The role of angular reflection in assessing elastic properties of bone by scanning acoustic microscopy.

For an assessment of the mechanical performance of bone, a quantitative description of its mechanical heterogeneity is necessary. Previously, scanning acoustic microscopy (SAM) was used as a non-destructive method to estimate bone stiffness on the micrometer scale. While up to now only the normal incidence of acoustic waves is taken into account, we extend in our study the evaluation procedure by considering the full opening of the acoustic lens. The importance of this technical aspect is demonstrated by determining the contrast in Young's modulus between newly formed osteons and the surrounding higher mineralized interstitial bone. Several regions of human cortical bone of a femur in cross-section were imaged. For all the regions quantitative backscattered-electron imaging (qBEI) to estimate the local mass density was combined with SAM measurements. These measurements reveal a non-monotonic dependence between acoustic reflectivity and Young's modulus, which shows that it is actually necessary to consider the lens opening in a quantitative way. This problem was experimentally and theoretically approached by using lenses with two different opening angles operated at different frequencies (52° at 400MHz and 80° at 820MHz) to image the same specimen. The mass density of bone in osteons was found to be 1930kg/m(3) on average, while the higher mineral content in interstitial bone results in a 9% increase of the density. The contrast in the effective Young's modulus E, as determined through SAM, is more pronounced, with an average value of 14GPa in osteons and a more than 60% increase in interstitial bone. Additionally, SAM maps show oscillations in E with a periodicity of the typical bone lamella thickness of approximately 7µm in both osteons and interstitial bone. This mechanical heterogeneity can be explained by the varying orientation of the mineralized collagen fibers.

In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E. coli: a feasible part of UV-mutagenesis.

We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or PyPy) in UV-irradiated E. coli deficient in uracil DNA glycosylase. The protocol consisted of UV-irradiation, holding in buffer to allow for deamination of cytosines in CPDs and photoreversal (PR) to establish uracils where cytosines in CPD deaminated. The deamination rate at TC photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MF(PR)) that developed as UV-irradiated cells were held before PR. Evidence suggested that an earlier study with this protocol under-estimated the deamination rate at sites producing the same mutations in an E. coli B/r strain. With a K12 strain, where the targeting apparently is principally by CPD and not (6-4) photoproducts, a larger rate of k = 0.0091 min(-1) at 42 degrees C resulted. The dark assay for MF also increased significantly with time for deamination consistent with a model for efficient mutation by translesion synthesis at uracil-containing CPD. In addition, we used a strain constructed by Cupples and Miller in which beta-galactosidase was inactive because -GGG- was at codon 461 and would revert to Lac(+) only when replaced by -GAG- or -GAA- for glutamate. CC photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD. MF(PR) for Lac(+) mutations increased and then decreased as a function of deamination time (at six temperatures 36-48 degrees C). Fitting an approximate model equation that distinguished two different deamination rates to these data suggested a first deamination producing Lac(+) at a rate about eight-fold less than a second deamination restoring the Lac(-) phenotype. We conclude that deamination, changing a cytosine-containing CPD to a uracil-containing CPD, could be an integral part of UV-induced C-to-T mutations.

Overexpression of Ogt reduces MNU and ENU induced transition, but not transversion, mutations in E. coli.

Studies of alkylation-induced mutations in Escherichia coli FX-11 revealed that both N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) produced tRNA suppressor mutations (G:C to A:T) but only ENU produced a significant number of backmutations (A:T to G:C, A:T to T:A and A:T to C:G). Further, the ENU-induced transversions were absent in a UmuC-defective strain. This suggested that transition mutations could result from alkylation of guanine or thymine at the O(6)- and O(4)-positions, respectively, but that transversions might result from alkylation of thymine at the O(2)-position. To test this idea, the gene encoding O(6)-alkylguanine-DNA methyltransferase (ogt) was recombined into a plasmid to overexpress the cellular levels of this enzyme. Ogt protein can de-alkylate O(6)-alkylguanine and O(4)-alkylthymine, but not O(2)-alkylthymine. Cells harboring the plasmid (or a control plasmid lacking the ogt gene) were exposed to different concentrations of MNU or ENU and the resulting mutations were analyzed. With either MNU or ENU, the frequency of GlnV(o) suppressors was reduced about 70-fold in the Ogt-overexpressing cells, suggesting that Ogt eliminated O(6)-alkylguanine. Similarly, GlnU(o) suppressor frequencies were substantially reduced. In contrast, the reduction in frequency for the backmutations was slight, only about 2.5-fold with MNU and less than two-fold for ENU. However, DNA sequence analysis of the backmutations showed that only A:T to G:C transitions were affected by overexpression of Ogt, suggesting repair of O(4)-alkylthymine. The frequency of transversions, in comparison, was essentially unaltered. These results implicate O(2)-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.

