Use of antibody bound to modified cellulose as an immunospecific adsorbent of antigens

Rabbit serum globulins were complexed with bromoacetyl cellulose. Under appropriate conditions 96 to 98% of the protein was chemically bound. Binding of the globulins required a preliminary physical adsorption followed by a chemical reaction. The solid globulin-cellulose complex, under the conditions used, adsorbed specific antigens from solution completely, up to a maximum amount which represented a ratio of antibody to antigen close to that found in a specific precipitate in the equivalence zone. Preliminary experiments are reported on the problems of the specificity of adsorption of homologous antigen compared to unrelated proteins, and of the elution of adsorbed antigen. Potential uses for this method in biochemical investigations are suggested.     

Synthesis of modified tetrasaccharide sequences of N-glycoproteins

The tetrasaccharides O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D- mannopyranosyl-(1----6)]-O-(4-deoxy-beta-D-lyxo-hexopyranosyl)-(1- ---4)-2- acetamido-2-deoxy-alpha, beta-D-glycopyranose (22) and O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl-(1----6)]-O- beta-D-talopyranosyl-(1----4)-2-acetamido-2-deoxy-alpha, beta-D- glucopyranose (37), closely related to the tetrasaccharide core structure of N-glycoproteins, were synthesized. Starting with 1,6-anhydro-2,3-di-O-isopropylidene-beta-D-mannopyranose, the glycosyl donors 3,6-di-O-acetyl-2-O-benzyl-2,4-dideoxy-alpha-D-lyxo- hexopyranosyl bromide (10) and 3,6-di-O-acetyl-2,4-di-O-benzyl-alpha-D-talopyranosyl bromide (30), were obtained in good yield. Coupling of 10 or 30 with 1,6-anhydro-2-azido-3-O-benzyl-beta-D-glucopyranose to give, respectively, the disaccharides 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2-O-benzyl-4 -deoxy- beta-D-lyxo-hexopyranosyl)-beta-D-glucopyranose and 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2,4-di-O-ben zyl- beta-D-talopyranosyl)-beta-D-glucopyranose was achieved with good selectivity by catalysis with silver silicate. Simultaneous glycosylation of OH-3' and OH-6' of the respective disaccharides with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl chloride yielded tetrasaccharide derivatives, which were deblocked into the desired tetrasaccharides 22 and 37.     

PCR-based gene disruption and recombinatory marker excision to produce modified industrial Saccharomyces cerevisiae without added sequences

The dominant selectable Kanr marker, which confers geneticin resistance in yeast, is extensively used for PCR based disruption of genes in functional analysis studies in laboratory strains of Saccharomyces cerevisiae. We have developed a gene disruption cassette, which incorporates the Kanr marker, and direct repeat sequences designed from the target gene to enable the deletion of the gene without the introduction of added DNA sequences. We report on the disruption of the HO gene as a test case, using the hodr-Kanr-hodr cassette. The cassette was shown to integrate at the HO locus and the Kanr marker excised by recombination between the two direct repeat sequences. The disruption/excision event resulted in the removal of one direct repeat and the coding sequence of the gene, and hence in this case loss of HO function, with the introduction of no foreign or additional sequences, including the Kanr marker. Having been derived from the target site, the remaining direct repeat sequence is native sequence in its native location. This design template has the potential to be adapted to other genes, and as such will be of advantage in instances such as the optimization of strains by recombinant DNA technology where the retention of minimal or no foreign sequences is desired.     

Viewing Malignant Melanoma Cells as Macrophage-Tumor Hybrids

Cutaneous malignant melanoma (CMM) begins in the epidermis as the clonal emergence of melanocytes having a deregulated mitotic cycle. In a manner not yet understood, some descendents of these cells loosen their adhesions in situ and migrate into the dermis, thus initiating the processes of invasion and metastasis. These cells look and act much like macrophage-melanoma hybrids created in the lab or arising in mice. But genetic proof for hybrids in human melanoma is still lacking. Nonetheless, should tumor cell hybridization account for the invasive phenotype, this would surely evoke new therapeutic approaches regarding mechanisms of cell fusion and hybrid-specific molecular signatures. Here are described some of the remarkable phenotypic similarities between experimental macrophage-melanoma hybrids and CMM. The results suggest that invasive and metastatic CMM might well arise through fusion and genomic hybridization between melanoma cells and migratory bone marrow-derived cells.Key words: tumor cell fusion, tumor macrophage hybrid, melanoma, metastasis, melanin, migration, adhesion, β1,6-branched oligosaccharides     

Low-molecular-weight Ia antigens in normal mouse serum

Low-molecular-weight Ia antigens can be detected in mouse serum. A procedure for isolating these antigens from serum in high yield is described. The Ia antigen preparation was found to be rich in carbohydrate, low in protein, and strongly bound to Concanavalin A andLotus lectins. Furthermore, the Ia antigenicity was destroyed by periodate oxidation and neuraminidase treatment, but was unaffected by pronase. These observations strongly suggest that the Ia antigens in serum are oligosaccharide in nature. Such a conclusion implies that at least some of the genes in theI region of theH-2 gene complex code for glycosyl transferase enzymes.     

Polymorphism in regulatory gene sequences

The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies.     

Polymorphism in regulatory gene sequences

The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies.     

Dinoflagellate phylogeny as inferred from heat shock protein 90 and ribosomal gene sequences

Background

Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages.

Methodology/Principal Findings

We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved.

Conclusions/Significance

The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression

The selective alteration of the genome using Cre recombinase to target the rearrangement of genes flanked by LOX recognition sequences has required the use of two separate genetic constructs in trans, one containing cre and the other containing the gene of interest flanked by LOX sites. We have developed a strategy in which both the cre recombinase gene and LOX recombination sites may be cloned within a single vector in cis. This method uses a modified form of Cre (CREM) that contains alterations to the 5′ region including the introduction of a Kozak consensus sequence and insertion of a functional intron. This system allows for the inducible, tissue-specific activation or inactivation of gene expression in a single vector and can be utilized for the 300-fold amplification of gene expression from a weak promoter. This approach can be applied to targeting strategies for generating genetically altered mice and gene therapy.     

VIP-derived sequences modified by N-terminal stearyl moiety induce cell death: the human keratinocyte as a model

Vasoactive intestinal peptide (VIP) is a recognized growth factor affecting many cell types. We have previously developed a series of lipophilic VIP analogues containing an N-terminal covalently attached stearyl moiety. The current studies identified stearyl-Nle(17)-VIP and stearyl-Nle(17)-neurotensin(6-11)VIP(7-28), acting at microM concentrations, as cytotoxic to human keratinocytes. The core C-terminal active VIP-derived peptide, stearyl-Lys-Lys-Tyr-Leu-NH(2) (St-KKYL-NH(2)), was identified as being responsible for the observed cytotoxicity. Cytotoxicity coincided with marked reduction in intracellular cyclic GMP and was abolished by co-treatment with the endonuclease inhibitor, aurine-tricarboxylic acid, suggesting apoptotic mechanisms. Stearyl-VIP derivatives thus offer lead compounds for future drug development against hyperproliferative skin conditions.     

Reagentless detection of DNA sequences on chemically modified electrodes

Microarray analysis requires complex optical instruments and numerous reagents. Several new electrochemical methods for creating sequence-selective microlocations are emerging; such approaches potentially facilitate electrochemical (electronic) readouts of microarrays. These developments parallel the migration of glucose sensing in diabetes monitoring from optical to electrochemical methods. Recent work provides a strategy that minimizes the use of added reagents and potentially produces a reusable sensor that can be applied to continuous monitoring applications.