bluuebunny/crossref_metadata_2025 huggingface dataset:
Knockdown of hypoxia‐inducible factor‐1α by siRNA inhibits C2C12 myoblast differentiation
Authors: Ono Yusuke , Sensui Hiroomi , Sakamoto Yuzuru , Nagatomi Ryoichi
Published: January 2006

We analyzed the role of Hypoxia‐inducible factor (HIF)‐1α in myoblast differentiation by examining the expression and regulation of HIF‐1α in proliferating and differentiating C2C12 myoblast, and by knocking down HIF‐1α of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF‐1α has been shown to be involved in differentiative process in non‐muscle tissues. Although HIF‐1α mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF‐1α protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF‐1α accumulated in the nuclei of myogenin‐positive myoblasts. The inhibition of proteasome in the growth phase led to HIF‐1α protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF‐1α, suppressed HIF‐1α accumulation. Therefore, we suggest that the level of HIF‐1α protein expression is regulated by a proteasome‐and chaperon‐dependent pathway in C2C12 myoblast. Knockdown of HIF‐1α effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF‐1α expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF‐1α is required for C2C12 myogenesis in vitro, and suggest that HIF‐1α may have an essential role in regenerative muscle tissue in vivo. J. Cell. Biochem. 98: 642–649, 2006. © 2006 Wiley‐Liss, Inc.
Global analyses of satellite‐derived vegetation index related to climatological wetness and warmth
Authors: Suzuki Rikie , Xu Jianqing , Motoya Ken
Published: January 2006

Wetness and warmth are the principal factors that control global vegetation distribution. This paper investigates climate–vegetation relationships at a global scale using the normalized difference vegetation index (NDVI), warmth index (WAI), and wetness index (WEI).

The NDVI was derived from a global, 20‐year Advanced Very High Resolution Radiometer (AVHRR) dataset with 4‐min resolution. The WEI was defined as the ratio of precipitation to potential evaporation. The WAI was defined as the cumulative monthly mean temperature that exceeds 5 °C annually. Meteorological data from the International Satellite Land‐Surface Climatology Project Initiative II (ISLSCP II) dataset were used to calculate the WEI and WAI. All analyses used annual values based on averages from 1986 to 1995 at 1 × 1 degree resolution over land. Relationships among NDVI, WEI, and WAI values were examined using a vegetation‐climate diagram with the WEI and WAI as orthogonal coordinates.

The diagram shows that large NDVI values correspond to areas of tropical and temperate forests and large WEI and WAI values. Small WEI and WAI values are associated with small NDVI values that correspond to desert and tundra, respectively.

Two major regimes are revealed by the NDVI vegetation‐climate diagram: wetness dominant and warmth dominant. Wetness dominates mid‐ and low latitudes. Warmth dominates high latitudes north of 60°N or elevated land such as the Tibetan Plateau. The boundary between the two regimes roughly corresponds to the vegetation boundary between taiga forest and southern vegetation. Over northern Eurasia, the boundary occurs in areas where the NDVI is large and the maximum monthly temperature is around 18 °C. Copyright © 2006 Royal Meteorological Society.
Synthesis and characterization of model diblock copolymers of poly(dimethylsiloxane) with poly(1,4‐butadiene) or poly(ethylene)
Authors: Ciolino Andrés , Sakellariou Giorgos , Pantazis Dimitris , Villar Marcelo A. , Vallés Enrique , Hadjichristidis Nikos
Published: January 2006

Model diblock copolymers of poly(1,4‐butadiene) (PB) and poly(dimethylsiloxane) (PDMS), PB‐b‐PDMS, were synthesized by the sequential anionic polymerization (high vacuum techniques) of butadiene and hexamethylciclotrisiloxane (D3) in the presence of sec‐BuLi. By homogeneous hydrogenation of PB‐b‐PDMS, the corresponding poly(ethylene) and poly(dimethylsiloxane) block copolymers, PE‐b‐PDMS, were obtained. The synthesized block copolymers were characterized by nuclear magnetic resonance (1H and 13C NMR), size‐exclusion chromatography (SEC), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and rheology. SEC combined with 1H NMR analysis indicates that the polydispersity index of the samples (Mw/Mn) is low, and that the chemical composition of the copolymers varies from low to medium PDMS content. According to DSC and TGA experiments, the thermal stability of these block copolymers depends on the PDMS content, whereas TEM analysis reveals ordered arrangements of the microphases. The morphologies observed vary from spherical and cylindrical to lamellar domains. This ordered state (even at high temperatures) was further confirmed by small‐amplitude oscillatory shear flow tests. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1579–1590, 2006
Self‐immolative dendrimers as novel drug delivery platforms
Authors: Shabat Doron
Published: January 2006

