Hidenori Hirose

Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi

ZW10, a dynamitin‐interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT‐1 (Rad50‐interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum‐localized t‐SNARE implicated in membrane trafficking. Like conventional syntaxin‐binding proteins, ZW10, RINT‐1 and p31 dissociated from syntaxin 18 upon Mg2+‐ATP treatment in the presence of NSF and α‐SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.

Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion

BNIP1, a member of the BH3‐only protein family, was first discovered as one of the proteins that are capable of interacting with the antiapoptotic adenovirus E1B 19‐kDa protein. Here we disclose a totally unexpected finding that BNIP1 is a component of the complex comprising syntaxin 18, an endoplasmic reticulum (ER)‐located soluble N‐ethylmaleimide‐sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE). Functional analysis revealed that BNIP1 participates in the formation of the ER network structure, but not in membrane trafficking between the ER and Golgi. Notably, a highly conserved leucine residue in the BH3 domain of BNIP1 plays an important role not only in the induction of apoptosis but also in the binding of α‐SNAP, an adaptor that serves as a link between the chaperone ATPase NSF and SNAREs. This predicts that α‐SNAP may suppress apoptosis by competing with antiapoptotic proteins for the BH3 domain of BNIP1. Indeed, overexpression of α‐SNAP markedly delayed staurosporine‐induced apoptosis. Our results shed light on possible crosstalk between apparently independent cellular events, apoptosis and ER membrane fusion.

Stress-induced Translation of ATF5 mRNA Is Regulated by the 5′-Untranslated Region

Activating transcription factor (ATF) 5 is a transcription factor belonging to the ATF/cAMP-response element-binding protein gene family. We previously reported that ATF5 mRNA expression increased in response to amino acid limitation. The ATF5 gene allows transcription of mRNAs with at least two alternative 5′-untranslated regions (5′-UTRs), 5′-UTRα and 5′-UTRβ, derived from exon1α and exon1β. 5′-UTRα contains highly conserved sequences, in which the upstream open reading frames (uORFs) uORF1 and uORF2 are found in many species. This study was designed to investigate the potential role of 5′-UTRs in translational control. These 5′-UTRs differentially determined translation efficiency from mRNA. The presence of 5′-UTRα or 5′-UTRβ represses translation from the downstream ATF5 ORF. Moreover, 5′-UTRα-regulated translational repression is released by amino acid limitation or NaAsO2 exposure. This release was not seen for 5′-UTRβ. Mutation of uAUG2 in the uORF2 of 5′-UTRα restored the basal expression and abolished the positive regulation by amino acid limitation or arsenite exposure. We demonstrated that phosphorylation of eukaryotic initiation factor 2α was required for amino acid limitation-induced translational regulation of ATF5. Furthermore, arsenite exposure activated the exogenously expressed heme-regulated inhibitor kinase and induced the phosphorylation of eukaryotic initiation factor 2α in nonerythroid cells. These results suggest that translation of ATF5 is regulated by the alternative 5′-UTR region of its mRNA, and ATF5 may play a role in protecting cells from amino acid limitation or arsenite-induced oxidative stress.

Dielectric Properties and DC Conductivity of Vacuum-Deposited SiO Films

Dielectric properties from 10-2 to 107 c/s and dc conductivity are investigated for vacuum-deposited SiO films. The conductivity decreases with increasing oxidation and the activation energy is estimated to be 8.6 kcal/mole. Two dielectric dispersions are found, the loss peaks of which are at 10-1 c/s (1f dispersion) and at above 107 c/s (hf dispersion), respectively. The strength of 1f dispersion decreases with increasing oxidation and metallic impurities have no appreciable effect on the dispersion. The activation energy of 1f dispersion is almost equal to that to dc conduction. The dc conduction and 1f dispersion are concluded to be attributed to diffusion of free Si ions. The hf dispersion is ascribed to local torsional vibration of SiO chains. A model of the molecular structure of SiO in the glassy state is suggested, which is well consistent with the electrical and optical properties of this material.

Non-Ohmic Conduction in Vacuum-Deposited SiO Films

The current through vacuum-deposited SiO films in the thickness range 1–4 µ is measured as a function of voltage and temperature between 10° and 80°C. Three regions are distinguished in the current-voltage relationship; (1) voltage below 0.1 V, (2) from 0.1 to 10 V and (3) above 10 V. The current-voltage characteristic is linear in the region (1) but becomes nonlinear in the regions (2) and (3). In the region (3), the current depends on voltage and thickness only through the electric field E, the activation energy decreases linearly with increasing E1/2 and the logarithmic current increases linearly with increasing E1/2. These results are quantitatively explained in terms of field enhanced thermal excitation of trapped electrons into the conduction band (Poole-Frenkel effect). The energy gap between trap level and conduction band is estimated to be 0.54 eV.

