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31.
We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling. 相似文献
32.
M C Harricane A Bonet-Kerrache C Cavadore D Mornet 《European journal of biochemistry》1991,196(1):219-224
An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction. 相似文献
33.
Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132 总被引:14,自引:0,他引:14 下载免费PDF全文
It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable. 相似文献
34.
Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus 总被引:12,自引:0,他引:12 下载免费PDF全文
The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection. 相似文献
35.
Rhonda C Foley Ling-Ling Gao Andrew Spriggs Lena YC Soo Danica E Goggin Penelope MC Smith Craig A Atkins Karam B Singh 《BMC plant biology》2011,11(1):59
Background
In legumes, seed storage proteins are important for the developing seedling and are an important source of protein for humans and animals. Lupinus angustifolius (L.), also known as narrow-leaf lupin (NLL) is a grain legume crop that is gaining recognition as a potential human health food as the grain is high in protein and dietary fibre, gluten-free and low in fat and starch. 相似文献36.
37.
A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao
Jorge MC Mondego Marcelo F Carazzolle Gustavo GL Costa Eduardo F Formighieri Lucas P Parizzi Johana Rincones Carolina Cotomacci Dirce M Carraro Anderson F Cunha Helaine Carrer Ramon O Vidal Raíssa C Estrela Odalys García Daniela PT Thomazella Bruno V de Oliveira Acássia BL Pires Carolina S Maria Rio Marcos Renato R Araújo Marcos H de Moraes Luis AB Castro Karina P Gramacho Marilda S Gonçalves José P Moura Neto Aristóteles Góes Neto Luciana V Barbosa Mark J Guiltinan Bryan A Bailey Lyndel W Meinhardt Julio CM Cascardo Gonçalo AG Pereira 《BMC genomics》2008,9(1):1-25
38.
A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples. 相似文献
39.
Brdjanovic D van Loosdrecht MC Hooijmans CM Alaerts GJ Heijnen JJ 《Biotechnology and bioengineering》1998,60(3):326-332
The methodology for determination of the minimally required aerobic sludge retention time (SRTminaer) in biological phosphorus removal (BPR) systems is presented in this article. Contrary to normal biological conversions, the BPR process is not limited by a SRTmin resulting from the maximum growth rate of the organisms. This is because the aerobic SRT should be long enough to oxidize the amount of poly-hydroxy-alkanoates (PHA) stored in the anaerobic phase. This means that the SRTminaer will primarily depend on the PHA conversion kinetics and the maximal achievable PHA content in the cell (storage capacity). The model for the prediction of the minimally required aerobic SRT as a function of kinetic and process parameters was developed and compared with experimental data used to evaluate several operational aspects of BPR in a sequencing batch reactor (SBR) system. The model was proved as capable of describing them satisfactorily.Copyright 1998 John Wiley & Sons, Inc. 相似文献
40.
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