Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells

To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e. g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals.     

Equal reduction in infarct size by ethylisopropyl-amiloride pretreatment and ischemic preconditioning in the in situ rabbit heart

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to provide functional protection during ischemia and reperfusion and to reduce infarct size in isolated rat hearts. In rat hearts, treatment with ethylisopropyl-amiloride (EIPA, a selective Na⁺/H⁺ exchange inhibitor) was additive to the protection afforded by ischemic preconditioning. In addition, such compounds have been demonstrated to reduce infarct size in in situ rabbit hearts. The aim of the present study was to determine to what extent preischemic treatment with EIPA could reduce infarct size in an in situ rabbit model of regional ischemia and reperfusion. We also wished to determine if this effect was additive to the infarct reducing effect of ischemic preconditioning. Anaesthetized, open chest rabbits, were subjected to 45 min of regional ischemia and 150 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by TTC staining. Four groups were investigated: control, ischemic preconditioned (IP) (5 min of ischemia followed by 10 min reperfusion), EIPA (0.65 mg/kg iv given preischemically) and EIPA + IP. The main results expressed as percent infarction of the risk zone ± S.E.M. for the different groups were: control 59.2 ± 3.3% (n = 6), IP 16.3 ± 2.1% (n = 6) (p < 0.001 vs. control), EIPA 16.9 ± 4.1% (n = 5) (p < 0.001 vs. control), EIPA + IP 22.5 ± 9.5% (n = 6) (p < 0.001 vs. control). In conclusion: EIPA, when administered prior to ischemia, caused a reduction in infarct size in the in situ rabbit heart which was similar to that seen with ischemic preconditioning, however, the effect was not additive to ischemic preconditioning.     

Epidermal growth factor receptor expression in human gliomas

Summary The expression of epidermal growth factor receptor (EGFR) was determined in cryosections of 42 human gliomas using biotinylated epidermal growth factor (B-EGF) and two monoclonal antibodies (mAb) against EGFR. All gliomas were found to express EGFR when examined with B-EGF, whereas 33 expressed EGFR when examined with the two mAbs. The highly malignant gliomas (glioblastomas and anaplastic astrocytomas) had a more heterogeneous staining pattern and a larger proportion of tumour cells staining strongly with B-EGF than did the low-grade gliomas (astrocytomas, oligodendrogliomas, mixed gliomas, and ependymomas). This indicates that high-grade gliomas contain more tumour cells rich in EGFR than do the low-grade gliomas. Reactive astrocytes, ependymal cells, and many types of nerve cells (cerebral cortical pyramidal cells, pyramidal and granular hippocampal cells, Purkinje cells, cerebellar granular cells and neurons in the molecular layer of the cerebellum) expressed EGFR, whereas small neurons and normal glial cells were not found to express EGFR.     

Porcine SINEs: Characterization and use in species-specific amplification

A porcine repetitive DNA sequence has been isolated from an intron of the glucose phosphate isomerase gene. The copy number of this and related sequences was estimated to be approximately 10(5) copies per genome. The sequence possesses all the characteristics of short interspersed elements (SINEs) described in other mammals: The repeat is 300 bp in length, has an poly(A)stretch, and contains insertion duplication sites. Homology to seven other porcine sequences, which also have the characteristics of SINEs, has been demonstrated. Primer oligonucleotides, based on conserved regions in the SINE sequences, have been synthesized. Using these primers, PCR-mediated specific amplification of porcine sequences was demonstrated from pig x mouse and pig x hamster hybrid cell lines. Cloning and sequencing of some amplified porcine sequences verify that the sites of priming are SINE sequences.     

Differential effects of some novel synthetic oestrogen analogs on oxidative PC12 cell death caused by serum deprivation

Oestrogens with no or reduced oestrogen receptor (ER) binding properties are reported to have neuroprotective functions. However, we have previously shown that the hormonally inactive isomer of 17β-estradiol (17β-E), 17α-estradiol (17α-E), down-regulates glutathione (GSH) synthesis, and fails to rescue serum deprivation-induced cell death in the rat pheochromocytoma cell line PC12 in micromolar concentration. The present study examined cellular protective effects of new 17β-E analogs and 2-methoxyestradiol (2-ME) analogs with no or little oestrogen activity. 17β-E, 17α-E, 2-ME, and an antagonist of the G protein-coupled oestrogen receptor (GPER), G36, were also included. Both 17α-E and 2-ME protected against deprivation-induced cell death in PC12 cells at 1?nM, but they enhanced the deprivation-induced cell death accompanied by caspase 3 activity and decreased intracellular GSH levels during deprivation at 10?µM. In addition, 10?μM 17α-E activated the p38 mitogen activated protein kinase pathway, which was linked to the enhanced death and reduced GSH levels. Analogs of 2-ME modified with a 6-isoquinoline moiety (6iq) protected against deprivation-induced cell death at 1?nM and did not interfere with the GSH levels nor increase p38 protein levels at 10?µM. The promoter activity of the catalytic subunit of the rate-limiting enzyme, glutamate cysteine ligase (GCLC) in GSH synthesis as well as protein levels of GCLC and Nrf2, increased with the 2-ME analogs at 10?µM. In conclusion, the steroids have differential protective effects, and modifying 2-ME may give the steroid more favourable properties than 17α-E, 2-ME, and G36 in regard to GSH regulation.     

A new sensor,the “filtration probe,” for quantitative characterization of the penicillin fermentation. I. Mycelial morphology and culture activity

In this article the filtration resistance of fungal (Penicillium) mycelia is shown to be quantitatively related to the morphology of the cells. On this basis, it was possible to determine the intracellular volume by simple filtration measurements. Since the dry weight of the cells can also be measured, a morphological variable-the “hyphae density” (dry weight hyphae/volume hyphae)-can be quantified. This variable typically is measured to have values in the range of 0.35–0.20 g/cm³; the lower values represent the final stages of the fermentation. It can be shown that this loss of cell material during fermentation is limited to cytoplasmic components, and that most (70%) of the components which disappear are proteins. Concurrent with and linearly related to this loss of proteins, the culture activity in terms of both cell maintenance energy (respiration activity) and penicillin synthesis also decreased. The hyphae density measured by filtration is therefore a direct measure of the metabolic potential of the organism.     

Epitope mapping of human aromatic L-amino acid decarboxylase

Autoimmune polyendocrine syndrome type I (APS I) is a rare hereditary condition considered a model disease for organ specific autoimmunity. A wide range of autoantibodies targeting antigens present in the affected organs have been identified. Autoantibodies against aromatic L-amino acid decarboxylase (AADC) are present in about 50% of APS I patients. In order to increase our understanding of autoantibody specificity in APS I, the aim of the present study was to localize target regions on AADC recognized by sera from APS I patients. Using several complementing strategies, we have shown that autoantibodies against AADC mainly recognize conformational epitopes. The major antigenic determinants were detected N-terminally to amino acid residue 237. Replacement of amino acids 227-230 (ERDK) with alanine residues reduced the reactivity towards AADC by >80% in all patient sera tested, suggesting that amino acids 227-230 are an important part of an immunodominant epitope.     

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within genes and 36 (90%) in gene introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related.