Exposure of Astrocytes to Hypoxia/Reoxygenation Enhances Expression of Glucose-Regulated Protein 78 Facilitating Astrocyte Release of the Neuroprotective Cytokine Interleukin 6

Abstract: Astrocytes exposed to hypoxia (H) or hypoxia/reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an ≈78-kDa polypeptide that demonstrated sequence identity with glucose-regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high-molecular-weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of anti-sense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R-induced astrocyte release of IL-6 by ≈50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL-6 under the stress of H/R.     

Genome-wide analysis of organ-preferential metastasis of human small cell lung cancer in mice

Although a number of molecules have been implicated in the process of cancer metastasis, the organ-selective nature of cancer cells is still poorly understood. To investigate this issue, we established a metastasis model in mice with multiple organ dissemination by i.v. injection of human small cell lung cancer (SBC-5) cells. We analyzed gene-expression profiles of 25 metastatic lesions from four organs (lung, liver, kidney, and bone) using a cDNA microarray representing 23,040 genes and extracted 435 genes that seemed to reflect the organ specificity of the metastatic cells and the cross-talk between cancer cells and microenvironment. Furthermore, we discovered 105 genes that might be involved in the incipient stage of secondary-tumor formation by comparing the gene-expression profiles of metastatic lesions classified according to size (<1 or >2 mm) as either "micrometastases" or "macrometastases." This genome-wide analysis should contribute to a greater understanding of molecular aspects of the metastatic process in different microenvironments, and provide indicators for new strategies to predict and prevent metastasis.     

Load‐sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor

The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild‐type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild‐type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their ‘unplugged’ forms were only half of the conductivity of the wild‐type channel. These results suggest that the D33E mutation induces a load‐dependent inactivation of the MotA/B complex. We propose that the stator element is a load‐sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co‐ordinated proton translocation.     

Soil organic matter dynamics in density and particle-size fractions following destruction of tropical rainforest and the subsequent establishment of Imperata grassland in Indonesian Borneo using stable carbon isotopes

Background and aims

Large portions of the deforested areas in Southeast Asia have been ultimately replaced by the invasive grass Imperata cylindrica, but the dynamics of soil organic matter (SOM) during such land transitions are poorly understood. This study presents SOM dynamics in density and particle-size fractions following rainforest destruction and the subsequent establishment and persistence of Imperata grassland.

Methods

We examined soil C stock and natural ¹³C abundance in these fractions to depths of 100 cm. We predicted future soil C storage and evaluated C turnover rates in these fractions using a simple exponential model. Because soil texture strongly affects soil C storage, two chronosequences of soils differing in soil texture were compared (n?=?1 in each chronosequence).

Results

The clay-associated SOM increased in all soil layers (0–100 cm) along the forest-to-grassland chronosequence, whereas light-fraction SOM in the surface soil layer (0–5 cm) decreased.

Conclusions

In the surface layer, all SOM fractions exhibited rapid replacement of forest-derived C to grassland-derived C, indicating fast turnover. Meanwhile, δ¹³C values of the light fraction in the surface layer indicated that forest-derived charcoal and/or occluded low-density organic matter constituted unexpectedly large proportions of the light fraction. Mathematical modelling (0–50 cm) showed that grassland-derived C in the clay and silt fractions in all soil layers increased almost linearly for at least 50 years after grassland establishment. In the meantime, the forest-derived C stock in the clay fraction constituted 82 % of the total stable C pool at 0–50-cm depths even under steady-state conditions (t = ∞), indicating that residue of forest-derived SOM associated with clay largely contributed to preserving the soil C pool. Comparing soils with different soil textures, clay and silt particles in coarse-textured soil exhibited a substantially higher degree of organo-mineral interactions per unit volume of clay or silt compared to fine-textured soils.     

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.     

Molecular characterization of a membrane-bound prenyltransferase specific for isoflavone from Sophora flavescens

Prenylated isoflavones are secondary metabolites that are mainly distributed in legume plants. They often possess divergent biological activities such as anti-bacterial, anti-fungal, and anti-oxidant activities and thus attract much attention in food, medicinal, and agricultural research fields. Prenyltransferase is the key enzyme in the biosynthesis of prenylated flavonoids by catalyzing a rate-limiting step, i.e. the coupling process of two major metabolic pathways, the isoprenoid pathway and shikimate/polyketide pathway. However, so far only two genes have been isolated as prenyltransferases involved in the biosynthesis of prenylated flavonoids, namely naringenin 8-dimethylallyltransferase from Sophora flavescens (SfN8DT-1) specific for some limited flavanones and glycinol 4-dimethylallyltransferase from Glycine max (G4DT), specific for pterocarpan substrate. We have in this study isolated two novel genes coding for membrane-bound flavonoid prenyltransferases from S. flavescens, an isoflavone-specific prenyltransferase (SfG6DT) responsible for the prenylation of the genistein at the 6-position and a chalcone-specific prenyltransferase designated as isoliquiritigenin dimethylallyltransferase (SfiLDT). These prenyltransferases were enzymatically characterized using a yeast expression system. Analysis on the substrate specificity of chimeric enzymes between SfN8DT-1 and SfG6DT suggested that the determinant region for the specificity of the flavonoids was the domain neighboring the fifth transmembrane α-helix of the prenyltransferases.     

