Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis.

Nanomaterials are frontier technological products used in different manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNT) are finding numerous applications in electronics, aerospace devices, computers, and chemical, polymer, and pharmaceutical industries. SWCNT are relatively recently discovered members of the carbon allotropes that are similar in structure to fullerenes and graphite. Previously, we (47) have reported that pharyngeal aspiration of purified SWCNT by C57BL/6 mice caused dose-dependent granulomatous pneumonia, oxidative stress, acute inflammatory/cytokine responses, fibrosis, and decrease in pulmonary function. To avoid potential artifactual effects due to instillation/agglomeration associated with SWCNT, we conducted inhalation exposures using stable and uniform SWCNT dispersions obtained by a newly developed aerosolization technique (2). The inhalation of nonpurified SWCNT (iron content of 17.7% by weight) at 5 mg/m(3), 5 h/day for 4 days was compared with pharyngeal aspiration of varying doses (5-20 microg per mouse) of the same SWCNT. The chain of pathological events in both exposure routes was realized through synergized interactions of early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition, and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.

Increased mutant frequencies in the HPRT gene locus of leukemia HL-60 cells treated with succinylacetone.

Succinyl acetone (SA) was initially identified in the urine of patients with tyrosinemia type I, an autosomally recessive inherited disease. SA has been used to downregulate the activity of myeloperoxidase (MPO) through its specific inhibition of heme biosynthesis and to investigate the biological properties of MPO in the human myeloid leukemic (HL-60) cell line. The goal of this study is to evaluate the mutagenic potential of SA by determining the frequencies of somatic mutations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) reporter gene in HL-60 cells following treatment with the chemical. Treatments of HL-60 cells with 500 micromol/L SA for 72 h, a condition generally used to inhibit the MPO activity, resulted in a significantly increased HPRT mutant frequency (HPRT-Mf), compared with the control of untreated cells (47.25 x 10(-6) versus 7.5 x 10(-6), respectively, p <0.01). Treatment of the cells with lower doses of SA also led to an increase in HPRT-Mf but this was significant only with 200 micromol/L (28.67 x 10(-6), p<0.05) and not with doses lower than 100 micromol/L (p0.05), compared with the control of untreated cells (7.5 x 10(-6)). These data show a dose-response increase in HPRT-Mf in HL-60 cells treated with SA, suggesting that this chemical causes mutations in the HPRT locus in these cells either directly or indirectly through its inhibition of the MPO activity.

Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustion emissions.

We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G --> T transversions at either KRAS (86%) or TP53 (76%), (b) clustered at the G-rich codons 153-158 of TP53 (33%), and (c) had 100% of the guanines of the G --> T transversions on the nontranscribed strand. This mutation spectrum is consistent with an exposure to polycyclic aromatic hydrocarbons, which are the primary component of the smoky coal emissions. These results show that mutations in the TP53 and KRAS genes can reflect a specific environmental exposure.

Topographic analysis of K- ras mutations in histologically normal lung tissues and tumours of lung cancer patients.

Mutations in the K- ras gene are very common in lung tumours and are implicated in the development of lung cancer, but the timing of their occurrence remains poorly understood. We investigated K- ras mutations in cell samples microdissected by laser capture microscopy at multiple sites from lung tissue sections representing tumour tissue and matched histologically normal tissue obtained from 48 lung cancer patients. K- ras mutations were detected in cell samples from 10 of 38 (26.3%) lung adenocarcinomas and in none of the histologically normal or tumour cell samples taken from 10 lung squamous cell carcinomas. Of the K- ras mutation-positive adenocarcinomas, in 4 cases a mutation was found in only the tumour tissue, in 1 case a mutation was found only in the histologically normal tissue, and in 5 cases mutations were found in both the tumour tissue and histologically normal tissue. Among these 5 cases, 2 had identical mutations in both the tumour tissue and histologically normal tissue, 2 had 1 mutation in the tumour tissue and 2 mutations in the histologically normal tissue, 1 of which was identical to the mutation found in the tumour, and 1 case had 2 codon 12 mutations in tumour tissue and 2 mutations, in codons 9 and 11, in histologically normal tissue. These results showed that K- ras mutations are frequent in histologically normal cells taken from outside lung adenocarcinomas and suggest that some of these mutations may represent early events which could pave the way of lung carcinogenesis.