Reduction of ENU-induced transversion mutations by the isoflavone genistein in Escherichia coli.

In studies of mutagenesis induced by the carcinogen N-ethyl-N-nitrosourea (ENU) in the bacterium Escherichia coli FX-11, it was observed that G:C to A:T transitions did not require the inducible umuDC gene products, while a portion of the A:T to G:C transitions and all transversion mutations were dependent on a functional umuC gene. This observation suggested that the different base substitutions may result from differential processing of specific DNA adducts produced by ENU. To further understand these processes, we have investigated the effect of the soybean isoflavone genistein on the production of ENU-induced mutations. This compound, in particular, has been shown to exhibit numerous effects including the inhibition of the growth or proliferation of a variety of cancers, inhibition of angiogenesis, inhibition of tyrosine protein kinases and anti-oxidant properties. In our experiments, tyrosine defective (TyrA(-)) E. coli were exposed to ENU and a portion of the ENU-treated cells were exposed to genistein. The results showed a three-fold reduction in the overall mutation frequency when cells were treated with genistein subsequent to ENU-exposure and this anti-mutagenic effect was dependent on the dose of genistein employed. However, only certain types of base substitution mutagenesis were affected. In particular, transversion mutations were reduced an average of about 8.5-fold, while transitions were not greatly affected. In addition, UV-mutagenesis was reduced about three-fold and induction of the SOS response (as monitored with a sulA-lacZ fusion) was decreased. These results suggest that genistein may interfere with expression of the SOS response, including the UmuC-mediated lesion bypass mechanism that is necessary for UV-mutagenesis and the generation of transversions by ENU in E. coli.

Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11.

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.

Reduction of toxicity of a vinca alkaloid by an anti-vinca alkaloid antibody.

Inadvertent oncolytic overdoses occur rarely, but can have serious consequences. We have investigated the possibility of using an antibody, 27.8.1A, reactive with vinca alkaloids, as a means of reducing the toxicity associated with overdose situations. In vitro cytotoxicity of a vinca derivative, 4-desacetyl- vinblastine-3-carbox-hydrazide (DAVLBHYD), with and without the addition of 27.8.1A, was determined. Using CCRF-CEM, a human acute lymphoblastic leukemia cell line, as a target in this assay, we observed a greater than 90% increase in cell viability using 100 micrograms/ml 27.8.1A with a 0.1 microgram/ml concentration of DAVLBHYD. 27.8.1A had no effect on cell viability when doxorubicin was used as a control drug in this assay. Similarly, the addition of an irrelevant antibody. EGFrL11, had no effect on the toxicity of DAVLBHYD. In an in vivo survival experiment, nude mice were injected with a toxic dose of DAVLBHYD and subsequently given four doses of 27.8.1A. All anti vinca antibody-treated mice survived, in contrast to the untreated group or irrelevant antibody-treated group in which only 25% and 10% of the mice survived, respectively.

The rex genes of lambda prophage modify ultraviolet light and N-methyl-N-nitrosourea-induced responses to DNA damage in Escherichia coli.

Inactivation and mutagenesis of Escherichia coli by ultraviolet light and N-methyl-N-nitrosourea (MNU) were examined in isogenic strains that were (i) free of lambda phage, (ii) lysogenic for lambda or (iii) contained the lambda rexAB operon on a plasmid (prex20). Inactivation was enhanced when rex genes were present and this enhancement was greater for MNU than for UV. Mutagenesis was not greatly affected by the presence of the rex genes with one exception; UV-induced glnU degrees suppressor mutations were reduced in the strain harboring prex20. Control experiments using a strain containing only plasmid pACYC184 showed no effect on inactivation. A small depression in UV-induced mutagenesis was observed, however. These data suggest that the lambda RexAB proteins interfere with processes necessary for recovery from DNA damage and for fixation of one example of UV-induced mutation. This and other recent observations of effects by rex genes are discussed.

N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli: multiple roles for UmuC protein.

