Ethylene and auxin interaction in the control of adventitious rooting in Arabidopsis thaliana.

Adventitious roots (ARs) are post-embryonic roots essential for plant survival and propagation. Indole-3-acetic acid (IAA) is the auxin that controls AR formation; however, its precursor indole-3-butyric acid (IBA) is known to enhance it. Ethylene affects many auxin-dependent processes by affecting IAA synthesis, transport and/or signaling, but its role in AR formation has not been elucidated. This research investigated the role of ethylene in AR formation in dark-grown Arabidopsis thaliana seedlings, and its interaction with IAA/IBA. A number of mutants/transgenic lines were exposed to various treatments, and mRNA in situ hybridizations were carried out and hormones were quantified In the wild-type, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) at 0.1 µM enhanced AR formation when combined with IBA (10 µM), but reduced it when applied alone; this effect did not occur in the ein3eil1 ethylene-insensitive mutant. ACC inhibited the expression of the IAA-biosynthetic genes WEI2, WEI7, and YUC6, but enhanced IBA-to-IAA conversion, as shown by the response of the ech2ibr10 mutant and an increase in the endogenous levels of IAA. The ethylene effect was independent of auxin-signaling by TIR1-AFB2 and IBA-efflux by ABCG carriers, but it was dependent on IAA-influx by AUX1/LAX3.Taken together, the results demonstrate that a crosstalk involving ethylene signaling, IAA-influx, and IBA-to-IAA conversion exists between ethylene and IAA in the control of AR formation.

Cleavage of the MLL gene by activators of apoptosis is independent of topoisomerase II activity.

Exposure to topoisomerase II inhibitors is linked to the generation of leukemia involving translocations of the MLL gene, normally restricted to an 8.3 kbp tract, the breakpoint cluster region (BCR). Using an in vitro assay, apoptotic activators, including radiation and anti-CD95 antibody, trigger site-specific cleavage adjacent to exon 12 within the MLL BCR and promote translocation of the MLL gene in cells that can survive. To explore the mechanism of cleavage and rearrangement in more detail, the entire MLL BCR was placed into the pREP4 episomal vector and transfected into human lymphoblastoid TK6 cells. Episomes containing either the MLL BCR, or deletion constructs of 367 bp or larger, were cleaved at the same position as genomic MLL after exposure to apoptotic stimuli. Further analysis of sequence motifs surrounding the cleaved region of MLL showed the presence of both a predicted nuclear matrix attachment sequence and a potential strong binding site for topoisomerase II, flanking the site of cleavage. Inactivation of topoisomerase II by the catalytic inhibitor merbarone did not inhibit MLL cleavage, suggesting that the initial cleavage step for MLL rearrangement is not mediated by topoisomerase II.

Apoptotic triggers initiate translocations within the MLL gene involving the nonhomologous end joining repair system.

Translocations involving the MLL gene at 11q23 are a frequent finding in therapy-related leukemia and are concentrated within a short, 8.3-kb tract of DNA, the breakpoint cluster region. In addition, a specific site adjacent to exon 12 within this region of MLL is cleaved in cells undergoing apoptosis. We show here, using human TK6 lymphoblastoid cells, that irradiation and the apoptotic trigger anti-CD95 antibody are each able to initiate translocations at the MLL exon 12 cleavage site. The translocation junctions produced contain regions of microhomology consistent with operation of the nonhomologous end joining (NHEJ) repair process. Participation of the NHEJ process is supported by the identification of the NHEJ component DNA-PKcs at the site of apoptotic cleavage. Suppression of DNA-PKcs function by the phosphatidylinositol 3-kinase inhibitor wortmannin compromises DNA end joining, increases site-specific cleavage within MLL, and eliminates MLL-restricted translocations. We propose that activation of apoptotic effector nucleases alone is sufficient to generate proleukemogenic translocations and raises the possibility that some of these may persist in cells that evade apoptotic execution and survive.

Comparative studies on cytotoxic and genotoxic effects of two organic mercury compounds in lymphocytes and gastric mucosa cells of Sprague-Dawley rats.

Human lymphocytes (HL) as well as lymphocytes (RL), hepatocytes (RH), and gastric mucosa cells (GM) of Sprague-Dawley rats were treated in vitro for 1 h with methylmercury chloride (MMC, 0.5-4 micrograms/ml) and dimethylmercury (DMM, 5-40 micrograms/ml). The cytotoxicity of the two organic mercury compounds was assessed by dye exclusion, and the extent of induced DNA fragmentation was measured with a single-cell microgel electrophoresis assay. Both MMC and DMM induced DNA damage and cytotoxicity in a dose-related manner in HL, RL, and GM. MMC was more effective in causing a significant increase in median DNA migration than DMM at doses yielding approximately the same degree of cytotoxicity. In rat hepatocytes the MMC-induced DNA damage was, however, lower than in the other cells. An analysis of repair kinetics following exposure to 2 micrograms/ml MMC was carried out in human lymphocytes obtained from an adult male donor. The bulk of DNA repair occurred 90 min after in vitro exposure, and it was about complete by 120 min following cessation of exposure. Finally, in order to have a basis for extrapolating to the human situation, in vivo studies were performed with Sprague-Dawley rats, also assessing the DNA damage and cytotoxicity in the lymphocytes and gastric mucosa cells. These in vivo results after oral exposure may be directly compared to the in vitro data obtained in the same cells.