Comparison of mutations in the p53 and K-ras genes in lung carcinomas from smoking and nonsmoking women.

Lung cancer incidence is increasing in women with little or no tobacco exposure, and the cause of this trend is not known. One possibility is increased sensitivity to environmental tobacco smoke in women nonsmokers diagnosed with lung cancer. To determine whether mutations associated with tobacco exposure are found in the lung tumors of women who are lifetime nonsmokers or occasional smokers, we compared the p53 and K-ras mutational spectra in lung carcinomas from 23 female nonsmokers, 2 female occasional smokers (< 10 pack-years), and 30 female long-term smokers (20-100 pack-years). We also looked for p53 and K-ras mutations in three carcinoid lung tumors, two from female nonsmokers and one from a female occasional smoker. For the p53 gene, exons 4-8 were examined for mutations; for the K-ras gene, exon 1 was examined. No mutations were found in the carcinoid tumors. In lung carcinomas, p53 mutations were identified in six (26.1%) of the cases from lifetime nonsmokers and consisted of five transitions (including three C to T, one G to A, and one T to C) and one T to A transversion. In comparison, p53 mutations were identified in 10 (31.3%) of the 32 lung carcinomas from short-term and long-term smokers and consisted of six transversions (four G to T, one A to T, and one G to C), one A to G transition, one C to T transition, and two deletions of one to four bp. Mutations in the p53 gene found in nonsmokers also occurred in either different codons or different positions within a codon compared with those seen in long-term smokers. K-ras mutations in codon 12 were identified in two lung carcinomas (8.7%) from lifetime nonsmokers. The K-ras mutations found were a G to T transversion and a G to A transition. Eight (25%) of the 32 lung carcinomas from smokers contained K-ras mutations in codons 12 and 13 (four G to T transversions and four G to A transitions). In addition, six silent mutations that are most likely polymorphisms were found in both smokers and nonsmokers. These results confirm that K-ras mutations are more frequent in smokers than in nonsmokers, but that the same type of mutation in the K-ras gene is found in both groups. In contrast, although the frequency of mutation in the p53 gene was similar in lifetime nonsmokers compared with long-term smokers, the types and spectra of mutation are significantly different. Two of the C to T transitions found in nonsmokers, but none of those found in smokers, occur at the C of a CpG site. These results suggest the mutagen(s) and/or mechanisms of p53 mutations in women nonsmokers are different from those responsible for p53 mutations in women smokers, which are probably largely induced by tobacco mutagens.

Impact of maternal lifestyle factors on newborn HPRT mutant frequencies and molecular spectrum--initial results from the Prenatal Exposures and Preeclampsia Prevention (PEPP) Study.