Backmutations of an ochre (UAA) nonsense defect in the tyrA gene of Escherichia coli were induced by N-ethyl-N-nitrosourea (ENU) in both UmuC+ and UmuC- strains. This site is particularly flexible to base substitution mutation and all but one (TAA-->TGA) substitution can be recovered using a reversion assay. Employing direct sequencing of polymerase chain reaction-amplified genomic DNA and/or colony-hybridization methods, the changes induced by several separate doses of ENU in a total of 587 independent backmutations were investigated. In the UmuC+ strain, all possible single-base substitutions were recovered. Different frequencies for individual single-base substitutions were obtained and correlations with the surrounding base sequence could be seen. Transitions occurred most frequently at thymine residues having a purine on the 5'-side while transversions occurred more frequently at thymine residues having a cytosine on the 5'-side. In the UmuC- strain, ENU failed to induce A:T-->T:A and A:T-->C:G transversions and the frequency of A:T-->G:C transitions was reduced. However, an unidentified class of extragenic mutations were induced to a greater extent. These results suggest distinct pathways for ENU-induced mutagenesis at ethylated thymine residues and delineate several separate functions for the UmuC protein.

Expression of beta-galactosidase from a hybrid promoter:operator element in Escherichia coli.

A hybrid trpPO:lacO regulatory sequence was cloned upstream of a promoterless lacZ gene and recombined onto a lambda bacteriophage. Escherichia coli lysogens representing the four possible phenotypes for lacI and trpR were constructed and the synthesis of beta-galactosidase was assayed under various growth conditions. The results illustrated that both control elements could be efficiently and independently regulated by the addition or omission of appropriate accessory molecules.

Induction of immunogenicity of monoclonal antibodies by conjugation with drugs.

Human anti-mouse antibody has been a nearly consistent result of human clinical trials utilizing murine antibodies. It is generally anticipated that the problem of human anti-mouse antibody will be reduced as genetically engineered, more human ("humanized") antibodies become available. It is not clear, however, what effect chemical modification of such "humanized" antibodies will have on their immunogenicity. The present studies utilize a mouse antibody and rat host model to explore aspects of this question. Rats injected with unmodified mouse monoclonal antibodies failed to mount anti-mouse immune responses, presumably due to their phylogenetic relatedness. In contrast, rats injected with a Vinca immunoconjugate mounted strong anticonjugate antibody responses that were directed primarily against the linker portion of the conjugate. The in vivo serum pharmacokinetics of 125I-labeled antibody and conjugates were evaluated in rats with existing anticonjugate antibody. The peak serum level attained was inversely correlated with the level of reactivity of the anticonjugate antibody with the injected compound. This model provides a potentially useful tool for exploration of the immunogenicity of drug, toxin, or radionuclide monoclonal antibody conjugates.

Uracil-DNA glycosylase activity affects the mutagenicity of ethyl methanesulfonate: evidence for an alternative pathway of alkylation mutagenesis.

Mutagenesis induced by the alkylating agent ethyl methanesulfonate (EMS) is thought to occur primarily via mechanisms that involve direct mispairing at alkylated guanines, in particular, O6-ethyl guanine. Recent evidence indicates that alkylation of guanine at the O-6 position might enhance the deamination of cytosine residues in the complementary strand. To determine whether such deamination of cytosine could play a role in the production of mutations by EMS, the efficacy of this agent was tested in uracil-DNA glycosylase deficient (Ung) strains of Escherichia coli. The Ung- strains showed a linear response with increasing doses of EMS. This response was independent of the umuC gene product. In contrast, the Ung+ strains yielded a dose-squared response that became linear at higher doses of EMS when the cells were defective for the umuC gene product. These results support a model for mutagenesis involving the deamination of cytosines opposite O6-alkylated guanines followed by an error-prone repair event.

Antitumor activity of L/1C2-4-desacetylvinblastine-3-carboxhydrazide immunoconjugate in xenografts.

The murine IgG3 monoclonal antibody L/1C2 is reactive with a high percentage of human carcinomas and has preferentially strong reactivity with tumors of squamous differentiation. This antibody was tested for antitumor activity in vitro and in xenograft models as a carbohydrate-linked immunoconjugate with the Vinca derivative 4-desacetylvinblastine-3-carboxhydrazide (DAVLBHYD). The conjugate retained good immunoreactivity and was highly active in a cytotoxicity assay. In human tumor nude mouse xenograft studies, L/1C2-DAVLBHYD antitumor activity was superior to that seen with free drug, free antibody, mixtures of free drug and free antibody, or control DAVLBHYD conjugates prepared with non-tumor-binding IgGs. With well-established tumors, potent antitumor activity was observed, including the ability to specifically regress greater than 400-mg tumors to 0 mg. In some cases, apparent long-term cures were effected. In studies using six different human tumor xenografts, the level of potency of L/1C2-DAVLBHYD was related to L/1C2 antigen expression, although the growth rate probably also contributes to the conjugate sensitivity of the tumors.

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein.

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Asymmetric cytosine deamination revealed by spontaneous mutational specificity in an Ung- strain of Escherichia coli.

