Occurrence of two β-tubulin isoforms with different polymerizing abilities in L5178Y cells

Mouse lymphoma L5178Y cells express at least two isoforms of β-tubulin, designated Mβ_I, and Mβ_II, as revealed by isoelectrofocusing, whereas two independently isolated normal T-cell clones, 3D10 and K23, express only Mβ_I. Mβ_II-tubulin is more acidic (pI, 5.10) than Mβ_I-tubulin (pI, 5.15). L5178Y cells were disrupted under the microtubule-stabilizing conditions, followed by centrifugation to separate fractions containing polymerized and unpolymerized tubulin. We found that a proportion of Mβ_II to total β-tubulins is larger in the fraction containing unpolymerized tubulin than in that containing polymerized tubulin. In addition, when tubulin was purified from extracts of L5178Y cells by repeated cycles of polymerization-depolymerization, the Mβ_II-tubulin isoform was gradually lost during the successive purification steps. The low recovery of Mβ_II-tubulin was observed, irrespective of the presence or absence of MAPs, and even in the presence of an excess amount of essentially polymerizable porcine brain tubulin. These results indicate that Mβ_II-tubulin is less able to polymerize than is Mβ_I-tubulin, both in vivo and in vitro.     

Intestinal tumorigenicity of mice carrying hemizygous Pax6, Pax6(Sey-4H).

The genotype-phenotype relationship in mice was examined experimentally using one of the small eye mutants, Pax6(Sey-4H), which deletes the chromosome 2 middle region, hemizygously. The genotyping indicated that the deleted region starts at a site 102.60 Mb from the centromere and has a length of 6.51 Mb, in which 12 known and 27 novel genes are located. Expecting the development of myeloid leukemia, gamma-irradiation was performed to female mutants at the age of 10 weeks. The mutants did not develop myeloid leukemia during the observation period of 18 months. Instead, they developed tumors in the alimentary tract spontaneously (56.0%). The tumor latency was shortened by the radiation exposure, but the tumor incidence of the gamma-irradiated group (62.5%) was as high as that of spontaneously developing tumors. Intraductal proliferation of the epithelium of the Wirsung duct was observed in the gamma-irradiated mutants (18.8%). Considering the results of the Pax6(Sey-4H) mutant together with those of another small eye mutant, Pax6(Sey-3H), the anomaly and the tumorigenicity of the intestinal tract were closely related to the hemizygosity of the 3.2 Mb segment of chromosome 2, where both mutants show a common deletion.     

Pathogenic involvement of heregulin-β(1) in anti-atherogenesis

Human heregulins are neuregulin-1 type I polypeptides that act as ligands of the ErbB family of receptor tyrosine kinases. These peptides play an essential role in the development of the cardiovascular system, including angiogenesis and compensation of cardiac function. Both heregulins and ErbB receptors are expressed at high levels in various types of vascular cells. The results of cell culture, animal, and clinical experiments have shown heregulin-β(1) to be a promising drug candidate for prevention of atherosclerosis. Various mechanisms have been suggested to be involved in this process, such as suppression of macrophage foam cell formation and vascular smooth muscle cell proliferation. Heregulin-β(1) retards pro-inflammatory responses by attenuating the expression of interleukin-1β, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinase-9, and cyclooxygenase-2 in monocytes. The peptide also has anti-oxidant and anti-apoptotic properties, and activates endothelial nitric oxide synthase in cardiomyocytes. Chronic infusion of heregulin-β(1) into apolipoprotein E-knockout mice suppresses the development of atherosclerotic lesions. In rat balloon injury models, heregulin-β(1) injection attenuates neointimal formation in the carotid artery. Clinical studies have shown that markedly reduced levels of heregulin-β(1) in the arterial wall and blood are closely associated with the progression of human coronary atherosclerotic lesions in patients with coronary artery disease. Therefore, these findings provide insight into the potential use of heregulin-β(1) as an extended therapeutic window for combating atherosclerosis and restenosis after coronary angioplasty.     

Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.     

Urinary liver-type fatty acid binding protein as a useful biomarker in chronic kidney disease

Background: We reported that urinary L-FABP reflected the progression of chronic kidney disease (CKD). This study is aimed to evaluate the clinical significance of urinary liver type fatty acid binding protein (L-FABP) as a biomarker for monitoring CKD. Methods: Urinary L-FABP was measured using human L-FABP ELISA kit (CMIC.Co., Ltd., Tokyo, Japan). The relations between urinary L-FABP and clinical parameters were evaluated in non-diabetic CKD (n = 48) for a year. In order to evaluate the influence of serum L-FABP derived from liver upon urinary L-FABP, both serum and urinary L-FABP were simultaneously measured in patients with CKD (n = 73). Results: For monitoring CKD, the cut-off value in urinary L-FABP was determined as 17.4 μg/g.cr. by using a receiver operating characteristics (ROC) curve. Renal function deteriorated significantly more in patients with ‘high’ urinary L-FABP (n = 36) than in those with ‘low’ L-FABP (n = 12). The decrease in creatinine clearance was accompanied by an increase in urinary L-FABP, but not in urinary protein. Serum L-FABP in patients with CKD was not correlated with urinary L-FABP. Conclusion: Urinary excretion of L-FABP increases with the deterioration of renal function. Serum L-FABP did not influence on urinary L-FABP. Urinary L-FABP may be a useful clinical biomarker for monitoring CKD.     

Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein

RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its β-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein. R12 forms an intramolecular parallel quadruplex. The quadruplex contains G:G:G:G tetrad and G(:A):G:G(:A):G hexad planes. Two quadruplexes form a dimer through intermolecular hexad–hexad stacking. Two lysine clusters of bPrP have been identified as binding sites for R12. The electrostatic interaction between the uniquely arranged phosphate groups of R12 and the lysine clusters is suggested to be responsible for the affinity of R12 to bPrP. The stacking interaction between the G:G:G:G tetrad planes and tryptophan residues may also contribute to the affinity. One R12 dimer molecule is supposed to simultaneously bind the two lysine clusters of one bPrP molecule, resulting in even higher affinity. The atomic coordinates of R12 would be useful for the development of R12 as a therapeutic agent against prion diseases and Alzheimer''s disease.     

Genetic screening for modifiers of the DREF pathway in Drosophila melanogaster: identification and characterization of HP6 as a novel target of DREF

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5′-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5′-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.     

Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. βPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-βPix-actin pathway for this mechanism.Alignment of the mitotic spindles with a predetermined axis, which confines the plane of cell division, occurs in many types of cells and is crucial for morphogenesis and embryogenesis. Cell geometry (30, 32, 47), cell polarity (6, 24, 35), and cell-cell adhesions (20, 22, 48) are proposed to be the determinants for the axis of the spindles. In most cases, spindle alignment along the predetermined axis requires both astral microtubules and the actin cytoskeleton and is believed to involve dynein-dependent microtubule pulling forces functioning at the cell cortex (4, 12, 31).We have previously shown that in nonpolarized adherent cells, such as HeLa cells, integrin-mediated cell-substrate adhesion orients the spindles parallel to the substratum, which ensures that both daughter cells remain attached to the substrate after cell division (42). This mechanism requires the actin cytoskeleton, astral microtubules, the microtubule plus-end-tracking protein EB1, and myosin X. Furthermore, our recent study has shown that the lipid second messenger phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] is also essential to this mechanism. PtdIns(3,4,5)P3 is accumulated in the midcortex of metaphase cells, which is important for the localized accumulation of dynactin, a dynein-binding partner, at the midcortex. We have proposed that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on the spindle to the midcortex and orients the spindle parallel to the substratum (43). However, the molecular mechanisms that regulate the actin cytoskeleton and PtdIns(3,4,5)P3 in the spindle orientation control remain unknown.The Rho family of GTPases, including Rho, Rac, and Cdc42, plays central roles in the regulation of not only the actin cytoskeleton but also microtubules in the control of various activities of cell motility, including cell adhesion, cell migration, and cell cycle progression (9, 33, 41). Rho family GTPases are also reported to regulate several mitotic events. RhoA plays a crucial role in contractile ring function and localizes to the cleavage furrow along with its effectors, ROCK, citron kinase, and mDia, during cytokinesis (18, 11). Cdc42 and its effector, mDia3, are reported to regulate the alignment of chromosomes during prometaphase and metaphase (49). Interestingly, Cdc42 is also required for proper spindle positioning in polarized cells such as budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans one-cell stage embryos, and mouse oocytes, which undergo asymmetric cell division (1, 23, 13, 28). However, how Cdc42 regulates spindle orientation and whether it has a role in spindle orientation in nonpolarized cells remain unknown.Here, we show that Cdc42 is required for the mechanism that orients the spindle parallel to the substratum in nonpolarized adherent cells. Moreover, our results show that Cdc42 regulates both PtdIns(3,4,5)P3 and the actin cytoskeleton through PI(3)K- and p21-activated kinase 2 (PAK2)/βPix-signaling pathways, respectively. Both pathways are required for the localized accumulation of dynein/dynactin complexes in the midcortex in metaphase cells and, thus, for the proper spindle orientation parallel to the substratum.     

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rha-(1→, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rha-(1→, which was identified in a previous study.     

Functional aberration of myofibrils by cardiomyopathy-causing mutations in the coiled-coil region of the troponin-core domain

Two cardiomyopathy-causing mutations, E244D and K247R, in human cardiac troponin T (TnT) are located in the coiled-coil region of the Tn-core domain. To elucidate effects of mutations in this region on the regulatory function of Tn, we measured Ca²⁺-dependent ATPase activity of myofibrils containing various mutants of TnT at these residues. The results confirmed that the mutant E244D increases the maximum ATPase activity without changing the Ca²⁺-sensitivity. The mutant K247R was shown for the first time to have the effect similar to the mutant E244D. Furthermore, various TnT mutants (E244D, E244M, E244A, E244K, K247R, K247E, and K247A) showed various effects on the maximum ATPase activity while the Ca²⁺-sensitivity was unchanged. Molecular dynamics simulations of the Tn-core containing these TnT mutants suggested that the hydrogen-bond network formed by the side chains of neighboring residues around residues 244 and 247 is important for Tn to function properly.