Self‐immolative dendrimers were recently developed and introduced as a potential platform for a single‐triggered multi‐prodrug. These unique structural dendrimers can release all of their tail units through domino‐like chain fragmentation, which is initiated by a single cleavage at the dendrimer core. The incorporation of drug molecules as the tail units and an enzyme substrate as the trigger generates a multi‐prodrug unit that is activated with a single enzymatic cleavage. We have demonstrated several examples of self‐immolative dendritic prodrug systems and have shown significant advantages with respect to the appropriate monomeric prodrug. We anticipate that single‐triggered, dendritic prodrugs will be exploited to further improve selective chemotherapeutic approaches in cancer therapy. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1569–1578, 2006
Divinylsiloxane‐bisbenzocyclobutene‐based polymer modified with polystyrene–polybutadiene–polystyrene triblock copolymers
Authors: So Ying‐Hung , Foster Pamela , Im Jang‐Hi , Garrou Philip , Hetzner Jack , Stark Edmund , Baranek Kayla
Published: January 2006

Divinylsiloxane‐bisbenzocyclobutene (DVS‐bisBCB) polymer has very low dielectric constant and dissipation factor, good thermal stability, and high chemical resistance. The fracture toughness of the thermoset polymer is moderate due to its high crosslink density. A thermoplastic elastomer, polystyrene–polybutadiene–polystyrene triblock copolymer, was incorporated into the matrix to enhance its toughness. The cured thermoset matrix showed different morphology when the elastomer was added to the B‐staged prepolymer or when the elastomer was B‐staged with the DVS‐bisBCB monomer. Small and uniformly distributed elastomer domains were detected by transmission electron micrographs (TEM) in the former case, but TEM did not detect a separate domain in the latter case. A high percentage of the polystyrene–polybutadiene–polystyrene triblock copolymer could be incorporated into the DVS‐bisBCB thermoset matrix by B‐staging the triblock copolymer with the BCB monomer. The elastomer increased the fracture toughness of DVS‐bisBCB polymer as indicated by enhanced elongation at break and increased K1c values obtained by the modified edge‐lift‐off test. Elastomer modified DVS‐bisBCB maintained excellent electrical properties, high Tg and good thermal stability, but showed higher coefficient of linear thermal expansion values. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1591–1599, 2006
Thermal and mechanical analysis of metallocene‐catalyzed ethene–α‐olefin copolymers: The influence of the length and number of the crystallizing side chains
Authors: Piel C. , Starck P. , Seppälä J. V. , Kaminsky W.
Published: January 2006

Copolymers of ethene and 1‐octene, 1‐dodecene, 1‐octadecene, and 1‐hexacosene were carried out with [Ph2C(2,7‐di‐tertBuFlu)(Cp)]ZrCl2/methylalumoxane as a catalyst to obtain short‐chain branched polyethylenes with branch lengths of 6–26 carbon atoms. This catalyst provided high activity and a very good comonomer and hydrogen response. In this study, the influence of the length and number of the side chains on the mechanical properties of the materials was investigated. The crystalline methylene sequence lengths of the copolymers and lamellar thicknesses were calculated after the application of a differential scanning calorimetry/successive self‐annealing separation technique. By dynamic mechanical analysis, the storage modulus as an indicator of the stiffness and the loss modulus as a measure of the effect of branching on the α and β relaxations were studied. The results were related to the measurements of the polymer density and tensile strength to determine the effect of longer side chains on the material properties. The hexacosene copolymers had side chains of 24 carbons and remarkable material properties very different from those of conventional linear low‐density polyethylenes. The side chains of these copolymers crystallized with one another and not only parallel to the backbone lamellar layer, depending on the hexacosene concentration in the copolymer. The side chains crystallized even at low hexacosene concentrations in the copolymer. A transfer of these results to 16 carbons side chains in ethene–octadecene copolymers was also possible. © 2006 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1600–1612, 2006
Preparation of block copolymers via Diels Alder reaction of maleimide‐ and anthracene‐end functionalized polymers
Authors: Durmaz Hakan , Colakoglu Bige , Tunca Umit , Hizal Gurkan
Published: January 2006

A number of diblock copolymers were successfully prepared by Diels–Alder reaction, between maleimide‐ and anthracene‐end functionalized poly (methyl methacrylate) (PMMA), polystyrene (PS), poly(tert‐butyl acrylate) (PtBA), and poly(ethylene glycol) (PEG) in toluene, at 110 °C. For this purpose, 2‐bromo‐2‐methyl‐propionic acid 2‐(3,5‐dioxo‐10‐oxa‐4‐azatricyclo[5.2.1.02,6]dec‐8‐en‐4‐yl)‐ethyl ester, 2, 9‐anthyrylmethyl 2‐bromo‐2‐methyl propanoate, 3, and 2‐bromo‐propionic acid 2‐(3,5‐dioxo‐10‐oxa‐4‐azatricyclo[5.2.1.02,6]dec‐8‐en‐4‐yl)‐ethyl ester, 4, were used as initiators in atom transfer radical polymerization, in the presence of Cu(I) salt and pentamethyldiethylenetriamine (PMDETA), at various temperatures. On the other hand, PEG with maleimide‐ or anthracene‐end functionality was achieved by esterification between monohydroxy PEG and succinic acid monoathracen‐9‐ylmethyl ester, 1, or 4‐maleimido‐benzoyl chloride. Thus‐obtained PMMA‐b‐PS, PEG‐b‐PS, PtBA‐b‐PS, and PMMA‐b‐PEG block copolymers were characterized by 1H NMR, UV, and GPC. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1667–1675, 2006
Permafrost existence in rock glaciers of the Argentera Massif, Maritime Alps, Italy
Authors: Ribolini Adriano , Fabre Denis
Published: January 2006

