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ABSTRACT: The influences of impeller types on morphology and protein expression were investigated in a submerged culture ofAspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells
that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation
grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production
for both the intracellular heterologous protein (β-glucuronidase) and the extracellularly homologous protein (α-amylase).
The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation
ofA. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted
in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller
agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged
fed-batch fermentation of recombinantA. oryzae.
Biotechnology and Bioprocess Engineering 04/2012; 9(3):184-190. · 1.28 Impact Factor
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ABSTRACT: In the present study, the effects of the whole skin of Venenum bufonis on apoptotic and anti-invasive activity in A549 human lung cancer cells were investigated. Treatment with extract of the whole skin of V. bufonis (SVB) resulted in a significant decrease in cell growth of A549 cells, depending on dosage, which was associated with apoptosis induction, as proved by chromatin condensation and accumulation of apoptotic fraction. SVB treatment induced expression of death receptor-related proteins, such as death receptor 4, which further triggered activation of caspase-8 and cleavage of Bid. In addition, the increase in apoptosis by SVB treatment was correlated with dysfunction of mitochondria, activation of caspase-9 and -3, downregulation of IAP family proteins, such as XIAP, cIAP-1 and cIAP-2, and concomitant degradation of activated caspase-3-specific target proteins, such as poly (ADP-ribose) polymerase and β-catenin proteins. However, z-DEVD-fmk, a caspase-3-specific inhibitor, blocked SVB-induced apoptosis and increased the survival rate of SVB-treated cells, indicating that activation of caspase-3 plays a key role in SVB-induced apoptosis. In addition, within concentrations that were not cytotoxic to A549 cells, SVB induced marked inhibition of cell motility and invasiveness. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 in AGS cells were dose-dependently inhibited by treatment with SVB, and this was also correlated with a decrease in expression of their mRNA and proteins, and upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expression. Further studies are needed; however, the results indicated that SVB induces apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways. Our data also demonstrated that MMPs are critical targets of SVB-induced anti-invasiveness in A549 cells.
International Journal of Oncology 12/2011; 40(4):1210-9. · 2.40 Impact Factor
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ABSTRACT: Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory β5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.
Journal of Biological Chemistry 11/2010; 286(5):3618-29. · 4.77 Impact Factor
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ABSTRACT: Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.
Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.
The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.
PLoS ONE 01/2010; 5(7):e11547. · 4.09 Impact Factor
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ABSTRACT: Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC(WT)-EGFP, pMYOC(P370L)-EGFP, pMYOC(1-367)-EGFP, pOPTN(WT)-EGFP, and pOPTN(E50K)-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN(WT)-EGFP and pOPTN(E50K)-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.
American Journal Of Pathology 12/2009; 176(1):343-52. · 4.89 Impact Factor
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ABSTRACT: Sp1, a transcription factor, is upregulated in keratoconus, a cornea-thinning disease. Keratoconus corneas have also been shown to contain increased levels of degradative enzymes such as cathepsin B and decreased proteinase inhibitors such as alpha1-proteinase inhibitor (alpha1-PI). We transfected cultured human corneal stromal cells to overexpress Sp1. The resulting effects on cathepsin B and alpha1-PI levels as well as the cellular proliferative and apoptotic activities were examined by Western blotting and cytochemical staining. It was found that the Sp1 transfected cells contained a greater amount of cathepsin B than did mock transfected controls. The activity of cathepsin B was also increased. By contrast, the protein level of alpha1-PI was lowered in corneal stromal cells upon Sp1 overexpression. The Sp1-induced alterations thus mimicked closely those observed in keratoconus, supporting the notion that Sp1 upregulation may be a key factor contributing directly to the disease development. Furthermore, the apoptotic activity was unaffected in Sp1 transfectants but the proliferation was inhibited, consistent with the idea that Sp1 may play a role in differentiation of corneal cells.
Genes to Cells 10/2009; 14(10):1133-9. · 2.68 Impact Factor
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ABSTRACT: The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
Biotechnology Journal 06/2008; 3(5):659-68.
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ABSTRACT: The alpha-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1Delta) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1Delta) and HpOCR1 (Hpocr1Delta) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1Delta were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1Delta is not in the initiation step but rather in the subsequent step of outer chain elongation by alpha-1,2-mannose addition. Most interestingly, Hpocr1Delta showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N- and O-linked glycosylation. In contrast, addition of the first alpha-1,6-mannose residue onto the core oligosaccharide Man8GlcNAc2 was completely blocked in Hpoch1Delta despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in Hpoch1Delta provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1Delta strain with the targeted expression of Aspergillus saitoi alpha-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man5GlcNAc2 oligosaccharide as a major N-glycan.
Journal of Biological Chemistry 04/2006; 281(10):6261-72. · 4.77 Impact Factor
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ABSTRACT: Growth at the restrictive temperature (42°C) of Aspergillus nidulans B120, carrying the conditional-lethal mutation sodVIC1, was partially improved by the addition of 1.0 M sorbitol to the medium. The mutant grown at 42°C, with osmotic stabilizer, showed abnormal hyphal morphology, a decrease in β-1,3-glucan synthase activity as well as cell wall sugar content, but an increase in chitin synthase activity and N-acetyl-glucosamine content. The mutation also affected the secretion of extracellular protease. The temperature-dependent osmo-sensitive phenotype of a Saccharomyces cerevisiaeα-COP mutation can be rescued by the A. nidulans sodVIC+ gene. These results indicate that the sodVIC1 mutation affects proper processing of secretory proteins destined for the surface of cells or beyond.
FEMS Microbiology Letters 01/2006; 208(2):253 - 257. · 2.04 Impact Factor
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ABSTRACT: Growth at the restrictive temperature (42 degrees C) of Aspergillus nidulans B120, carrying the conditional-lethal mutation sod(VI)C1, was partially improved by the addition of 1.0 M sorbitol to the medium. The mutant grown at 42 degrees C, with osmotic stabilizer, showed abnormal hyphal morphology, a decrease in beta-1,3-glucan synthase activity as well as cell wall sugar content, but an increase in chitin synthase activity and N-acetyl-glucosamine content. The mutation also affected the secretion of extracellular protease. The temperature-dependent osmo-sensitive phenotype of a Saccharomyces cerevisiae alpha-COP mutation can be rescued by the A. nidulans sod(VI)C(+) gene. These results indicate that the sod(VI)C1 mutation affects proper processing of secretory proteins destined for the surface of cells or beyond.
FEMS Microbiology Letters 04/2002; 208(2):253-7. · 2.04 Impact Factor
