Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein

Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms

Effective impairment of myeloma cells and their progenitors by hyperthermia

Multiple myeloma (MM) remains incurable, and MM-initiating cells or MM progenitors are considered to contribute to disease relapse through their drug-resistant nature. In order to improve the therapeutic efficacy for MM, we recently developed novel superparamagnetic nanoparticles which selectively accumulate in MM tumors and extirpate them by heat generated with magnetic resonance. We here aimed to clarify the therapeutic effects on MM cells and their progenitors by hyperthermia. Heat treatment at 43°C time-dependently induced MM cell death. The treatment upregulated endoplasmic reticulum (ER) stress mediators, ATF4 and CHOP, while reducing the protein levels of Pim-2, IRF4, c-Myc and Mcl-1. Combination with the proteasome inhibitor bortezomib further enhanced ER stress to potentiate MM cell death. The Pim inhibitor SMI-16a also enhanced the reduction of the Pim-2-driven survival factors, IRF4 and c-Myc, in combination with the heat treatment. The heat treatment almost completely eradicated “side population” fractions in RPMI8226 and KMS-11 cells and suppressed their clonogenic capacity as determined by in vitro colony formation and tumorigenic capacity in SCID mice. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells and enhance their susceptibility to chemotherapeutic drugs

Transcription and Translation Products of the Cytolysin Gene psm-mec on the Mobile Genetic Element SCCmec Regulate Staphylococcus aureus Virulence

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus

Effect of amine structure on CO2 capture by polymeric membranes

Poly(amidoamine)s (PAMAMs) incorporated into a cross-linked poly(ethylene glycol) exhibited excellent CO2 separation properties over H2. However, the CO2 permeability should be increased for practical applications. Monoethanolamine (MEA) used as a CO2 determining agent in the current CO2 capture technology at demonstration scale was readily immobilized in poly(vinyl alcohol) (PVA) matrix by solvent casting of aqueous mixture of PVA and the amine. The resulting polymeric membranes can be self-standing with the thickness above 3 μm and the amine fraction less than 80 wt%. The gas permeation properties were examined at 40 °C and under 80% relative humidity. The CO2 separation performance increased with increase of the amine content in the polymeric membranes. When the amine fraction was 80 wt%, the CO2 permeability coefficient of MEA containing membrane was 604 barrer with CO2 selectivity of 58.5 over H2, which was much higher than the PAMAM membrane (83.7 barrer and 51.8, respectively) under the same operation conditions. On the other hand, ethylamine (EA) was also incorporated into PVA matrix to form a thin membrane. However, the resulting polymeric membranes exhibited slight CO2-selective gas permeation properties. The hydroxyl group of MEA was crucial for high CO2 separation performance

Effect of amine structure on CO₂ capture by polymeric membranes

<p>Poly(amidoamine)s (PAMAMs) incorporated into a cross-linked poly(ethylene glycol) exhibited excellent CO₂ separation properties over H₂. However, the CO₂ permeability should be increased for practical applications. Monoethanolamine (MEA) used as a CO₂ determining agent in the current CO₂ capture technology at demonstration scale was readily immobilized in poly(vinyl alcohol) (PVA) matrix by solvent casting of aqueous mixture of PVA and the amine. The resulting polymeric membranes can be self-standing with the thickness above 3 μm and the amine fraction less than 80 wt%. The gas permeation properties were examined at 40 °C and under 80% relative humidity. The CO₂ separation performance increased with increase of the amine content in the polymeric membranes. When the amine fraction was 80 wt%, the CO₂ permeability coefficient of MEA containing membrane was 604 barrer with CO₂ selectivity of 58.5 over H₂, which was much higher than the PAMAM membrane (83.7 barrer and 51.8, respectively) under the same operation conditions. On the other hand, ethylamine (EA) was also incorporated into PVA matrix to form a thin membrane. However, the resulting polymeric membranes exhibited slight CO₂-selective gas permeation properties. The hydroxyl group of MEA was crucial for high CO₂ separation performance.</p

Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q^{245}TVTKK^{250} on SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV

Karyopherin-β1 Regulates Radioresistance and Radiation-Increased Programmed Death-Ligand 1 Expression in Human Head and Neck Squamous Cell Carcinoma Cell Lines