Thermal and geo‐electrical investigations were carried out on five rock glaciers in the Argentera Massif to detect the presence of permafrost and to define its existence and activity elevation limits. Ground surface temperature was monitored using digital dataloggers, and sub‐surface ice occurrence was inferred from one‐dimensional geo‐electrical soundings. Bottom temperatures of snow cover, extracted from winter recordings, indicate possible or probable presence of permafrost for some rock glaciers. Geo‐electrical soundings reveal the existence of sediments rich and super‐saturated with ice for thicknesses often reaching several tens of metres. The limits for permafrost existence and for rock glacier activity can thus be fixed respectively at about 2500 m and 2600 m ASL. The results from 110 earlier geo‐electrical soundings, carried out on rock glaciers in different sectors of the southwestern Alps, were processed and compared with the results of this work. A general classification of rock glaciers into active, inactive and relict was possible for the Argentera Massif. Inactive and relict rock glaciers probably originated during colder Holocene and Late‐glacial periods. Copyright © 2006 John Wiley & Sons, Ltd.
Laryngeal carcinoma: five-year survival and patterns of failure in 202 consecutive patients treated with primary or post-operative radiotherapy in Hong Kong
Authors: Vlantis Alexander C , Yu Brian K H , Tsang Raymond K Y , Kam Michael K M , Lo Phoebe S Y , van Hasselt C Andrew
Published: January 2006

Objectives: We aimed to conduct a retrospective analysis of patients treated with radiotherapy for laryngeal carcinoma at a single institution.

Methods: We analysed data from 202 consecutive patients treated with primary or post-operative radiotherapy for laryngeal carcinoma over a 10-year period.

Results: Sixty-nine patients had a T1, 65 a T2, 39 a T3 and 29 a T4 lesion. Forty-one patients were node-positive. The clinical stage was I in 67 patients, II in 53, III in 36 and IV in 46. Primary radiotherapy was given to 152 patients. The median follow up was 60 months. The five-year overall local control rate was 86 per cent, the ultimate local control rate was 93 per cent, the five-year regional control rate was 96 per cent, the five-year relapse-free survival rate was 82 per cent and the five-year overall survival rate was 69 per cent.

Conclusions: Patients with laryngeal carcinoma treated with primary or post-operative radiotherapy had a five-year overall survival rate of 69 per cent.
A stastistical system for detecting Salmonella outbreaks in British livestock
Authors: KOSMIDER R. , KELLY L. , EVANS S. , GETTINBY G.
Published: January 2006

Worldwide, early detection systems have been used in public health to aid the timely detection of increases in disease reporting that may be indicative of an outbreak. To date, their application to animal surveillance has been limited and statistical methods to analyse human health data have not been viewed as being applicable for animal health surveillance data. This issue was investigated by developing an early detection system for Salmonella disease in British livestock. We conclude that an early detection system, as for public health surveillance, can be an effective tool for enhanced surveillance. In order to implement this system in the future and extend it for other data types, we provide recommendations for improving the current data collection process. These recommendations will ensure that quality surveillance data are collected and used effectively to monitor disease in livestock populations.
Altered States of Consciousness and Palaeoart: an Alternative Neurovisual Explanation
Authors: Hodgson Derek
Published: January 2006

There has been much controversy recently regarding Lewis-Williams's assertion that altered states of consciousness and shamanism can explain Palaeolithic art. Evidence now seems to be accumulating that this account is unable to provide a sustainable explanation for Upper Palaeolithic depictions. This proposition will be explored and substantiated by examining further weaknesses contained therein. Additionally, in response to claims by those defending altered states that no alternative explanation for palaeoart has been proposed as a viable alternative, it will be shown that such a description does exist but has not been given the attention it deserves because of a misplaced concern for shamanism.
Iconography of Termites' Nests and Termites: Symbolic Nuances of Formlings in Southern African San Rock Art
Authors: Mguni Siyakha
Published: January 2006