Epidemiological studies have demonstrated associations between maternal tobacco smoke exposure and consumption of alcohol during pregnancy and increased risk of pediatric malignancies, particularly infant leukemias. Molecular evidence also suggests that somatic mutational events occurring during fetal hematopoiesis in utero can contribute to this process. As part of an ongoing multi-endpoint biomarker study of 2000 mothers and newborns, the HPRT T-lymphocyte cloning assay was used to determine mutant frequencies (Mf) in umbilical cord blood samples from an initial group of 60 neonates born to a sociodemographically diverse cohort of mothers characterized with respect to age, ethnicity, socioeconomic status, and cigarette smoke and alcohol exposure. Non-zero Mf (N = 47) ranged from 0.19 to 5.62 x 10(-6), median 0.70 x 10(-6), mean +/- SD 0.98 +/- 0.95 x 10(-6). No significant difference in Mf was observed between female and male newborns. Multivariable Poisson regression analysis revealed that increased HPRT Mf were significantly associated with maternal consumption of alcohol at the beginning [Relative Rate (RR) = 1.84, 95% CI = 0.99-3.40, P = 0.052) and during pregnancy (RR = 2.99, 95% CI = 1.14-7.84, P = 0.026). No independent effect of self-reported active maternal cigarette smoking, either at the beginning or throughout pregnancy, nor maternal passive exposure to cigarette smoke was observed. Although based on limited initial data, this is the first report of a positive association between maternal alcohol consumption during pregnancy and HPRT Mf in human newborns. In addition, the spectrum of mutations at the HPRT locus was determined in 33 mutant clones derived from 19 newborns of mothers with no self-reported exposure to tobacco smoke and 14 newborns of mothers exposed passively or actively to cigarette smoke. In the unexposed group, alterations leading to specific exon 2-3 deletions, presumably as a result of illegitimate V(D)J recombinase activity, were found in five of the 19 mutants (26.3%); in the passively exposed group, two exon 2-3 deletions were present among the seven mutants (28.6%); and in the actively exposed group, six of the seven mutants (85.7%) were exon 2-3 deletions. Although no overall increase in HPRT Mf was observed and the number of mutant clones examined was small, these initial results point to an increase in V(D)J recombinase-associated HPRT gene exon 2-3 deletions in cord blood T-lymphocytes in newborns of actively smoking mothers relative to unexposed mothers (P = 0.011). Together, these results add to growing molecular evidence that in utero exposures to genotoxicants result in detectable transplacental mutagenic effects in human newborns.

Effects of bulky polycyclic aromatic hydrocarbon adducts on DNA replication by exonuclease-deficient T7 and T4 DNA polymerases.

In vitro DNA replication by exonuclease-deficient T7 DNA polymerase (Sequenase) and an exonuclease deficient T4 DNA polymerase was examined on a 244-nucleotide DNA template treated with three electrophilic polycyclic aromatic hydrocarbon (PAH) metabolites: racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE), trans-2,3-dihydroxy-anti-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene (FADE), or 3,4-epoxy-3,4-dihydrocyclopenta[cd]pyrene (CPPE). The DNA replication terminated opposite template guanines and, to a lesser extent, at template adenines, as expected, as purines were modified preferentially by the chemical treatments. Analysis of the products synthesized on the damaged templates indicated that bypass replication by Sequenase proceeded in three steps: (1) replication first terminated one base 3' to each adduct; (2) a nucleotide was then incorporated opposite the PAH-modified base; and (3) replication continued at some sites to give full bypass of the lesions. The rate of lesion bypass was affected by the type of chemical adduct, the sequence context of the adduct, and the concentration of deoxynucleoside triphosphates. Short DNA repeats appeared to facilitate translesion replication.

Prognostic value of specific KRAS mutations in lung adenocarcinomas.

Adenocarcinomas of the lung remain a significant public health problem. Locally defined (stage I) tumors are considered amenable to resection with curative intent. However, only about 45% of these patients survive for 5 years. The median survival for more advanced tumors is drastically lower. Much research has been focused on identifying a valid genetic biomarker of prognosis. Mutations of the proto-oncogene KRAS have been identified by some groups as being a valid prognostic indicator for adenocarcinoma of the lung. To evaluate the effect of KRAS gene mutation on the survival of patients with lung adenocarcinoma, 181 archival tumors were examined by PCR and denaturing gradient gel electrophoresis. Mutations in either codon 12 or 13 were found in 31.5% of the samples. The most common mutation was a G-->T transversion in codon 12, representing 66.7% of the mutations. No difference was observed in the survival of patients with a KRAS mutation versus those whose tumors contained wild-type KRAS. This lack of difference was also observed when the analysis was restricted to those with stage I tumors or when patients with stage I or II disease were grouped together. However, certain amino acid substitutions, including cysteine, arginine, and aspartate, indicated a significantly poorer prognosis, whereas hydrophobic amino acid substitutions showed a significantly better prognosis than wild-type (P = 0.04). Sample sizes were small for this analysis due to the number of possible mutations. As expected, the stage of tumor at resection was the most significant predictor of outcome. Based on this study of 181 patients from two major medical centers located in different cities, we conclude that KRAS mutation status is not a satisfactory predictor of prognosis in lung adenocarcinoma, but the substitution of a polar or charged amino acid for the wild-type glycine residue may be a negative prognostic indicator.

