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11.
A new approach to high sensitivity differential hybridization 总被引:4,自引:0,他引:4
We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in λgt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with 35S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization. 相似文献
12.
Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C4 species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C4 types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C3 plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C3 mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C4 plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983) 相似文献
13.
Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C4 plants was reported to be mimicked by either of the twotypes of cation jump: H+-jump in maize and phylogenically relatedspecies (H+-type) and Na+-jump in all the other C4 species tested(Na+-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C4 mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H+-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na+-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H+ consumption showed good correlation with[14C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H+ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H+and pyruvate anion into mesophyll chloroplasts of H+-type C4species in the light. (Received January 5, 1994; Accepted May 6, 1994) 相似文献
14.
Takeshi Tabira Jun-ichi Inobe Keiichi Nakahara Mitsuhiro Osame Takashi Yamamura 《Neurochemical research》1994,19(8):1067-1071
To understand the immune mechanism suggested in HTLV-I-associated myelopathy (HAM/TSP), we investigated T cell response to proteolipid protein (PLP). Because of high autologous proliferative response (APR) of peripheral blood mononuclear cells (PBMC) in culture, the lymphocyte proliferation assay was not useful in this disease. Unexpectedly, however, APR was profoundly (70–98%) suppressed in 6 of 9 cases when PLP peptide 105-124 was added in the culture. PLP peptide 85-104 or 145-159 also suppressed APR in a few cases. Time course study showed that the peptide-mediated suppression became apparent after day 4 in culture. The results can be interpreted as that suppressor cells recognizing the PLP peptides were present in the PBMC of HAM/TSP patients and suppressed the APR as the consequence of antigen specific response. This may indicate that a T cell response to certain PLP determinants is involved in the pathomechanism of HAM/TSP at least in part. Molecular mimicry between PLP and HTLV-I mayaccount for the T cell sensitization to PLP in HAM/TSP.Special issue dedicated to Dr. Marjorie B. Lees. 相似文献
15.
Abstract: The effect of phloretin on prostaglandin (PG) F2α -induced phosphoinositide hydrolysis and elevation of intracellular Ca2+ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC50 value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF2α at 1 μ M transiently increased astrocytic intracellular Ca2+ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF2α receptor linked to phospholipase C in astrocytes. 相似文献
16.
Tamio Ohno Jun-ichi Katoh Yoshiaki Kikkawa Hiromichi Yonekawa Masahiko Nishimura 《Experimental Animals》2003,52(5):415-417
To develop SMXA recombinant inbred (RI) strains as more valuable genetic resources, 302 microsatellite (Mit) loci were added to the strain distribution patterns (SDP) reported previously. The improved SDP were constructed in a total of 1085 loci containing 484 Mit markers, 571 restriction landmark genomic scanning (RLGS) spot markers and 30 others. This substantially improved SDP can be freely accessed on our homepage (http://www.med.nagoya-u.ac.jp/sisetu/SDP.htm). 相似文献
17.
Kohei Murata Masato Sakon Jun-ichi Kambayashi Masaki Okuyama Toshiharu Hase Takesada Mori 《Journal of cellular biochemistry》1995,57(1):120-126
Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin. 相似文献
18.
Klaus W. Neumann Jun-ichi Tamura Tomoya Ogawa 《Bioorganic & medicinal chemistry》1995,3(12):1637-1650
The glycopeptides 1 and 2 ), carrying the core structure of serine-linked cell-surface proteoglycans were synthesized in a stereocontrolled manner. The carbohydrate key imidate xylosyl donors 3 and glycotetraosyl donors 4 and 5, as well as a tetrapeptide glycosyl acceptor 6, were coupled in the crucial glycosylation step. In these reactions, the application of either trimethylsilyl trifluoromethanesulfonate (TMSOTf) or borontrifluoride etherate (BF3-Et2O) as catalysts proved to be highly efficient. The serine linked glycopeptides 34, 36 and 37 thus obtained yielded target compounds 1 and 2 on complete deprotection. 相似文献
19.
20.
Prolonged administration of dipropylacetic acid, a branched-chain fatty acid, reduced the glycine cleavage activity in the liver of rat to about one-third of the activity in the control rat. The reduction of the activity appeared to be due mainly to reduction of the level of P-protein, a pyridoxal phosphate enzyme, which is responsible for the first step of the glycine cleavage, although dipropylacetic acid was also found to inhibit the activity of P-protein in vitro in a noncompetitive but partially competitive manner with respect to glycine. The rat treated with dipropylacetic acid may provide an experimental approach for the biochemical study of hyperglycinemia which accompanies to metabolic disorders of branchedchain keto acids. In the dipropylacetic acid-treated rat, however, the glycine concentration in the serum was not appreciably elevated and this may be accounted for by the fact that the activities of both the glycine cleavage system and serine dehydratase are considerably higher in the rat liver as compared with those in other animals including human. 相似文献