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141.
Sinha J Inan M Fanders S Taoka S Gouthro M Swanson T Barent R Barthuli A Loveless BM Smith LA Smith T Henderson I Ross J Meagher MM 《Journal of biotechnology》2007,127(3):462-474
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis. 相似文献
142.
Kumai T Takeba Y Matsumoto N Nakaya S Tsuzuki Y Yanagida Y Hayashi M Kobayashi S 《Life sciences》2007,81(15):1193-1198
We investigated the effects of fasudil, a Rho kinase inhibitor, on hypertension in spontaneously hypertensive rats and on the catecholamine synthetic pathway. Ten-week-old male SHR and Wistar-Kyoto rats were administered fasudil (10 mg/kg/day s.c.) for 4 days. Systolic blood pressure was measured using the tail-cuff method. Catecholamine levels were measured with high-performance liquid chromatography-ECD methods. Tyrosine hydroxylase protein levels were measured in Western blot analysis. The tyrosine hydroxylase mRNA level was measured using real-time PCR methods. Fasudil significantly decreased systolic blood pressure in spontaneously hypertensive rats, but not in Wistar-Kyoto rats. Fasudil also significantly decreased catecholamine, tyrosine hydroxylase protein, and tyrosine hydroxylase mRNA levels in the adrenal medulla of spontaneously hypertensive rats. These results suggest that the depressor effects of fasudil on hypertension in spontaneously hypertensive rats may be related to inhibition of the catecholamine synthetic pathway. 相似文献
143.
Yoshida K Yoshioka D Inoue K Takaichi S Maeda I 《Applied microbiology and biotechnology》2007,76(5):1043-1050
The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several
green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated
bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510–570 nm.
Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type
strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its
wild type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to
red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors. 相似文献
144.
Hatazawa R Tanaka A Tanigami M Amagase K Kato S Ashida Y Takeuchi K 《American journal of physiology. Gastrointestinal and liver physiology》2007,293(4):G788-G797
We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression. 相似文献
145.
146.
Ohta T Eguchi R Suzuki A Miyakaze S Ayuzawa R Kaji K 《Journal of cellular physiology》2007,211(3):673-681
In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown. 相似文献
147.
A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells. 相似文献
148.
Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart
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The mouse homeobox gene Otx2 plays essential roles at each step and in every tissue during head development. We have previously identified a series of enhancers that are responsible for driving the Otx2 expression in these contexts. Among them the AN enhancer, existing 92 kb 5' upstream, directs Otx2 expression in anterior neuroectoderm (AN) at the headfold stage. Analysis of the enhancer mutant Otx2(DeltaAN/-) indicated that Otx2 expression under the control of this enhancer is essential to the development of AN. This study demonstrates that the AN enhancer is promoter-dependent and regulated by acetylated YY1. YY1 binds to both the AN enhancer and promoter region. YY1 is acetylated in the anterior head, and only acetylated YY1 can bind to the sequence in the enhancer. Moreover, YY1 binding to both of these two sites is essential to Otx2 expression in AN. These YY1 binding sites are highly conserved in AN enhancers in tetrapods, coelacanth and skate, suggesting that establishment of the YY1 regulation coincides with that of OTX2 function in AN development in an ancestral gnathostome. 相似文献
149.
Senda M Ito A Tsuchida A Hagiwara T Kaneda T Nakamura Y Kasama K Kiso M Yoshikawa K Katagiri Y Ono Y Ogiso M Urano T Furukawa K Oshima S Furukawa K 《The Biochemical journal》2007,402(3):459-470
Although disialyl glycosphingolipids such as GD3 and GD2 have been considered to be associated with malignant tumours, whether branched-type disialyl glycosphingolipids show such an association is not well understood. We investigated the sialyltransferases responsible for the biosynthesis of DSGG (disialylgalactosylgloboside) from MSGG (monosialylgalactosylgloboside). Among six GalNAc:alpha2,6-sialyltransferases cloned to date, we focused on ST6GalNAc III, V and VI, which utilize sialylglycolipids as substrates. In vitro enzyme analyses revealed that ST6GalNAc III and VI generated DSGG from MSGG with V(max)/K(m) values of 1.91 and 4.16 respectively. Transfection of the cDNA expression vectors for these enzymes resulted in DSGG expression in a renal cancer cell line. Although both ST6GalNAc III and VI genes were expressed in normal kidney cells, the expression profiles of ST6GalNAc VI among 20 renal cancer cell lines correlated clearly with those of DSGG, suggesting that the sialyltransferase involved in the synthesis of DSGG in the kidney is ST6GalNAc-VI. ST6GalNAc-VI and DSGG were found in proximal tubule epithelial cells in normal kidney tissues, while they were downregulated in renal cancer cell lines and cancer tissues. All these findings indicated that DSGG was suppressed during the malignant transformation of the proximal tubules as a maturation arrest of glycosylation. 相似文献
150.