K-ras gene mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients.

K-ras gene mutations have been reported as early events in colorectal tumorigenesis, but their role in tumor initiation and development is still unclear. To analyze and compare K-ras mutational patterns between colorectal tissues at different stages of tumor progression in individual patients, 65 colorectal tissue samples, including carcinoma, adenoma, histologically normal mucosa, submucosal muscularis propria, and histologically normal mucosa distant from tumor, were obtained from 13 patients with colorectal cancer. In addition, normal mucosal tissues obtained from four normal individuals were analyzed. Each of the 13 tumors was shown previously to harbor a mutation in either codon 12 or 13 of the K-ras gene by direct sequencing. These tissues were reanalyzed, using the recently established mutant allele enrichment + denaturing gradient gel electrophoresis method, which can detect one mutant allele in 10(4)-10(5) normal alleles, thus allowing for the analysis of infrequent cells bearing mutations against the background of wild-type cells. No K-ras codon 12 mutation was detected by this method in the histologically normal mucosal tissues sampled at the margin of resection distant from the tumor or in those obtained from four normal individuals. On the other hand, these mutations were detected in 9 of 10 adenoma and 6 of 10 mucosa samples from 10 patients with known K-ras codon 12 mutations, and also in 2 of 3 carcinoma, 2 of 3 adenoma, and 1 of 3 mucosa samples obtained from 3 patients with known K-ras codon 13 mutations. Thus, K-ras codon 12 mutations were found to occur with a high frequency (53.8%) in histologically normal mucosa adjacent to tumors of patients with K-ras mutation-positive colorectal cancer, suggesting that they may be useful biomarkers for early detection of colorectal cancer. Furthermore, multiple K-ras mutations were found in tissues of nearly half of the 13 patients, indicating that distinct evolutionary subclones may be involved in the development of tumor in some patients with colorectal cancer.

Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues.

A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.

Detection of low-fraction K-ras mutations in primary lung tumors using a sensitive method.

Mutations in the K-ras gene are often identified in lung tumors and are implicated in the development of lung cancer. We used a sensitive method to analyze low-fraction mutations occurring in codon 12 of the K-ras gene in 114 primary lung tumors, including 77 adenocarcinomas, 31 squamous cell carcinomas and 6 adenosquamous carcinomas, which had previously been shown to be negative for codon 12 K-ras mutation in a first screening using less sensitive methods. Sixteen of these tumors were found to contain a low-fraction mutation, including 9 mutations among the adenocarcinomas, six mutations among the squamous cell carcinomas and one mutation among the adenosquamous carcinomas. Our study also showed that the occurrence of low-fraction mutation was associated with a positive smoking history, as was previously found for the occurrence of high-fraction mutation. Patients with low-fraction mutations were younger (mean age 58.8 years) than those with either high-fraction mutations (63.2 years) or no mutation (66 years). Patients with low-fraction mutations were more often stage 1 (8 of 10) than patients with either high fraction mutations (22 of 44) or no mutation (33 of 71). Moreover, the overall survival was better for the group with a low-fraction mutation than both the high-fraction mutation group and the group with no K-ras mutation, but due to small sample size, the difference was not statistically significant. Our results suggest that using highly sensitive methods of K-ras mutant detection in tumor DNA could obscure differences between patients in whom the mutation is found throughout the tumor, those in whom the mutation is only present in a small subpopulation and those who have no mutation.

