Altered DNA repair capacity and bleomycin sensitivity as risk markers for non-small cell lung cancer.

DNA repair capacity in human peripheral blood lymphocytes was monitored by the repair rate of bleomycin-induced DNA damage using an alkaline single-cell gel electrophoresis assay (comet assay). DNA repair capacity, after 15 min repair time, in lymphocytes of non-small cell lung cancer patients (n = 160) and controls (n = 180) was 67% and 79.3%, respectively (p < 0.0004). Bleomycin sensitivity defined as the tail moment of bleomycin-treated peripheral blood lymphocytes, without allowing time for DNA repair, was significantly higher in lung cancer patients than in tumor-free hospital controls (p < 0.0001). There was no correlation, in either patient or control group, between the bleomycin sensitivity and DNA repair capacity with age or gender. The median values of DNA repair capacity and sensitivity in controls were used as the cut-off points for calculating odds ratios (OR). After adjustment for age, gender and smoking status, the cases vs. controls had reduced DNA repair capacity (OR = 2.1; 95% confidence limit [CL] 1.1-4.0) and increased bleomycin sensitivity (OR = 4; 95% CL 2.2-7.4). For current smokers, the adjusted risk associated with bleomycin sensitivity was 2.3 (95% CL 1.1-4.9). We conclude that our standard comet assay as a phenotypical repair test has sufficient sensitivity and rapidity allowing application to both native and cryopreserved lymphocytes. Bleomycin sensitivity and DNA repair capacity were found to be 2 independent susceptibility markers for non-small cell lung cancer, confirming similar investigations with different marker end points. The latter were much more time consuming than the method used in our study. Thus, the comet assay is more suitable for screening large numbers of individuals in epidemiological studies. Validation of this assay in large prospective studies for the identification of subjects at high risk for non-small cell lung cancer is now warranted.

Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes.

Individual susceptibility to carcinogens, an important determinant of disease risk, is influenced by host factors such as the ability to repair DNA lesions. In order to identify subjects who are at high risk, we have developed a microgel electrophoresis assay for use in molecular epidemiological studies. The assay was validated in a pilot case-control study: Peripheral blood lymphocytes were collected from 100 patients with lung cancer and 110 control patients without cancer and from the same hospital, and stored at -80 degrees C. After thawing, phytohaemagglutinin-stimulated cells were treated with bleomycin at 20 microg/ml for 30 min and the extent of DNA damage and DNA repair capacity were determined by microgel electrophoresis. Peripheral blood lymphocytes from patients with lung cancer were significantly more sensitive to mutagens than those from controls and showed reduced DNA repair capacity (both P < 0.001). Both endpoints were independent risk factors for smoking-related lung cancer. Repeated analysis of peripheral blood lymphocytes from the same individual demonstrated good reproducibility of the assay. Cryopreservation of the lymphocytes for less than or = 12 months did not significantly affect their sensitivity. Our standardized microgel electrophoresis assay is suitable for determining individual sensitivity to mutagens and DNA repair capacity: it is sensitive and faster than cytogenetic assays, and can be applied to native and cryopreserved peripheral blood lymphocytes.

Quantitative assessment of bleomycin-induced poly(ADP-ribosyl)ation in human lymphocytes by immunofluorescence and image analysis.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is catalytically activated by DNA strand interruptions. It catalyses the covalent modification of proteins with ADP-ribose polymers, using NAD(+) as precursor. Here, we have studied the DNA damage-induced formation of poly(ADP-ribose) in intact human peripheral blood lymphocytes (PBL) by in-situ immunofluorescence detection. The response of PBL to bleomycin (BLM), which is known to induce DNA single and double strand breaks, was investigated with regard to polymer formation. For this purpose, a quantitative approach was developed to assess more accurately the immunostaining of polymer formation by computerised image analysis. As an application of this new method, we have determined the polymer formation following BLM treatment in quiescent human PBL versus mitogen activated cells. Quiescent human PBL showed a similar basal immunostaining for the polymer compared to phytohemagglutinin (PHA)-activated cells, expressed as relative mean pixel intensity (RMPI) (1.3+/-0.8 and 2.2+/-0.9, respectively; P<0.3). After BLM treatment, there was a clear-cut enhancement of polymer immunostaining, with PHA-activated cells showing significantly higher RMPI than non-activated cells (9.2+/-1.4 and 4.2+/-1.0, respectively; P<0.005). As expected, in the presence of the ADP-ribosylation inhibitor 3-aminobenzamide (3-AB), the RMPI of immunostained polymer was decreased in both quiescent and PHA-activated PBL to 1.2+/-0.7 and 1.5+/-0.9, respectively. Our findings reveal (i) that mitogen-stimulated, intact lymphocytes show enhanced polymer formation following BLM treatment, and (ii) that our new quantitative immunofluorescence assay coupled with computerised image analysis is reliable and sensitive enough to detect changes in polymer formation rate.

Clonidine-induced rhythmic activity in rabbit anococcygeus muscle.

1. Clonidine 0.5 mM induced an extremely regular rhythmic activity in isolated rabbit anococcygeus muscle. The movements were resistant to tetrodotoxin effect. 2. Prazosin (5 x 10(-8)M-5 x 10(-6)M) and yohimbine (1.5 x 10(-7)M-5 x 10(-4)M) showed no remarkable effect on clonidine-induced rhythmic activity. 3. The clonidine-induced contractions were dependent on extracellular calcium and could be inhibited by the omission of calcium from medium or the introduction of verapamil (IC50 = 1.3 x 10(-7)M) or nifedipine (IC50 = 7.5 x 10(-8)M). 4. Pretreatment of animals with reserpine made the preparations 2800-fold more sensitive to this action of clonidine. 5. It can be concluded from this study that clonidine is able to induce rhythmic activity in rabbit anococcygeus muscle through a mechanism that increases intracellular concentration of Ca++ via membrane calcium channels.

Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli.

Ureaplasma urealyticum is a pathogenic ureolytic mollicute which colonizes the urogenital tracts of humans. A genetic polymorphism between the two biotypes of U. urealyticum at the level of the urease genes was found. The urease gene cluster from a biotype 1 representative of U. urealyticum (serotype I) was cloned and sequenced. Seven genes were found, with ureA, ureB, and ureC encoding the structural subunits and ureE, ureF, ureG, and a truncated ureI) gene encoding accessory proteins. Urease expression was not obtained when the plasmid containing these genes was incorporated into an opal suppressor strain of Escherichia coli, although this enzymatic activity was found in the same E. coli strain transformed with pC6b, a plasmid with previously cloned urease genes from the U. urealyticum T960 strain of biotype 2 (serotype 8). Although there are 12 TGA triplets encoding tryptophan within urease genes, the level of expression obtained was comparable to the levels reported for other bacterial genes expressed in E. coli. Nested deletion experiments allowed us to demonstrate that ureD is necessary for urease activity whereas another open reading frame located downstream is not. The promoter for ureA and possibly other urease genes was identified for both serotypes.

Different calcium dependencies of contractile activity of prostatic and epididymal portions of rat vas deferens.

1. The effects of some organic calcium entry blockers and different concentrations of extracellular calcium on electrically-evoked contractions of isolated epididymal and prostatic portions of rat vas deferens were investigated. 2. Both epididymal and prostatic parts of rat vas deferens responded to single pulse or train electrical field stimulation, with twitch contractions of submaximal amplitude. 3. Verapamil showed a biphasic action on the contractions produced by single pulse electrical stimulation. In concentrations < 10(-5) M, it potentiated the responses of both portions, but at higher concentrations, the excitatory action was overcome by a concentration-dependent inhibitory effect. 4. Nifedipine reduced the amplitude of electrically-evoked contractions of both portions in a concentration-dependent manner. The ED50 of nifedipine was 3.6 x 10(-8) M and 2.1 x 10(-6) M in prostatic and epididymal portions, respectively. 5. Dantrolene sodium reduced the amplitude of electrically-evoked contractions of both portions in a concentration-dependent manner. The ED50 of dantrolene was 1.55 x 10(-4) M and 9.1 x 10(-4) M in prostatic and epididymal portions, respectively. 6. Reduction of Ca2+ concentration in medium reduced the amplitude of contractions of both portions significantly. This calcium dependence was more apparent in low frequencies of electrical stimulation.

The role of calcium and alpha-adrenoceptors in contractile response of chick expansor secundariorum muscle to field stimulation.

1. The effects of some alpha-adrenergic agonists and antagonists on electrically-evoked contractions and tension of chick expansor secundariorum muscle (ESM), and dependence of these events on extracellular calcium was investigated. 2. Both train and continuous electrical stimulation can produce regular contractions in preparations obtained from 40-60 day old chicks. 3. Clonidine had a biphasic action on the contractions produced by train electrical stimulation. In concentrations ranging from 10(-8) to 3 x 10(-7) M, clonidine decreased the contraction amplitude, but in higher concentrations, it caused an increase in both the muscle tension and the contraction amplitude. These effects were reversed by application of yohimbine although yohimbine by itself had no effect on the contractions. 4. Introduction of calcium free isotonic high potassium medium decreased muscle tone which was followed by further dose-dependent increase in tension, along with the addition of cumulative doses of CaCl2 (ED50 = 2.8 x 10(-3) M). 5. Nifedipine reduced the amplitude of ESM contractions produced by continuous electrical stimulation in a dose dependent manner (IC50 = 6.7 x 10(-7) M). 6. Methoxamine induced a completely dose dependent increase in muscle tension which was dependent on extracellular calcium and was inhibited by nifedipine. In the presence of 10(-8) M nifedipine, ED50 of methoxamine stimulatory effect increased from the control value of 2.2 x 10(-7) to 8.4 x 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Methoxamine-induced rhythmic activity in rabbit anococcygeus muscle.

1. Methoxamine 0.5 microns induced an extremely regular rhythmic activity in isolated rabbit anococcygeus muscle. 2. Prazosin had an inhibitory effect on methoxamine-induced rhythmic contractions. IC50 of prazosin was 7.94 nM. 3. The methoxamine-induced contractions are dependent on extracellular calcium and can be inhibited by the omission of calcium from media or the introduction of verapamil (IC50 = 0.11 microM) or nifedipine (IC50 = 0.21 microM). 4. Application of reserpine made the preparations 40-fold more sensitive to methoxamine. 5. It can be concluded that rhythmic contractions produced by methoxamine are mediated through stimulatory action of methoxamine on alpha-I adrenoceptors and depend on extracellular calcium. 6. Lithium made the muscle more sensitive to methoxamine action. In preincubated muscles with 1, 3 and 5 mM lithium the initiation of contractions occurred at 1.5 x 10(-7), M, 5 x 10(-8) M and 1.5 x 10(-8) M of methoxamine, respectively.

Phylogenetic analysis of Mycoplasma penetrans, isolated from HIV-infected patients.

A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.