ABA inhibits entry into stomatal‐lineage development in Arabidopsis leaves

The number and density of stomata are controlled by endogenous and environmental factors. Despite recent advances in our understanding of stomatal development, mechanisms which prevent stomatal‐lineage entry remain unclear. Here, we propose that abscisic acid (ABA), a phytohormone known to induce stomatal closure, limits initiation of stomatal development and induces enlargement of pavement cells in Arabidopsis cotyledons. An ABA‐deficient aba2‐2 mutant had an increased number/proportion of stomata within a smaller cotyledon, as well as reduced expansion of pavement cells. This tendency was reversed after ABA application or in an ABA over‐accumulating cyp707a1cyp707a3 doublemutant. Our time course analysis revealed that aba2‐2 shows prolonged formation of meristemoids and guard mother cells, both precursors of stoma. This finding is in accordance with prolonged gene expression of SPCH and MUTE, master regulators for stomatal formation, indicating that ABA acts upstream of these genes. Only aba2‐2 mute, but not aba2‐2 spch double mutant showed additive phenotypes and displayed inhibition of pavement cell enlargement with increased meristemoid number, indicating that ABA action on pavement cell expansion requires the presence of stomatal‐lineage cells.     

Long-term effects of peginterferon alfa-2a therapy in Japanese patients with chronic hepatitis B virus infection

Background

There is no information on the long-term effects of peginterferon (PEG-IFN) alfa-2a therapy for chronic hepatitis B (CHB) in Japan. This double-blind, randomized trial investigated the efficacy of PEG-IFN therapy.

Methods

We analyzed 22 Japanese patients with CHB (hepatitis B e antigen [HBeAg]-positive: 17, HBeAg-negative: 5) treated with PEG-IFN alfa-2a and followed-up posttreatment for 5 years. Responders represented patients who showed persistent normalization of alanine transferase (ALT) levels, HBeAg clearance, and low hepatitis B virus (HBV) DNA levels (HBeAg-positive patient; <5 log copies/mL, HBeAg-negative patient; <4.3 log copies/mL) at end of treatment, and at 1, 2, 3, 4 and 5 years posttreatment. In addition, baseline HBeAg-positive patients who showed sustained normalization of ALT level, HBeAg clearance, and low HBV DNA level for more than 6 months until at 1, 2, 3, 4, and 5 years after completion of PEG-IFN were also classified as “triple responders” and the proportion of triple responders relative to all patients was termed the “triple response rate”.

Results

The response rates among HBeAg-positive patients were 13 %, 25 %, 14 %, 21 % and 21 % at end of treatment, and at 1, 2, 3, 4, and 5 years, respectively. The response rate tended to be higher in patients treated for 48 than 24 weeks. The respective response rates among HBeAg-negative patients were 0 %, 20 %, 20 %, 20 % and 25 %. During the treatment period, hepatitis B surface antigen (HBsAg) clearance at 3.5 years was noted in one patient, who was 37-year-old, male, had genotype C and received PEG-IFN alfa-2a at 90 μg for 48 weeks.

Conclusion

At 5 years after completion of PEG-IFN, the triple response rate in HBeAg-positive patients and combined response rate in HBeAg-negative patients were 21 % (3/14) and 25 % (1/4), respectively. The triple response was seen in three patients who had all been treated with PEG-IFN for 48 weeks.

     

Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan

We performed chromosomal analysis on 540 mouse embryonic stem (ES) cell lines obtained during 2001 to 2004 from 20 institutions in Japan. Overall, 66.5% of the ES cell lines showed normal chromosomal numbers, but 15.9%, 9.1%, and 2.8% showed modal chromosomal numbers of 41, 42, and 39, respectively. When we karyotyped 88 ES cell lines selected arbitrarily from the 540 lines, 53 (60.2%) showed normal diploid karyotypes; the sex chromosome constitution of 52 lines was XY, with the remaining 1 being XX. Among 35 ES cell lines showing abnormal karyotypes, trisomy of chromosome 8 (41, XY, +8) was dominant (51.4%), 14.3% had trisomy 8 with loss of one sex chromosome (40, XO, +8), and 11.4% had trisomy 8 together with trisomy 11 (42, XY, +8, +11). Karyotypic abnormalities including trisomy 8 and trisomy 11 occurred in 88.6% and 17.1% of ES cell lines, respectively. The XO sex chromosome constitution was observed in 25.7% of all abnormal ES cell lines. Of the 88 selected ES cell lines, 60 lines were established from strain 129 animals, 17 from F1 progeny of C57BL/6J x CBA (called TT2 in this study), and 11 from C57BL/6J mice. Normal diploid karyotypes were observed in 58.3% of lines derived from 129, 58.8% of those from TT2, and 72.7% of C57BL/6J. The relatively high incidence of abnormalities in chromosomal number and karyotype in ES cell lines used in Japan suggests the importance of chromosomal analysis of ES cells for successful establishment of new animal models through germline transmission.