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.     

Isolation and culture of cells derived from human cerebral microvessels

Summary Microvessels were isolated from non-neoplastic human cerebral cortical fragments resected for treatment of intractable seizure disorder. The microvessels were incubated in modified Lewis medium with 20 or 30% fetal bovine serum. Within 1–2 weeks, two cell populations emerged from the isolates. One type of cells had polygonal morphology, showed density-dependent contact inhibition at confluence in vitro, showed lectin-binding characteristics of endothelium (but only moderate positivity for factor VIII antigen), demonstrated induction of -glutamyl trans-peptidase when exposed to astrocyte-conditioned media, and responded to insulin by a pronounced increase in DNA synthesis. The other variety of cells grew in vitro more slowly in irregular strands separated by clear zones, showed ultrastructural features of smooth muscle, and isoelectric focusing of cell proteins revealed the presence of smooth-musclespecific -isoactin. Both types of cells could be serially subcultured. The ability to isolate and grow the two cell types, tentatively identified as human cerebral microvascular endothelium and smooth muscle, may facilitate studies of human blood-brain barrier function as well as the pathogenesis of cerebral microangiopathies unique to the human brain.Funded by Canadian Heart Foundation, Heart and Stroke Foundation of Ontario and UCLA Biomedical Research Support Grant     

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.     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

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.