Cyclic responding by pigeons on the peak timing procedure.

The present experiment examined whether discrimination learning shapes the single-peaked response distributions usually obtained with the peak procedure. Two sources of learning in pigeons were disclosed: learning to respond near the time of reinforcement on fixed interval (FI) trials and learning to withhold responding once the FI duration had elapsed on peak interval (PI) trials. Pigeons also produced a highly unexpected second peak in responding on nonreinforced PI trials at 3 times the FI duration. Follow-up experiments showed that a 1:4 FI:PI duration ratio supported double peaks, but only 1 peak was obtained with a 1:8 FI:PI duration ratio. Finally, 4 peaks could be observed on extra-long PI trials under a 1:4:8 FI:PI:PI ratio procedure. The multiple-peaked response distributions are an unprecedented finding that present a major challenge to any theory of time perception.

Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes.

The genotoxicity of methyl mercury chloride (MMC, 0-25 x 10(-6) M) and dimethyl mercury (DMM, 0-434 x 10(-6) M) was evaluated by chromosome metaphase analysis in human lymphocytes treated in vitro for 24 h. Structural (CA) and numerical (AN) chromosomal aberrations were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered a monitor for induced cellular toxicity. MMC induced CA and AN in a dose-related manner at doses exceeding 0.6 x 10(-6) M, and the proportion of cells with CA was constantly and significantly higher than that of cells with AN. DMM was able to induce both effects as well, although to a lesser extent than MMC, CA and AN being induced at doses exceeding 43.4 x 10(-6) M and 1.73 x 10(-6) M, respectively. MMC was 6-fold more effective in inducing CA than DMM at equivalent toxic doses. On the other hand, no significant difference was observed between the two compounds in inducing AN. Therefore MMC was much more clastogenic than DMM, whereas mitotic spindle disturbances appeared to be almost equally induced by both compounds.

Liquid holding effects on methylmercury genotoxicity in human lymphocytes.

Methylmercury chloride (MMC) treatment of resting (G0) human lymphocytes resulted in the formation of chromosome and chromatid aberrations. This treatment also induced aneuploidy in second metaphases, suggesting that MMC produces stable damage involved in chromosome segregation errors. The storage of treated cells (liquid holding for 48 h before cell proliferation) did not result in an important recovery from induced cell toxicity or chromosome damage. Therefore, MMC seems to be an X-ray-like agent, able to produce long-lasting damages giving rise to both structural and numerical chromosome abnormalities.

Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects.

The comet test (single cell gel electrophoresis, SCGE) appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. Previously, we analyzed the degree of DNA damage in peripheral human lymphocytes from 100 healthy subjects living in Pisa (Italy) taking into account age, gender and smoking habit, and we also reported some results aiming at the assessment of the comet test (Betti el al., 1994). In addition, SCE analysis was carried out in order to compare the two endpoints. Because of the interesting results obtained, the present study was extended to 200 individuals, and data analyzed included information concerning number of cigarettes smoked a day, tar/cigarette and job. Data obtained confirmed that the SCGE is more sensitive than SCE in revealing smoking habit effects but comet induction did not seem to be related to the amount of cigarette tar inhaled. Moreover, sampling time was found to play a greater role in the comet assay as compared to SCE. Job position did not significantly influence SCE mean/subject or comet length mean/subject.

Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects.

Microscopic examination of individual human lymphocytes embedded in agarose, subjected to electrophoresis and stained with a fluorescent DNA-binding dye, provides a novel way of measuring DNA damage as extent of migration of DNA fragments, mainly single-strand breaks. With this relatively simple method, DNA damage arising as a consequence of smoking, age and other factors was examined in peripheral human lymphocytes from 100 healthy individuals living in Pisa (Italy). The extent of DNA migration was found to be significantly increased by smoking. It is noteworthy that the effect of smoking was more significant in men than in women and that DNA migration was similar in the young and in the older people. SCE analysis did not reveal any significant effect of smoking, sex or age in the same population, suggesting a higher responsiveness of the comet test to DNA-damaging agents.

Genotoxicity of two metabolites of benzene: phenol and hydroquinone show strong synergistic effects in vivo.

