Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2′-modified-4′-thionucleosides

AbstractGene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2′-modified-4′-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2′-fluoro-4′-thionucleoside and 2′-fluoronucleoside units as well as only 2′-fluoronucleoside units, while those prepared as combination of 2′-OMe nucleoside/2′-OMe-4′-thionucleoside and 2′-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity

Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses

Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts

AbstractGM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses

Traceless synthesis of protein thioesters using enzyme-mediated hydrazinolysis and subsequent self-editing of cysteinyl prolyl sequence

A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The hydrazinolysis followed by thioesterification affords cysteinyl prolyl thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide thioesters. The developed protocol was successfully applied to conversion of recombinant proteins to thioesters

A new heterozygous compound mutation in the CTSA gene in galactosialidosis

Galactosialidosis is an autosomal recessive lysosomal storage disease caused by the combined deficiency of lysosomal β-galactosidase and neuraminidase due to a defect in the protective protein/cathepsin A. Patients present with various clinical manifestations and are classified into three types according to the age of onset: the early infantile type, the late infantile type, and the juvenile/adult type. We report a Japanese female case of juvenile/adult type galactosialidosis. Clinically, she presented with short stature, coarse facies, angiokeratoma, remarkable action myoclonus, and cerebellar ataxia. The patient was diagnosed with galactosialidosis with confirmation of impaired β-galactosidase and neuraminidase function in cultured skin fibroblasts. Sanger sequencing for CTSA identified a compound heterozygous mutation consisting of NM_00308.3(CTSA):c.746 + 3A>G and c.655-1G>A. Additional analysis of her mother’s DNA sequence indicated that the former mutation originated from her mother, and therefore the latter was estimated to be from the father or was a de novo mutation. Both mutations are considered pathogenic owing to possible splicing abnormalities. One of them (c.655-1G>A) is novel because it has never been reported previously

Novel pH-dependent regulation of human cytosolic sialidase 2 (NEU2) activities by siastatin B and structural prediction of NEU2/siastatin B complex

Human cytosolic sialidase (Neuraminidase 2, NEU2) catalyzes the removal of terminal sialic acid residues from glycoconjugates. The effect of siastatin B, known as a sialidase inhibitor, has not been evaluated toward human NEU2 yet. We studied the regulation of NEU2 activity by siastatin B in vitro and predicted the interaction in silico. Inhibitory and stabilizing effects of siastatin B were analyzed in comparison with DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) toward 4-umbelliferyl N-acetylneuraminic acid (4-MU-NANA)- and α2,3-sialyllactose-degrading activities of recombinant NEU2 produced by E. coli GST-fusion gene expression. Siastatin B exhibited to have higher competitive inhibitory activity toward NEU2 than DANA at pH 4.0. We also revealed the stabilizing effect of siastatin B toward NEU2 activity at acidic pH. Docking model was constructed on the basis of the crystal structure of NEU2/DANA complex (PDB code: 1VCU). Molecular docking predicted that electrostatic neutralization of E111 and E218 residues of the active pocket should not prevent siastatin B from binding at pH 4.0. The imino group (1NH) of siastatin B can also interact with D46, neutralized at pH 4.0. Siastatin B was suggested to have higher affinity to the active pocket of NEU2 than DANA, although it has no C7–9 fragment corresponding to that of DANA. We demonstrated here the pH-dependent affinity of siastatin B toward NEU2 to exhibit potent inhibitory and stabilizing activities. Molecular interaction between siastatin B and NEU2 will be utilized to develop specific inhibitors and stabilizers (chemical chaperones) not only for NEU2 but also the other human sialidases, including NEU1, NEU3 and NEU4, based on homology modeling

The Latent Risk of Acidosis in Commercially Available Total Parenteral Nutrition (TPN) Products: a Randomized Clinical Trial in Postoperative Patients

To evaluate the latent risk of acidosis in commercially available total parenteral nutrition (TPN) products, three types of commercially available TPN products were compared in postoperative patients. Sixty-four hospitalized patients with gastro-intestinal disease who undertook curative gastro intestinal resection were studied prospectively and administered with TPN solutions. Three types of commercially available TPN products were assigned randomly to eligible patients. Serial studies of blood acid-base status, serum electrolytes, and urinary acid-base status were conducted in the three groups administered with different TPN solutions. Patients received appropriate electrolytic solutions on the operation day and TPN solution from 2 to 7 days after operation. There were no differences among any of the serum electrolytes in the three groups. In one group, urinary pH decreased slightly and urinary net acid excretion (NAE) increased significantly after administration. This TPN product contains about 40 mEq/L of non-metabolizable acid to avoid the Maillard reaction that produces a complex of glucose and amino acids. Urinary NAE did not change in the other two groups. These TPN products do not use non-metabolizable acid to adjust pH. The present results suggest that the non-metabolizable acid may be a risk factor of metabolic acidosis

Proteomic alteration in gastic adenocarcinomas from Japanese patients

BACKGROUND: Gastric adenocarcinomas comprise one of the common types of cancers in Asian countries including Japan. Comprehensive protein profiling of paired surgical specimens of primary gastric adenocarcinomas and nontumor mucosae derived from Japanese patients was carried out by means of two-dimensional gel electrophoresis (2D-EP) and liquid chromatography-electrospray ionic tandem mass spectrometry (LC-ESI-MS) to establish gastric cancer-specific proteins as putative clinical biomarkers and molecular targets for chemotherapy. RESULTS: Relatively common alterations in protein expression were revealed in the tumor tissues. Increases in manganese dismutase and nonhistone chromosomal protein HMG-1 (HMG-1) were observed, while decreases in carbonic anhydrases I and II, glutatione-S-transferase and foveolin precursor (gastrokine-1) (FOV), an 18-kDa stomach-specific protein with putative tumor suppressor activity, were detected. RT-PCR analysis also revealed significant down-regulation of FOV mRNA expression in tumor tissues. CONCLUSION: A possible pathological role for down-regulation of FOV in gastric carcinogenesis was demonstrated. Evaluation of the specific decreases in gene and protein expression of FOV in patients may be utilized as clinical biomarkers for effective diagnosis and assessment of gastric cancer

Agesasines A and B, Bromopyrrole Alkaloids from Marine Sponges Agelas spp.

Exploration for specialized metabolites of Okinawan marine sponges Agelas spp. resulted in the isolation of five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9-hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5). Their structures were elucidated on the basis of spectroscopic analyses. Agesasines A (1) and B (2) were assigned as rare bromopyrrole alkaloids lacking an aminoimidazole moiety, while 3–5 were elucidated to be linear bromopyrrole alkaloids with either aminoimidazolone, aminoimidazole, or N-methylated aminoimidazole moieties