Mitochondrial alternative oxidase contributes to successful tardigrade anhydrobiosis.

Anhydrobiosis can be described as an adaptation to lack of water that enables some organisms, including tardigrades, to survive extreme conditions, even some that do not exist on Earth. The cellular mechanisms underlying anhydrobiosis are still not completely explained including the putative contribution of mitochondrial proteins. Since mitochondrial alternative oxidase (AOX), described as a drought response element in plants, was recently proposed for various invertebrates including tardigrades, we investigated whether AOX is involved in successful anhydrobiosis of tardigrades. Milnesium inceptum was used as a model for the study. We confirmed functionality of M. inceptum AOX and estimated its contribution to the tardigrade revival after anhydrobiosis of different durations. We observed that AOX activity was particularly important for M. inceptum revival after the long-term tun stage but did not affect the rehydration stage specifically. The results may contribute to our understanding and then application of anhydrobiosis underlying mechanisms.

Behavior of Caenorhabditis elegans in a nicotine gradient modulated by food.

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 botanical nematicides by Meloidogyne incognita and Caenorhabditis elegans.

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 (

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

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.

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.

[Absorption, metabolism and excretion of nicotine in humans]. / Wchlanianie, przemiany metaboliczne i wydalanie nikotyny u czlowieka.

Nicotine is an alkaloid present in many plants of Solanaceae family. The levorotatory enantiomer (S) is a naturally occurring form. Nicotine enters the human body as a component of tobacco smoke. In alkaline environment the rate of nicotine permeation through biological membranes is increased. Almost 90% of nicotine absorbed by the body is metabolized in the liver. Nicotine may also be metabolized in the kidneys, lungs, brain, and respiratory epithelium membranes. The nicotine undergoes many transformations. Key role in the metabolism of nicotine is played by cytochrome P450 oxidases (mainly CYP2A6). Apart from them, UDP-glucuronosyltransferases, cytosolic aldehyde oxidase, amine N-methyltransferase, and flavin-containing monooxygenase 3 are involved in the decomposition of nicotine. Six major metabolites of nicotine have been identified. One of the most important metabolite is cotinine, from which is formed of trans-3'-hydroxycotinine--the compound which is excreted in the largest amount within the urine. The rate of nicotine metabolism is affected by diversified activity of polymorphic enzymes involved in this process, diet, gender and physiological condition of the organism.

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.

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 microM (-)-nicotine. At concentrations of 1 and 10 microM, 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 microM of nicotine. At 100 microM 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 microM) 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.

Proteins induced by cadmium in soybean cells.

The cadmium (Cd)-induced changes in protein pattern and identification of metal-stimulated polypeptides were analyzed in soybean cell suspension culture. The cell cultures were treated with various concentrations of Cd(2+) (3-10microM) for 24, 48 and 72h. The synthesis of [(35)S]-labeled proteins and their accumulation were analyzed by SDS-PAGE, whereas the identification of selected protein bands was performed by mass spectrometry. It is shown that Cd induced the appearance of the following proteins in soybean cells: superoxide dismutase, histone H2B, chalcone synthase and glutathione transferase.

Genotoxicity of lead in lupin root cells as evaluated by the comet assay.

This paper presents the results of a study on the influence of lead (Pb(+2)) on DNA integrity on plant cells. The study was performed on the root tips of lupin (Lupinus luteus cv. Juno) seedlings treated with two selected concentrations of Pb(NO3)2: 150 and 350 mg l(-1), which were found to inhibit root growth by 50% and 70%, respectively [Rucinska et al. Plant Physiol. Biochem. 37 (1999) 37187-37194]. Roots exposed to those external lead concentrations took up about 50 and 70 mg l(-1) Pb(+2) g(-1) fresh weight (FW) over 48 h of incubation. A dose-dependent increase in the degree of root injury was observed in the presence of both tested concentrations. The genotoxicity of lead in lupin root cells was analysed using a mild alkaline comet assay at pH 12.3, which allows the detection of single strand breaks. The quantity of the DNA fragments migrating away from the nuclear remnant (tail area) increased proportionally to the lead content inside the roots, and was positively correlated with the degree of root injury. At 150 mg l(-1) Pb(+2), a high frequency distribution of nuclei having large values of tail lengths and moments was observed. By contrast, the number of nuclei with minimum values of these parameters increased at 350 mg l(-1) Pb(+2). This data suggests that lead at low concentrations induces the formation of short, rapidly migrating DNA fragments, whereas at higher concentrations, lead probably causes other changes to DNA that result in slower DNA migration in the electric field.

Cadmium-induced changes in antioxidant enzymes in suspension culture of soybean cells.

Cadmium (Cd), similarly to other heavy metals, inhibits plant growth. We have recently showed that Cd(2+) either stimulates (1-4 microM) or inhibits (>or= 6 microM) growth of soybean (Glycine max L.) cells in suspension culture (Sobkowiak & Deckert, 2003, Plant Physiol Biochem. 41: 767-72). Here, soybean cell suspension cultures were treated with various concentrations of Cd(2+) (1-10 microM) and the following enzymes were analyzed by native electrophoresis: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APOX). We found a significant correlation between the cadmium-induced changes of soybean cell culture growth and the isoenzyme pattern of the antioxidant enzymes. The results suggest that inhibition of growth and modification of antioxidant defense reactions appear in soybean cells when Cd(2+) concentration in culture medium increases only slightly, from 4 to 6 microM.