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21.
Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta 下载免费PDF全文
Hiromi Akeboshi Yasunori Chiba Yoshiko Kasahara Minako Takashiba Yuki Takaoka Mai Ohsawa Youichi Tajima Ikuo Kawashima Daisuke Tsuji Kohji Itoh Hitoshi Sakuraba Yoshifumi Jigami 《Applied microbiology》2007,73(15):4805-4812
Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed. 相似文献
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Shinji Kawasaki Masahiro Nagasaku Tsuyoshi Mimura Hitomi Katashima Susumu Ijyuin Takumi Satoh Youichi Niimura 《Applied microbiology》2007,73(23):7796-7798
This report investigates the requirement for CO2 for colony formation by Bifidobacterium species in both anoxic and oxic environments. All tested Bifidobacterium species exhibited difficulty in developing colonies in an atmosphere of 100% N2 but developed well when 1% CO2 was present. In the presence of CO2, the oxygen tolerance of the tested species was not improved. In the absence of CO2, only B. boum, a microaerophilic species, could develop colonies under an N2-based 5% O2 atmosphere, indicating that while CO2 is not an essential factor for colony development, both CO2 and O2 have stimulatory effects on B. boum colony development. 相似文献
23.
Ujifuku Kenta Fujimoto Takashi Sato Kei Morofuji Yoichi Muto Hideki Masumoto Hiroshi Nakagawa Shinsuke Niwa Masami Matsuo Takayuki 《Cellular and molecular neurobiology》2022,42(4):997-1004
Cellular and Molecular Neurobiology - Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood–brain barrier (BBB) implication is supposed to... 相似文献
24.
Dubouzet JG Maeda S Sugano S Ohtake M Hayashi N Ichikawa T Kondou Y Kuroda H Horii Y Matsui M Oda K Hirochika H Takatsuji H Mori M 《Plant biotechnology journal》2011,9(4):466-485
Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots. 相似文献
25.
The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions. 相似文献
26.
Kaneaki?EdoEmail author Youichi?Kawaguchi Masanori?Nunokawa Hiloshi?Kawamula Seigo?Higashi 《Environmental Biology of Fishes》2005,74(1):1-7
Synopsis We describe a total of 25 anadromous Sakhalin taimen Hucho perryi collected on the coast of Sarufutsu, northern Hokkaido, Japan in June 1997 and 1999. We examined morphology, stomach contents
and growth of three anadromous taimen (one male and two females) in detail, which were preserved in good condition. The three
taimen were aged and one female still had some eggs retained in her abdomen. The stomach contents of the three taimen consisted
of sand lance Ammodytes personatus Girard and sculpin Triglops sp. On the basis of scale analyses, the growth rate of the three taimen was estimated by using the back-calculation method,
and the highest rates were observed at young ages. Guanine pigmentation was present at the base of the caudal fin of each
taimen and is considered as a potential morphological trait to differentiate anadromous from fluvial specimens. Another anadromous
taimen, of which some individuals had reared for more than 3 years in seawater are also reported. For the conservation of
this rare and endangered species, their migration route between rivers and sea should be protected 相似文献
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Fujisawa Y Nagai Y Miyatake A Miura K Shokoji T Nishiyama A Kimura S Abe Y 《American journal of physiology. Heart and circulatory physiology》2006,290(3):H1120-H1127
Recently, a new member of the calcitonin gene-related peptide (CGRP) family, adrenomedullin 2 (AM2) or intermedin (IMD), was identified. AM2/IMD has been shown to have a vasodilator effect in mice and rats and an effect on urine formation in rats. In the present study, we investigated the effects of intravenously infused rat AM2 (rAM2) on blood pressure (BP), heart rate (HR), renal sympathetic nerve activity (RSNA), and renal blood flow (RBF) in conscious unrestrained rats relative to the effects of rat adrenomedullin (rAM) and proadrenomedullin NH2-terminal 20 peptide (rPAMP). Intravenous infusion of rAM2 (5 nmol/kg) significantly decreased BP and increased HR, RSNA, and RBF. These hypotensive and sympathoexcitatory effects diminished after 20 min, and HR returned to control levels 30 min after cessation of the infusion. In contrast, a significant increase in RBF was still evident 60 min after cessation of the peptide infusion. The duration of BP, HR, and RSNA responses was longer with rAM (5 nmol/kg) than with rAM2 infusion, whereas the increases in RBF induced by rAM2 and rAM were similar in their amplitude and duration. Infusion of rPAMP (200 nmol/kg) increased HR and RSNA but had no effect on RBF. Baroreceptor denervation suppressed, but did not diminish, the increases in HR and RSNA to rAM2. These findings indicate that the physiological roles of rAM2 and rAM are similar and that rAM2 also has a long-lasting vasodilator action on the renal vascular bed. 相似文献