Sporadic and Thorotrast-induced angiosarcomas of the liver manifest frequent and multiple point mutations in K-ras-2.

Hepatic angiosarcoma (HA) is an uncommon neoplasm associated with known etiologic factors in 25% to 42% of cases. It is, however, one of the most common sarcomas found in the liver. The aim of this study was to find was to find mutations in the K-ras-2 oncogene in sporadic and Thorotrast (TT)-induced HA. Point mutations in K-ras-2 were sought in archival, formalin-fixed tissue blocks from 24 patients with angiosarcoma. Of these, 19 cases were sporadic and 5 were TT-induced. Mutational analysis was performed by topographic microdissection with PCR amplification followed by genotyping. Specific mutations were determined by two independent methods: (a) direct sequencing of the PCR product confirmed by rePCR and by using a different sequencing primer, and (b) PCR-based selective enrichment of mutant DNA by endonuclease digestion followed by heteroduplex DNA analysis using denaturing gradient gel electrophoresis. Eleven K-ras-2 point mutations were detected in 7 of 24 (29%) tumors, including 5 of 19 (26%) sporadic HA and 2 of 5 (40%) TT-induced HA. There were seven G:C > A:T and four G:C > T:A mutations. All seven mutated tumors contained a codon 12-aspartate amino acid substitution. In addition, a second codon 12-cysteine mutant cell population was present in one of two codon 12-aspartate mutated TT-induced HA and in three of five codon 12-aspartate sporadic tumors. Of these four tumors, three contained both aspartate and cysteine mutations and were composed of multiple nodules; the fourth was a single mass. Seventeen tumors had multiple nodules; whereas 5 had a K-ras-2 mutation, 12 were wild-type. The molecular pathology of both sporadic and TT-induced HA is characterized by a high rate of K-ras-2 mutations characteristic of oxidative damage (ie, G:C > A:T and G:C > T:A mutations) resulting in two mutated population sets: codon 12 GGT > GAT and GGT > TGT (glycine to aspartic acid and cysteine). This is, to date, the first study to characterize the K-ras-2 gene mutations within human sporadic and TT-induced HA by direct sequence analysis and denaturing gradient gel electrophoresis. These data further support the hypothesis linking adduct-forming vinyl chloride exposure to HA containing a much higher frequency of K-ras-2 mutations and a mutational spectrum characteristic of chloroethylene oxide, a carcinogenic metabolite of vinyl chloride.

Fidelity and predominant mutations produced by deep vent wild-type and exonuclease-deficient DNA polymerases during in vitro DNA amplification.

Denaturing gradient gel electrophoresis (DGGE) was used to examine error rates and mutations induced by native (wt) and exonuclease-deficient (exo-) Deep Vent DNA polymerases during DNA amplification by polymerase chain reaction (PCR), in the presence or absence of the T4 bacteriophage gene 32 protein (gp32).gp32 was found to decrease the error rate of the wt, but not that of the exo-, Deep Vent. The average errors per base duplication for the native form were 8.0 x 10(-5) and 6.0 x 10(-5) in the absence and presence of gp32, respectively. For the exo- form, the error rates were 2.0 x 10(-4) and 2.2 x 10(-4) errors per base duplication in the absence and presence of gp32, respectively. Examination of mutations produced by native Deep Vent showed that A/T to G/C transition predominated, consistent with the results of our earlier studies with DNA polymerases derived from other thermophilic bacteria. These results indicate that PCR with high fidelity can be achieved by using wt Deep Vent in combination with gp32.

Detection of K-ras mutations in lung carcinomas: relationship to prognosis.