Formlings, now better understood to depict termitaria (termites' nests) and termites, are a pervasive category of San (Bushman) rock art north of the River Limpopo. This article investigates the associations of termites' nests in San thought, belief, and ritual, in an attempt to explain formling symbolism and why termites' nests, and not other subjects, were chosen for depiction. Unequivocal ethnographic testimonies of San spiritual world-view are compounded with iconographic analysis to show nuances of San understanding and perception of the spirit world. In turn, this ethnographic hermeneutic reveals a significant but previously unexplored facet of spirit-world imagery which evokes notions of creative and transformative power. This newly highlighted vignette of San cosmology unlocks aspects of San imagery, such as the interface between the natural and the metaphysical, that have hitherto been less understood.
Reviewing dispute settlement at the World Trade Organization: a time to reconsider the role/s of compensation?
Authors: DAVIES ARWEL
Published: January 2006

The World Trade Organization provides a forum for the settlement of trade disputes arising between its 148 Members. Should consultations fail, the parties may choose to initiate formal proceedings in Geneva, and must do so in preference to taking unilateral action. The dispute settlement rules are presently under review with a view to their clarification and improvement, making this a natural time to ask whether the appropriate strategy has been identified. This article focuses on the functions of compensation in the overall context of WTO remedies. Particular attention is given to the prospects for new disciplines and increased practice connected with the granting of both trade compensation and financial compensation. Also considered is the extent to which financial compensation can and should be linked to reparation in the sense of correcting the injury caused by WTO violations. The discussion is informed by the general international law position, by proposals made during the on-going review process and by emerging dispute settlement practice.
Participation of NGOs before the WTO and EC tribunals: which court is the better friend?
Authors: SLOTBOOM MARCO M.
Published: January 2006

The dispute settlement system of the European Community (EC) is undeniably much more elaborate than that of the World Trade Organization (WTO). The reasons for this are evident. First, unlike the WTO the EC, being a supranational organization, has the competence to adopt legislative and administrative acts in its own right. Democratic principles require that the legality of these acts can be examined by a judicial body. Second, legislative and administrative acts of the EC frequently have a direct impact on EC citizens. It would be contrary to democratic principles if citizens affected by legislative or administrative acts of the EC were not able to have the legality of such measures examined by a judicial body. By way of contrast, the inter-State nature of WTO proceedings is such that its dispute settlement system is exclusively open to WTO Members. Third, the EC institutions are capable of beginning legal proceedings against each other and against EC Member States. In particular, if the EC Commission considers that an EC Member State has failed to fulfill an EC Treaty obligation, it may bring the matter before the ECJ. In WTO law, there is no opportunity for inter-institutional proceedings or proceedings between the WTO and its Members.
Impact of chemically enhanced diffusion on dissolved inorganic carbon stable isotopes in a fertilized lake
Authors: Bade Darren L. , Cole Jonathan J.
Published: January 2006

At high pH the chemical reaction of CO2 with OH− can significantly increase the mass transfer of CO2 between air and water. The reaction of CO2 with OH− strongly fractionates carbon isotopes in comparison to simple diffusion. These processes, chemically enhanced diffusion (CED) and chemically enhanced fractionation (CEF), greatly influence the carbon budgets and carbon isotope ratios for water bodies with high pH. Using floating chambers, we estimated mass transfer coefficients for CO2 and a nonreactive gas, CH4, in an experimentally eutrophied lake. The mass transfer coefficient estimated from CH4 flux did not vary greatly between measurements (k600 = 1.83 ± 0.33 cm h−1; mean ±1 SD) and agreed well with other independent estimates of mass transfer. The mass transfer coefficient of CO2, however, was chemically enhanced by 3.5‐ to 7.5‐fold. This enhancement was related to pH and temperature but was slightly higher than predictions from an existing model. We determined the role of CEF by modifying a model of CED to include both carbon isotopes (12C and 13C). A whole‐lake addition of inorganic 13C to Peter Lake created dynamics in δ13C‐dissolved inorganic carbon (DIC) and provided a test of the new model. The value of δ13C‐DIC decreased from approximately −9‰ to −21‰, a result that was well predicted by the model including CEF but could not be duplicated when CEF was omitted. Thus CED and CEF influenced the mass balance of air‐water CO2 exchange and had isotopic consequences for DIC. Although CEF is considered inconsequential for mean oceanic conditions, this model could be applied to marine systems for inorganic carbon modeling in areas where pH is elevated or physical mass transfer is limited because of low turbulence.
Solar wind speed within 20 RS of the Sun estimated from limb coronal mass ejections
Authors: Nakagawa Tomoko , Gopalswamy Nat , Yashiro Seiji
Published: January 2006

