Identifying patterns of DNA for tumor diagnosis using capillary electrophoresis-amplified fragment length polymorphism (CE-AFLP) screening

Amplified Fragment Length Polymorphism (AFLP) screening is a genome-wide genotyping strategy that has been widely used in plants and bacteria, but little has been reported concerning its use in humans. We investigated if the AFLP procedure could be coupled with high-throughput capillary electrophoresis (CE) for use in tumor diagnosis and classification. Using CE-AFLP, a series of molecular 'fingerprints' were generated for a set of gastric tumor and normal genomic DNA samples. The CE-AFLP procedure was qualitatively and quantitatively robust, and a variety of clustering tools were used to identify a specific DNA marker 'pattern' of 20 features that classified the tumor and normal samples to reasonable degrees of accuracy (Sensitivity 95%, Specificity 80%). The CE-AFLP-based approach also correctly classified 16 tumor samples, which in a previous study had exhibited no detectable genomic aberrations by comparative genome hybridization (CGH). This is the first reported application of CE-AFLP screening in tumor diagnosis. As the procedure is relatively inexpensive and requires minimal prior sequence knowledge and biological material, we suggest that CE-AFLP-based protocols may represent a promising new approach for DNA-based cancer screening and diagnosis.     

Acetylcholinesterase in central vocal control nuclei of the zebra finch (<Emphasis Type="Italic">Taeniopygia guttata</Emphasis>)

The distribution of acetylcholinesterase (AChE) in the central vocal control nuclei of the zebra finch was studied using enzyme histochemistry. AChE fibres and cells are intensely labelled in the forebrain nucleus area X, strongly labelled in high vocal centre (HVC) perikarya, and moderately to lightly labelled in the somata and neuropil of vocal control nuclei robust nucleus of arcopallium (RA), medial magnocellular nucleus of the anterior nidopallium (MMAN) and lateral magnocellular nucleus of the anterior nidopallium (LMAN). The identified sites of cholinergic and/or cholinoceptive neurons are similar to the cholinergic presence in vocal control regions of other songbirds such as the song sparrow, starling and another genus of the zebra finch (Poephila guttata), and to a certain extent in parallel vocal control regions in vocalizing birds such as the budgerigar. AChE presence in the vocal control system suggests innervation by either afferent projecting cholinergic systems and/or local circuit cholinergic neurons. Co-occurrence with choline acetyltransferase (ChAT) indicates efferent cholinergic projections. The cholinergic presence in parts of the zebra finch vocal control system, such as the area X, that is also intricately wired with parts of the basal ganglia, the descending fibre tracts and brain stem nuclei could underlie this circuitry’s involvement in sensory processing and motor control of song.     

Peritoneovenous shunt - modification with the use of long saphenous vein

The authors describe their own initial experience with saphenoperitoneal modification of the peritoneovenous shunt in intractable ascites solution. Their findings with this easy type of permanent ascites drainage using the "patient's own resources" are puzzling.     

Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo

We report the targeted mutagenesis of the murine iron regulatory protein (IRP)-1 and IRP2 genes, respectively, with a classical gene trap construct. Insertion of the targeting cassette into the second intron of either gene by homologous recombination interrupts their open reading frames near the N termini. Mice that are homozygous for the correctly modified IRP1 or IRP2 alleles, respectively, display a strong reduction (90%, IRP1(-/-)) or nondetectable levels (IRP2(-/-)) of the targeted proteins. Interestingly, the pre-mRNAs transcribed from the identical targeting cassettes are processed differently within the two different contexts. Detailed analysis of the respective products identifies the choice of alternative splice and 3' end processing sites in the same tissues in vivo. We discuss the implications for the understanding of RNA processing and for targeting strategies for functional genomics in the mouse.     

Disseminated lethal Encephalitozoon cuniculi (genotype III) infections in cotton-top tamarins (Oedipomidas oedipus)--a case report

For the first time, Encephalitozoon (E.) cuniculi genotype III ('dog strain') was verified in two cotton-top tamarins (Oedipomidas oedipus) by light microscopy, immunohistochemistry, electron microscopy, PCR and sequencing. The animals had a disseminated lethal infection with this protist. In earlier reports, genotype III had been found only in domestic dogs, man, emperor tamarins (Saguinus imperator) and golden lion tamarins (Leontopithecus rosalia). This investigation establishes now that the 'dog strain' can occur in cotton-top tamarins too. This is further evidence for the zoonotic potential of E. cuniculi. Furthermore, free E. cuniculi spores were identified also in blood vessels of several tissues. These findings indicate that during a disseminated infection E. cuniculi spores can occur in peripheral blood, too. We propose that blood should also be included in the investigations for the detection of microsporidia, so that a possible disseminated course of an infection can be detected.     

