Characterisation of stroma-dependent blast colony-forming cells in human marrow

Human bone marrow contains a population of haemopoietic progenitor cells that can be distinguished by their ability to adhere to preformed stromal layers (cultured in the presence of methylprednisolone [MP+] and form blast cell colonies. The stromal layers function in the colony assay after they have been heavily irradiated but not after they have been passaged. The binding of the progenitor cells to the stromal cells is complete after 2 hours of coincubation, and stromal layers of 9.6 cm2 can provide adhesion sites for at least 2,000 blast colony-forming cells. The blast colony-forming cells were shown by micromanipulation to self-renew as well as to give rise to multipotential and lineage-committed colony-forming progenitor cells.     

Polymorphisms of DQ β genes in HLA-DR4 haplotypes from healthy and diabetic individuals

The restriction fragment length polymorphism (RFLP) of DQ was assessed in a panel of control and insulin-dependent diabetes (IDD) patients who were serologically typed as HLA-DR4 homozygotes or HLA-DR3, DR4 heterozygotes. Digestions of genomic DNA with Barn HI, Bg1 II, Pst I, Xba I, and Hind III revealed a total of 15 RFLPs in the panel of 71 HLA-DR4 chromosomes. These RFLPs were organized into six allelic groups on the basis of segregation analysis in families. Complete RFLP haplotypes for the 5 restriction enzymes could be constructed for 42 of the HLA-DR4 chromosomes. This analysis revealed 18 RFLP haplotypes of DQ associated with the DR4 chromosomes tested. Two of these haplotypes, designated DQ3.DR4.a and DQ3.DR4.b, accounted for over 50 % of the DR4 chromosomes analyzed. These two haplotypes were antithetical for the RFLPs detected by all five enzymes, indicating that they represent very distinct forms of DQ . The remaining 16 haplotypes were infrequent or unique and were closely related to either a DQ3.DR4.a or DQ3.DR4.b. Two of the RFLPs detected, a 5.8 kb Bg1 II fragment and a 10.5 kb Barn HI fragment, had increased frequencies in disease-associated chromosomes. However, none of the RFLPs we detected exhibited a statistically significant increase in IDD or control populations. In contrast, the DQ3.DR4.b DQ haplotype was significantly decreased in IDD-associated DR4 chromosomes. (P=0.04). These results suggest that the DQ3.DR4.b DQ allele may be protective for the development of IDD.     

Age at maturity in landlocked and anadromous Atlantic salmon parr from two Québec rivers

Synopsis Age at maturity in male Atlantic salmon parr from landlocked populations in the Watshishou and Musquaro Rivers is significantly greater than in anadromous populations from the same rivers. We conclude that high post-smolt mortality in anadromous stocks is conducive to male parr maturity at an early age. We also suggest that the lower proportion of maturing male parr in landlocked stocks may be related to competition among males for mates and the smaller ultimate size of spawning adult landlocked salmon.     

A miniaturised parallel-plate shearing apparatus for the measurement of cell adhesion

The design and construction of a miniaturised shearing apparatus is described. Cultures (24 h) of an established epithelial cell line (BEB) were exposed to flow conditions in the shearing chamber at 37 degrees C, and subsequently glass coverslip cultures were prepared for photography. The critical shear radius (CSR) was determined by densitometry from a negative film and the minimum distraction force (MDF) at the CSR calculated using predetermined viscosity values of the flow medium. The mean calculated MDF of BEB cells ranged from 1.04-1.36 Nm-2, and was independent of the culture inoculation density (9 to 37 X 10(4) cells cm-2) and the time (5-20 min) of exposure to shearing conditions. The MDF was increased to 3.3 Nm-2 by a 30 min exposure of cultures to concanavalin A (50 micrograms ml-1), and this effect was abolished by treating with mannose (0.05 M). The results demonstrated that the radial flow chamber principle is applicable to the measurement of cell to substratum adhesion of cultured mammalian cells.     

Flow cytometric analysis of protein thiol groups in relation to the cell cycle and the intracellular content of glutathione in rat hepatocytes

The relationship between protein thiols (PSH) and cell proliferation was examined in ethanol-fixed rat hepatocytes. A new protocol was developed for simultaneous measurement of protein thiol vs. DNA content by flow cytometry. The fluorescent dye o-phthalaldehyde (OPT) was used for flow cytometric measurements of protein thiol groups. The influence of nonprotein thiols was examined by monitoring the cell cycle of cells in which the glutathione content (GSH) was modified by treatment with buthionine sulphoximine (BSO). Three rat liver cell lines (IAR 20, IAR 6.1, IAR 6.1RT7) were used: these cell lines possess different growth characteristics and degrees of tumorigenicity, which made it possible to analyse changes in PSH during normal and deranged cell proliferation. The effects on the cell cycle of the changes in PSH due to the depletion of GSH were measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry. The data obtained can be summarised as follows: a) OPT fluorescence increases with increasing DNA content in all rat liver cell lines examined; b) the greatest variation in PSH content occurs in G1. There is a smaller variation in G2 + M, and PSH levels are relatively invariant throughout S-phase; c) a higher content of PSH is found in the tumorigenic cell lines; d) the amount and distribution of PSH is not affected by BSO treatment; e) kinetic studies indicate that BSO treatment has no effect on the ability of the IAR rat liver cell lines to progress through the cycle.     

Hydrogen peroxide cytotoxicity. Low-temperature enhancement by ascorbate or reduced lipoate.

The principal mechanism of H2O2 toxicity is thought to involve the generation of hydroxyl (HO.) radicals through its interactions with Fe2+ ions by the Fenton reaction. Of particular interest has been the demonstration by Ward, Blakely & Joner [(1985) Radiat. Res. 103, 383-392] that the cytotoxicity of H2O2 is diminished at low temperature. We have now examined this phenomenon further with a mammalian epithelial cell line (CNCMI-221). Resistance of these cells to 100 microM-H2O2 added extracellularly exhibits a transition in the temperature range between 27 degrees C and 22 degrees C. We have found that the low-temperature resistance to cytotoxic concentrations of H2O2 is abolished by preincubation of cells with reductants such as ascorbate or reduced lipoic acid. This implies that the low-temperature resistance to H2O2 cytotoxicity may be due to inhibition of cellular reductive processes. The restoration of the cytotoxic action of H2O2 at 4 degrees C by ascorbate is prevented by pre-exposure of cells to desferrioxamine. This is evidence that transition-metal ions (such as iron ions) are involved in the cytotoxicity and is consistent with a mechanism of cell damage that depends on the Fenton reaction and a metal ion in the reduced state. Restoration of H2O2 cytotoxicity at low temperature by ascorbate is consistent with the artificial production of an intracellular reducing environment that at normal temperatures is sustained by cellular metabolism.     

Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.