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Minute Samples (minute + sample)
Selected AbstractsFe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2007Shun Feng Abstract Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+ -IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of ,-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (,RPLC,nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12,,g mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with ,RPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Effects of Ethanol on Extracellular Levels of Adenosine in the Basal Forebrain: An In Vivo Microdialysis Study in Freely Behaving RatsALCOHOLISM, Issue 5 2010Rishi Sharma Background:, Adenosine is implicated to play a pivotal role in mediating many neuronal responses to ethanol. While in vitro studies performed in cell culture have demonstrated that acute ethanol exposure increases extracellular adenosine levels, this effect has not been demonstrated, in vivo, in the brain. We performed an in vivo microdialysis study to examine the effects of local ethanol perfusion on extracellular levels of adenosine in the basal forebrain (BF). Methods:, Under sterile conditions and using a standard surgical protocol, adult male Sprague,Dawley rats were implanted with unilateral microdialysis guide cannula targeted toward the BF. Following postoperative recovery, the microdialysis probe was inserted. After allowing at least 12 to 16 hours for probe insertion recovery, the experiment was begun. Artificial cerebrospinal fluid (aCSF) was perfused (0.7 ,l/min) for 80 minutes, and 4 × 20-minute pre-ethanol baseline samples were collected. Subsequently, 30, 100, and 300 mM doses of ethanol were perfused. Each ethanol dose was perfused for 80 minutes, and 4 × 20-minute samples were collected. Finally, aCSF was perfused, and 4 × 20 postethanol samples were collected. Adenosine in the microdialysate was separated and measured with HPLC coupled with an UV detector. On completion, the animals were euthanized, brain removed and processed for histology. Results:, Local ethanol perfusion in the BF produced a significant increase in extracellular adenosine with the highest dose of 300 mM ethanol producing a 4-fold increase. Cresyl violet (Nissl) staining did not indicate any toxic damage in the area surrounding the probe tip. Choline acetyltransferase immunohistochemistry revealed that all microdialysis probe sites were localized in the BF. Conclusion:, Our study is the first to demonstrate that ethanol acts directly in the brain to increase extracellular adenosine. [source] Time-dependent Variations in Ischemia-modified Albumin Levels in Mesenteric IschemiaACADEMIC EMERGENCY MEDICINE, Issue 6 2009Abdulkadir Gunduz MD Abstract Objectives:, The objective was to determine the value of ischemia-modified albumin (IMA) in the diagnosis of mesenteric embolism. The authors investigated whether or not plasma IMA levels rose in the acute period in a rat model of mesenteric ischemia and the related time-dependent changes. Methods:, In this randomized, controlled, nonblinded trial, 36 mature female Wistar rats were divided into six groups: three control (Groups I, III, and V) and three ischemia (Groups II, IV, and VI). In the control groups, blood was sampled at 30 minutes (Group I), 2 hours (Group III), and 6 hours (Group V) following a simple laparotomy. In the ischemia groups, following laparotomy, the superior mesenteric artery (SMA) was clamped using a bulldog clamp, and blood samples were taken at 30 minutes (Group II), 2 hours (Group IV), and 6 hours (Group VI). Results:, Plasma IMA levels in the ischemia groups were significantly higher compared to those of the control groups (p < 0.004). In addition, levels were higher in the 6-hour blood samples of the ischemia group than in the 2-hour and 30-minute samples (p < 0.001). Serum IMA was also higher in the 2-hour blood samples of the ischemia group than in the 30-minute samples (p < 0.001). Conclusions:, These preliminary findings suggest that serum IMA levels may represent a significant parameter in the early diagnosis of acute mesenteric ischemia and that further studies are necessary. [source] THE TREASURE OF GUARRAZAR: TRACING THE GOLD SUPPLIES IN THE VISIGOTHIC IBERIAN PENINSULA*ARCHAEOMETRY, Issue 1 2007M. F. GUERRA The treasure of Guarrazar, found in the 19th century in Spain, is the most important illustration of the high level of Visigothic jewellery in the Iberian Peninsula. The votive crowns and crosses of this treasure are an arrangement of pierced gold in a Byzantine,Germanic style, decorated with emeralds, garnets, sapphires and other materials. In order to establish the provenance of the gold, we analysed a group of 46 minute samples from the most important pieces kept in Spain for major and trace elements. The combination of PIXE and PIGE with an external 3 MeV proton µ-beam was used to analyse the samples. Considering the gold sources cited by Pliny the Elder and the composition of contemporary Visigothic coins, we suggest the exploitation of south Iberian mines. Using the same set-up, we complemented these results with the analysis of 11 emeralds inlaid in items from the Guarrazar jewellery that is kept in France. We suggest the use of European sources unknown to the Romans for these gemstones. [source] | ||||||||||