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81.
JP Herv s J. Martí -Clú a A. Mu oz-Garcí a MC Santa-Cruz 《Biotechnic & histochemistry》2002,77(1):27-35
We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account. 相似文献
82.
83.
Castagnone-Sereno P; Semblat JP; Leroy F; Abad P 《Molecular biology and evolution》1998,15(9):1115-1122
A highly abundant satellite DNA comprising 20% of the Meloidogyne fallax
(Nematoda, Tylenchida) genome was cloned and sequenced. The satellite
monomer is 173 bp long and has a high A + T content of 72.3%, with frequent
runs of A's and T's. The sequence variability of the monomers is 2.7%,
mainly due to random distribution of single-point mutations. A search for
evidence of internal repeated subunits in the monomer sequence revealed a
6-bp motif (AAATTT) for which five degenerated repeats, differing by just a
single base pair, could be identified. Pairwise comparison of the M. fallax
satellite with those from the sympatric species Meloidogyne chitwoodi and
Meloidogyne hapla revealed a high sequence similarity (68.39%) with one
satellite DNA subfamily in M. chitwoodi, which indicated an unexpected
close relationship between them. Given the high copy number and the extreme
sequence homogeneity among monomeric units, it may be assumed that the
satellite DNA of M. fallax could have evolved through some recent and
extensive amplification burst in the nematode genome. In this case, its
relatively short life would not yet have allowed the accumulation of random
mutations in independent amplified repeats. Considering the morphological
resemblance between the two species and their ability to produce
interspecific fertile hybrids under controlled conditions, these results
indicate that M. fallax may share a common ancestor with M. chitwoodi, from
which it could have diverged recently. All these data suggest that M.
fallax could be the result of a recent speciation process and show that
Meloidogyne satellite DNAs may be of interest to resolve phylogenetic
relationships among closely related species from this genus.
相似文献
84.
85.
Vincent Ranwez Frédéric Delsuc Sylvie Ranwez Khalid Belkhir Marie-Ka Tilak Emmanuel JP Douzery 《BMC evolutionary biology》2007,7(1):241
Background
Molecular sequence data have become the standard in modern day phylogenetics. In particular, several long-standing questions of mammalian evolutionary history have been recently resolved thanks to the use of molecular characters. Yet, most studies have focused on only a handful of standard markers. The availability of an ever increasing number of whole genome sequences is a golden mine for modern systematics. Genomic data now provide the opportunity to select new markers that are potentially relevant for further resolving branches of the mammalian phylogenetic tree at various taxonomic levels. 相似文献86.
J P Whitelegge P Jewess M G Pickering C Gerrish P Camilleri J R Bowyer 《European journal of biochemistry》1992,207(3):1077-1084
Photosystem-2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]-azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron-acceptor site of photosystem 2. SDS/PAGE of the 14C-labelled reaction centres followed by fluorography revealed photoaffinity-labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS-stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8-kDa photoaffinity-labelled peptide, generated by proteolysis of the reaction-centre material with trypsin, was isolated and purified to apparent homogeneity using reverse-phase and size-exclusion HPLC techniques. The amino terminus of the photoaffinity-labelled peptide was determined to be Leu-Gly-Met-Arg-Pro-Xaa-Ile-Ala-Val-Ala-Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu137-Arg225. Chymotrypsin/trypsin digestion of photoaffinity-labelled reaction centres followed by reverse-phase HPLC was used to isolate a smaller photoaffinity-labelled peptide. On Edman sequencing, Ser-Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser-Ala at the amino terminus in the region Leu137-Arg225 are Ser148-Ala-Pro and Ser212-Ala-Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine. 相似文献
87.
Rachna Agarwal Stanislav Zakharov S. Saif Hasan Christopher M. Ryan Julian P. Whitelegge William A. Cramer 《FEBS letters》2014
Compared to thylakoid and inner membrane proteins in cyanobacteria, no structure–function information is available presently for integral outer-membrane proteins (OMPs). The Slr1270 protein from the cyanobacterium Synechocystis 6803, over-expressed in Escherichia coli, was refolded, and characterized for molecular size, secondary structure, and ion-channel function. Refolded Slr1270 displays a single band in native-electrophoresis, has an α-helical content of 50–60%, as in E. coli TolC with which it has significant secondary-structure similarity, and an ion-channel function with a single-channel conductance of 80–200 pS, and a monovalent ion (K+:Cl−) selectivity of 4.7:1. The pH-dependence of channel conductance implies a role for carboxylate residues in channel gating, analogous to that in TolC. 相似文献
88.
Alexander Kotrschal Eva JP Lievens Josefin Dahlbom Andreas Bundsen Svetlana Semenova Maria Sundvik Alexei A Maklakov Svante Winberg Pertti Panula Niclas Kolm 《Evolution; international journal of organic evolution》2014,68(4):1139-1149
Animal personalities range from individuals that are shy, cautious, and easily stressed (a “reactive” personality type) to individuals that are bold, innovative, and quick to learn novel tasks, but also prone to routine formation (a “proactive” personality type). Although personality differences should have important consequences for fitness, their underlying mechanisms remain poorly understood. Here, we investigated how genetic variation in brain size affects personality. We put selection lines of large‐ and small‐brained guppies (Poecilia reticulata), with known differences in cognitive ability, through three standard personality assays. First, we found that large‐brained animals were faster to habituate to, and more exploratory in, open field tests. Large‐brained females were also bolder. Second, large‐brained animals excreted less cortisol in a stressful situation (confinement). Third, large‐brained animals were slower to feed from a novel food source, which we interpret as being caused by reduced behavioral flexibility rather than lack of innovation in the large‐brained lines. Overall, the results point toward a more proactive personality type in large‐brained animals. Thus, this study provides the first experimental evidence linking brain size and personality, an interaction that may affect important fitness‐related aspects of ecology such as dispersal and niche exploration. 相似文献
89.
Solveig Tangedal Marianne Aanerud Louise JP Persson Karl A Brokstad Per S Bakke Tomas M Eagan 《Respiratory research》2014,15(1)
Background
Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD).Methods
433 COPD patients aged 40–76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman’s rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant.Results
Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured.Conclusion
We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum. 相似文献90.
Puppione DL Yam LM Bassilian S Souda P Castellani LW Schumaker VN Whitelegge JP 《Biochimica et biophysica acta》2006,1764(8):1363-1371
Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides. 相似文献