The K-ras mutation is one of the most common genetic alterations found in human lung cancer. To evaluate the prognostic value of ras gene alterations in lung cancer in a U.S. population, we have screened 173 human lung tumors, which included 127 adenocarcinomas, 37 squamous carcinomas, and 9 adenosquamous carcinomas, for mutations in the K-ras gene using the combination of the PCR and denaturing gradient gel electrophoresis. Forty-three tumors contained K-ras mutations. Of these, 41 were identified among the adenocarcinomas (32%), 1 among the squamous carcinomas (2.7%), and 1 among the adenosquamous carcinomas (11%). Forty of these mutations were found in codon 12 and consisted of 24 G to T transversions, 12 G to A transitions, 2 G to C transversions, and 1 double GG to TT mutation. Two other G to T transversions were found in codon 13, and 1 A to C transversion was found in codon 61. The data showed that gender did not seem to affect the incidence and the types of the K-ras mutations or amino acid changes. Examination of the mutations in adenocarcinomas in relation to overall survival showed no difference in adenocarcinomas with K-ras mutations compared with K-ras-negative adenocarcinomas. However, the substitution of the wild-type GGT (glycine) at codon 12 with a GTT (valine) or a CGT (arginine) showed a strong trend (P = 0.07) toward a poorer prognosis compared with wild-type or other amino acid substitutions. Substitution of the wild-type glycine for aspartate (GAT) showed a strong trend (P = 0.06) for a better outcome than the valine or arginine substitution. Although these trends will require larger patient populations for verification, these data suggest that the prognostic significance of K-ras mutations may depend on the amino acid substitution in the p21(ras) protein.

In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene.

Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. In order to acquire an understanding of the mutagenic pathways of CPP, we studied mutations induced by this chemical in human cells. Four independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with CPP, and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance. The kinds and positions of the mutations were analyzed using the combination of high-fidelity polymerase chain reaction (hifi-PCR) and denaturing gradient gel electrophoresis (DGGE). The third exon of the HPRT gene was amplified from the 6TG-resistant cells using the hifi-PCR and the amplified fragment was subsequently analyzed by DGGE to separate mutant sequences from the wild-type sequence. Mutant bands were excised from the gel, amplified using PCR and sequenced. Sixteen different mutations were identified and consisted mostly of the G to T and A to T transversions. Other mutations identified included G to A and A to G transitions, a G to C transversion, and a single G deletion. Of these mutations, six occurred within a run of six guanines. The predominance of transversions involving a guanine or an adenine observed with CPP is similar to the data previously reported for the racemic mixtures of benzo[a]pyrene (B[a]P), suggesting that the mechanisms of mutation induced by CPP may be similar to those induced by B[a]P.

Resolution and conservation of mismatches in DNA end joining.

DNA end joining is a major pathway for the elimination of double-strand breaks from chromosomal DNA of higher eucaryotic cells. Extracts of Xenopus laevis eggs rejoin such breaks even when their short single-stranded termini are expected to form imperfectly matched overlaps. However, end-joined products cloned in Escherichia coli, necessarily give rise to perfectly matched products. Therefore it has not been possible to determine whether the end joining process creates mismatched products, perfectly matched (resolved) products or both. To investigate whether mismatch resolution was the result of the X. laevis end joining process or of activities of the bacterial host we used denaturing gradient gel electrophoresis to analyse joined products. We found that the end joining process does include mismatch resolution, the degree of which varies with regard to the nature of the original overlap structure. Mismatches 3' to a gap are completely resolved, mismatches 3' to a nick and 5' to a nick or gap are resolved to some extent but are generally conserved. Mismatches between base matches are always conserved. These findings suggest competing processes of ligation, DNA fill-in synthesis or exonucleolytic excision of mismatched bases next to a gap or nick. At mismatches 3' to a nick the probability of ligation is greater than that of excision while at mismatches 3' to a gap the probability of excision is greater than elongation of a given mismatch. At mismatches 5' to nicks or gaps it appears that ligation or elongation and ligation, respectively, are the most probable pathways but products resulting from mismatch excision, elongation and ligation are also detected.

Use of anti-TNF-alpha antiserum to investigate toxic alveolitis arising from cotton dust exposure.

