Alterations of K-ras and p53 mutations in colorectal cancer patients in Central Europe.

Many molecular investigations of colorectal cancer (CRC) have suggested that the accumulation of specific mutations in proto-oncogenes and tumor suppressor genes regulating cell growth via signal transduction trigger the stagewise progression to malignancy. In this study, the frequency, location, and type of mutations of the K-ras proto-oncogene exon I and p53 tumor suppressor gene exons 5-8 were analyzed in colorectal carcinomas of 65 patients from Central Europe, using polymerase chain reaction (PCR)-cold single-strand conformation polymorphism (SSCP) screening and direct sequencing. The incidence of K-ras activating mutations in these Central European samples was lower (25%) compared to that obtained in American and western European populations (40-50% at least), while the incidence of p53 inactivating mutations was similar (58%). These results suggest that some other genetically linked mechanisms may play a role in CRC development and progression, and hence K-ras and p53 mutations cannot be considered to be universal genetic markers for CRC.

Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution.

Combinatorial library selections through the systematic evolution of ligands by exponential enrichment (SELEX) technique identify so-called nucleic acid aptamers that bind with high-affinity and specificity to a wide range of selected molecules. However, the modest chemical functionality of nucleic acids poses some limits on their versatility as binders and catalysts, and, furthermore, the sensitivity of pure RNA- and DNA-based aptamers to nucleases restricts their use as therapeutic and diagnostic agents. Here we review synthetic chemistries for modifying nucleotides that have been developed to enhance the affinity of aptamers for targets and to increase their stability in biological fluids. Implementation of in vitro selections with modified nucleotides promises to be an elegant technique for the creation of ligands with novel physical and chemical properties and is anticipated to have a significant impact on biotechnology, diagnostics and drug development. The current molecular designs and applications of modified nucleotides for in vitro selections are reviewed, along with a discussion of future developments expected to further the utility of this approach in both practical and theoretical terms.

p53 mutations in hairy cell leukemia.

We have studied the frequency of p53 mutations in genomic DNA extracted from peripheral blood or the spleen of 61 patients with hairy cell leukemia using PCR-SSCP and automated cycle sequencing. We identified exon 5-8 mutations in 17 cases, corresponding to a frequency of 28%. In four cases, mutations were localized in exon 5; one patient with atypical HCL had a mutation in exon 6 at the 3' boundary; five cases showed mutations in exon 7, while exon 8 was found to be mutated in seven cases. The mutations found could be divided into three major categories: structural (n=9), inactivating (n= 6), and neutral (n= 2) mutations. None of the three transitions found occurred at CpG dinucleotides. The rate of p53 mutations found in this large cohort of HCL patients is unexpectedly high as in other non-Hodgkin lymphomas p53 mutations predict for poor treatment outcome. The character of the mutations we have found is entirely different from that described in other hematologic malignancies.

A 3 milliTesla 60 Hz magnetic field is neither mutagenic nor co-mutagenic in the presence of menadione and MNU in a transgenic rat cell line.

The mechanisms by which an electromagnetic field (EMF) influences biological material are poorly understood. One potentially important model suggests that a magnetic field can stabilize free radicals in such a way as to permit their dispersement rather than their return to the ground state (Okazaki et al., 1988; Scaiano, 1995). We have tested this hypothesis by examining mutagenesis in the E. coli lacI gene target carried in the Big Blue rat embryo fibroblast cell line, R2 lambda LIZ. Mutant frequencies were determined in cells exposed to a magnetic field, cells pretreated with the mutagens N-methylnitrosourea (MNU) or 2-methyl-1,4-naphthoquinone (menadione), prior to being held in a 60 Hz 3 milliTesla (mT) magnetic field and cells concurrently exposed to the mutagens and the magnetic field. Menadione was selected because its mutagenic mechanism involves the formation of free radicals, while MNU is an alkylating agent not thought to act through radical formation. According to the radical stabilization hypothesis the application of a magnetic field to menadione treated cells would accentuate the mutagenic effects. Our results failed to indicate that the magnetic field affects mutagenesis by the oxygen-radical mediated mutagen, menadione.

Increased hprt mutant frequencies in Brazilian children accidentally exposed to ionizing radiation.