A collection of 164 spontaneous lacI- mutations were recovered from a uracil-DNA glycosylase deficient (Ung-) strain of Escherichia coli and analyzed by DNA sequencing. As predicted by genetic studies, G:C----A:T transitions predominated among base substitution events. However, DNA sequence analysis indicated that these events did not occur at random. Of the 31 G:C----A:T transitions recovered, 24 involved cytosine residues located in the nontranscribed strand of the gene and 15 of the 31 transitions occurred at cytosines located on the 3' side of 3 or more A:T base pairs. These differentials likely reflect the more single-stranded character of the non-transcribed strand of the gene and of regions rich in A:T base pairs. In addition, mutation at the frameshift hotspot was altered in the Ung- strain, suggesting a role for DNA repair in the formation of structural intermediates that potentiate these events. Also, the analysis of non-hotspot frameshifts, deletions and duplications showed that many involved local DNA sequence. Specifically, several of the frameshift, deletion and duplication mutations occurred near the sequence 5'-CTGG-3'. Thus, DNA sequence analysis of mutational specificity in an Ung- strain has provided evidence that gene expression, DNA repair and DNA context can all potentially influence the classes and frequencies of spontaneous mutation.

Thermal resistance to photoreactivation of ultraviolet light induced mutations in the lacI gene of E. coli ung.

Ultraviolet light (UV) induced mutations in the lacI gene of Escherichia coli are thought to be targeted by DNA photoproducts. A number of reports suggest that both cyclobutyl pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts may be involved. To investigate the potential contribution of each of these DNA photoproducts to mutagenesis in the lacI gene, we held UV-irradiated cells at a temperature of 44 degrees C for 75 min and then exposed them to photoreactivating light (PR). This protocol is expected to preferentially deaminate specifically those cytosines that are contained in cyclobutyl dimers and subsequently monomerize the dimers to yield uracils in the DNA. In a strain deficient for uracil-DNA glycosylase (Ung-), these uracils would not be removed and a G:C----A:T transition would result at the site of the dimer. This protocol resulted in the enhancement of amber nonsense mutations that result from transitions at potential cytosine-containing dimer sites. The enhanced mutation frequencies resulting from this procedure were used to estimate the probability of dimer formation at the individual sites. A comparison of the dimer distribution with the mutation frequencies following UV alone suggests that both cyclobutyl dimers and (6-4) photoproducts contribute to UV-mutagenesis in the lacI gene. In addition, we conclude that the frequency of mutation at any particular site not only reflects the occurrence of DNA damage, but also the action of metabolic processes that are responsible for DNA repair and mutagenesis.

DNA sequence analysis of spontaneous mutation in a PolA1 strain of Escherichia coli indicates sequence-specific effects.

The sequences of a collection of 261 spontaneous lacI- mutants recovered in a PolA- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5'-GTGG-3' sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5'-GTGG-3' sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5'-GTGG-3' sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.

Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung- strain of Escherichia coli.

In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E. coli was examined. In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34. In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites. This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase. Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced. This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition. Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence. It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.

Thermal resistance of UV-mutagenesis to photoreactivation in E. coli B/r uvrA ung: estimate of activation energy and further analysis.

Ultraviolet light (UV) induced mutations in the glnU and glnVa tRNA genes in Escherichia coli are thought to be targeted by UV photoproducts. In a previous study with a uracil-DNA glycosylase deficient strain, UV-induced glnU0 and glnV0 tRNA suppressor mutations became resistant to photoreactivation (PR) following thermal treatment. It was proposed that deamination of cytosine in the cytosine-containing cyclobutyl dimers at the sites of these suppressor mutations produced uracil residues in sequence upon PR. In the absence of glycosylase, the C----U conversion yielded the requisite G:C----A:T transitions. In the present study, this thermal resistance of UV-mutagenesis to PR is characterized. It is dependent on the initial UV-fluence and temperature of holding but not on the UmuC+ gene product. The data obtained yield an estimate of an activation energy of 17 +/- 3 kcal/mol for the deamination of cytosines contained in dimers. This compares to 29 kcal/mol for unaffected cytosines in DNA. In addition, an estimate of the probability of cyclobutyl dimer formation at the target sites for glnU0 and glnV0 suppressor mutations indicate that these lesions can not entirely account for the mutation frequencies recovered in the absence of PR. This is interpreted as an indication that, in addition to thymine-cytosine cyclobutyl dimers, other UV-induced lesions, possibly Thy(6-4)Cyt photoproducts, may also target glnU0 and glnV0 suppressor mutations.