The genetic toxicity of time: importance of DNA-unwinding time to the outcome of single-cell gel electrophoresis assays.

Single-cell gel electrophoresis assays (comet assays) are described in which DNA damage is assessed in mouse skin keratinocytes treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL) either in vitro or in vivo. The positive results observed under both conditions of test encourage the further development of the mouse skin comet assay as a screen for direct-acting in vivo genotoxins. From the outset of the present experiments we were struck by the compacted nature of the DNA in mouse skin keratinocytes. Under similar conditions of assay, rodent hepatocytes presented a uniform 'unwound' distribution of DNA over the whole nuclear region. In order to study this effect we varied what seemed to be the most obviously related assay parameter: the DNA-unwinding time. A series of experiments was conducted in which control and MNNG-treated cells were exposed to a range of alkaline DNA-unwinding times (0.3-18 h) followed by measurement of the three comet tail parameters (length, DNA content, and their product, tail moment). Each of these parameters increased with increasing time of unwinding such that the tails observed for MNNG-treated cells with 0.3 h of DNA unwinding were similar in length to the tails of control cells exposed to an 8 h DNA-unwinding time. It is concluded that DNA-unwinding time is a critical parameter of the comet assay and that it may require optimisation for each tissue/cell type studied. Further, the data alert to the prospect that agents that uniquely affect chromosomal protein superstructure may increase comet tail length/DNA content in the absence of chemically induced DNA damage. Thus, there may be two discrete classes of chemical interaction with chromosomal DNA that yield identical comet assay results, but which have different implications for the genetic toxicity of the test agent. Similar effects were observed for rat hepatocytes or mouse lymphoma cells exposed to an 18 h DNA-unwinding time, but no comet tails were produced by exposure of cells to the lysis conditions (pH 10.0) for 18 h.

Mutagenicity to the mouse bone marrow by the mouse germ cell mutagen N-propyl-N-nitrosourea.

N-Propyl-N-nitrosourea (PNU) is shown to be active in male mouse bone marrow micronucleus assays when dosed at either 100 or 200 mg/kg in saline. Activity was observed following either intraperitoneal (i.p.) injection or oral gavage. This observation is consistent with the demonstration by Murota and Shibuya of the specific-locus mutagenicity caused by PNU in male mouse spermatogonia when dosed at 200 mg/kg by i.p. injection. These data strengthen further the observation that rodent germ cell mutagens are also mutagenic to rodent somatic cells.

Contribution of T-cell receptor-contacting and peptide-binding residues of the class II molecule HLA-DR4 Dw10 to serologic and antigen-specific T-cell recognition.

The relative contributions of putative T-cell receptor (TCR)-contacting and peptide-binding residues of a major histocompatibility complex (MHC) class II restriction element to serologic and antigen-specific T-cell recognition were investigated by site-specific mutagenesis. Amino acids 70 and 71 in the DR beta 1 domain of DR4 Dw10 are uniquely differnet from the other Dw subtypes of DR4. Residue 70 is predicted to be located at the membrane-distal surface of the class II molecule, where it may influence T-cell recognition by a direct interaction with a TCR. Residue 71 is predicted to form part of the antigen-binding groove where its influence on T-cell recognition may be mediated indirectly via an effect on peptide binding. Transfected murine L cells were produced expressing the products of DR4 Dw10B genes in which the codons for residues 70 and 71 had been mutated towards DR4 Dw14. Support for the predicted orientations of beta-chain residues 70 and 71 was lent by the observation that only residue 70 plays an important role in the formation of a serologic determinant. Mutation of this residue was sufficient to produce recovery of recognition by a human monoclonal antibody, NI, which has specificity for all the DR4 subtypes with the exception of DR4 Dw10. The human T-cell clone HA1.7, specific for influenza virus hemagglutinin (HA) peptide 307-319 and restricted by DR1 Dw1, exhibits degeneracy of MHC restriction on the DR4 Dw subtypes with the exception of DR4 Dw10.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of a cytotoxic human monoclonal antibody with specificity for HLA-DR4 and -DRw10 by cells derived from a highly sensitized kidney recipient.