We have examined the effects of ionizing radiation on somatic mutations in vivo, using the hprt clonal assay. The study was performed on blood samples obtained from children exposed during a radiological accident that happened in 1987, in Goiânia, Brazil. The group of children exposed to ionizing radiation includes six males and four females ranging in age from 6 to 14 years at the time of exposure. The radiation doses ranged from 15 to 70 cGy. A Brazilian control group, not exposed to ionizing radiation, was also analyzed under similar conditions. the mean hprt mutant frequency for the exposed group was 4.6 times higher than the control group, although the cloning efficiency from the exposed group was significantly reduced. Linear regression analysis of the mutant frequency and ionizing radiation dose did not show a significant relationship between these two parameters. However, a reliable inverse relationship was demonstrated when the regression analysis was performed with nonselective cloning efficiency and ionizing radiation dose. It was demonstrated that nonselective cloning efficiency diminishes as ionizing radiation dose increases. To correct mutant frequencies for clonal events, the clonal relationship between the hprt mutant clones was examined by T-cell receptor analysis. The majority of the mutants analyzed represented individual clones, thus validating the observed mutant frequencies.

Mutational specificity and cancer chemoprevention.

Mutational specificity describes the composite of all of the genetic alterations in a collection of mutations arising from a specific treatment. The information includes not only the nature of the genetic change (e.g., a base substitution or a frameshift), but also information about nucleotide position and hence the DNA context. As both the type of DNA damage and its position can be expected to reflect the nature of the chemical and physical mutagen, mutational specificity can be expected to provide insights into mechanisms of mutation. Conversely, mutational spectra should also provide insights into the identity of the mutagen. Indeed, the pioneering work on mutational specificity in Escherichia coli indicates that each physical or chemical treatment produces a unique spectrum of mutations. With the application of biotechnology to the field of genotoxicology, the database of sequenced mutations has become quite substantial. Both in vitro and in vivo data has been obtained following exposure to a variety of agents. In this communication we will critically assess whether the reality of mutational specificity has fulfilled the expectations and to examine what potential remains to be explored, especially in the area of monitoring human populations. The usefulness of both mutational spectra analysis and population monitoring with regards to chemoprevention are discussed.

Detection of p53 mutations in benign and dysplastic nevi.

We have examined melanocytic cells derived directly from fresh biopsy tissue for the presence of p53 mutations. Using selective media that permits growth of melanocytes and inhibits growth of fibroblasts and keratinocytes, we established short-term, primary cultures of melanocytes from skin biopsies of common acquired nevi, dysplastic nevi, and from metastatic melanoma. Using PCR-single-stranded conformational polymorphism analysis, we have detected p53 mutations in 2 of 11 benign compound nevi and 2 of 5 dysplastic nevi. All nevi positive for p53 mutations were derived from patients who previously had cutaneous moles and three of the four had a family and/or personal history of melanoma.

Beta-lactam-induced bacteriolysis of amino acid-deprived Escherichia coli is dependent on phospholipid synthesis.

The penicillin tolerance of amino acid-deprived relA+ Escherichia coli is attributed to the stringent response; i.e., relaxation of the stringent response suppresses penicillin tolerance. The beta-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic sn-glycerol 3-phosphate dehydrogenase). Therefore, beta-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. The lysis process during amino acid deprivation can be experimentally dissociated into two stages designated the priming stage (during which the interaction between the beta-lactam and the penicillin-binding proteins occurs) and the beta-lactam-independent lysis induction stage. Both stages were shown to require phospholipid synthesis. It has been known for some time that the inhibition of phospholipid synthesis is among the plethora of physiological changes resulting from the stringent response. These results indicate that the inhibition of peptidoglycan synthesis and the penicillin tolerance associated with the stringent response are both secondary consequences of the inhibition of phospholipid synthesis.

p53 mutations in human bladder cancer.

