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11.
High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge 总被引:2,自引:1,他引:2
The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 107 cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement. 相似文献
12.
Kimihiko Shibata Atsuko Tarui Natsumi Todoroki Shinjiro Kawamoto Shouji Takahashi Yoshio Kera Ryo-hei Yamada 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,130(4)
The presence of N-methyl-
-aspartate (NMLA) was demonstrated in bivalves, Corbicula sandai and Tapes japonica. To our knowledge, this is the first report on the occurrence of NMLA in animal tissues. NMLA in bivalve tissues was identified according to the following findings; (a) its derivatives with (+)- and (−)- 1-(9-fluorenyl)ethyl chloroformate (FLEC) behaved identically with those of authentic NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) its derivatives with (+)- and (−)- FLEC behaved identically with (−)- and (+)-FLEC derivatives of authentic N-methyl-
-aspartate (NMDA), respectively, on HPLC and (c) its behavior on thin-layer chromatography was the same as those of authentic NMLA. We also describe the distribution of NMDA, and
- and
-aspartate, to which N-methylaspartate enantiomers are structurally related. NMDA was more widely dirtributed than NMLA in bivalves. These bivalves containing NMLA showed lower
-aspartate contents and
/(
+
) ratios of aspartate, than the bivalves containing NMDA. 相似文献
13.
Furuhata S Ando K Oki M Aoki K Ohnishi S Aoyagi K Sasaki H Sakamoto H Yoshida T Ohnami S 《Molecular and cellular biochemistry》2007,298(1-2):125-138
Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted
particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their
potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular
disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs
to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human
umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray
and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three
differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells
(LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical
for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis
in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes
Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than
the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal
liver at E11.5 and E14.5 of mouse development. 相似文献
14.
Y Nakayama N Nakamura S Oki M Wakabayashi Y Ishihama A Miyake N Itoh A Kurosaka 《The Journal of biological chemistry》2012,287(38):32222-32235
We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294-3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc. 相似文献
15.
Peroxisome proliferator-activated receptor beta/delta regulates very low density lipoprotein production and catabolism in mice on a Western diet 总被引:5,自引:0,他引:5
Akiyama TE Lambert G Nicol CJ Matsusue K Peters JM Brewer HB Gonzalez FJ 《The Journal of biological chemistry》2004,279(20):20874-20881
The results of recent studies using selective agonists for peroxisome proliferator-activated receptor beta (PPARbeta) suggest that this receptor may have a role in regulating levels of serum lipids in animal models of obesity and insulin resistance. To further examine this possibility, serum lipid profiles of mice lacking a functional PPARbeta receptor were determined. PPARbeta-null mice maintained on either normal chow or a 10-week high fat (HF) diet, a condition that has been shown to induce insulin resistance and obesity in mice, have elevated levels of serum triglycerides primarily associated with very low density lipoprotein (VLDL) with no difference in either total cholesterol or phospholipids. Consistent with this finding, PPARbeta-null mice on a HF-diet were shown to have an increased rate of hepatic VLDL production as well as lowered lipoprotein lipase activity in serum compared with wild-type controls. The latter parallels an increase in the hepatic expression of the genes encoding angiopoietin-like proteins 3 and 4 in PPARbeta-null mice on a HF diet, both proteins of which have recently been shown to inhibit lipoprotein lipase (LPL) activity in vivo. Consistent with elevated VLDL production, a marked increase in plasma VLDL apoB48, -E, -AI, and -AII, as well as a sharp depletion of the hepatic lipid stores was also found in PPARbeta-null mice. In addition, PPARbeta-null mice on a HF diet were shown to have increased adiposity, despite lower total body weight. Together, these results indicate a clear role for PPARbeta in regulating levels of serum triglycerides in mice on a high fat Western diet by modulating both VLDL production and LPL-mediated catabolism of VLDL-triglycerides and also suggest a potential therapeutic role for PPARbeta in the improvement of serum lipids in the setting of metabolic syndrome. 相似文献
16.
Koji Sugiyama Shinji Kawanishi Yasuhiro Oki Marin Kamiya Ryosuke Hanada Masahiro Egi Shuji Akai 《Bioorganic & medicinal chemistry》2018,26(7):1378-1386
One-pot sequential reactions using the acyl moieties installed by enzymatic dynamic kinetic resolution of alcohols have been little investigated. In this work, the acryloyl moiety installed via the lipase/oxovanadium combo-catalyzed dynamic kinetic resolution of a racemic dienol [4-(cyclohex-1-en-1-yl)but-3-en-2-ol or 1-(cyclohex-1-en-1-yl)but-2-en-1-ol] with a (Z)-3-(phenylsulfonyl)acrylate underwent an intramolecular Diels–Alder reaction in a one-pot procedure to produce an optically active naphtho[2,3-c]furan-1(3H)-one derivative (98% ee). This method was successfully applied to the asymmetric total synthesis of (?)-himbacine. 相似文献
17.