An estimation of the solar wind speed in the vicinity of the Sun is carried out using the initial speed and acceleration of coronal mass ejections (CMEs) that appeared close to the solar limb. A linear relationship was found between the initial acceleration and the speed of the limb CMEs. It appears that a dragging force is acting on the CMEs, depending on the speed difference between the CMEs and the ambient plasma. The ambient solar wind speed within 20 solar radii estimated from low‐latitude CMEs during 1998–2003 ranged from 100 to 700 km s−1, while the solar wind speed measured at 1 AU ranged from 300 to 700 km s−1. The estimated solar wind speeds in the vicinity of the Sun sometimes agreed with the simultaneous in situ measurements at 1 AU, but in other periods they were slower than the speeds measured at 1 AU. It is suggested that most of the time the low‐latitude solar wind completes accelerating within 20 solar radii, but occasionally additional acceleration is present beyond 20 solar radii.
Parallel and oblique proton fire hose instabilities in the presence of alpha/proton drift: Hybrid simulations
Authors: Hellinger Petr , Trávníček Pavel
Published: January 2006

Parallel and oblique proton fire hose instabilities are investigated in the presence of a small abundance of alpha particles with a nonzero drift velocity with respect to protons. Both instabilities scatter protons and alpha particles in the perpendicular direction with respect to the ambient magnetic field and decelerate both species with respect each other; especially the oblique fire hose effectively diffuses ions owing to its non‐quasi‐linear evolution. Linear Vlasov theory predicts that the presence of the alpha/proton drift enhances the maximum growth rates of the two instabilities in a similar way and that the parallel fire hose is typically the dominant instability. On the other hand, the oblique fire hose has often the maximum growth rate comparable to that of the parallel one and hybrid simulations show that the oblique instability may be active even when the parallel one is marginally stable. Consequently, both instabilities are relevant in the solar wind context.
Observations of viscous magnetization in multidomain magnetite
Authors: Muxworthy Adrian R. , Williams Wyn
Published: January 2006

The viscous behavior of multidomain magnetite has been directly observed in both natural and synthetic samples using Bitter pattern imaging. A computer‐controlled fully automated microscope fitted with a heating stage, field coils, and digital camera was used to record viscous acquisition and decay sequences as a function of time, temperature, and field. Domain walls (DW) were observed to move continuously through a series of quasi‐static states over many hours rather than instantaneously. Viscosity was observed only on grains oriented near to the {111} surface. Generally, DWs moved perpendicular to their surface in the direction of the applied field; however, because observed domains respond primarily to the movement of main domains underneath the surface, occasionally, DWs moved in the opposite direction to the applied field. Small variations in temperature were found to strongly influence the viscosity, supporting the idea that viscosity is thermally activated. Viscous and nonviscous domain structures were examined using magnetic force microscopy. These images revealed that the domains displaying viscous behavior tended to be narrow (∼2 μm in width). Larger domains on grains oriented near the {111} surface did not display viscosity, reflecting the greater energy required to move larger domain structures. This may explain why no viscosity was observed on the {110} surfaces, as the domains were wider, that is, ∼6–10 μm. A complex spiraling vortex‐like magnetic domain structure was imaged. Etch pit analysis found a corresponding dislocation pit at the same location. It is suggested that this corresponds to the microstructure around a screw dislocation line.
Effects of extremely‐low‐frequency pulsed electromagnetic fields on collagen synthesis in rat skin
Authors: Ahmadian Shahin , Zarchi Saeed Rezaei , Bolouri Bahram
Published: January 2006

To investigate the effects of extremely‐low‐frequency PEMFs (pulsed electromagnetic fields) on the synthesis of epidermal collagen, six groups of animals each consisting of eight mature male rats were selected randomly: one group for the control and five for the test. Using a parallel set of Helmholtz coils, a uniform field intensity of 2 mT at different frequencies of 25, 50 and 100 Hz yielded the most effective frequency to be 25 Hz. Then, at this frequency, two different field intensities of 1 and 4 mT were applied. The treatment time of 2.5 h/day lasted for 8 days, keeping the same procedure for the control group, except with the field turned off. On the ninth day, the rats were killed and skin samples from the dorsal region were taken for collagen assessment by measuring hydroxyproline content using the Stegemann–Stalder [(1967) Clin. Chim. Acta 8, 267–273] method. The results indicated that a PEMF of 2 mT at 25 Hz increased the collagen synthesis (P<0.05). The other intensities and frequency setting did not have any noticeable effect; however, at a frequency of 25 Hz at 4 mT, collagen increase was also noticed. It was concluded that at 25 Hz under a field setting of 2 mT for the duration of 8 days, stimulation of skin at 2.5 h/day would cause increase in collagen synthesis in rat skin.
Generation and characterization of a high‐affinity chimaeric antibody against hepatitis B surface antigen
Authors: Bose Biplab , Khanna Navin , Acharya Subrat K. , Sinha Subrata
Published: January 2006

Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the VH (heavy‐chain variable region) and VL (light‐chain variable region) genes of this mouse antibody, and fused them with CH1 (heavy‐chain constant domain 1) of human IgG1 and CL (light‐chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab‐CMV), which has a modular cassette coding for part of the hinge, CH2 and CH3 of human IgG1. The recombinant construct was transfected in CHO (Chinese‐hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum‐free medium and the full‐length chimaeric anti‐HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate (≈5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody.
Effect of lobe pumping on human albumin: development of a lobe pump simulator using smoothed particle hydrodynamics1
Authors: Gomme Peter T. , Prakash Mahesh , Hunt Ben , Stokes Nick , Cleary Paul , Tatford Owen C. , Bertolini Joseph
Published: January 2006

Using SPH (smoothed particle hydrodynamics), the motion of a lobe pump under load was simulated in order to p2redict the level of shear stress experienced by a protein solution. By varying the gap size between the lobes and pump housing, variations in pump efficiency and shear stress were determined. The simulations indicated that pump shear was dependent on gap size, with shear stress levels (0–40 Pa) correlating with those estimated in previous albumin‐pumping studies. As gap size increased, the number of fluid particles experiencing low shear (<10 Pa) increased, whereas those experiencing high shear (>20 Pa) showed a decreasing trend. The pump efficiency, however, decreased with gap size, requiring more lobe revolutions to pass a unit volume. Taken together, these observations indicate that lobe pumps operated with increased gaps between the rotors and the housing result in larger number of particles within the fluid experiencing shear stresses. Moreover, the simulations indicate that it is best to use larger lobe pumps operated at high efficiency to transfer protein‐containing solutions.
Identification of a keratinocarcinoma cell line expressing AQP3
Authors: Nakakoshi Masamichi , Morishita Yoshiyuki , Usui Keitaro , Ohtsuki Mamitaro , Ishibashi Kenichi
Published: January 2006

Background information. AQP3 (aquaporin 3) in the skin is important for skin moisture as demonstrated by the studies of AQP3‐null mice, which have accelerated skin drying. Prevention of dry skin is important not only from a cosmetic but also from a clinical point of view. Primary keratinocyte cultures are cumbersome for screening substances that modulate AQP3 expression.

Results. A human keratocarcinoma cell line was found to express AQP3 mRNA and protein, which responded to hypertonic stimulation with sorbitol, suggesting that the AQP3 expression is normally regulated in this cell line. This cell line also expressed the type 1 keratinocyte transglutaminase gene. The AQP3 expression was unaffected by all‐trans‐retinoic acid up to 10−6 M. Similarly, the retinoic acid did not increase the AQP3 expression up to 1$ concentration in rat skin.

Conclusion. This cell line is useful for the screening of candidate substances that modulate AQP3 expression.
Adult murine CNS stem cells express aquaporin channels
Authors: La Porta Caterina A.M. , Gena Patrizia , Gritti Angela , Fascio Umberto , Svelto Maria , Calamita Giuseppe
Published: January 2006

Background information. Fluid homoeostasis is of critical importance in many functions of the CNS (central nervous system) as indicated by the fact that dysregulation of cell volume underlies clinical conditions such as brain oedema and hypoxia. Water balance is also important during neurogenesis as neural stem cells move considerable amounts of water into or out of the cell to rapidly change their volume during differentiation. Consistent with the relevance of water transport in CNS, multiple AQP (aquaporin) water channels have been recognized and partially characterized in brain cell function. However, the presence and distribution of AQPs in CNS stem cells has not yet been assessed.

Results. In the present study, we investigate the expression and subcellular localization of AQPs in murine ANSCs (adult neural stem cells). Considerable AQP8 mRNAs were found in ANSCs where, as expected, the transcript of two additional AQPs, AQP4 and AQP9, was also detected. Immunoblotting with subcellular membrane fractions of ANSCs showed predominant expression of AQP8 in the mitochondria‐enriched fraction. This result was consistent with the spotted immunoreactivity profile encountered within the ANSCs by confocal immunofluorescence.

Conclusions. AQP8 may have a role in mitochondrial volume regulation during ANSC differentiation. Recognition of AQPs in ANSCs is a step forward in our knowledge of water homoeostasis in the CNS and provides useful information for the purposes of stem cell technology.
Distinct roles for de novo versus hydrolytic pathways of sphingolipid biosynthesis in Saccharomyces cerevisiae
Authors: Cowart L. Ashley , Okamoto Yasuo , Lu Xinghua , Hannun Yusuf A.
Published: January 2006