Mutations in the tumor suppressor gene p53 play an important role in carcinogenesis and tumor progression. To assess the status of p53 from genomic DNA from bladder cancer samples a two stage polymerase chain reaction was employed. The technique provided material for subsequent detection of mutations by Single Strand Conformation Polymorphism (SSCP) analysis followed by DNA sequence analysis. SSCP analysis of exons 5 to 9 of p53 was performed using fragments from PCR end-labeled with 32P followed by autoradiography using an electrophoresis system with temperature control. This SSCP method improved resolution of mutations in exons 5, 7, and 8 and the sharpness of bands in exons 6 and 9. Bands with altered migration patterns were excised from the dried SSCP gels, reamplified by PCR, and sequenced. Mutations in conserved exons 5, 6, 7, 8, and 9 of the p53 gene were analyzed from bladder tumor biopsies. Our results are consistent with the literature in that mutations in p53 are predominantly found in high grade bladder cancer (Odds Ratio = 4.05, Fisher Exact P = 0.104); however, the results were not statistically significant due to small numbers. Eight of 35 (23%) tumor samples examined showed mutations in p53 (including two double mutations). Six of 13 (46%) grade III and IV tumors had p53 mutations vs. 2 of 17 (12%) grade I and II tumors. Normal individuals carried no p53 mutations. We found no correlation between pack years of smoking and mutation in p53. The spectrum of mutations confirmed a high proportion of G:C C:G transversions as well as the occurrence of double mutations.

Sensitive two-stage PCR of p53 genomic DNA exons 5-9.

To assess the status of the tumor suppressor gene p53 from samples with low levels (sub-nanogram amounts) of genomic DNA (e.g., from exfoliated cells, skin, small biopsies, mucosa), a technique based on two successive rounds of PCR was developed. In the first round, a 1.84-kb fragment spanning exons 5-9 was generated using a "touchdown" protocol. After purification by spun-column chromatography, this fragment was used as a template for the amplification of the individual exons 5-9 with inner primer sets specific for the adjacent intronic regions. Using this nested primer approach, several micrograms of each individual exon were obtained starting from as little as 62.5 pg of total genomic DNA. This material proved ideal for future analyses by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing.

Decay of the ampicillin-induced lysis process in amino acid-deprived Escherichia coli.

The ability to induce the ampicillin-mediated lysis of amino acid-deprived Escherichia coli by relaxing the stringent response decreased progressively during the course of amino acid deprivation, apparently because of a time-dependent decay in a key lysis event. The decay of this labile activity was not apparent when ampicillin treatment was initiated early and maintained continuously throughout the amino acid starvation period.

Effects of aminoglycosides and spectinomycin on the synthesis and release of lipopolysaccharide by Escherichia coli.

The effects of aminoglycosides and spectinomycin on lipopolysaccharide (LPS) synthesis and release by Escherichia coli were studied. LPS synthesis was previously reported to be regulated by the stringent control mechanism. In agreement with this, the control of LPS synthesis in amino acid-deprived relA+ cells was relaxed by spectinomycin, a proven stringent control antagonist, but not by kanamycin, an agent which is ineffective as a stringent control antagonist. The other stringent control antagonists tested (gentamicin, tobramycin, and, to a lesser extent, amikacin) unexpectedly failed to relax the control of LPS synthesis, and this was subsequently shown to be due to their inhibitory action on LPS synthesis. The release of LPS by nongrowing (amino acid-deprived and antibiotic-treated) bacteria was stimulated only under conditions in which the control of LPS synthesis was relaxed.

Suppression of mutations conferring penicillin tolerance by interference with the stringent control mechanism of Escherichia coli.

Mutations in Escherichia coli previously reported (R. E. Harkness and E. E. Ishiguro, J. Bacteriol. 155:15-21, 1983; L. C. Shimmin, D. Vanderwel, R. E. Harkness, B. R. Currie, A. Galloway, and E. E. Ishiguro, J. Gen. Microbiol. 130:1315-1323, 1984) as conferring a temperature-dependent tolerance to lysis induced by inhibitors of peptidoglycan synthesis were suppressed by treatment with inhibitors of the stringent response or by introduction of a relA mutation. The relA+ derivatives of the mutants exhibited a stringent response at the nonpermissive temperature. The consequent inhibition of the autolytic enzyme system (W. Kusser and E. E. Ishiguro, J. Bacteriol. 164:861-865, 1985) was apparently responsible for the lysis-tolerant phenotypes of these mutants.

Temperature sensitivity of the penicillin-induced autolysis mechanism in nongrowing cultures of Escherichia coli.