Cotton dust has been associated with byssinosis and toxic alveolitis. A murine animal model has been developed with which to investigate the pathogenesis of these disorders. Studies with the model have reproduced the neutrophilic inflammation characteristic of the alveolitis, and have shown the presence of tumor necrosis factor-alpha (TNF-alpha) in the bronchoalveolar lavage (BAL) fluid. The current study investigated the role of TNF-alpha in the inflammatory response by use of a polyclonal antiserum to recombinant murine TNF-alpha. Following a 4-h exposure to cotton dust, experimental animals showed a 40-fold increase in BAL cells with 92% neutrophils. There was a 24-fold increase in TNF-alpha in the BAL fluid. Up regulation of TNF-alpha mRNA expression was detected in BAL cells. Mice pretreated with anti-TNA-alpha antiserum displayed a marked attenuation of the neutrophilic inflammation; however, the level of TNF-alpha mRNA expression was not reduced in these mice. These studies support a major role of TNF-alpha in the toxic alveolitis induced by cotton dust inhalation.

Effects of the T4 bacteriophage gene 32 product on the efficiency and fidelity of DNA amplification using T4 DNA polymerase.

Two bacteriophage DNA polymerases (Pol), T4 Pol and modified T7 Pol, were used to catalyze DNA amplification in vitro by PCR, and their efficiency and fidelity in DNA amplification were examined in the presence and absence of the T4 bacteriophage gene 32-encoded protein (SSB32). The SSB32 protein significantly improved the efficiency of amplification by T4 Pol. Examination of the amplified DNA by denaturing gradient gel electrophoresis (DGGE) revealed that the protein also reduced the rates of error produced by T4 Pol during PCR, from 6.3 x 10(-6) to 2.0 x 10(-6) errors per base duplication after 10(11)-fold amplification. This protein also improved, but to a lesser extent, the fidelity of modified T7 Pol, from 1.80 x 10(-5) to 1.15 x 10(-5) errors per base duplication. High fidelity polymerase chain reaction (hifi-PCR) is needed for studies requiring isolation of mutant sequences present as only a small fraction of the wild type in the amplified DNA. Although several thermostable Pol are currently available for use in automated PCR, their fidelity was found to be significantly lower than that of the thermosensitive T4 Pol. Therefore, T4 Pol is useful for studies requiring hifi-PCR, although this enzyme needs to be added in the reaction mixture during every cycle of PCR.

Predominant mutations induced by the Thermococcus litoralis, vent DNA polymerase during DNA amplification in vitro.

We have analyzed the predominant mutations created during DNA amplification by PCR utilizing a DNA polymerase isolated from the Thermococcus litoralis (Vent DNA polymerase). Exon 3 of the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene was amplified using conditions optimized for efficiency of DNA amplification. The resulting PCR product was subjected to denaturing gradient gel electrophoresis (DGGE) to separate polymerase-induced mutant sequences from correctly amplified sequences. The nature of induced mutations was determined by isolating and sequencing the mutant sequences from the gel. Eighteen predominant mutations were found in the 104-bp low temperature melting domain of exon 3 and consisted of 16 A/T to G/C transitions, a G/C to T/A transversion and a complex 4-bp deletion. Thus, the Vent exonuclease proofreading activity seems to affect all misincorporation events with apparently equal probability (i.e., by a factor of five). The comparison of the error rates between analogues of Vent DNA polymerase proficient and deficient in the proofreading 3'-->5' exonuclease activity indicates that the lack of proofreading resulting in an approximate five-fold increase in induced error rate. However, the similarity of the patterns of the mutant sequences observed in DGGE suggested that both enzymes created predominantly the same kinds of mutations and at the same positions in this DNA template under the in vitro reaction conditions studied. This predominance of A/T to G/C transition is also a characteristic of the Taq DNA polymerase, although the positions of most errors induced by both enzymes are not identical.