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221.
Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser502 , Ser528 , and Ser536 as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser502 nor Ser536 has been identified previously as NF-M phosphorylation sites. 相似文献
222.
The paper is concerned with expected type I errors of some stepwise multiple test procedures based on independent p‐values controlling the so‐called false discovery rate (FDR). We derive an asymptotic result for the supremum of the expected type I error rate(EER) when the number of hypotheses tends to infinity. Among others, it will be shown that when the original Benjamini‐Hochberg step‐up procedure controls the FDR at level α, its EER may approach a value being slightly larger than α/4 when the number of hypotheses increases. Moreover, we derive some least favourable parameter configuration results, some bounds for the FDR and the EER as well as easily computable formulae for the familywise error rate (FWER) of two FDR‐controlling procedures. Finally, we discuss some undesirable properties of the FDR concept, especially the problem of cheating. 相似文献
223.
Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome ofAspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in theAspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.Abbreviations CM
complete medium
- Gal-T
UDP-Gal:GlcNAc -1,4-galactosyltransferase (EC 2.4.1.38/90)
- GnT I
UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)
- HPLC
high performance liquid chromatography
- M3-octyl
Man1-6[Man1-3]Man-octyl
- PAGE
polyacrylamide gel electrophoresis
- MES
2-(N-morpholino)ethane sulfonate
- PCR
polymerase chain reaction
- PEG
polyethylene glycol
- PMSF
phenyl methyl sulfonyl fluoride
- SDS
sodium dodecyl sulfate
- SSC (1×)
0.15m NaCl/0.015m sodium citrate (pH 7.0)
- STC
1.2m sorbitol, 100mm Tris-HCl, pH 7.4, and 10mm CaCl2
- STET
0.1m NaCl, 10mm Tris-HCl, pH 8.0, 1mm EDTA, pH 8.0, 5% Triton-X-100
Deceased. This paper is dedicated to the memory of Lorne S. Reid. 相似文献
224.
Hideki Sezutsu Eiji Nitasaka Tsuneyuki Yamazaki 《Molecular & general genetics : MGG》1995,249(2):168-178
LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission. 相似文献
225.
H. Ikezawa K. Tameishi A. Yamada H. Tamura K. Tsukamoto Y. Matsuo K. Nishikawa 《Amino acids》1995,9(3):293-298
Summary Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites ofBacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced inBacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G towardp-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP andp-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities. 相似文献
226.
227.
Dinuclear copper(I) complexes with bridging bis(dicyclohexylphosphino)methane (dcpm) or bis(diphenylphosphino)methane (dppm) and 2,2′-bipyridine or 2-[N-(2-pyridyl)methyl]amino-5,7-dimethyl-1,8-naphthyridine (L), [Cu2(bpy)2(dppm)2](BF4)2 (1), [Cu2(bpy)2(dcpm)](BF4)2 (2), [Cu2(L)(dppm)](BF4)2 (3) and [Cu2(L)(dcpm)](BF4)2 (4) were prepared, and their structures were determined by X-ray crystal analysis. Two-, three-, and four-coordinate copper(I) centers are found in these complexes. Compounds 3 and 4 show close CuI?CuI separations of 2.664(3) and 2.674(1) Å, respectively, whereas an intramolecular copper-copper distance of 3.038 Å is found in 2 having only dcpm as an additional bridge. Powdered samples of 1, 3, and 4 display intense and long-lived phosphorescence with λmax at 533, 575, and 585 nm at room temperature, respectively. In the solid state, 2 exhibits only a weak emission at 555 nm. The time-resolved absorption and emission spectra of these complexes were investigated. The difference in the emission properties among complexes 1-4 suggests that both CuI?CuI distances and coordination environment of the copper(I) centers affect the excited-state properties. 相似文献
228.
Daisuke Suzuki Keiichi Inoue Makoto Sakai Hideki Kandori Yuki Sudo 《Journal of molecular biology》2009,392(1):48-4922
Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl−-dependent manner with a Km of 307 ± 56 mM, showing that Cl−-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO3), implying that I−, Br−, and NO3− can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl−-binding site. In light of these results, we discuss the effects of the Cl− binding to SRI and the roles of Cl− binding in its function. 相似文献
229.
The FtsH2 protease, encoded by the slr0228 gene, plays a key role in the selective degradation of photodamaged D1 protein during the repair of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. To test whether additional proteases might be involved in D1 degradation during high rates of photodamage, we have studied the synthesis and degradation of the D1 protein in ΔPsbO and ΔPsbV mutants, in which the CaMn4 cluster catalyzing oxygen evolution is less stable, and in the D1 processing mutants, D1-S345P and ΔCtpA, which are unable to assemble a functional cluster. All four mutants exhibited a dramatically increased rate of D1 degradation in high light compared to the wild-type. Additional inactivation of the ftsH2 gene slowed the rate of D1 degradation dramatically and increased the level of PSII complexes. We conclude that FtsH2 plays a major role in the degradation of both precursor and mature forms of D1 following donor-side photoinhibition. However, this conclusion concerned only D1 assembled into larger complexes containing at least D2 and CP47. In the ΔpsbEFLJ deletion mutant blocked at an early stage in PSII assembly, unassembled D1 protein was efficiently degraded in the absence of FtsH2 pointing to the involvement of other protease(s). Significantly, the ΔPsbO mutant displayed unusually low levels of cellular chlorophyll at extremely low-light intensities. The possibilities that PSII repair may limit the availability of chlorophyll for the biogenesis of other chlorophyll-binding proteins and that PsbO might have a regulatory role in PSII repair are discussed. 相似文献
230.
Ana-Cristina Sotomayor Pérez Marilyne Davi Daniel Ladant Alexandre Chenal 《Journal of molecular biology》2010,397(2):534-17004
Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel β-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a β-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed. 相似文献