Plasmid condensation induced by cationic compounds: hydrophilic polylysine and amphiphilic cationic lipid

The construction of an efficient carrier for genetic material is a major research objective that needs to be achieved before gene therapy can become a viable pharmacological approach. Artificial aggregates containing nucleic acids are one of the options for the systemic delivery of genetic information. The diversity of functions the aggregate is expected to fulfill necessitates its complex architecture. In order to obtain a complex supramolecular aggregate, formed from elements that are themselves complex molecules, appropriate procedures based on the detailed understanding of processes at the molecular level are required. In this study, we investigated how the various properties of cationic compounds affect nucleic acid condensation. The combination of two condensing agents, differing in their affinity towards water, when mixed with plasmids, resulted in aggregates which are resistant to enzymatic digestion and which form particles with well-defined size distributions. Such uniform and well-defined complexes may subsequently be further modified in order to obtain a fully functional genetic material carrier.     

Characteristics and a comparison of three classes of microsatellite-based markers and their application in plants

Microsatellites (SSR--simple sequence repeats, STR--short tandem repeats, SSLP--simple sequence length polymorphism, VNTR--variable number of tandem repeats) are the class of repetitive DNA sequences present in all living organisms. Particular characteristics of microsatellites, such as their presence in the genomes of all living organisms, high level of allelic variation, co-dominant mode of inheritance and potential for automated analysis make them an excellent tool for a number of approaches like genotyping, mapping and positional cloning of genes. The three most popular types of markers containing microsatellite sequences that are presently used are: (1) SSR (simple sequence repeats), generated by amplifying in a PCR reaction with the use of primers complementary to flanking regions; (2) ISSR (inter-simple sequence repeats), based on the amplification of regions between inversely oriented closely spaced microsatellites; and (3) SAMPL (selective amplification of microsatellite polymorphic loci), which utilises AFLP (amplified fragment-length polymorphism) methodology, with one exception--for the second amplification, one of the starters is complementary to the microsatellite sequence. The usefulness of the three above-mentioned markers for numerous purposes has been well documented for plants.     

Influence of the extraction mode on the yield of some furanocoumarins from Pastinaca sativa fruits

Analysis of plant material is an important task in chemotaxonomical investigations, in search of plants with pharmacological activity or in standardisation of plant drugs. The choice of optimal conditions for the analysis of plant material and effect of extraction method on the yield of furanocoumarins from Pastinaca sativa fruits were examined. The following extraction methods were used in experiments: exhaustive extraction in Soxhlet apparatus, ultrasonification (USAE) at 25 and 60 degrees C, microwave-assisted solvent extraction in open and closed system (MASE) and accelerated solvent extraction (ASE). In most cases, the yield of furanocoumarins was highest by use of ASE method as well as by ultrasonification at 60 degrees C.     

CFP10 discriminates between nonacetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction

ESAT-6 (the 6 kDa early secreted antigenic target) protein species in short-term culture filtrate of Mycobacterium tuberculosis were separated in a 4-5 narrow range pI gradient two-dimensional gel electrophoresis (2-DE). Eight ESAT-6 protein species were analyzed in detail by peptide mass fingerprinting matrix-assisted laser desorption/ionization-mass spectrometry as well as by electrospray ionization-tandem mass spectrometry. An N-terminal Thr acetylation was identified in four species and a C-terminal truncation was identified in two species. In 2-DE blot overlay assays, the recombinant 10 kDa culture filtrate protein (CFP10) discriminated N-terminal acetylated and nonacetylated ESAT-6 by differential interaction, whereas removal of the C-terminal 11 residues of ESAT-6 had no effects thereon. This example shows that the access to the protein species level can be a prerequisite to understand regulation of protein-protein interaction.     

Novel water-soluble photosensitizers from dextrans

Novel water-soluble polymeric photosensitizers based on the natural polymer dextran were synthesized and studied. The modified dextran contained photoactive anthracene (An) chromophores. They were soluble in water with the solubility decreasing with an increase in the number of An moieties bound to the polymeric chain. In aqueous solutions, the macromolecules adopted a compact conformation which resulted in the formation of hydrophobic microdomains. The properties of these domains were characterized with molecular probes such as perylene and pyrazolo-quinoline derivative. The polymer absorbed in the UV/vis region and photosensitized reactions mediated by energy and/or electron transfer from electronically excited An to the molecules of organic compounds solubilized in polymeric microdomains or resided in water.