Saccharomyces cerevisiae produces the sphingolipid ceramide by de novo synthesis as well as by hydrolysis of complex sphingolipids by Isc1p (inositolphosphoceramide-phospholipase C), which is homologous with the mammalian neutral sphingomyelinases. Though the roles of sphingolipids in yeast stress responses are well characterized, it has been unclear whether Isc1p contributes to stress-induced sphingolipids. The present study was undertaken in order to distinguish the relative roles of de novo sphingolipid biosynthesis versus Isc1p-mediated sphingolipid production in the heat-stress response. Ceramide production was measured at normal and increased temperature in an ISC1 deletion and its parental strain (ISC1 being the gene that codes for Isc1p). The results showed that Isc1p contributes specifically to the formation of the C24-, C24:1- and C26-dihydroceramide species. The interaction between these two pathways of sphingolipid production was confirmed by the finding that ISC1 deletion is synthetically lethal with the lcb1-100 mutation. Interestingly, Isc1p did not contribute significantly to transient cell-cycle arrest or growth at elevated temperature, responses known to be regulated by the de novo pathway. In order to define specific contributions of ISC1, microarray hybridizations were performed, and analyses showed misregulation of genes involved in carbon source utilization and sexual reproduction, which was corroborated by defining a sporulation defect of the isc1Δ strain. These results indicate that the two pathways of ceramide production in yeast interact, but differ in their regulation of ceramides of distinct molecular species and serve distinct cellular functions.
Biochemical characterization of androgen receptor-interacting protein 4
Authors: Domanskyi Andrii , Virtanen Katja T. , Palvimo Jorma J. , Jänne Olli A.
Published: January 2006

ARIP4 [AR (androgen receptor)-interacting protein 4] is a member of the SNF2-like family of proteins. Its sequence similarity to known proteins is restricted to the centrally located SNF2 ATPase domain. ARIP4 is an active ATPase, and dsDNA (double-stranded DNA) and ssDNA (single-stranded DNA) enhance its catalytic activity. We show in the present study that ARIP4 interacts with AR and binds to DNA and mononucleosomes. The N-terminal region of ARIP4 mediates interaction with AR. Kinetic parameters of the ARIP4 ATPase are similar to those of BRG-1 and SNF2h, two members of the SNF2-like protein family, but the specific activity of ARIP4 protein purified to >90% homogeneity is approximately ten times lower, being 120 molecules of ATP hydrolysed by an ARIP4 molecule per min in contrast with approx. 1000 ATP molecules hydrolysed per min by ATP-dependent chromatin remodellers. Unlike other members of the SNF2 family, ARIP4 does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. ARIP4 is covalently modified by sumoylation, and mutation of six potential SUMO (small ubiquitin-related modifier) attachment sites abolished the ability of ARIP4 to bind DNA, hydrolyse ATP and activate AR function. We conclude that, similar to its closest homologues in the SNF2-like protein family, ATRX (α-thalassemia, mental retardation, X-linked) and Rad54, ARIP4 does not seem to be a classical chromatin remodelling protein.
Bile-salt-stimulated lipase and mucins from milk of ‘secretor’ mothers inhibit the binding of Norwalk virus capsids to their carbohydrate ligands
Authors: Ruvoën-Clouet Nathalie , Mas Eric , Marionneau Séverine , Guillon Patrice , Lombardo Dominique , Le Pendu Jacques
Published: January 2006

Breast-feeding-associated protection against calicivirus diarrhoea is associated with the presence of high levels of 2-linked oligosaccharides in mother's milk, and human calicivirus strains including the NV (Norwalk virus) use gut 2-linked fucosylated glycans as receptors, suggesting the presence of decoy receptors in milk. Our aim was to analyse the ability of human milk to inhibit the attachment of rNV VLPs (recombinant NV-like particles) to their carbohydrate ligands and to characterize potential inhibitors found in milk. Milk from women with the secretor phenotype was strongly inhibitory, unlike milk from women that are non-secretors, which is devoid of 2-linked fucosylated structures. At least two fractions in human milk acted as inhibitors for the NV capsid attachment. The first fraction corresponded to BSSL (bile-salt-stimulated lipase) and the second to associated mucins MUC1 and MUC4. These proteins present tandem repeat O-glycosylated sequences that should act as decoy receptors for the NV, depending on the combined mother/child secretor status.
A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44
Authors: Stoeck Alexander , Keller Sascha , Riedle Svenja , Sanderson Michael P. , Runz Steffen , Le Naour Francois , Gutwein Paul , Ludwig Andreas , Rubinstein Eric , Altevogt Peter
Published: January 2006

Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-β-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor α-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.
Interactions between Hairy/Enhancer of Split-related proteins and the pancreatic transcription factor Ptf1-p48 modulate function of the PTF1 transcriptional complex
Authors: Ghosh Bidyut , Leach Steven D.
Published: January 2006

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transciption factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix–loop–helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.
Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae
Authors: Elbing Karin , McCartney Rhonda R. , Schmidt Martin C.
Published: January 2006

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of β and γ subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its β and γ subunits.
Interactive domains between pore loops of the yeast K+ channel TOK1 associate with extracellular K+ sensitivity
Authors: Johansson Ingela , Blatt Michael R.
Published: January 2006

