Novel effect of azide on sodium channel of Xenopus oocytes

Bath application of a few mM of sodium azide to a voltage clamped oocyte activated a voltage dependent Na+ current. Characteristics of the azide-induced current were the same as those of already described Na+ current induced by prolonged depolarization. The Na+ current induced either by azide or depolarization was suppressed by the application of Ca2+ channel blockers (Diltiazem and La3+). Azide is known to affect some metabolic processes (ATP and cGMP production etc.), but the present effect could not be attributed to metabolic actions.     

KIF1Bbeta functions as a haploinsufficient tumor suppressor gene mapped to chromosome 1p36.2 by inducing apoptotic cell death

Deletion of the distal region of chromosome 1 frequently occurs in a variety of human cancers, including aggressive neuroblastoma. Previously, we have identified a 500-kb homozygously deleted region at chromosome 1p36.2 harboring at least six genes in a neuroblastoma-derived cell line NB1/C201. Among them, only KIF1Bbeta, a member of the kinesin superfamily proteins, induced apoptotic cell death. These results prompted us to address whether KIF1Bbeta could be a tumor suppressor gene mapped to chromosome 1p36 in neuroblastoma. Hemizygous deletion of KIF1Bbeta in primary neuroblastomas was significantly correlated with advanced stages (p = 0.0013) and MYCN amplification (p < 0.001), whereas the mutation rate of the KIF1Bbeta gene was infrequent. Although KIF1Bbeta allelic loss was significantly associated with a decrease in KIF1Bbeta mRNA levels, its promoter region was not hypermethylated. Additionally, expression of KIF1Bbeta was markedly down-regulated in advanced stages of tumors (p < 0.001). Enforced expression of KIF1Bbeta resulted in an induction of apoptotic cell death in association with an increase in the number of cells entered into the G(2)/M phase of the cell cycle, whereas its knockdown by either short interfering RNA or by a genetic suppressor element led to an accelerated cell proliferation or enhanced tumor formation in nude mice, respectively. Furthermore, we demonstrated that the rod region unique to KIF1Bbeta is critical for the induction of apoptotic cell death in a p53-independent manner. Thus, KIF1Bbeta may act as a haploinsufficient tumor suppressor, and its allelic loss may be involved in the pathogenesis of neuroblastoma and other cancers.     

Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.     

Differentiation in the status of self-incompatibility among Calibrachoa species (Solanaceae)

 The overall status of self-incompatibility, as assessed by the rate of capsule-set after self-pollination, was investigated in the genus Calibrachoa (Solanaceae). Thirty-two species were surveyed using a total of 655 individuals collected in 102 different native populations in Argentina, Brazil, Mexico, and Uruguay. The rate of capsule-set in 278 voucher specimens collected from the same native habitats was also measured to obtain additional information to assess the degree of self-(in)compatibility. Only one species, Calibrachoa parviflora, was self-compatible (SC, autogamous) and the other 31 species were found to be self-incompatible (SI). A mixed population (SI and SC individuals in the same population) was not found. The differentiation of C. parviflora as an autogamous species is associated with a successful occupation of different (riparian) habitats within a larger range of geographic distribution compared to the rest of the species in the principally SI genus of Calibrachoa. Received: March 21, 2001 / Accepted: December 25, 2001     

Discovery of pyridone-containing imidazolines as potent and selective inhibitors of neuropeptide Y Y5 receptor

A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuropeptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of the imidazoline ring led to identification of 1-(difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7m). Compound 7m displayed statistically significant inhibition of food intake in an agonist-induced food intake model in SD rats and no adverse cardiovascular effects in anesthetized dogs. In addition, markedly higher brain penetrability and a lower plasma Occ90 value were observed in P-gp-deficient mdr1a (?/?) mice compared to mdr1a (+/+) mice after oral administration of 7m.     

Visuomotor Response to Object Expansion in Free-Flying Bumble Bees

The flight control systems of flying insects enable many kinds of sophisticated maneuvers, including avoidance of midair collisions. Visuomotor response to an approaching object, received as image expansion on insects’ retina, is a complex event in a dynamic environment where both animals and objects are moving. There are intensive free flight studies on the landing response in which insects receive image expansion by their own movement. However, few studies have been conducted regarding how freely flying insects respond to approaching objects. Here, using common laboratory insects for behavioral research, the bumblebee Bombus ignitus, we examined their visual response to an approaching object in the free-flying condition. While the insect was slowly flying in a free-flight arena, an expanding stripe was projected laterally from one side of the arena with a high-speed digital mirror device projector. Rather than turning away reported before, the bumble bees performed complex flight maneuvers. We synchronized flight trajectories, orientations and wing stroke frequencies with projection parameters of temporal resolution in 0.5 ms, and analyzed the instantaneous relationship between visual input and behavioral output. In their complex behavioral responses, we identified the following two visuomotor behaviors: increasing stroke frequency when the bumble bees confront the stripe expansion, and turning towards (not away) the stripe expansion when it is located laterally to the bee. Our results suggested that the response to object expansion is not a simple and reflexive escape but includes object fixation, presumably for subsequent behavioral choice.     

A simple and highly successful C-terminal sequence analysis of proteins by mass spectrometry

A simple and efficient method for C-terminal sequencing of proteins has long been pursued because it would provide substantial information for identifying the covalent structure, including post-translational modifications. However, there are still significant impediments to both direct sequencing from C termini of proteins and specific isolation of C-terminal peptides from proteins. We describe here a highly successful, de novo C-terminal sequencing method of proteins by employing succinimidyloxycarbonylmethyl tris (2,4,6-trimethoxyphenyl) phosphonium bromide and mass spectrometry.     

Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases

Homozygosity for the naturally occurring Delta32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted approximately 50% of CCR5 alleles in a pool of primary human CD4(+) T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4(+) T cells had lower viral loads and higher CD4(+) T-cell counts than mice engrafted with wild-type CD4(+) T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4(+) T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4(+) T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.