Nuclear transport receptors, such as karyopherin-&beta;1 (KPNB1), play important roles in the nuclear-cytoplasmic transport of macromolecules. Recent evidence indicates the involvement of nuclear transport receptors in the progression of cancer, making these receptors promising targets for the treatment of cancer. Here, we investigated the anticancer effects of KPNB1 blockage or in combination with ionizing radiation on human head and neck squamous cell carcinoma (HNSCC). HNSCC cell line SAS and Ca9-22 cells were used in this study. Importazole, an inhibitor of KPNB1, or knockdown of KPNB1 by siRNA transfection were applied for the blockage of KPNB1 functions. The roles of KPNB1 on apoptosis induction and cell surface expression levels of programmed death-ligand 1 (PD-L1) in irradiated HNSCC cells were investigated. The major findings of this study are that (i) blockage of KPNB1 specifically enhanced the radiation-induced apoptosis and radiosensitivity of HNSCC cells; (ii) importazole elevated p53-upregulated modulator of apoptosis (PUMA) expression via blocking the nuclear import of SCC-specific oncogene &Delta;Np63 in HNSCC cells; and (iii) blockage of KPNB1 attenuated the upregulation of cell surface PD-L1 expression on irradiated HNSCC cells. Taken together, these results suggest that co-treatment with KPNB1 blockage and ionizing radiation is a promising strategy for the treatment of HNSCC

高感度酵素抗体法を用いたRett症候群および他の神経疾患における髄液ガングリオシド分析

年齢2～10歳のRett症候群患者12名と,同年齢の髄液検査が必要であった他疾患患者19名,および正常対照例7名において,脳脊髄液中の5つの主要なガングリオシドGM1, GD1a, GD1b, GT1bおよびGQ1bを感度の高い酵素抗体法で測定した.我々は,この方法を用いて標準曲線を作成し,これらを測定できた.本法は,高感度な検査法であることを証明した.対象症例における脳脊髄液中の総ガングリオシド量は,脳脊髄液1ml当たり,正常対照例で100～900ng,神経疾患患者で25ngから最大5,000ngの値をとっていた.Rett症候群を含め,どの神経疾患でも特別なパターンは認められなかった.過去にRett症候群においては,あるガングリオシドが大脳および小脳で低下しているという所見の報告があったが,この所見と関連する結果は髄液では得られなかった.また,我々が,髄液における5つの主要なガングリオシドの値をRett症候群患者と正常および疾患対照例と比較した所では,そのデータからRett症候群を予測させるような低下パターンを見出すことはできなかった.但し,12例中5例でGD1aが, 4例でGT1bおよびGQ1bの合計値が正常対照例の下限値よりも低下していた.Rett症候群の独歩可能な患児と不可能な患児における髄液ガングリオシド値の比較検討では差がなく,また異った臨床ステージの患者間の比較検討でも差がなかった.しかしながら,髄膜炎の急性期,熱性けいれんの直後,またRett症候群のけいれんの多い児2名を含み,てんかん患児の一部で髄液中のガングリオシド,主として,GD1b,GT1b値が上昇していた.The cerebrospinal fluid (CSF) gangliosides GM1, GDla, GDlb, GTlb and GQlb were measured using a highly sensitive enzyme-immunostaining technique, in 12 patients with Rett syndrome (RS) ranging in age from 2y to 10y, in age-matched patients with various neurologic diseases (n=19) and in normal control subjects (n=7). The method used proved to be highly sensitive; a standard curve was established and we were able to obtain reliable assay results. Total CSF gangliosides varied widely among normal controls (100～900 ng/ml CSF) as well as in those with neurologic diseases (25 to 5,000 ng/ml CSF). No specific ganglioside pattern was found in any of the disease groups, including RS. Although previous reports have shown reductions in the levels of gangliosides in the RS brain, we found no evidence of a predictable and constant pattern of reduced levels of any of the five major CSF gangliotetraose series gangliosides in this syndrome; however, the amount of GDla was reduced in five of the 12 RS patients, and the amount of GQ1b+GT1b was reduced in four as compared with the minimum value obtained in the control group. Analysis of CSF ganglioside levels in RS patients revealed no differences between ambulant and non-ambulant patients, nor were there any differences among patients in different clinical stages. There was, however, a positive correlation between CSF ganglioside levels, mainly the b-pathway gangliosides, and the presence and frequency of seizures in some patients, including the one with febrile convulsion, those in the acute stage of meningitis, two of the RS patients and some of those with epileptic syndromes