A human monoclonal antibody which reacts preferentially with HLA-DR4 and -DRw10 B-cell targets has been produced. A human B-cell line, secreting antibody which reacted preferentially with DR4 and DR1 targets, was derived from a highly sensitized kidney recipient who had rejected two grafts. This line was fused with the mouse myeloma P3X63Ag8.653 and a selected hybridoma cloned. The clones secrete IgM(lambda), which reacts strongly with HLA-DR4 and -DRw10 and more weakly with -DRw14 and a proportion of -DR1 B cells in cytotoxicity assays. Using B-cell lines as targets in cytotoxicity and enzyme-linked immunosorbent assays, the antibody gives a broader pattern of reaction, reacting with HLA-DR1, -DR4, -DR9, -DRw10, -DRw14, and some -DR2 targets. The antibody (NI) is currently in use as a reagent for tissue typing.

MicroELISA assays of anti-HLA activity and isotype of human monoclonal antibodies.

Both monoclonal human antibodies to HLA-DR antigens and supernatants from oligoclonal B-cell lines can be conveniently screened for activity by microELISA assays which use 1/10 of the volume of reagents used in conventional ELISA assays. Target cells are fixed to the bases of wells in Terasaki plates, 5 microliters volumes of supernatants incubated in these wells, and target bound antibody detected by peroxidase-conjugated anti-immunoglobulin followed by the substrate ABTS(2,2'-azino-di-(3-ethylbenzthiazoline sulphonate). The plates are read on a micro EIA reader. Supernatants can also be assayed for immunoglobulin content and isotype in Terasaki plates by coating the wells with isotype-specific anti-immunoglobulin, adding test supernatant and developing with appropriate peroxidase-conjugated anti-immunoglobulin sera and ABTS. When assaying for immunoglobulin content, the plates can be read either with a reader or by eye. Advantages and modifications of these procedures are discussed. There are no apparent practically important disadvantages to these procedures as compared with more conventional ELISA assays.

Monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein. Localization of the C-terminus of the protein to the cytoplasmic side of the red cell membrane and distribution of the protein in some human tissues.

(1) We have prepared murine monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein (band 3). (2) All of these antibodies react with regions of the protein located at the cytoplasmic surface of the red cell. (3) One of the antibodies reacts with an epitope present on a cytoplasmic loop of the protein located between the C-terminus and a point 168 amino acids from the C-terminus. The other antibodies recognize different epitopes on the C-terminal tail of the protein and the sequences likely to be involved in these epitopes are defined. (4) Our results show that the C-terminus of the red-cell anion transport protein is located on the cytoplasmic side of the red-cell membrane. (5) None of the antibodies inhibited sulphate exchange transport when introduced into resealed red-cell membranes; however, the bivalent form of one of the antibodies reduced the inhibitory potency of 4-acetamido-4'-isothiocyanatostilbene disulphonate on sulphate exchange transport in resealed erythrocyte membranes. (6) Immunostaining of human kidney sections with the antibodies showed strong staining of the basolateral membrane of some but not all of the epithelial cells of distal tubules and the initial connecting segment of collecting tubules. With human liver, only the haematopoeitic cells of fetal liver reacted with all the antibodies.

The occurrence of profilaggrin and its processing in cultured keratinocytes.

An affinity-purified antibody to rat filaggrin detects filaggrin and profilaggrin in extracts of newborn rat epidermis, and a monoclonal antibody to human filaggrin, HF-1, detects the two proteins in extracts of human epidermis. Immunohistologic studies show that HF-1 reacts with keratohyaline granules of human epidermis and those seen in cultured human keratinocytes. Immunoblotting studies have demonstrated that profilaggrin is synthesized in both cultured human keratinocytes and in a long-lived line of cultured rat keratinocytes, but only in the latter is the protein processed to a product of the molecular weight of filaggrin.