Possible interactions between hydroquinone (HQ) and phenol (PHE), 2 known benzene metabolites, in inducing micronuclei in mouse bone marrow cells were investigated. HQ and PHE administered alone gave weak and negative results, respectively, at the doses tested. However, simultaneous administration of both compounds caused a considerable increase in the induction of micronuclei as well as an increase in bone marrow toxicity. Using 3 different statistical methods, it was shown that the observed joint effect was significantly higher than additive interaction, and was close to multiplicative interaction. These findings bring further support to the hypothesis that the toxic and genotoxic effects of benzene are produced by several metabolites acting synergistically.

Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis.

Single cell gel electrophoresis (SCGE), or comet assay, appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. A follow-up study of 90 smokers who ceased smoking was undertaken to determine the possible decrease of DNA damage in their leukocytes. Before beginning the trial, volunteers smoked on average 26.1 +/- 8.4 cigarettes/day. Comet length did not correlate with the number of cigarettes/day or with the condensate tar content. At the end of the study, 28 volunteers had abandoned the trial, 40 volunteers relapsed into smoking at different times, but with a reduced number of cigarettes/day, whereas 22 fully succeeded in smoking cessation. Throughout the 5 sampling times, a great variability of comet length at individual level was found. However, after 1 year of follow-up, comet length means were found to be significantly shorter (p < 0.0001) in those volunteers who completely quit smoking compared to those who relapsed into smoking (27.2 +/- 1.6 vs. 31.9 +/- 5.1 microns, respectively), irrespective of the amount of cigarettes previously smoked. No effect of age or sex was found. Six months later, these results were confirmed by a further study carried out on a reduced sample of volunteers. The present data strongly suggest that, in spite of the great variability observed, 1 year of smoking cessation is associated with a significant reduction of DNA damage in circulating leukocytes.

Extensive investigation on colonic motility with pharmacological testing is useful for selecting surgical options in patients with inertia colica.

Three women with idiopathic severe chronic constipation and inertia colica, who failed to respond to medical treatment, were extensively investigated for gut motor function, especially that of the colon. Twenty-four-hour manometric recordings disclosed that motility was severely reduced throughout the entire colon and response to eating was minimal. One of the patients also was tested for esophageal, gastric, and small bowel motor activity, which gave normal results. Edrophonium chloride stimulation (10 mg iv) provoked no increase in colonic contractile activity in any patient. On these grounds, the patients were submitted to surgical intervention (total colectomy with ileorectal anastomosis two, and left hemicolectomy the other) with fairly good results at follow-up. These results indicate the wisdom of carrying out extensive functional investigations in severely constipated patients before surgery is contemplated.

Prolonged (24-hour) manometric recording of rectal contractile activity in patients with slow transient constipation.

A periodic motor activity, named the rectal motor complex, has been recently described in the healthy human rectum. We studied the rectal contractile activity for 24 h by a low compliance manometric system in a group of 10 women with slow transit constipation. Analysis of the 24-hour manometric recordings showed that these subjects: (1) had overall scarce rectal motility; (2) display few rectal motor complexes (average, 3.3 +/- 1.3/subject/24 h) which are irregularly distributed over time, and (3) respond weakly to ingestion of a standard meal (average duration of the motor response 19 +/- 6 min). The observations suggest that an underlying neuropathic process may be involved in the pathogenesis of the impaired rectal motility in patients with slow transit constipation.

[Changes in upper gastrointestinal motility during scleroderma]. / Alterazioni della motilità gastrointestinale superiore in corso di sclerodermia.

Scleroderma (progressive systemic sclerosis) is a systemic collagen disease in which the upper gut is frequently involved. In particular, most patient show altered esophageal motility, which frequently result in severe esophagitis, often resistant to therapeutic measures. The small bowel is also frequently involved by the disease, especially in the late stage of scleroderma. Small bowel alterations are sometimes clinically silent, but can also be the origin of malabsorption syndrome, small intestine perforation, pneumatosis cystoides or chronic intestinal pseudo-obstruction. The occurrence of an altered gastrointestinal motility in scleroderma can be detected by means of manometric techniques; their use in the wide area of collagenopathies may help understanding the pathophysiology of the altered gastrointestinal function frequently existing in these diseases.

Manometric evaluation of jejunal limb after total gastrectomy and Roux-Orr anastomosis for gastric cancer.

Total gastrectomy with Roux-Orr anastomosis is frequently performed for gastric cancer. Since intestinal motility of the Roux limb has never been evaluated after this operation, pressure activity was investigated in the Roux limb of ten patients (aged 51-77 years) who had undergone total gastrectomy and Roux-Orr reconstruction. Investigations were carried out during a 6-h fast and 3 h after a 605 kcal mixed meal. During fasting only two patients had activity fronts and these were abnormal. All ten patients displayed non-propagating bursts of contractions and three had discrete clustered contractions and high amplitude jejunal contractions. The fed state was characterized by a severely reduced motor activity pattern and other abnormalities. Total gastrectomy with Roux-Orr anastomoses provokes a relatively severe disturbance in intestinal activity.