The effect of incubation temperature on the ampicillin-induced autolysis of nongrowing Escherichia coli was determined. The autolysis mechanisms in amino acid-deprived relA mutant cells treated with chloramphenicol were temperature sensitive. This temperature-sensitive autolysis was demonstrated in three independent ways: turbidimetric determinations, viable cell counts, and solubilization of radiolabeled peptidoglycan.

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis.

Lysis of nongrowing Escherichia coli by combinations of beta-lactam antibiotics and inhibitors of ribosome function.

It is generally assumed that only actively growing bacteria are killed by inhibitors of peptidoglycan synthesis. Several exceptional examples are described here. As expected, ampicillin did not lyse nongrowing, amino acid-deprived cultures of relA+ strains of Escherichia coli, but the subsequent addition of several ribosome inhibitors (chloramphenicol, tetracycline, gentamicin, and kanamycin) caused various degrees of lysis in such ampicillin-treated cultures. Of the antibiotics tested, only streptomycin was ineffective in this regard. Peptidoglycan synthesis has been shown to be inhibited in amino acid-deprived relA+ bacteria by the stringent control mechanism (E. E. Ishiguro and W. D. Ramey, J. Bacteriol. 127:1119-1126, 1976), and the ribosome inhibitors tested here relaxed peptidoglycan synthesis to various degrees under these conditions. The relative lysis-inducing activities of the ribosome inhibitors on ampicillin-treated, amino acid-deprived bacteria were directly correlated to their relative activities as stringent control antagonists. This phenomenon was not dependent on amino acid deprivation. Cultures treated with growth inhibitory levels of the various ribosome inhibitors alone were lysed by ampicillin, apparently because peptidoglycan synthesis continues uninhibited when growth is arrested by treatment with ribosome inhibitors. These results indicate that autolysis can be triggered by the inhibition of peptidoglycan synthesis occurring in the absence of wall expansion; i.e., active cell growth is unnecessary.

Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis.

It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria. An exceptional case is reported here. The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. This phenomenon is termed chloramphenicol-dependent lysis. To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher). Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective. Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis. Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control. Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria. This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.

Characteristics of the binding of aminoglycoside antibiotics to teichoic acids. A potential model system for interaction of aminoglycosides with polyanions.

The binding of the aminoglycoside antibiotic dihydrostreptomycin to defined cell-wall teichoic acids and to lipoteichoic acid isolated from various gram-positive eubacteria was followed by equilibrium dialysis. Dihydrostreptomycin was used at a wide range of concentration under different conditions of ionic strength, concentration of teichoic acid, presence of cationic molecules like Mg2+, spermidine, other aminoglycoside antibiotics (gentamicin, neomycin, paromomycin). Interaction of dihydrostreptomycin with teichoic acid was found to be a cooperative binding process. The binding characteristics seem to be dependent on structural features of teichoic acid and are influenced by cationic molecules. Mg2+, spermidine and other aminoglycosides antibiotics inhibit the binding of dihydrostreptomycin to teichoic acid competitively. The binding of aminoglycosides to teichoic acids is considered as a model system for the interaction of aminoglycoside antibiotics with cellular polyanions. Conclusions of physiological significance are drawn.

A novel glycerophosphodiesterase from Bacillus pumilus.

A novel glycerophosphodiesterase activity was detected in extracts from phosphate-starved Bacillus pumilus DSM27 cells. The enzyme had a substrate specificity for glycerophosphodiester bonds and the reaction product formed with partially purified enzyme was (sn)-glycero-3-phosphate. Purified cell wall teichoic acid of the polyglycerophosphate type, as well as deacylated, unsubstituted lipoteichoic acid of the polyglycerophosphate type, di(glycerophospho)glycerol (deacylated cardiolipin) and mono(glycerophospho)glycerol (deacylated phosphatidylglycerol) served as substrates for the enzyme. Their native counterparts, however, cell wall-bound polyglycerophosphate, lipoteichoic acid (D-alanine substituted and dealanylated), cardiolipin and phosphatidylglycerol were poor or no substrates, respectively. Enzyme activity was inhibited by purified cell walls and by heparin. The enzyme was partially purified using a column of Heparin-Sepharose.