosomes with the immobilized RTA were virtually the same, while no considerable interaction was observed for Escherichia coli ribosomes. The interaction was not influenced by the presence of 5 mM adenine, which is higher than the reported dissociation constant (1 mM) for the adenine-RTA complex. These results demonstrate that binding of the target adenine with the active site of RTA does not contribute much to the total interaction of ribosomes and RTA. Global analyses of association and dissociation data with several binding models, taking account of mass transport, allowed us to conclude that the data were unable to fit a simple 1:1 binding model, but were best described by a model including a conformational change involved in high affinity complex formation.