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961.
充气和搅动对球形棕囊藻生长及囊体形成的影响 总被引:1,自引:1,他引:1
球形棕囊藻生活史中包含游离单细胞和球形囊体两种生活形态,但是实验室中培养的球形棕囊藻经常无法形成囊体。研究通过向培养基中泵入过滤空气,以及给培养基提供不同程度的搅动,研究了充气和搅动对球形棕囊藻生长及囊体形成的影响。充气和搅动均显著提高了囊体的数量,并且提高了囊体内细胞的生长速率。但是充气对于囊体直径及囊体内细胞密度并无显著影响。搅动则明显的提高了囊体直径和囊体内细胞数量。然而,尽管充气以及搅动有利于球形棕囊藻囊体的形成,但是培养的囊体直径依然小于自然海区中囊体的大小。 相似文献
962.
Zheng Guo Tianwen Zhang Xia Li Qi Wang Jianzhen Xu Hui Yu Jing Zhu Haiyun Wang Chenguang Wang Eric J Topol Qing Wang Shaoqi Rao 《BMC bioinformatics》2005,6(1):1-12
Background
Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method.Results
We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end.Conclusions
De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods. 相似文献963.
Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes 总被引:1,自引:0,他引:1
Conan K. Wang 《Biophysical journal》2009,97(5):1471-1481
Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 × 103 M−1 and 6.0 × 103 M−1, respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a β-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides. 相似文献
964.
New ternary transition metal complexes of formulations [Ni(bpa)(p-AB)Cl]n · 3nH2O (bpa = 2,2′-bipyridylamine, p-AB = aminobenzenecarboxylic acid) (1), [Cu(bpa)(p-AB)Cl] · H2O (2), [Zn(bpa)(p-AB)2] · H2O (3) are prepared, their structural features are characterized by crystal structural studies, and their DNA binding propensity has been evaluated by fluorescence and viscosity method. In complex 2 and 3, both bpa and p-AB act as the bidentate N and O-donor ligand, respectively. While in complex 1, p-AB acts as a rare tridentate ligand. In the packing pattern of each complex, π-π interaction in their solid state is also described. The complexes show the competitive inhibition of ethidium binding to DNA, and the DNA binding propensity can be reflected as the relative order: 1 > 2 > 3. 相似文献
965.
Mohammad Mahfuzul Haque Mohammed Fadlalla Zhi-Qiang Wang Sougata Sinha Ray Koustubh Panda Dennis J. Stuehr 《The Journal of biological chemistry》2009,284(29):19237-19247
Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize l-Arg to nitric oxide (NO) and l-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (kr), ferric heme-NO dissociation (kd), and ferrous heme-NO oxidation (kox). We found that point mutation (E762N) of a conserved residue on the enzyme''s FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of l-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower kr. During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ∼50–70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that kox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on kox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower kr and faster kox of E762N nNOS combine to lower its apparent Km,O2 for NO synthesis by at least 5-fold, which in turn increases its V/Km value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the kox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity.Nitric oxide (NO)2 is a biological mediator that is produced in animals by three NO synthase isozymes (NOS, EC 1.14.13.39): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1, 2). The NOS are modular enzymes composed of an N-terminal oxygenase domain and a C-terminal flavoprotein domain, with a calmodulin (CaM)-binding site connecting the two domains (3). During NO synthesis, the flavoprotein domain transfers NADPH-derived electrons through its FAD and FMN cofactors to a heme located in the oxygenase domain. The FMN-to-heme electron transfer enables heme-dependent oxygen activation and a stepwise conversion of l-Arg to NO and citrulline (4, 5). Heme reduction also requires that CaM be bound to NOS and is rate-limiting for NO biosynthesis (6–9).NOS enzymes operate under the constraint of having their newly made NO bind to the ferric heme before it can exit the enzyme (10). How this intrinsic heme-NO binding event impacts NOS catalytic cycling is shown in Fig. 1 and has previously been discussed in detail (10–13). The l-Arg to NO biosynthetic reaction (FeIII to FeIIINO in Fig. 1) is limited by the rate of ferric heme reduction (kr), because all biosynthetic steps downstream are faster than kr. However, once the ferric heme-NO complex forms at the end of each catalytic cycle, it can either dissociate to release NO into the medium (at a rate kd as shown in Fig. 1) or become reduced by the flavoprotein domain (at a rate k′r in Fig. 1; equal to kr) to form the enzyme ferrous heme-NO species (FeIINO), which releases NO very slowly (11, 12). Consequently, two cycles compete during steady-state NO synthesis (Fig. 1); NO dissociation from the ferric heme (kd) is part of a “productive cycle” that releases NO and is essential for NOS bioactivity, whereas reduction of the ferric heme-NO complex (kr′) channels the enzyme into a “futile cycle” that actually represents a NO dioxygenase activity. The rate of futile cycling is also determined by the rate of O2 reaction with the ferrous heme-NO complex (at a rate kox in Fig. 1), which regenerates the ferric enzyme. Surprisingly, NOS enzymes have evolved to have a broad range of kr (varies 40×), kox (varies 15×), and kd (varies 30×) values (Table S1) (12). This causes each NOS to distribute quite differently during steady-state NO synthesis and gives each NOS a unique catalytic profile (12).Open in a separate windowFIGURE 1.Global kinetic model for NOS catalysis. Ferric enzyme reduction (kr) is rate-limiting for the biosynthetic reactions (central linear portion). kcat1 and kcat2 are the conversion