Primary Research: Short Communication: Evidence Supporting Rare AIDS-Kaposi's Sarcoma Metastasis In Keeping With Their Vascular Endothelial Evolution

BACKGROUND: It is postulated that the unusual manifestations of Kaposis's sarcoma cells in nonendothelial brain tissues and on eyeballs in advanced acquired immune deficiency syndrome (AIDS) cases are metastasized AIDS-Kaposi's sarcoma cells arising from vascular endothelial cells. METHODS: Experiments were performed to explore the above hypothesis by testing for intercellular adhesion molecule-1 (CD54 antigens) on cutaneous AIDS-Kaposi's sarcoma cells as well as on AIDS-Kaposi's sarcoma cells isolated from eyeballs as studies have illustrated that, unlike localized Kaposi's sarcoma cells of primary lesions, proliferating Kaposi's sarcoma cells in proximity to primary lesions express a negative or diminished phenotype when evaluated for identical surface antigens. Parallel CD54 antigen tests were done on vascular endothelial cells and monocytes/macrophages as endothelial cells are considered evolutionarily related to Kaposi's sarcoma cells and monocytes/macrophages are ideal CD54 antigen positive controls. RESULTS: Our data showed that only AIDS-Kaposi's sarcoma cells of the eyes did not express CD54 antigens. CONCLUSIONS: We therefore report that our findings support the postulation suggesting AIDS-Kaposi's sarcoma dissemination in advanced AIDS patients in keeping with their vascular endothelial heredity.     

Trends in protein evolution inferred from sequence and structure analysis

Complementary developments in comparative genomics, protein structure determination and in-depth comparison of protein sequences and structures have provided a better understanding of the prevailing trends in the emergence and diversification of protein domains. The investigation of deep relationships among different classes of proteins involved in key cellular functions, such as nucleic acid polymerases and other nucleotide-dependent enzymes, indicates that a substantial set of diverse protein domains evolved within the primordial, ribozyme-dominated RNA world.     

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release,caspase activation,and mitochondrial dysfunction

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.     

The COG database: an updated version includes eukaryotes

Background

The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.

Results

We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.

Conclusion

The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.     

Suspension arrays for high throughput, multiplexed single nucleotide polymorphism genotyping

BACKGROUND: Genetic diversity can help explain disease susceptibility and differential drug response. The most common type of variant is the single nucleotide polymorphism (SNP). We present a low-cost, high throughput assay for SNP genotyping. METHODS: The assay uses oligonucleotide probes covalently attached to fluorescently encoded microspheres. These probes are hybridized directly to fluorescently labeled polymerase chain reaction (PCR) products and the results are analyzed in a standard flow cytometer. RESULTS: The genotypes determined with our assay are in good agreement with those determined by TaqMan. The range of G/C content for oligonucleotide probes was 23.5-65% in the 17 bases surrounding the SNP. Further optimization of probe length and target concentration is shown to dramatically enhance the assay performance for certain SNPs. Using microspheres which have unique fluorescent signatures, we performed a 32-plex assay where we simultaneously determined the genotypes of eight different polymorphic genes. CONCLUSIONS: We demonstrate, for the first time, the feasibility of multiplexed genotyping with suspension arrays using direct hybridization analyses. Our approach enables probes to be removed from or added to an array, enhancing flexibility over conventional chips. The ability to multiplex both the PCR preparation and the hybridization should enhance the throughput, cost, and speed of the assay.     

Ceruloplasmin and cardiovascular disease

Transition metal ion–mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His⁴²⁶, is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.     

Low-Substrate Regulated Microaerophilic Behavior as a Stress Response of Aquatic and Soil Bacteria

Low-substrate regulated microaerophilic behavior (LSRMB) was observed in 10–54% of the bacteria isolated from several fresh-water lakes or ponds, subsurface soils, activated sludge, and Antarctic dry valley soils. Five Pseudomonas and two Bacillus type species showed LSRMB. A subsurface Pseudomonas jessenii strain was used as a model to show the metabolic interaction between substrate and oxygen concentrations, cell band movement, and the appearance of unique stress lipids and proteins. When the oxygen in the P. jessenii culture medium was increased from 11% to 100% saturation under atmospheric condition, the concentration of 17:0 cyclopropane fatty acid, a stress indicator, increased five-fold, and four unique proteins were also detected. This stress response occurred only in low-substrate media. It is our hypothesis that LSRMB is a common but under-appreciated trait of many aquatic and soil bacteria. Received: 8 December 1999 / Accepted: 9 February 2000     

Comparison of five rep-PCR genomic fingerprinting methods for differentiation of fecal Escherichia coli from humans, poultry and wild birds

The development of a methodology to identify the origin of fecal pollution is important both for assessing the degree of risk posed to public health and for developing strategies to mitigate the environmental loading of pathogens associated with waterborne disease transmission. Five rep-PCR genomic fingerprinting methods, such as rep-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, ERIC2-PCR, BOX-PCR and (GTG)(5)-PCR, were assessed for their potential in differentiation of 232 fecal Escherichia coli isolates obtained from humans, poultry (chicken, duck and turkey) and wild birds (Canada goose and gull). Based on the results of cluster analysis and discriminant function analysis, (GTG)(5)-PCR was found to be the most suitable method for molecular typing of fecal E. coli, followed by BOX-PCR, REP-PCR, ERIC-PCR and ERIC2-PCR. A discriminant function analysis of (GTG)(5)-PCR fingerprints showed that 94.1%, 79.8%, 80.5%, 74.4%, 86.7% and 88.6% of turkey, chicken, duck, Canada goose, gull and human E. coli isolates were classified into the correct host group, respectively. Subsequently, (GTG)(5)-PCR was tested for its ability to track the origin of 113 environmental E. coli isolated from natural pond water. In conclusion, the (GTG)(5)-PCR genomic fingerprinting method can be considered as a promising genotypic tool for epidemiological surveillance of fecal pollution in aquatic environments.     

Evaluation of repetitive extragenic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic- and wild-animals

The objective of this study was to investigate the potential of repetitive extragenic palindromic anchored polymerase chain reaction (rep-PCR) in differentiating fecal Escherichia coli isolates of human, domestic- and wild-animal origin that might be used as a molecular tool to identify the possible source(s) of fecal pollution of source water. A total of 625 fecal E. coli isolates of human, 3 domestic- (cow, dog and horse) and 7 wild-animal (black bear, coyote, elk, marmot, mule deer, raccoon and wolf) species were characterized by rep-PCR DNA fingerprinting technique coupled with BOX A1R primer and discriminant analysis. Discriminant analysis of rep-PCR DNA fingerprints of fecal E. coli isolates from 11 host sources revealed an average rate of correct classification of 79.89%, and 84.6%, 83.8%, 83.3%, 82.5%, 81.6%, 80.8%, 79.8%, 79.3%, 77.4%, 73.2% and 63.6% of elk, human, marmot, mule deer, cow, coyote, raccoon, horse, dog, wolf and black bear fecal E. coli isolates were assigned to the correct host source. These results suggest that rep-PCR DNA fingerprinting procedures can be used as a source tracking tool for detection of human- as well as animal-derived fecal contamination of water.