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

The presence of subdomains in the endoplasmic reticulum (ER) enables this organelle to perform a variety of functions, yet the mechanisms underlying their organization are poorly understood. In the present study, we show that syntaxin 18, a SNAP (soluble NSF attachment protein) receptor localized in the ER, is important for the organization of two ER subdomains, smooth/rough ER membranes and ER exit sites. Knockdown of syntaxin 18 caused a global change in ER membrane architecture, leading to the segregation of the smooth and rough ER. Furthermore, the organization of ER exit sites was markedly changed concomitantly with dispersion of the ER-Golgi intermediate compartment and the Golgi complex. These morphological changes in the ER were substantially recovered by treatment of syntaxin-18-depleted cells with brefeldin A, a reagent that stimulates retrograde membrane flow to the ER. These results suggest that syntaxin 18 has an important role in ER subdomain organization by mediating the fusion of retrograde membrane carriers with the ER membrane.

Glass Transition Phenomena and Rheological Properties of Petroleum Asphalt

Volume dilatometry was made for nine kinds of asphalt in a wide range of asphaltene content X . The glass transition temperature T g becomes higher with increase of X . The discontinuity in thermal expansion coefficient at T g decreases with X and T g can no more be observed for X >70%. The complex shear modulus and shear creep compliance of five samples of asphalt were measured. Above T g , asphalt is viscoelastic. The temperature-time reducibility applies very well to asphalt. The temperature dependence of retardation times as well as steady flow viscosity obeys to Williams-Landel-Ferrys equation, the standard temperature involved in this equation being 56° higher than T g for all the samples. The distribution function of retardation times is approximately of a wedge shape when plotted in logarithmic scales, the slope being 0.5 or less according to X . The steady flow viscosity and the maximum retardation time reduced to 20°C markedly increase with X . These facts allow us some predictions about molecu...

Volume Viscoelasticity of Polymers and Other Highly Dissipative Materials

A new method for determining the real and imaginary parts of bulk modulus of polymers and other solid materials is represented. The method, as is called “suspension method”, consists of measurements of sound velocity and attenuation in a suspension of powders and is superior in experimental accuracy to the existing method which essentially consists of the comparison of longitudinal and shear wave measurements. The new method has also a merit in the applicability to the sample in powdery state. The results are given for styrene-butadiene rubber, natural rubber, polyethylene, polypropylene, polytrifluoromonochloroethylene, paraffin and yeast cell, among which styrene-butadiene rubber is most fully investigated. From these results, the followings are concluded: In the primary dispersion of amorphous polymers, the relaxation strength is of the same order of magnitude for bulk and shear moduli, and moreover, relaxation times, their distribution and temperature dependence have no appreciable discrepancy between...

Viscoelastic Behavior of Copolymers of Methyl Methacrylate and n-Butyl Methacrylate and Glass Polishing by Use of Them

In the first part of the work, shear creep compliance is measured for six kinds of copolymers of methyl methacrylate and n-butyl methacrylate above the respective glass transition temperatures. Shift factors and retardation spectra are calculated therefrom. The molecular weight between entanglement points which is obtained from the maximum of spectrum increases with the increase in n-butyl methacrylate fraction. This effect is interpreted in terms of the plasticizer effect of side chains. The maxima of the spectra of the copolymers are proportional to the square root of the corresponding retardation times if the spectra are reduced to the corresponding state in which the wedge portions of the spectra coincide with each other. In the second part, the results by Orioka who polished optical glass pieces by use of the copolymers are semi-quantitatively explained assuming that the surface of the glass polisher is heated by friction up to the temperature at which the shear creep compliance J(t) at t=polishing time is above 10-7cm2/dyn.

Mechanical Dispersions in Polycarbonate

Viscoelastic behavior of poly (bisphenol-A carbonate) above the glass transition temperature is studied by a creep test. In the primary dispersion region, the retardation spectrum increases with increase of retardation time with a slope of 1/2 in a doubly logarithmic plot as predicted by the Rouse theory, and then reaches a maximum. The molecular weight between entanglements is estimated as 2300 from this maximum value. The temperature dependence of retardation times obeys the WLF equation. The local mode dispersion below the glass transition temperature is studied by use of several dynamic methods. Dielectric measurement is made for comparison. The activation energy is obtained as 11 kcal/mode. Distribution of retardation times of the local mode dispersion is attributed to non-uniformity in force constant and friction coefficient to each molecular unit.