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.     

A practical test for in vitro evaluation of photosensitization: Assessment with hematoporphyrin derivative (HPD)

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities.The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.Abbreviations DHE Dihematoporphyrin Ether - FCS Fetal Calf Serum - HPD Hematoporphyrin Derivative - PDT Photodynamic Therapy     

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.     

Additional mapping of mouse chromosome 2 genes

The purpose of this work was to elucidate the genetic fine structure of the central portion of mouse chromosome (Chr) 2. Seven Chr 2 congenic mouse strains [B10.PA(L)-pa we un a ^t , B10.PA(L)-pa A ^w , B10.PA(L)-we un a ^t , B10.PA(J)-pa a, B10.FS-we A ^w , B10.C-we A ^w , and B10.YBR-a] were produced. Breeding studies were carried out using strains B10.PA(L)-pa we un a ^t and B10.LP-H-13 ^b to accurately determine the recombination frequencies between marker genes pa and we (1.9%±0.3), we and un (8.8%±0.5), and un and a ^t (4.5%±0.4) of strain B10.PA(L)-pa we un a ^t . These strains and other Chr 2 congenic strains were typed for immunologically defined loci using monoclonal antibody (mAb) C23 reactive with the gene product of B2m ^b T-lymphocyte clone C1 reactive with the gene product of H-3 ^a and H-3 ^c , and lymphocyte clone H1.8 reactive with the gene product of Hd-1 ^a . B2m and H-3 typing located a recombinational event separating [pa B2m H-3] from we (the order of bracketed genes is not known). Hd-1 typing indicated that Hd-1 maps distal to [H-42, H-44] and proximal to un. The gene order [pa, B2m, H-3], we, [H-42, H-45], Hd-1, un, H-13, a ^t , with H-44 mapping centromeric to Hd-1, is indicated by the data. Address correspondence and offprint requests to: R. J. Graff.     

Characterization of the central region containing the X-inactivation center and terminal region of the mouse X Chromosome using irradiation and fusion gene transfer hybrids

The irradiation and fusion gene transfer (IFGT) procedure provides a means of isolating subchromosomal fragments for use in the mapping of loci and for cloning probes from a particular area of a chromosome. Using this procedure, two large panels of somatic cell hybrids that contain mouse X Chromosome (Chr) fragments have been generated. These hybrid panels were generated by irradiating the monochromosomal mouse-hamster hybrid HYBX, which retains the mouse X Chr, with either 10 K or 50 K rads of X-irradiation followed by fusion with a recipient Chinese hamster cell line. IFGT hybrids retaining mouse material were generated at high frequency. These hybrids were used to orient loci in the X-inactivation center region that had not been resolvable in our interspecies backcross panel and also to map, within the terminal region of the X Chr, repeat elements detected by the probe p15-4. These hybrids not only complement existing interspecies meiotic mapping panels for the detailed analysis of specific regions of particular chromosomes, but also provide a potential source of material for chromosome-specific probe isolation.     

The anatomy of tissue cultured red raspberry prior to and after transfer to soil

The leaf, petiole, stem and root anatomy of an aseptically cultured red raspberry clone (Rubus idaeus L.) was studied before and 5 weeks after transfer to soil under controlled environmental conditions. Tissues persistent from culture showed little or no change with time in soil; they grew minimally and slight secondary wall deposition occurred. New organs formed in successive weeks after transplantation showed a graded increase in potential size and development. Some features, such as collenchyma formation, rapidly returned to control levels; this was seen in new leaves expanding in the first week after transplantation. Other features, such as sclerenchyma formation, did not occur in leaves expanding during the first 2 weeks after transplantation, even when these were a month or more in age. Some sclerenchyma was seen in leaves expanding in the third week after transplantation, increasing in later-formed leaves. Increasing the light intensity of transplant accelerated the return to control-type organ size and appearance. During acclimatization transitional forms of leaves, petioles, stems and roots develop that ranged anatomically from culture-to control-type. This trend is analagous to the normal developmental sequence of organ formation as it affects the potential for development of successily formed organs.     

Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.