Gating of the outward-rectifying K+ channel TOK1 of Saccharomyces cerevisiae is controlled by membrane voltage and extracellular K+ concentration. Previous studies identified two kinetically distinct effects of K+, and site-mutagenic analysis associated these K+-dependencies with domains of the extracellular turrets of the channel protein. We have mapped the TOK1 pore domains to extant K+ channel crystal structures to target additional residues contributing to TOK1 gating. Leu270, located in the first pore domain of TOK1, was found to be critical for gating and its K+ sensitivity. Analysis of amino acid substitutions indicated that spatial position of the polypeptide backbone is a primary factor determining gating sensitivity to K+. The strongest effects, with L270Y, L270F and L270W, led to more than a 30-fold decrease in apparent K+ affinity and an inversion in the apparent K+-dependence of voltage-dependent gating compared with the wild-type current. A partial rescue of wild-type gating was obtained on substitution in the second pore domain with the double mutant L270D/A428Y. These, and additional results, demarcate extracellular domains that are associated with the K+-sensitivity of TOK1 and they offer primary evidence for a synergy in gating between the two pore domains of TOK1, demonstrating an unexpected degree of long-distance interaction across the mouth of the K+ channel.
IRES-dependent regulation of FGF-2 mRNA translation in pathophysiological conditions in the mouse
Authors: Gonzalez-Herrera I.G. , Prado-Lourenco L. , Teshima-Kondo S. , Kondo K. , Cabon F. , Arnal J.-F. , Bayard F. , Prats A.-C.
Published: January 2006

The mRNA coding for FGF-2 (fibroblast growth factor 2), a major angiogenic factor, is translated by an IRES (internal ribosome entry site)-dependent mechanism. We have studied the role of the IRES in the regulation of FGF-2 expression in vivo, under pathophysiological conditions, by creating transgenic mice lines expressing bioluminescent bicistronic transgenes. Analysis of FGF-2 IRES activity indicates strong tissue specificity in adult brain and testis, suggesting a role of the IRES in the activation of FGF-2 expression in testis maturation and brain function. We have explored translational control of FGF-2 mRNA under diabetic hyperglycaemic conditions, as FGF-2 is implied in diabetes-related vascular complications. FGF-2 IRES is specifically activated in the aorta wall in streptozotocin-induced diabetic mice, in correlation with increased expression of endogenous FGF-2. Thus, under hyperglycaemic conditions, where cap-dependent translation is blocked, IRES activation participates in FGF-2 overexpression, which is one of the keys of diabetes-linked atherosclerosis aggravation. IRES activation under such pathophysiological conditions may involve ITAFs (IRES trans-acting factors), such as p53 or hnRNP AI (heterogeneous nuclear ribonucleoprotein AI), recently identified as inhibitory or activatory ITAFs respectively for FGF-2 IRES.
The large spectrum of eIF2B-related diseases
Authors: Fogli A. , Boespflug-Tanguy O.
Published: January 2006

eIF2B (eukaryotic initiation factor 2B) is a GEF (guanine nucleotide-exchange factor) that plays, with its substrate eIF2, a key regulatory role in the translation initiation phase of protein synthesis. The importance of correct control of eIF2 and eIF2B for normal physiology is underlined by the recent involvement of the five genes that encode the five eIF2B subunits in a severe autosomal recessive neurodegenerative disease, described in young children as CACH (childhood ataxia with central nervous system hypomyelination)/VWM (leukoencephalopathy with vanishing white matter) syndrome. The syndrome is characterized by episodes of rapid deterioration during febrile illnesses or following head trauma and symmetrical demyelination of the brain white matter with cavitation aspects, leading to a progressive vanishing of the white matter replaced by CSF (cerebrospinal fluid). However, a wide clinical spectrum has been observed in the 148 patients presently reported, from congenital forms with rapid death to adult-onset forms with slow mental decline and progressive motor dysfunction, sometimes associated with congenital eye abnormalities or ovariodysgenesis. So far, 77 different mutations in each of the five EIF2B genes (EIF2B1–5), encoding subunits eIF2Bα–ϵ, have been found, with two-thirds affecting the eIF2Bϵ subunit. The correlation found between the level of GEF activity of eIF2B in the mutated white blood cells and the age at disease onset suggests a direct role of the abnormal translation control in the pathophysiology of the disease.
Decapping the message: a beginning or an end
Authors: Liu H. , Kiledjian M.
Published: January 2006

Removal of the mRNA 5′ cap is an important step in the regulation of mRNA stability. mRNAs are degraded by at least two distinct exonucleolytic decay pathways, one from the 5′ end, and the second from the 3′ end. Two major cellular decapping enzymes have been identified, and each primarily functions in one of the two decay pathways. The Dcp2 decapping enzyme utilizes capped mRNA as substrate and hydrolyses the cap to release m7GDP (N7-methyl GDP), while a scavenger decapping enzyme, DcpS, utilizes cap dinucleotides or capped oligonucleotides as substrate and releases m7GMP (N7-methyl GMP). In this review, we will highlight the function of different decapping enzymes and their role in mRNA turnover.