Self-cross-linking polyelectrolyte complexes for therapeutic cell encapsulation

Self-cross-linking polyelectrolytes are used to strengthen the surface of calcium alginate beads for cell encapsulation. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), containing 30 mol % 2-aminoethyl methacrylate, and poly(sodium methacrylate), containing 30 mol % 2-(methacryloyloxy)ethyl acetoacetate, were prepared by radical polymerization. Sequential deposition of these polyelectrolytes on calcium alginate films or beads led to a shell consisting of a covalently cross-linked polyelectrolyte complex that resisted osmotic pressure changes as well as challenges with citrate and high ionic strength. Confocal laser fluorescence microscopy revealed that both polyelectrolytes were concentrated in the outer 7-25 microm of the calcium alginate beads. The thickness of this cross-linked shell increased with exposure time. GPC studies of solutions permeating through analogous flat model membranes showed molecular weight cut-offs between 150 and 200 kg/mol for poly(ethylene glycol), suitable for cell encapsulation. C 2C 12 mouse cells were shown to be viable within calcium alginate capsules coated with the new polyelectrolytes, even though some of the capsules showed fibroid overcoats when implanted in mice due to an immune response.     

GeneOrder: comparing the order of genes in small genomes

MOTIVATION: The recent rapid rise in the availability of whole genome DNA sequence data has led to bottlenecks in their complete analysis. Specifically, there is a need for software tools that will allow mining of gene and putative gene data at a whole genome level. These new tools will complement the current set already in use for studying specific aspects of individual genes and putative genes in detail. A key software challenge is to make them user-friendly, without losing their flexibility and capability for use in research. RESULTS: The creation of GeneOrder-a web-based interactive, computational tool-allows researchers to compare the order of genes in two genomes. It has been tested on full genome sequence data for viruses, mitochondria and chloroplasts that were obtained from the NCBI GenBank database. It is accessible at http://www.bif.atcc.org/GENEOrder/index.html. GeneOrder prepares the comparison in table form, listing the order of similar genes. Hyperlinks are provided from this output; these lead to the 'Protein Coding Regions' in the NCBI database.     

Evaluation of hematological and hepatorenal functions of methanolic extract of Moringa oleifera Lam. root treated mice

Methanolic extract of M. oleifera root was found to contain some alkaloids (total alkaloid 0.2%). Effects of multiple weekly (35, 46, 70 mg/kg) and daily therapeutic (3.5, 4.6, 7.0 mg/kg) i.p. doses of the crude extract (CE) on liver and kidney functions and hematological parameters in mice were studied. No alteration in hematological and biochemical parameters at low and moderate dose level of daily and low dose level of weekly treatment of the extract was observed. However, the extract at moderate dose level in weekly treatment changed serum aminotransferase and plasma cholesterol levels significantly. High dose in addition to the above parameters changed total bilirubin, non protein nitrogen, blood urea and plasma protein. High dose of daily treatment and moderate and high dose of weekly treatment of CE increased WBC count and decreased clotting time significantly. The results indicate that the weekly moderate and high dose (> 46 mg/kg body wt.) and daily/therapeutic high dose (7 mg/kg) of CE affects liver and kidney functions and hematological parameters whereas the weekly dose (3.5 mg/kg) and low and moderate daily/therapeutic dose (3.5 and 4.6 mg/kg) did not produce adverse effects on liver and kidney functions.     

Generation of tumor cell lysate-loaded dendritic cells preprogrammed for IL-12 production and augmented T cell response

Dendritic cells (DC) loaded with tumor associated antigens (TAA) are often used for the vaccination of cancer patients; however methodologies for the vaccine preparation have not yet been standardized. The purpose of this work was to optimize the ex-vivo production of functional TAA-loaded DC that would produce interleukin-2 (IL-12) and enhance the T cell response. We generated ex-vivo DC from human monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4, and whole necrotic tumor cells (cell lysates) of cancer cell lines were used as model TAA. DC were loaded with lysates without or with additional tumor necrosis factor-alpha (TNF-alpha), or cytokine combination treatments and tested for functional ability in vitro. Tumor cell lysates alone did not fully mature DC either phenotypically or functionally. After antigen uptake additional maturation signals were necessary. TNF-alpha matured DC phenotypically, but additional interferon-gamma (IFN-gamma) treatment was necessary to achieve functional maturation, the production of significant amounts of IL-12. Since IL-12 production by DC increased during the first 24 h of maturation and declined by 48 h, proper timing of the ex-vivo DC treatment was crucial for the generation of functionally mature antigen-loaded DC. Our results suggest that after allowing 4 h of tumor lysate uptake by immature DC, further treatment with TNF-alpha and IFN-gamma for 24 h provides the optimal conditions to obtain functional TAA-loaded DC. These TAA-loaded cytokine pretreated DC then prime na?ve T cells, and enhance both T helper 1 (Th1), Th2 and cytotoxic T lymphocyte (CTL) responses, that are necessary to achieve an effective, specific anti-tumor response.     

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 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.