Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6- alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector- transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane- derived species to alkylate Trx.      

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Otoacoustic emissions as a screening test for hearing impairment in children recovering from acute bacterial meningitis

OBJECTIVES: To study the efficacy of otoacoustic emissions (OAEs) as a screening test for hearing impairment in children with acute bacterial meningitis. Hearing tests were performed before discharge from the hospital in an attempt to improve coverage and avoid delays in the diagnosis of postmeningitic hearing loss. METHODS: Children with bacterial meningitis were recruited from 21 centers. In the 48 hours before discharge from the hospital, all patients underwent a thorough audiologic assessment consisting of transient evoked OAEs, auditory brainstem responses (ABRs), otoscopy, and tympanometry. Hearing loss was defined as ABR threshold >/=30 dB. The results of OAE screening were compared with the gold standard of ABR threshold. RESULTS: Of 124 children recruited, we were able to perform both OAEs and ABRs on 110 children. Seven (6.3%) of the 110 children had ABR threshold >/=30 dB; 2 had sensorineural hearing loss and 5 had conductive hearing loss. At follow-up, hearing loss persisted in both cases of sensorineural hearing loss and no new cases were identified. All 7 children with hearing loss failed the OAE screening test. Ninety-four children with normal hearing thresholds passed the test, and 9 failed. Thus, the screening test had a sensitivity of 1.00 (95% confidence interval, 0.59 to 1.00), a specificity of 0.91 (0.85 to 0.97), a positive predictive value of 0. 44 (0.20 to 0.70), and a negative predictive value of 1.00 (0.96 to 1.00). CONCLUSIONS: OAE screening in children recovering from meningitis was found to be feasible and effective. The test was highly sensitive and reasonably specific. Inpatient OAE screening should allow early diagnosis of postmeningitic hearing loss and prompt auditory rehabilitation.     

Cutaneous metastases from adenocarcinoma of unknown primary

Cutaneous manifestations of malignancy are not uncommon, especially in advanced disease. They may also occur early in malignant disease or they may even signify recurrence particularly if they are paraneoplastic in nature. Clinical diagnosis can be difficult because of the wide spectrum of appearance of these lesions, and, in many cases, because of the lack of an identifiable underlying primary. Presented here is the case of a 65-year-old woman with multiple inflammatory cutaneous metastases, which were sclerodermoid in nature. These appeared 14 months after initial diagnosis of adenocarcinoma of unknown primary (ACUP) and signified the beginning of a rapid deterioration in her condition. The coexistence of limited systemic sclerosis (scleroderma) and ACUP initially raised several interesting diagnostic possibilities. Adenocarcinoma of unknown primary and the sclerodermoid reaction in malignancy are discussed.     

Familial protein S deficiency with a variant protein S molecule in plasma and platelets

A protein S deficient family presenting a variant protein S molecule in plasma and platelets is described. The propositus, age 20, and two brothers suffered from venous thrombotic disease. The propositus, the only family member studied while taking oral anticoagulants, had a protein S antigen (ag) level of 17% and undetectable activity. As demonstrated by immunoblotting both the propositus and one clinically affected brother (42% ag, 7% activity) presented variant protein S molecules of 65,000 molecular weight (mol wt) while the other clinically affected brother (64% ag, 11% activity) had only protein S with normal electrophoretic mobility of 70,000 mol wt. The mother had normal protein S levels (93% ag, 100% activity) but had both normal and variant protein S molecules and based on her functional protein S data a normal anticoagulant activity of the variant molecule is suggested. One asymptomatic but protein S deficient sister (68% ag, 9% activity) as well as the asymptomatic protein S deficient father (59% ag, 10% activity) had only protein S molecules of 70,000 mol wt. The variant protein S bound to C4b-binding protein in plasma, and differed from normal protein S in carbohydrate content. Platelets of each family member contained the same immunoblotting pattern of normal and variant protein S forms as found in plasma, consistent with the hypothesis that protein S gene expression involves codominant expression of two alleles that is similar in cells that control the synthesis of both platelet and plasma forms of protein S.     

Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells

Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T- cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL-4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40-signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL.