The effect of monofunctional or difunctional platinum adducts and of various other associated DNA damage on the expression of transfected DNA in mammalian cell lines sensitive or resistant to difunctional agents

The effects of introducing various DNA damage into pSV2gpt DNA on the subsequent expression of xanthine guanine phosphoribosyltransferase (XGPRT), after its transfection into two Walker 256 cell lines, one which is inherently sensitive only to difunctional agents while the other shows a normal sensitivity, have been examined. Both the sensitive (WS) and the relatively resistant (WR) cell lines were shown to be equally capable of both ligation of DNA double-strand breaks (although the efficiency varied with the actual site of the break) introduced into pSV2gpt and homologous recombination of pSV2gpt fragments (recombination events are thought to be important in the repair of DNA-DNA interstrand crosslinks). Reacting the plasmid with either the difunctional platinum compound, Cisplatin, or the monofunctional reacting Pt(Dien) caused a dose-dependent decrease in the subsequent expression of XGPRT. This decrease was about the same with either agent in either cell line when expressed as a function of dose of drug. However, when the actual binding of platinum to DNA by these compounds was measured, a large difference (due to the higher specific binding of Pt(Dien) to DNA) in the effects of the difunctional, as opposed to the monofunctional agent, was apparent and this was a reflection of the relative cytotoxicities of these compounds towards mammalian cells. Although at doses of Cisplatin equitoxic to WS and WR cells 20-fold less Pt is bound to the DNA of WS cells, no significant difference was seen on the expression of pSV2gpt, reacted with this agent, between WS or WR cells. Based upon a knowledge of the proportions of adducts formed in DNA reacted with Cisplatin, the lesion that inactivates expression of XGPRT was probably the intrastrand crosslink and it was calculated that due to the size of the plasmid, the interstrand crosslink was unlikely to be present at these inactivating doses. It is suggested that the inherent sensitivity of WS cells only to difunctional agents is due to their response to such relatively rare lesions such as a DNA-DNA interstrand crosslink.     

Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products.

We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.     

Homozygous paracentric inversion 12 in a mentally retarded boy: a case report and review of the literature

Summary A mentally retarded male was found to be homozygous for a paracentric inversion of the long arm of chromosome 12(inv(12)(q21.1q23.2)). His parents, who are first cousins, and his phenotypically normal younger brother are inversion heterozygotes. Homozygous structural rearrangements are discussed and cases of paracentric inversions, including a further nine previously unpublished, are reviewed.     

A natural hybrid newt, Triturus helveticus×T. vulgaris, from a pond in mid-Wales

The first unequivocal example of a natural Trilurus helvelicus × T. vulgaris hybrid is described. The specimen was a male and discriminant analysis of physical characters indicated that it was morphologically intermediate between the parent species. A karyotype confirmed that the hybrid bore a haploid set of chromosomes from T. helveticus and a haploid set from T. vulgaris. Examination of the sex chromosomes showed that it was the result of mating between a male T. helveticus and a female T. vulgaris. As numerous mature sperm bundles were observed in both testes, the hybrid was therefore potentially fertile.     

The response of murine intestinal crypts to short-range promethium-147 beta irradiation: deductions concerning clonogenic cell numbers and positions

An exteriorized loop of mouse intestine was exposed to 147Pm low-energy electrons, where the dose rate decreased by a factor of 5 from the base of the crypt to the top of the proliferative zone. A crypt survival curve was obtained, expressed in terms of exposure time. The shape of the curve was interpreted in terms of survival parameters for colony-forming cells (clonogens) derived using 137Cs gamma rays and the depth-dose curve measured for 147Pm electrons. It is concluded that the shape of the crypt survival curve using 147Pm electrons is inconsistent with the notion of either the presence of a large number of clonogens or a small number near the top of the proliferative zone. A computer fitting procedure showed that the best agreement between predicted and observed curves was achieved with 2.7 +/- 0.5 clonogens at cell position 5.6 +/- 0.6, in the putative stem-cell zone.     

A redetermination of the concentration of alpha 2-macroglobulin in human plasma

The concentration of alpha 2-macroglobulin in human plasma has been remeasured utilizing a carefully isolated and characterized sample of alpha 2-macroglobulin as a standard. A highly purified sample of alpha 2-macroglobulin with a total trypsin binding capacity of 1.7 mol trypsin/mol alpha 2-macroglobulin was used as a standard for both a radial immunodiffusion and a rocket immunoelectrophoresis technique. With this preparation as a standard, the concentration of alpha 2-macroglobulin in a normal plasma pool over 10,000 donors was found to be about 1.2 mg/ml. A similar concentration (1.3 mg/ml) was found when using a functional trypsin binding assay. This concentration is considerably less than the usually accepted mean of the normal range for alpha 2-macroglobulin.     

Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage.

We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.     

Phosphate promotes glycation of antithrombin III which interferes with heparin binding

Nonenzymatic glycation of antithrombin III has been reported to cause the reduction of heparin-catalyzed thrombin-inhibiting activity in diabetes. The effect of in vitro nonenzymatic glycation of pure antithrombin III on heparin binding and heparin-potentiated activity under a variety of buffers and pH values was studied to further clarify the physiological significance of this reaction. The extent of glycation, measured by the fructosamine assay and [14C]glucose binding, was enhanced by the presence of phosphate ion (pH 7.45, 8.5 and 9.5) and increased linearly with increasing phosphate ion concentration from 0.01 to 0.2 M phosphate. Conversely, the heparin-catalyzed antithrombin activity decreased from 93.1% of controls for 0.01 M phosphate to 73.5% for 0.2 M phosphate as the extent of glycation increased. The increase in intrinsic fluorescence induced by binding of heparin to antithrombin III was also moderated by glycation of antithrombin III in a dose-dependent manner with a negative correlation coefficient of -0.94. Direct measurement of the heparin binding by affinity chromatography showed a decrease in the heparin-binding fraction which correlated with the degree of glycation and the decrease in heparin-catalyzed activity. These studies suggest that nonenzymatic glycation may be responsible for the reduction in antithrombin III activity observed in some diabetics.