Single-nucleotide polymorphism of the urokinase-plasminogen activator gene during aging and transformation of human diploid kidney cell cultures

Single-nucleotide polymorphisms (SNPs) are differences in the nucleotide sequence of a specific gene from different individuals. The frequency at which SNPs occur varies among individuals, is gene dependent, and may be influenced by the aging process or by mechanisms that result in cell transformation. Urokinase-plasminogen activator (uPA) is a serine protease that is important in embryonic development, aging, and the onset of pathogenic conditions. The frequency of SNP and the stability of the SNPs in the uPA gene have not been defined with regard to processes that are associated with cellular aging or transformation. In this study, the complete nucleotide sequence has been determined for the gene encoding uPA from 26 human diploid kidney cell lines. The frequency of SNP occurrence within the uPA gene and whether this frequency changed during cellular aging, or after cell transformation, were determined. The results demonstrated three donor-dependent SNPs. One SNP was located at base pair 422, which is in the region of the gene responsible for encoding the high-molecular weight domain of uPA (HMW-uPA). The other SNPs were located at base pairs 691 and 822, both of which are in the region of the gene responsible for encoding the low-molecular weight domain of uPA (LMW-uPA). Single-nucleotide polymorphisms were not detected in the portion of the gene responsible for encoding the uPA secretion signal. Leucine or proline would be encoded at amino acid 141 of HMW-uPA as the result of an SNP at base pair 422. The SNP detected at base pair 691 would encode for lysine or glutamine at amino acid 231 of LMW-uPA. The SNP detected at base pair 822 would not change the encoded asparagine located at position 274 of the protein. The SNPs identified in this study were donor dependent and were not altered during cellular aging, or by changes in karyology due to spontaneous transformation of the cell line. These results demonstrate that the integrity of the uPA gene is stable and not subject to alterations that accompany cell aging or transformation.     

Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: EAV virions contain six different envelope proteins. The glycoprotein GP(5) (previously named G(L)) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP(2b) (previously named G(S)), GP(3), and GP(4) are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP(5) and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP(2b), GP(3), and GP(4) proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP(2b) protein in the assembly of the GP(2b)/GP(3)/GP(4) complex. Open reading frames encoding cysteine-to-serine mutants of the GP(2b) protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP(2b) forms an intermolecular cystine bridge with one of the cysteines of the GP(4) protein, while the cysteine residues at positions 48 and 137 of GP(2b) are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP(4) protein is responsible for the covalent association of GP(3) with the disulfide-linked GP(2b)/GP(4) heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.     

Translational physiology: porcine models of human coronary artery disease: implications for preclinical trials of therapeutic angiogenesis.

"Therapeutic angiogenesis" describes an emerging field of cardiovascular medicine whereby new blood vessels are induced to grow to supply oxygen and nutrients to ischemic cardiac or skeletal muscle. Various methods of producing therapeutic angiogenesis have been employed, including mechanical means, gene therapy, and the use of growth factors, among others. The use of appropriate large-animal models is essential if these therapies are to be critically evaluated in a preclinical setting before their use in humans, yet little has been written comparing the various available models. Over the past decade, swine have been increasingly used in studies of chronic ischemia because of their numerous similarities to humans, including minimal preexisting coronary collaterals as well as similar coronary anatomy and physiology. Consequently, this review describes the most commonly used swine models of chronic myocardial ischemia with special attention to regional myocardial blood flow and function and critically evaluates the strengths and weaknesses of each model in terms of utility for preclinical trials of angiogenic therapies.     

Chemical ionization mass spectrometric determination of acrolein in human breast cancer cells

A selected ion flow tube-chemical ionization mass spectrometric method is presented for the first determination of acrolein metabolically produced in biological tissues. Acrolein in aqueous samples (2.5 ml) is preconcentrated by distillation and directly analyzed using gas-phase proton transfer from H3O+. This method provides sensitive detection of acrolein with the method detection limit of 15 nM at the 99% confidence level. Detection is linear up to the highest concentration studied (13.5 microM, R2 = 0.998). Acrolein levels are determined in doxorubicin-sensitive (MCF-7) and doxorubicin-resistant (MCF-7/Adr) human breast cancer cells in vitro. The intracellular acrolein concentrations differ insignificantly: 0.61 microM for sensitive cells and 0.54 microM for resistant cells. Treatment with a physiological concentration of doxorubicin (0.5 microM) for 24 h at 37 degrees C increased acrolein levels by factors of 2.6 and 1.9 for MCF-7 and MCF-7/Adr cells, respectively. The differential enhancement observed is consistent with the lower levels of enzymes that neutralize oxidative stress in sensitive MCF-7 cells and overexpression of an active drug efflux pump P-170 glycoprotein in resistant MCF-7/Adr cells.     

Attenuation of lipid peroxidation by antioxidants in rat-1 fibroblasts: comparison of the lipid peroxidation reporter molecules cis-parinaric acid and C11-BODIPY(581/591) in a biological setting

Lipid peroxidation is a major factor in the pathogenesis of many disease states. To detect the initial stages of lipid peroxidation or evaluate antioxidant efficacy, cis-parinaric acid (cis-PnA) has been successfully used and thoroughly validated. However, cis-PnA is not very well suited for medium throughput screening of antioxidants in living cells. We recently introduced and validated a lipid peroxidation reporter molecule, C11-BODIPY(581/591). To further explore this probe, we evaluated the protective effect of 12 natural antioxidants in rat-1 fibroblasts subjected to 50 microM cumene-hydroperoxide using both probes. The same pecking order for the individual antioxidant efficacies was obtained: alpha-tocopherol approximately gamma-tocopherol > quercetin approximately lycopene > kaempferol > palm oil > hydroxy-tyrosol > > alpha-carotene = beta-carotene = lutein = tyrosol = chlorogenic acid. This validates the accuracy of the C11-BODIPY(581/591) method and shows that this assay is an accurate and highly flexible method for indexing lipid peroxidation or determining antioxidant efficacy in living cells in a medium throughput scenario. The antioxidant efficacy was compared with their one-electron reduction potential, hydrophobicity and Trolox C equivalent antioxidant capacity. Our results show that although these parameters are valuable for determining structure-function relationships, they have limited predictive value for antioxidant efficacy in vivo.     

