Minichromosome maintenance 2 bound with retroviral Gp70 is localized to cytoplasm and enhances DNA-damage-induced apoptosis

The interaction of viral proteins with host-cellular proteins elicits the activation of cellular signal transduction pathways and possibly leads to viral pathogenesis as well as cellular biological events. Apoptotic signals induced by DNA-damage are remarkably up-regulated by Friend leukemia virus (FLV) exclusively in C3H hosts; however, the mechanisms underlying the apoptosis enhancement and host-specificity are unknown. Here, we show that C3H mouse-derived hematopoietic cells originally express higher levels of the minichromosome maintenance (MCM) 2 protein than BALB/c- or C57BL/6-deriverd cells, and undergo more frequent apoptosis following doxorubicin-induced DNA-damage in the presence of the FLV envelope protein gp70. Dual transfection with gp70/Mcm2 reproduced doxorubicin-induced apoptosis even in BALB/c-derived 3T3 cells. Immunoprecipitation assays using various deletion mutants of MCM2 revealed that gp70 bound to the nuclear localization signal (NLS) 1 (amino acids 18-24) of MCM2, interfered with the function of NLS2 (amino acids 132-152), and suppressed the normal nuclear-import of MCM2. Cytoplasmic MCM2 reduced the activity of protein phosphatase 2A (PP2A) leading to the subsequent hyperphosphorylation of DNA-dependent protein kinase (DNA-PK). Phosphorylated DNA-PK exhibited elevated kinase activity to phosphorylate P53, thereby up-regulating p53-dependent apoptosis. An apoptosis-enhancing domain was identified in the C-terminal portion (amino acids 703-904) of MCM2. Furthermore, simultaneous treatment with FLV and doxorubicin extended the survival of SCID mice bearing 8047 leukemia cells expressing high levels of MCM2. Thus, depending on its subcellular localization, MCM2 plays different roles. It participates in DNA replication in the nucleus as shown previously, and enhances apoptosis in the cytoplasm.     

Diffuse large B-cell lymphoma presenting with neurolymphomatosis and intravascular lymphoma: a unique autopsy case with diverse neurological symptoms

ABSTRACT: A 78-year-old Japanese male noticed a difficulty in the beginning of standing up, followed by a progressive numbness of extremities with pain, Bell's palsy, dysarthria, and difficulty in swallowing. A clinician had suspected cancer of unknown primary origin, accompanied by the diverse and elusive neurological symptoms, likely presenting as painful mononeuropathy simplex and cranial neuropathy. He developed dysbasia over weeks and died 1 month after the symptom onset. At autopsy, an ill-defined large and soft tumor mass in the right lobe of the liver with direct invasion into the right adrenal gland was observed. The left adrenal gland or right iliopsoas muscle was also involved. Microscopic findings showed a monotonous proliferation of medium-sized to large atypical lymphoid cells, which were diffusely positive for CD20 in immunohistochemistry, consistent with diffuse large B-cell lymphoma (DLBL). Furthermore, the lymphoma cells aggressively infiltrated endoneurial and subperineurial spaces not only in the peripheral nerves and plexuses, but partly in the spinal nerve roots, and intravascular spaces in various tissues. Therefore, systemic lymphoma (DLBL) complicated with neurolymphomatosis (NL) and intravascular lymphoma (IVL) was diagnosed. Very early diagnosis and treatment are necessary for the NL patients with poor prognosis. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5862472377020448.     

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N-acetyl-L-Met, N-acetyl-L-Gln and N-acetyl-L-Leu, but had a lower hydrolytic activity towards N-acetyl-L-Phe. The kinetic parameters K(m) and k(cat) were determined to be 24.6 mm and 370 s(-1), respectively, for N-acetyl-l-Met at 90 degrees C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn(2+), Mn(2+) and Ni(2+)), and Mn(2+) addition caused an alteration in substrate specificity. Site-directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole-3-acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal-binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.     

Prostaglandin E2-activated Epac promotes neointimal formation of the rat ductus arteriosus by a process distinct from that of cAMP-dependent protein kinase A

We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2'-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.     

Stabilization of water-in-oil emulsion by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 and its application to biotransformation

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml⁻¹, respectively, by 12 h.     

Diverging Understandings of Forest Management in Matsutake Science

Diverging Understandings of Forest Management in Matsutake Science. As high-value gourmet mushrooms, the matsutake complex of the genus Tricholoma has been the subject of extensive research. This article reviews two trajectories of matsutake research, showing how distinctive regional nodes may develop within a cosmopolitan modern science. The global center of matsutake research is in Japan, where problems of artificial cultivation and the “orchard-style” enhancement of production under forest conditions stimulate basic research. U.S. Pacific Northwest research forms a contrasting regional node, with a focus on sustainable yields in the context of timber production. Regional differences in research design and results point to the importance of distinctive scientific legacies, in this case formed in relation to divergent histories of forest management. Attention to regional distinctions in the framing of scientific problems is particularly important as scientific frameworks are exported to new places; for example, both Japanese and American forms of matsutake science have been extended to China. 高価なグルメきのこであるマツタケとその近縁種群のTricholoma属は広範囲に渡る科学的研究の対象となってきた。本論では二つの地域特徴的なマツタケ研究の軌跡を概観し、文化的差異を超えて世界的に通用する近代科学においても地域固有の関心に応じて特徴のある知識が結節し発展することを示す。マツタケ研究の世界的な中心地である日本では人工増殖やマツタケを殖やすための「果樹園的」な山林作りへの関心が基礎研究の方向性に刺激を与えてきた。一方日本とは対照的に、米国北西岸州では木材の持続的産出に主眼をおいた山林管理の流れの中で研究が進んできた。こうした研究計画や結果的に得られる知識の違いは、地域ごとに特徴のある科学的遺産 - 本件の場合は森林管理の歴史が多様に枝分かれしていること - に注目することが重要であることを知らせてくれる。近年日本や米国で発展したマツタケ研究の方法や成果が中国での研究にも影響を与えているが、特に新しい研究の場を広げる場合には科学的な関心、問題がどのような枠組で組み立てられるか地域によって多様であることを考慮することが重要である。     

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF₄^- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.     

Enzyme inactivation and quality preservation of sake by high-pressure carbonation at a moderate temperature

The effect of a high-pressure carbonation treatment on the change in quality of sake during storage was investigated. Measurements of the amino acidity and isovaleraldehyde content of carbonated sake (20 MPa pressure at 40, 45 and 50 degrees C for 7, 21 and 33 min, respectively) as well as of heat-treated sake (reaching temperature of 65 degrees C and immediately cooled) were almost unchanged during storage at 3 and 20 degrees C. Glucose in the sake subjected to these treatments was retained at an almost constant under the same storage conditions, except for the sake carbonated at 40 degrees C and stored at 20 degrees C. In contrast, the amino acidity, and glucose and isovaleraldehyde contents of non-pasteurized (fresh) sake increased during storage at both temperatures. The sake samples subjected to the carbonation treatment and heat treatment both gave better sensory scores than the fresh sake sample after 6 month of storage at 3 and 20 degrees C, especially at 3 degrees C for the flavor. These results suggest that the high-pressure carbonation treatment is an effective new technique for preserving the quality of sake.