Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

2D-NMR studies of the unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG).

The unnatural oligonucleotide alpha-d(TCTAAAC) was synthesized and was found more resistant towards endonucleases than its beta-analog. 2D-NMR experiments allowed the assignment of all non-exchangeable aromatic and sugar protons except for the overlapping 5' -5" resonances, as well as the exchangeable imino protons of the parallel hybrid duplex alpha-d (TCTAAAC)-beta-d(AGATTTG). NMR studies show that the strength of the association between the alpha-strand and the beta parallel strand is equivalent to that between their anti-parallel complementary beta-analogs beta-d(CAAATCT) and beta-d(AGATTTG). NOE data provide evidence that both duplexes form stable right-helical duplexes with an anti-conformation on the glycosyl linkages and a Watson-Crick pairing. NOESY and COSY spectra allowed us to determine that alpha and beta deoxyriboses adopt a 3' -exo conformation.     

An acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome mRNAs kills cultured parasites

Anti-messenger oligodeoxynucleotides covalently linked to an intercalating agent were tested for their ability to inhibit translation of Trypanosoma brucei mRNAs in a cell-free system. The sequence of these oligodeoxynucleotides was complementary to part of the 35-nucleotide (nt) sequence which is present at the 5' end of all trypanosome mRNAs (the so-called mini-exon sequence). In a rabbit reticulocyte lysate, a nonadeoxynucleotide linked to an acridine derivative, specifically inhibited protein synthesis from T. brucei mRNAs much more efficiently than unmodified oligodeoxynucleotides of similar length. These oligodeoxynucleotides were tested on cultured trypanosomes. The acridine-linked nonadeoxynucleotide had a lethal effect on the parasites. No effect was observed with the homologous unmodified 9-mer nor with those 9-mers linked to the acridine derivative which were not complementary to the mini-exon sequence. These effects are probably a result of hybrid formation between the anti-messenger and mini-exon sequence. Trypanocidal activity of the acridine-modified nonadeoxynucleotide is most likely due to (i) increased affinity for its target, (ii) improved resistance to 3' exonucleases, and (iii) promoted membrane penetration of living parasites.     

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.     

Periodic cleavage of poly(dA) by oligothymidylates covalently linked to the 1,10-phenanthroline-copper complex

1,10-Phenanthroline (OP) was covalently attached to the 3'-terminus of two oligothymidylates via different linkers [abbreviated as T8-(OP) and T6-(OP)]. In the presence of Cu2+ and 3-mercaptopropionic acid (MPA), these reagents induce a hybridization-dependent cleavage of poly(dA) and of a 27 nucleotide long oligodeoxynucleotide containing an A8 sequence. The principal cleavage sites on the 27-mer span four residues located near the 3'-terminal phosphate group of T8-(OP). When poly(dA) was degraded by T6-(OP) and T8-(OP), a series of bands were obtained corresponding to a repeat unit of six and eight nucleotides, respectively. This periodicity reflects the cooperative binding of oligothymidylate-OP to the polynucleotide matrix and the localized nicking sites.     

Chemical synthesis of natural and modified oligodeoxynucleotides

This paper reports chemical synthesis of natural and modified oligodeoxynucleotides. In the first part after a short review of protective groups for the deoxynucleosides and for the phosphate groups, the different phosphorylating and coupling methods are listed and also the application fields for the different techniques (phosphotriester method in solution or solid-phase synthesis). The second part involves oligodeoxynucleotides in which phosphodiester groups are partially or fully replaced by alkylphosphotriester, phosphonate, phosphoramidate, phosphorothioate or phosphorothiolate groups. The last part describes synthesis of oligodeoxynucleotides bearing chemically reactive groups or an intercalating agent.     

Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.