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Selected AbstractsProtein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): Recalcitrant tissue containing high levels of viscous polysaccharidesELECTROPHORESIS, Issue 3 2008Kouhei Nagai Abstract Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI,s were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds. [source] A quantitative study of factors affecting in vivo bioluminescence imagingLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 5 2008Kemi Cui Abstract In vivo bioluminescence imaging (BLI) has the advantages of high sensitivity and low background. By counting the number of photons emitted from a specimen, BLI can quantify biological events such as tumour growth, gene expression and drug response. The intensities and kinetics of the BL signal are affected by many factors and may confound the quantitative results acquired from consecutive imaging sessions or different specimens. We used three different mouse models of tumours to examine whether anaesthetics, positioning and tumour growth may affect the consistency of the BL signal. The results showed that BLI signal could be affected by different anaesthetics and repetitive positioning. Using the same anaesthetics produced consistent peak times, while other factors were held constant. However, as the tumours grew the peak times shifted and the time course of BL signals had different shapes, depending on the positioning of the mice. The data indicate that a carefully designed BLI experiment is required to generate optimal and consistent results. Copyright © 2008 John Wiley & Sons, Ltd. [source] Solid-phase microextraction combined with surface-enhanced laser desorption/ionization introduction for ion mobility spectrometry and mass spectrometry using polypyrrole coatingsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2004Yan Wang The successful application of polypyrrole (PPY) solid-phase microextraction (SPME) coatings as both an extraction phase and a surface to enhance laser desorption/ionization (SELDI) of analytes is reported. This SPME/SELDI fiber integrates sample preparation and sample introduction on the tip of a coated optical fiber, as well as acting as the transmission medium for the UV laser light. Using ion mobility spectrometry (IMS) detection, the signal intensity was examined as a function of extraction surface area and concentration of analyte. The linear relationship between concentration and signal intensity shows potential applicability of this detection method for quantitative analysis. Extraction time profiles for the fiber, using tetraoctylammonium bromide as test analyte, illustrated that equilibrium can be reached in less than one minute. To investigate the performance of the PPY coating, the laser desorption profile was studied. The fiber was also tested using a quadrupole time-of-flight (Q-TOF) mass spectrometer with leucine enkephalin as test analyte. Since no matrix was used, mass spectra free from matrix background were obtained. This novel SPME/SELDI fiber is easy to manufacture, and is suitable for studying low-mass analytes because of the intrinsic low background. These findings suggest that other types of conductive polymers could also be used as an extraction phase and surface to enhance laser desorption/ionization in mass spectrometry. Copyright © 2003 John Wiley & Sons, Ltd. [source] C-fiber (Remak) bundles contain both isolectin B4-binding and calcitonin gene-related peptide-positive axonsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2005Beth Brianna Murinson Abstract Unmyelinated nerve fibers (Remak bundles) in the rodent sciatic nerve typically contain multiple axons. This study asked whether C-fiber bundles contain axons arising from more than one type of neuron. Most small neurons of the lumbar dorsal root ganglion (DRG) are either glial cell line-derived neurotrophic factor dependent or nerve growth factor dependent, binding either isolectin B4 (IB4) or antibodies to calcitonin gene-related peptide (CGRP), respectively. Injection of IB4-conjugated horseradish peroxidase into a lumbar DRG resulted in intense labeling of IB4 axons, with very low background. Visualized by confocal fluorescence, IB4-binding and CGRP-positive nerve fibers orginating from different DRG neurons came together and remained closely parallel over long distances, suggesting that these two types of axon occupy the same Remak bundle. With double-labeling immunogold electron microscopy (EM), we confirmed that IB4 and CGRP axons were distinct and were found together in single Remak bundles. Previous studies indicate that some DRG neurons express both CGRP and IB4 binding. To ensure that our immunogold results were not a consequence of coexpression, we studied large populations of unmyelinated axons by using quantitative single-label EM. Tetramethylbenzidine, a chromogen with strong intrinsic signal amplification of IB4-horseradish peroxidase, labeled as many as 52% of unmyelinated axons in the dorsal root. Concomitantly, 97% of the Remak bundles with more than one axon contained at least one IB4-labeled axon. Probabilistic modeling using binomial distribution functions rejected the hypothesis that IB4 axons segregate into IB4-specific bundles (P < 0.00001). We conclude that most Remak bundle Schwann cells simultaneously support diverse axon types with different growth factor dependences. J. Comp. Neurol. 484:392,402, 2005. © 2005 Wiley-Liss, Inc. [source] Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplificationTHE PLANT JOURNAL, Issue 6 2001Ludmila I. Khrustaleva Summary The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single-copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal-amplification method termed tyramide-FISH (Tyr-FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome-conjugated tyramide. With Tyr-FISH it was possible to detect target T-DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr-FISH method very suitable for routine application in plant cytogenetic research. With Tyr-FISH we analysed the position of T-DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T-DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less-condensed prophase and interphase chromosomes revealed that the T-DNA was integrated outside the satellite DNA-hybridization region in a more proximal euchromatin region. [source] Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studiesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Isabelle Petit-Haertlein Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor ,-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D2O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293,K using LADI-III at ILL showed in a few 24,h exposures a very low background and clear small spots up to a resolution of 1.80,Å from a crystal of dimensions 1.60 × 0.38 × 0.38,mm corresponding to a volume of 0.23,mm3. [source] Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze proteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Isabelle Petit-Haertlein The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D2O by the sitting-drop method. Preliminary neutron Laue diffraction at 293,K using LADI-III at ILL showed that with a few exposures of 24,h a very low background and clear small spots up to a resolution of 1.85,Å were obtained using a `radically small' perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35,mm, corresponding to a volume of 0.13,mm3. [source] | |||||||||||||