Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.     

Rotifers in aging research: use of rotifers to test various theories of aging

Four theories of aging are discussed to examine how effectively they might explain the aging process in rotifers. One of the early theories, the rate of living theory of aging can perhaps be discounted. Although the theory predicts that increased biological energy expenditure, in the form of increased activity or reproduction, would lead to a shorter lifespan, these predictions are not born out by experimental evidence. At the whole animal level, a case can be made for a theory of programmed aging, where the end of reproduction signals the end of the lifespan. Support for this view comes from the observation that lifespan is positively correlated with reproductive parameters, that treatments that extend lifespan usually act to extend the reproductive period, and that the end of reproduction is associated with high mortality and senescent biochemical changes. Two molecular theories of aging are also discussed; the free radical theory of aging and the calcium theory of aging. These theories point to the fact that molecular damage accumulates and that calcium influx increases in the course of aging. When free radical buildup or calcium homeostasis is reduced, lifespan is extended. A molecular explanation of aging does not necessarily exclude the idea of programmed aging. It is probable that an eventual understanding of the aging process will rest on both a physiological and molecular basis.     

Effect of α-Tocopherol and Culture Method on Reproduction of Turbatrix aceti

The effect of α-tocopherol on the reproductive capacity of the free-living nematode Turbatrix aceti was determined using three different culture methods: mass culture, pair culture. and single culture. Significant differences were observed between control and α-tocopherol cultured nematodes for all reproductive parameters measured. The reproductive period started at a significantly earlier time and the length of the reproductive period was significantly longer in α-tocopherol cultured nematodes. The average number of offspring was 34 in control cultures as compared to 55 in α-tocopherol cultures. The eggs of α-tocopherol cultured females showed a more regular outline and uniform distribution of yolk than did eggs from control females.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures: Cell Characterization and Their Utilization of 1-Alkyl-Glycerophosphoethanolamine

Abstract: Primary cultures prepared from newborn rat brain were grown for 16 or 17 days in culture. Addition of brain extract from newborn rats to the medium stimulated the maturation of astrocytes and the development of oligodendrocytes. Both cell types were characterized by morphology and by immunohistochemistry. The phospholipid composition of these cells was estimated. Incubations were performed with l-[³H]alkyl- sn -glycerophosphoethanolamine in varying concentrations for 3 h. About one-third of the substrate supplied was internalized by the cells. Several enzymic reactions were observed. The acylating enzyme system was the most active one—a K _m was determined with 5 nmol intracellular 1-alkyl- sn -glycerophosphoethanolamine/mg cell protein. Plasmalogen formation was rather low. 1-Alkyl- sn -glycerol, a hydrolysis product, was found in small amounts. Some radioactivity was also incorporated into the phosphatidylcholine fraction.     

A novel strategy for the identification of protein-DNA contacts by photocrosslinking and mass spectrometry

Photochemical crosslinking is a method for studying the molecular details of protein-nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe3+-IMAC (immobilized metal affinity chromatography) purification of peptide-oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein-DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide-oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe3+-IMAC. A combination of MS analysis of the peptide-nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys209 of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys209 is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.     

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency

BACKGROUND: Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. METHODS: To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. RESULTS: AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 +/- 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1-AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. CONCLUSIONS: Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction.