Robert Sobkowiak

Cadmium-induced changes in growth and cell cycle gene expression in suspension-culture cells of soybean

Abstract The toxic effects of cadmium on growth and development of living organisms are well documented. However, the molecular mechanisms responsible for the inhibition of plant growth by cadmium are still not completely understood. We determined the effects of cadmium concentrations in the range of 1–11 μM on the growth of Glycine max L. cv. Navico suspension-culture cells, as well as on the expression of two cell cycle genes: cyclin B1 and cyclin-dependent type A kinase (CDK-A). There was no detectable decrease in cell viability at any tested Cd 2+ concentrations. The lower concentrations of Cd 2+ (1–4 μM) stimulated cell culture growth; however, this did not correspond with increased expression of cell cycle genes. The inhibition of cell growth was observed at concentration of Cd 2+ higher than 6 μM. Interestingly, it correlated well with the decreased cyclin B1 mRNA levels, but had no significant effect on the levels of CDK-A mRNA.

Genotoxicity of nicotine in cell culture of Caenorhabditis elegans evaluated by the comet assay

To assess the genotoxicity of nicotine, its DNA-damaging effect on Caenorhabditis elegans cells was tested with the alkaline single-cell microgel electrophoresis (comet) assay. The degree of DNA migration (a measure of possible DNA single-strand breaks, alkali-labile sites, and incomplete excision repair sites) was expressed as the head DNA%, tail length, and Olive tail moment. Large differences were found between experimental variants: 0, 1, 10, and 100 μM (-)-nicotine. At concentrations of 1 and 10 μM, no damages were detected by the comet assay, and the Olive tail moment and tail length were significantly lower than in the control (P < 0.001). The highest head DNA% and the lowest tail length and Olive tail moment were observed in the presence of 1 μM of nicotine. At 100 μM of nicotine, a significant increase (P < 0.001) was observed in Olive tail moment and tail length (up to 2.7- and 3-fold, respectively, compared to the control). The results are consistent with the lowest head DNA% among the three tested variants. This study demonstrated that nicotine treatment had dose-dependent effects on the level of DNA damage. Generally, a high dose of nicotine (100 μM) is genotoxic, while a reasonably low concentration has a protective effect. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine in C. elegans is discussed.

Concentration- and time-dependent behavioral changes in Caenorhabditis elegans after exposure to nicotine.

Nicotine induces profound behavioral responses in the model organism Caenorhabditis elegans. We tested the effect of a broad range of concentrations of nicotine (from 0.001 mM to 30 mM in nematode growth medium) on C. elegans locomotor behavior. We also followed the time-course influence on the sensitivity of C. elegans to nicotine (from 0 min to 300 min). A low concentration (0.001 mM) of this alkaloid causes a reduction of the speed of movement. By contrast, moderate concentrations (0.01 and 0.1 mM) induced acceleration of the mean speed of locomotion of C. elegans. High doses of nicotine (above 1 mM) induced slowing down of the movements and, finally, paralysis. Time-dependent analysis revealed that the stimulating effect of nicotine abolished the slowing down of C. elegans in control experiments after 30 min in the presence of 0.001, 0.1 and 10 mM nicotine. In the presence of 0.1 mM nicotine, the stimulation phase lasted up to 70 min. The evidence indicates that nicotine can have dual effects on the speed of locomotion, which is dependent on differences in its dosage and treatment time.

A simultaneous localization and tracking method for a worm tracking system

Abstract The idea of worm tracking refers to the path analysis of Caenorhabditis elegans nematodes and is an important tool in neurobiology which helps to describe their behavior. Knowledge about nematode behavior can be applied as a model to study the physiological addiction process or other nervous system processes in animals and humans. Tracking is performed by using a special manipulator positioning a microscope with a camera over a dish with an observed individual. In the paper, the accuracy of a nematode’s trajectory reconstruction is investigated. Special attention is paid to analyzing errors that occurred during the microscope displacements. Two sources of errors in the trajectory reconstruction are shown. One is due to the difficulty in accurately measuring the microscope shift, the other is due to a nematode displacement during the microscope movement. A new method that increases path reconstruction accuracy based only on the registered sequence of images is proposed. The method Simultaneously Localizes And Tracks (SLAT) the nematodes, and is robust to the positioning system displacement errors. The proposed method predicts the nematode position by using NonParametric Regression (NPR). In addition, two other methods of the SLAT problem are implemented to evaluate the NPR method. The first consists in ignoring the nematode displacement during microscope movement, and the second is based on a Kalman filter. The results suggest that the SLAT method based on nonparametric regression gives the most promising results and decreases the error of trajectory reconstruction by 25% compared with reconstruction based on data from the positioning system

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells

Abstract Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)–MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer’s disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

In vitro genoprotective and genotoxic effect of nicotine on human leukocytes evaluated by the comet assay.

Abstract The comet assay was used to measure the DNA damage induced in vitro by nicotine in human leukocytes as the extent of DNA migration in the comet head area, tail length, percent DNA in the tail, and Olive tail moment. Samples of whole blood were collected and blood cells were challenged with acute doses of 0.1, 1 and 10 µM of (−)-nicotine for 60 minutes. We found that nicotine treatment had dose-dependent effects on the level of DNA damage. At 1 and 10 µM of nicotine, both Olive tail moment and percent DNA in the tail significantly increased (p < 0.001), compared to the control. In the presence of 10 µM of nicotine, the shortest tail length and the smallest head area were detected. At a concentration of 0.1 µM, surprisingly, DNA damage detected by the comet assay was lower than in the control, which was proved by the observed significantly (p < 0.001) lower Olive tail moment and percent DNA in the tail as well as larger head area. The results suggest that nicotine, at a reasonably low concentration (0.1 µM), comparable to those found in the blood of habitual smokers, may have a protective effect, whereas higher doses of nicotine (1 and 10 µM) are genotoxic. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine is discussed.

Behavior of Caenorhabditis elegans in a nicotine gradient modulated by food

Abstract Nicotine decreases food intake, and smokers often report that they smoke to control their weight. To see whether similar phenomena could be observed in the model organism Caenorhabditis elegans, we challenged drug-naïve nematodes with a chronic low (0.01 mM) and high (1 mM) nicotine concentration for 55 h (from hatching to adulthood). After that, we recorded changes in their behavior in a nicotine gradient, where they could choose a desired nicotine concentration. By using a combination of behavioral and morphometric methods, we found that both nicotine and food modulate worm behavior. In the presence of food (E. coli OP50) the nematodes adapted to the low nicotine concentration, when placed in the gradient, chose a similar nicotine concentration like C. elegans adapted to the high nicotine concentration. However, in the absence of food, the nematodes adapted to the low nicotine concentration, when placed in the gradient of this alkaloid, chose a similar nicotine concentration like naïve worms. The nematodes growing up in the presence of high concentrations of nicotine had a statistically smaller body size, compared to the control condition, and the presence of food did not cause any enhanced slowing movement. These results provide a platform for more detailed molecular and cellular studies of nicotine addiction and food intake in this model organism.

Chemoreception of Meloidogyne incognita and Caenorhabditis elegans on botanical nematicidals

Plant–parasitic nematodes cause serious damage to various agricultural crops worldwide, and their control necessitates environmentally safe measures. Plant secondary metabolites of botanical origin are tested here–in to study their effect in Meloidogyne incognita locomotion, being this an important factor affecting host inoculation inside the soil. We compare the effect to the respective behavioral responses of the model organism Caenorhabditis elegans. The tested botanical nematicidals, all reported of activity against Meloidogyne sp. in our previous works, belong to different chemical groups of small molecular weight molecules encompassing acids, alcohols, aldehydes and ketones. Specifically we report on the attractant or repellent properties of trans–anethole, (E,E)–2,4–decadienal, (E)–2–decenal, fostiazate, and 2–undecanone. The treatments for both nematode species were made at sublethal concentration levels, namely 1mM (<EC50), and the chemical control used for the experiment was the commercial nematicide fosthiazate and oxamyl. According to our results, trans–anethole, decenal, and oxamyl act as C. elegans attractants. 2–undecanone strongly attracts M. incognita. These findings can be of use in the development of nematicidal formulates, contributing to the disruption of nematode chemotaxis to root systems.

Chemoreception of botanical nematicides by Meloidogyne incognita and Caenorhabditis elegans

ABSTRACT Plant-parasitic nematodes, such as Meloidogyne incognita, cause serious damage to various agricultural crops worldwide, and their control necessitates environmentally safe measures. We have studied the effects of plant secondary metabolites on M. incognita locomotion, as it is an important factor affecting host inoculation inside the soil. We compared the effects to the respective behavioral responses of the model saprophytic nematode Caenorhabditis elegans. The tested botanical nematicides, all reported to be active against Meloidogyne sp. in our previous works, are small molecular weight molecules (acids, alcohols, aldehydes, and ketones). Here, we specifically report on the attractant or repellent properties of trans-anethole, (E,E)-2,4-decadienal, (E)-2-decenal, fosthiazate, and 2-undecanone. The treatments for both nematode species were made at sublethal concentration levels, namely, 1 mM (<EC50), and the chemical controls used for the experiments were the commercial nematicides fosthiazate and oxamyl. According to our results, trans-anethole, decenal, and oxamyl attract C. elegans, while 2-undecanone strongly attracts M. incognita. These findings can be of use in the development of nematicidal formulations, contributing to the disruption of nematode chemotaxis to root systems.

Caenorhabditis elegans as an emerging model for studying the basic biology of anorectic effects of nicotine

Nicotine decreases food intake, and smokers often report that they smoke to control their weight. To see whether similar phenomena could be observed in the model organism Caenorhabditis elegans, we challenged drug-naϊve nematodes with a chronic low (0.01 mM) and high (1 mM) nicotine concentration for 55 h (from hatching to adulthood). After that, we recorded changes in their behavior in a nicotine gradient, where they could choose a desired nicotine concentration. By using a combination of behavioral and morphometric methods, we found that both nicotine and food modulate worm behavior. In the presence of food the nematodes adapted to the low nicotine concentration, when placed in the gradient, chose a similar nicotine concentration like C. elegans adapted to the high nicotine concentration. However, in the absence of food, the nematodes adapted to the low nicotine concentration, when placed in the gradient of this alkaloid, chose a similar nicotine concentration like naive worms. The nematodes growing up in the presence of high concentrations of nicotine had a statistically smaller body size, compared to the control condition, and the presence of food did not cause any enhanced slowing movement. These results provide a platform for more detailed molecular and cellular studies of nicotine addiction and food intake in this model organism.