Physical and functional interaction between the XPF/ERCC1 endonuclease and hRad52

The XPF/ERCC1 heterodimer is a DNA structure-specific endonuclease that participates in nucleotide excision repair and homology-dependent recombination reactions, including DNA single strand annealing and gene targeting. Here we show that XPF/ERCC1 is stably associated with hRad52, a recombinational repair protein, in human cell-free extracts and that these factors interact directly via the N-terminal domain of hRad52 and the XPF protein. Complex formation between hRad52 and XPF/ERCC1 concomitantly stimulates the DNA structure-specific endonuclease activity of XPF/ERCC1 and attenuates the DNA strand annealing activity of hRad52. Our results reveal a novel role for hRad52 as a subunit of a DNA structure-specific endonuclease and are congruent with evidence implicating both hRad52 and XPF/ERCC1 in a number of homologous recombination reactions. We propose that the ternary complex of hRad52 and XPF/ERCC1 is the active species that processes recombination intermediates generated during the repair of DNA double strand breaks and in homology-dependent gene targeting events.     

Using large-scale climate indices in climate change ecology studies

Recently, climate change research in ecology has embraced the use of large-scale climate indices in long-term, retrospective studies. In most instances, these indices are related to large-scale teleconnection and atmospheric patterns of which over a dozen have been identified. Although most of these relate to different geographical areas, many are related and interact. Consequently, even the simple task of selecting one to use in ecological research has become complicated, despite our ability to disentangle the results from analyses involving large-scale climate indices. Leaning upon recent reviews of the definition and functioning of large-scale climate indices, as well as reviews on the relationship between these and concomitant changes in ecological variables, we focus here on the usefulness of large-scale climate indices in different aspects of climate change ecology. By providing a general framework for using climate indices, we illustrate the potential advantages of their utility by integrating three case histories focusing on two groups of evolutionarily distinct organisms: birds and mammals.     

CTP:phosphocholine cytidylyltransferase alpha is a cytosolic protein in pulmonary epithelial cells and tissues

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in de novo synthesis of phosphatidylcholine (PC). The lung requires a steady synthesis of PC for lung surfactant of which disaturated PC is the essential active agent. Surfactant synthesis occurs in alveolar type II cells. Studies with non-pulmonary cells have suggested that CCT is both a nuclear and cytoplasmic protein. The unusual requirements of the lung for PC synthesis and, therefore, CCT activity suggest a unique mechanism of regulation and possibly localization of CCT. The localization of CCT alpha in lung epithelial cells and, of greater consequence, lung tissues are yet unknown. Three isoforms of CCT have been identified. Herein we investigated the localization of the ubiquitously expressed CCT alpha isoform. To ascertain CCT alpha localization in lungs and lung-related epithelial cells, we employed a number of localization methods. Immunogold electron microscopy using polyclonal antibodies raised to either the carboxyl terminus, catalytic domain, or amino terminus of CCT alpha localized CCT alpha mostly to the exterior plasma membrane or regions of the endoplasmic reticulum (ER) in both A549 and MLE-15 epithelial lung cell lines and primary cultures of fetal rat lung epithelial cells. In contrast to other studies, little or no nuclear labeling was observed. Indirect immunofluorescence of these cells with anti-CCT alpha antibodies resulted in a similar distribution. Indirect visualization of both hemagglutinin- and FLAG-tagged CCT alpha as well as direct visualization of enhanced green fluorescence protein-CCT alpha fusion protein corroborated a cytoplasmic localization of CCT alpha in pulmonary cells. Moreover, analysis of lung tissue from fetal and adult mouse by either immunogold electron microscopy or indirect immunofluorescence yielded a strong cytoplasmic CCT alpha signal with virtually no nuclear localization in epithelial cells lining the airways. The cytoplasmic localization of CCT alpha in type II cells was further substantiated with transgenic mice overexpressing FLAG-tagged CCT alpha using the lung-specific human surfactant protein C (SP-C) promoter. We conclude that CCT alpha does not localize to the nucleus in pulmonary tissues, and, therefore, nuclear localization of CCT alpha is not a universal event.     

Genome ploidy in different stages of the Giardia lamblia life cycle

The early diverging eukaryotic parasite Giardia lamblia is unusual in that it contains two apparently identical nuclei in the vegetative trophozoite stage. We have determined the nuclear and cellular genome ploidy of G. lamblia cells during all stages of the life cycle. During vegetative growth, the nuclei cycle between a diploid (2N) and tetraploid (4N) genome content and the cell, consequently, cycles between 4N and 8N. Stationary phase trophozoites arrest in the G₂ phase with a ploidy of 8N (two nuclei, each with a 4N ploidy). On its way to cyst formation, a G₁ trophozoite goes through two successive rounds of chromosome replication without an intervening cell division event. Fully differentiated cysts contain four nuclei, each with a ploidy of 4N, resulting in a cyst ploidy of 16N. The newly excysted cell, for which we suggest the term 'excyzoite', contains four nuclei (cellular ploidy 16N). In a reversal of the events occurring during encystation, the excyzoite divides twice to form four trophozoites containing two diploid nuclei each. The formation of multiple cells from a single cyst is likely to be one of the main reasons for the low infectious doses of G. lamblia .