Keith L. Constantine Mark S. Friedrichs David Detlefsen Maki Nishio Mitsuaki Tsunakawa Tamotsu Furumai Hiroaki Ohkuma Toshikazu Oki Susan Hill Robert E. Bruccoleri Pin-Fang Lin Luciano Mueller 《Journal of biomolecular NMR》1995,5(3):271-286
Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys1 with Cys13 and Cys7 with Cys19, and the side chain of Asp9 forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H2O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR
adopted basis Newton Raphson
- AIDS
acquired immunodeficiency syndrome
- CW
continuous wave
- DMSO
dimethylsulfoxide
- DQF-COSY
two-dimensional double-quantum-filtered correlation spectroscopy
- HIV
human immunodeficiency virus
- HSQC
heteronuclear single-quantum coherence
- NOE
nuclear Overhauser enhancement
- NOESY
two-dimensional nuclear Overhauser enhancement spectroscopy
- ppm
parts per million
- P.E.-COSY
two-dimensional primitive exclusive correlation spectroscopy
- REDAC
redundant dihedral angle constraint
- rf
radio frequency
- rmsd
root-mean-square difference
- SIV
simian immunodeficiency virus
- sw
spectral width
- m
mixing time
- TOCSY
two-dimensional total correlation spectroscopy
- TSP
trimethylsilyl-2,2,3,3-2H4-propionate
- 2D
two-dimensional 相似文献
18.
Subrina Jesmin Nobutake Shimojo Naoto Yamaguchi Chishimba Nathan Mowa Masami Oki Sohel Zaedi Sayeeda Nusrat Sultana Arifur Rahman Majedul Islam Atsushi Sawamura Satoshi Gando Satoru Kawano Takashi Miyauchi Taro Mizutani 《Life sciences》2014
Aims
Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.Main methods
Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.Key findings
An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.Significance
The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide. 相似文献19.
Hirokazu Sakan Kimihiko Nakatani Osamu Asai Akihiro Imura Tomohiro Tanaka Shuhei Yoshimoto Noriyuki Iwamoto Norio Kurumatani Masayuki Iwano Yo-ichi Nabeshima Noboru Konishi Yoshihiko Saito 《PloS one》2014,9(1)
Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)2 vitamin D3 (1,25VitD3). Disruption of FGF23-α-KL signaling is thought to be an early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL expression and a reciprocal rise in serum FGF23. It remains unclear, however, whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated α-KL expression in renal biopsy samples and measured levels of several parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and urinary samples from CKD patients (n = 236). We found that although renal α-KL levels were significantly reduced and serum FGF23 levels were significantly elevated in early and intermediate CKD, serum phosphate levels remained within the normal range. Multiple regression analysis showed that the increases in FGF23 were significantly associated with reduced renal function and elevated serum phosphate, but were not associated with loss of renal α-KL. Moreover, despite falling renal α-KL levels, the increase in FGF23 enhanced urinary fractional excretion of phosphate and reduced serum 1,25VitD3 levels in early and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell significantly over the course of CKD, and renal α-KL was a significant independent determinant of sKL. These results demonstrate that FGF23 levels rise to compensate for renal failure-related phosphate retention in early and intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate balance to be maintained despite the reduction in α-KL. In advanced CKD, however, renal α-KL declines further. This disrupts FGF23 signaling, and serum phosphate levels significantly increase, stimulating greater FGF23 secretion. Our results also suggest the serum sKL concentration may be a useful marker of renal α-KL expression levels. 相似文献
20.
A Double-Strand Break Repair Component Is Essential for S Phase Completion in Fission Yeast Cell Cycling 下载免费PDF全文
Kimihiko Suto Akihisa Nagata Hiroshi Murakami Hiroto Okayama 《Molecular biology of the cell》1999,10(10):3331-3343
Fission yeast rad22(+), a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22(+), designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolated rad22(+) and its novel homologue rti1(+) as multicopy suppressors of this mutant. rti1(+) suppresses all the defects of cells lacking rad22(+). Mating type switch-inactive heterothallic cells lacking either rad22(+) or rti1(+) are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture of rad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However, rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation. rad22(+) genetically interacts with rad11(+), which encodes the large subunit of replication protein A. Deletion of rad22(+)/rti1(+) or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4-6 degrees C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A. 相似文献