Molecular mechanism of ligand recognition by membrane transport protein,Mhp1

The hydantoin transporter Mhp1 is a sodium‐coupled secondary active transport protein of the nucleobase‐cation‐symport family and a member of the widespread 5‐helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site‐directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5‐substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5‐substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5‐(2‐naphthylmethyl)‐L‐hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.     

Spontaneous and induced chromosomal aberrations and gene mutations in human lymphoblasts: mitomycin C, methylnitrosourea, and ethylnitrosourea

The concentration-dependent mutagenic, clastogenic, and cytocidal activities of mitomycin C (MC), methylnitrosourea (MNU), and ethylnitrosourea (ENU) were measured in the human lymphoblast cell line TK6. For treatments resulting in fewer than 2 lethal hits, MNU, ENU, and MC gave rise to apparently linear dose-response curves for gene mutations (hgprt and tk genes) as well as for chromosomal aberrations. The numbers of induced mutants at the tk and hgprt loci were similar between the two loci for each compound. However, the ratio of mutagenic activity relative to the clastogenic activity (aberrations/cell) was lowest for mitomycin C, intermediate for methylnitrosourea, and highest for ethylnitrosourea. These results confirm in human cells the general observation that the processes of mutagenesis and clastogenesis are nonidentical: compounds vary independently in their mutagenic and clastogenic potentials.     

Variance estimation in single-cell mutation assays: comparison to experimental observations in human lymphoblasts at 4 gene loci

The performance of mutation assays with single cells involves a series of separate steps beginning with the induction of mutant cells and ending with the counting of mutant and wild-type colonies. The vatiation among identically treated cultures is here modeled as arising from 3 sources: (1) the number of mutant cells surviving treatment, (2) the number of mutant cells sampled in steps of sampling and growth required in assays involving phenotypic lag, and (3) the number of mutant and nonmutant colonies actually observed.

The arithmetical statements describing the expectation of variance from each step are presented and used to provide means to calculate an expected overall variance for typical experiments.

The model is then tested by comparing its predictions with the observed mutant fractions in human lymphoblastoid cells at the loci coding for 6-thioguanine, ouabain, podophyllotoxin, and 5,6-dichlororibofuranosyl benzimidazole resistances. The model is found to have excellent predictive qualities and should be useful in experimental design of studies involving induction of rare variants in single-cell systems.     

Internal standards for survival: increasing the accuracy for human cell mutation assays

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation or transformation assays and is a necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.     

Comparison of toxicity and mutagenicity of methylnitrosourea, methylnitronitrosoguanidine and ICR-191 among human lymphoblast lines

The toxic and mutagenic effects of the alkylating agents methylnitrosourea (MNU) and methylnitronitrosoguanidine (MNNG) and of the frameshift mutagen, ICR-191 were compared among 3 human diploid lymphoblast lines, MIT-2, WI-L2 and GM 130. The MIT-2 and WI-L2 lines were both sensitive to the toxic and mutagenic effects of all 3 agents tested. The WI-L2 line was more sensitive to the toxic effects of MNU and MNNG than the MIT-2 line, while it was somewhat less sensitive to the mutagenic effects of these alkylating agents. The GM 130 line was strikingly resistant to both the toxic and mutagenic effects of the alkylating agents. The order of sensitivity to the toxic effect of ICR-191 was MIT-2 > WI-L2 > GM 130, while the order of sensitivity to the mutagenic effects of this frameshift mutagen was GM 130 > MIT-2 > WI-L2. These results point to the importance of accounting possible variations in mutability among individuals when extrapolating from any single mutagenicity assay for human risk assessment.     

A human cell line sensitive to mutation by particle-borne chemicals

A human lymphoblastoid cell line with ability to perform oxidative metabolism of various chemicals is mutated by the direct addition of an intact particulate soot. This experiment demonstrates that materials associated with combustion-generated particulates are biologically available and able to cause genetic changes in metabolically competent human cells.     

Long-term, low-dose benzo[a]pyrene-induced mutation in human lymphoblasts competent in xenobiotic metabolism

We have studied the mutagenic effects of benzo[a]pyrene (BP) administered in a long-term, low-dose fashion to metabolically competent human lymphoblastoid cells. A continuous dose as low as 0.02 microM for 20 days produced a significant increase in mutant fraction at the 6TG-resistance (HGPRT) locus. The slope of the mutant fraction over time in the 0.02 microM BP-treated culture was twice that observed in the untreated concurrent control; 0.02 microM therefore represents the doubling dose of BP for gene mutation in this cell line. For higher doses of 0.1, 0.5 or 1 microM BP, the rate (or efficiency) of induced mutation was considerably higher for the first 5 or 6 days of exposure than for the last 14-15. This did not appear to be due to a growth disadvantage against early-arising mutants. Comparison to previously published data in the same cell system (Crespi and Thilly, 1984) revealed that the long-term , low-dose protocol (0-1 microM for up to 20 days) was significantly more efficient at inducing mutations than a short-term, high-dose protocol (0-10 microM for 1 day).     

Cytotoxicity and mutagencity of hyperthermia for diploid human lymphoblasts

Time dependent survival of diploid human lymphoblasts has been measured at 42, 43, 44, 45, 46 and 47°C. D_O was found to be 1500, 23, 4, 2, 1 and 0.6 min, respectively. Mutation to 6-thioguanine resistance was measured at 45°C and found to increase monotonically with time. Thus, relatively mild heat trauma can result in genetic change in somatic human cells.