rates of the enzyme FeIIO2 species to products in the l-Arg and Nω-hydroxy-l-arginine (NOHA) reactions, respectively. The ferric heme-NO product complex (FeIIINO) can either release NO (kd) or become reduced (k′r) to a ferrous heme-NO complex (FeIINO), which reacts with O2 (kox) to regenerate ferric enzyme. Enzyme partitioning and NO release are determined by the relative rates of kr, kox, and kd. This figure is adapted from Ref. 12.The enzyme physical and electronic factors that may set and regulate each of the three kinetic parameters (kr, kox, and kd) in NOS enzymes remain to be fully described. At present, the composition of the NOS flavoprotein domain and CaM appear to be primarily responsible for determining the kr (14–17), whereas the composition of the NOS oxygenase domain is presumed to determine the kd and kox (18, 19). Indeed, our recent point mutagenesis study identified a patch of electronegative residues on the FMN subdomain that are required to maintain a normal kr and NO synthesis activity in nNOS, suggesting that subdomain electrostatic interactions are important in the process (20). We found particularly large effects when the negative charge at Glu762 was neutralized via mutation to Asn. Remarkably, the NO synthesis activity of E762N nNOS was double that of wild-type nNOS, despite the mutant displaying a slow kr that was half of wild type. In the current report, we show that the E762N mutation has an additional, unsuspected effect on the kox kinetic parameter of nNOS. How this effect alters distribution of the nNOS enzyme during steady-state catalysis, impacts the apparent Km,O2, and leads to hyperactive NO synthesis is described. Our finding that the nNOS flavoprotein domain can tune a key kinetic parameter that defines the rate of a heme-based reaction in the nNOS oxygenase domain is unusual and suggests a means by which protein-protein interactions could regulate the catalytic behavior of nNOS. 相似文献
966.
The lack of replication of model-free linkage analyses performed on complex diseases raises questions about the robustness of these methods to various biases. The confounding effect of population stratification on a genetic association study has long been recognized in the genetic epidemiology community. Because the estimation of the number of alleles shared identical by descent (IBD) does not depend on the marker allele frequency when founders of families are observed, model-free linkage analysis is usually thought to be robust to population stratification. However, for common complex diseases, the genotypes of founders are often unobserved and therefore population stratification has the potential to impair model-free linkage analysis. Here, we demonstrate that, when some or all of the founder genotypes are missing, population stratification can introduce deleterious effects on various model-free linkage methods or designs. For an affected sib pair design, it can cause excess false-positive discoveries even when the trait distribution is homogeneous among subpopulations. After incorporating a control group of discordant sib pairs or for a quantitative trait, two circumstances must be met for population stratification to be a confounder: the distributions for both the marker and the trait must be heterogeneous among subpopulations. When this occurs, the bias can result in either a liberal, and hence invalid, test or a conservative test. Bias can be eliminated or alleviated by inclusion of founders' or other family members' genotype data. When this is not possible, new methods need to be developed to be robust to population stratification. 相似文献
967.
DNA甲基化是一种相对稳定且可遗传的表观遗传标记,在植物和动物细胞中均发现有DNA主动去甲基化现象,其机制在植物中已基本得到阐释,但在哺乳动物中尚未鉴定出一种有效的DNA去甲基化酶,并且DNA主动去甲基化途径也存在争议。文章综合分析了近期的文献资料,阐述了哺乳动物中发生DNA主动去甲基化的时空特异性,并从细胞和组织特异性角度介绍DNA主动去甲基化的可能通路和机制,即5-甲基胞嘧啶的氧化作用、5-甲基胞嘧啶脱氨基以及DNA修复等,旨在为破译表观遗传重编程过程提供理论依据。 相似文献
968.
马铃薯是淀粉生产中重要的农作物之一,而可溶性淀粉合成酶SSⅢ是可溶性淀粉合成酶的主要活性成分,通过基因工程的手段来研究SSⅢ基因在淀粉合成中的功能可以用于改良马铃薯淀粉的品质.本研究采用根癌农杆菌介导法将强组成型表达启动子CaMV 35S驱动的可溶性淀粉合成酶SSⅢ基因的RNA干扰表达载体导入马铃薯栽培品种克新1号和克新4号中,获得了65株卡那霉素抗性植株.对抗性植株PCR检测结果表明,SSⅢ基因的干扰片段已整合到马铃薯基因组中,RT-PCR检测表明SSⅢ基因在转录水平上受到了明显抑制.该研究为马铃薯淀粉品质的改良奠定了基础. 相似文献
969.
Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,
which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the
human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from
the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved
by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of
(R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity
in soluble proteins extracted from striatum and substantia nigra. 相似文献
970.
NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with
a particular difficulty in unambiguous identification of domain–domain interactions. Segmental labeling is a NMR strategy
that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral
overlaps and allows for quick identification of domain–domain interaction. Here, a novel segmental labeling strategy is presented
for detection of inter-domain NOEs. To identify domain–domain interactions in human apolipoprotein E (apoE), a multi-domain,
299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples
in which the N-terminal (NT-) domain was 2H(99%)/15N-labeled whereas the C-terminal (CT-) domain was either 15N- or 15N/13C-labeled. 3-D 15N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between
the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits
unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT-
and the CT-domains and determination of the NMR structure of apoE. 相似文献