| 1. |
- Ahmad, Faiyaz, et al.
(författare)
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Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B
- 2005
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. ; 307, s. 93-107
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.
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| 2. |
- Alexandersson, Erik, et al.
(författare)
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Purification and proteomic analysis of plant plasma membranes.
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 432, s. 161-173
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Tidskriftsartikel (refereegranskat)abstract
- All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.
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| 3. |
- Allen, Marie, et al.
(författare)
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Mitochondrial D-loop and coding sequence analysis using pyrosequencing
- 2005
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Ingår i: Methods in Molecular Biology. - New Jersey : Humana Press. - 1064-3745 .- 1940-6029. ; 297, s. 179-196
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Tidskriftsartikel (refereegranskat)abstract
- In forensic casework analysis, mitochondrial deoxyribonucleic acid (DNA) often is used when the evidence material contains scarce amounts of DNA. Here, a mitochondrial DNA typing system for D-loop and coding region analysis based on pyrosequencing is described. Pyrosequencing is a real-time, single-tube sequencing-by-synthesis method, in which a cascade of enzymatic reactions yields detectable light. This pyrosequencing system has a higher resolution than the D-loop analysis performed routinely today as it also covers informative positions in the mitochondrial coding region. The system is composed of 16 polymerase chain reaction (PCR) fragments and 24 pyrosequencing reactions with a turn around time for a 96-well plate of less than 3 h after PCR.
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| 4. |
- Allen, Marie, et al.
(författare)
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Universal tag arrays in forensic SNP analysis.
- 2005
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 297, s. 141-154
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Tidskriftsartikel (refereegranskat)abstract
- Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.
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| 5. |
- Andersson, Jan O.
(författare)
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Horizontal gene transfer between microbial eukaryotes.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 532:4, s. 473-487
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Tidskriftsartikel (refereegranskat)abstract
- Comparative genomics have identified two loosely defined classes of genes: widely distributed core genes that encode proteins for central functions in the cell and accessory genes that are patchily distributed across lineages and encode taxa-specific functions. Studies of microbial eukaryotes show that both categories undergo horizontal gene transfer (HGT) from prokaryotes, but also between eukaryotic organisms. Intra-domain gene transfers of most core genes seem to be relatively infrequent and therefore comparatively easy to detect using phylogenetic methods. In contrast, phylogenies of accessory genes often have complex topologies with little or no resemblance of organismal relationships typically with eukaryotes and prokaryotes intermingled, making detailed evolutionary histories difficult to interpret. Nevertheless, this suggests significant rates of gene transfer between and among the three domains of life for many of these genes, affecting a considerably diversity of eukaryotic microbes, although the current depth of taxonomic sampling usually is insufficient to pin down individual transfer events. The occurrence of intra-domain transfer among microbial eukaryotes has important implications for studies of organismal phylogeny as well as eukaryote genome evolution in general.
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| 6. |
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| 7. |
- Belting, Mattias, et al.
(författare)
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Developments in macromolecular drug delivery.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 480, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Macromolecular drugs hold great promise as novel therapeutics of several major disorders, such as cancer and cardiovascular disease. However, their use is limited by lack of efficient, safe, and specific delivery strategies. Successful development of such strategies requires interdisciplinary collaborations involving researchers with expertise on, e.g., polymer chemistry, cell biology, nanotechnology, systems biology, advanced imaging methods, and clinical medicine. This not only poses obvious challenges to the scientific community but also provides opportunities for the unexpected at the interface between different disciplines. This introductory chapter summarizes and gives references to studies on macromolecular delivery that should be of interest to a broad scientific audience involved in macromolecular drug synthesis as well as in vitro and in vivo drug delivery studies.
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| 8. |
- Björkbacka, Harry
(författare)
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Microarray Experiments to Uncover Toll-Like Receptor Function.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 517, s. 1-23
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Tidskriftsartikel (refereegranskat)abstract
- This chapter is intended as a handbook for anyone interested in using microarrays to study Toll-like receptor (TLR) function or any other biological question. Although microarray technology has developed into a standard tool at many laboratories disposal, most of the actual microarray processing is done by core facilities using highly specialized equipment. This chapter only briefly describes these methods in principle and instead focus on the parts that investigators themselves can influence, such as the experimental design, RNA isolation, statistical analysis, cluster analysis, data visualization, and biological interpretation.
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| 9. |
- Büki, Andras, 1966-, et al.
(författare)
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Clinical and model research of neurotrauma
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 566, s. 41-55
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Tidskriftsartikel (refereegranskat)abstract
- Modeling traumatic brain injury represents a major challenge for neuroscientists - to represent extremely complex pathobiological processes kept under close surveillance in the most complex organ of a laboratory animal. To ensure that such models also reflect those alterations evoked by and/or associated with traumatic brain injury (TBI) in man, well-defined, graded, simple injury paradigms should be used with clear endpoints that also enable us to assess the relevance of our findings to human observations. It is of particular importance that our endpoints should harbor clinical significance, and to this end, biological markers ultimately associated with the pathological processes operant in TBI are considered the best candidate. This chapter provides protocols for relevant experimental models of TBI and clinical materials for neuroproteomic analysis.
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| 10. |
- Dahlgren, Claes, 1949, et al.
(författare)
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Measurement of respiratory burst products generated by professional phagocytes
- 2007
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Ingår i: Neutrophil Methods and Protocols. Mark T. QuinnFrank R. DeLeoGary M. Bokoch (red.). - Totowa, NJ : Springer. - 1064-3745 .- 1940-6029. - 9781588297884 ; , s. 349-63
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Bokkapitel (refereegranskat)abstract
- Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The burst of 02 consumption is utilized by an NADPH-oxidase to generate highly-reactive oxygen species (ROS) starting with one and two electron reductions to generate superoxide anion (O2-) and hydrogen peroxide (H2O2), respectively. ROS are strongly bactericidal but may also cause tissue destruction and induce apoptosis in other immune competent cells of both the innate and the adaptive immune systems. Thus, the development of basic techniques to measure/quantify ROS generation/release by phagocytes during activation of the respiratory burst is of great importance, and a large number of techniques have been used for this purpose. Three of these techniques, chemiluminescence amplified by luminol/ isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of p-hydroxyphenylacetate, are described in detail in this chapter. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically-oriented research on innate immune mechanisms and inflammation.
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