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During cultivation, wheat is exposed to several abiotic and/or biotic stress conditions that may adversely impact the wheat yield and quality. The impact of abiotic stress caused by nitrogen deficiency and biotic stress caused by phytopathogenic fungus Fusarium culmorum on biomarkers of oxidative stress in the flag leaf of nine winter wheat varieties (Ficko, U-1, Galloper, BC Mandica, BC Opsesija, Ingenio, Isengrain, Felix, and Bezostaya-1) was analyzed in this study. Hydrogen peroxide concentration and lipid peroxidation level were measured as indicators of oxidative stress, while the antioxidant response was determined by measuring the concentration of phenolic compounds and activities of antioxidant enzymes. Wheat variety and nitrogen treatment had a significant effect on all examined biomarkers of oxidative stress in the flag leaf, while the impact of Fusarium treatment was less pronounced. The most significant impact on the measured stress biomarkers had a low nitrogen level, which mainly increased hydrogen peroxide concentration and lipid peroxidation level and decreased activities of antioxidant enzymes in most varieties. The obtained results were discussed and compared with the previous study in which biochemical analyzes were performed on the wheat spike. There was no significant strong correlation between flag leaf and spike response in the measured parameters, which, in addition to the variety-specific response, also indicates a tissue-specific antioxidant response.

The nicotinamide derivates represent the promising candidate in the field of pesticides due to significant fungicidal activity, insecticidal activity, herbicidal activity, plant growth regulator activity and bactericidal activity. The series of nicotinamide-bromides with various alkyl chain lengths (from ethyl to octadecyl) were synthesized, and the toxicity of aqueous solutions towards Fusarium graminearum, Sclerotinia sclerotiorum, and Botrytis cinerea was examined. The obtained data were compared with DFT calculations results to understand the structural origin of fungicidal behavior and correlate it with the structural properties of investigated molecules. A significant correlation was established between the lipophilicity of the molecules (obtained from DFT calculations) and the growth rate of the examined phytopathogenic fungi. The results had suggested that Sclerotinia sclerotiorum is most sensitive to nicotinamide compounds exposure, while F. graminearum is most resistant to the change of lipophilicity of compounds. For all investigated compounds, the growth rate was decreased with the increase of carbon atoms in the side-chain until [C14Nic] [Br]. Further prolongation increased the growth rate of fungus, as it was noted for [C16Nic] [Br] and [C18Nic][Br]. This behavior was explained by the distinguished hydrophobic and hydrophilic surfaces in [C14Nic][Br] due to interactions between keto oxygen and bromide anion absent in the case of nicotinamides with a longer chain.

Botulinum toxin type A (BoNT-A) is a potent presynaptic neurotoxin and a standard therapy in hyperkinetic movement disorders, presumably due to its local muscular anticholinergic effect. However, recent experimental data point to possible central effects in the CNS. The present aim was to examine the contribution of the transcytosis-dependent central toxin action on the long term muscular function recovery in rats, as well as TeNT-evoked spastic paralysis. Rats were bilaterally injected with BoNT-A into the gastrocnemius muscle (2 U/kg) or sciatic nerve (5 U/kg). The following day, the animals were injected intrathecally (i.t.) with BoNT-A- neutralizing antitoxin. On day 62, animals were injected i.m. with tetanus neurotoxin (TeNT). In different motor tests (gait ability score, digit abduction score, rota-rod, beam walking and swimming performance), i.t. antitoxin significantly accelerated the flaccid paralysis and motor performance recovery. BoNT-A reduced the lower hind-limb diameter and muscle size without significant recovery during the entire experiment, which resulted in reduction of CMAP and H reflex. The TeNT-evoked increase in muscle tone was reduced by BoNT-A dependently on its central effect. However, the H-reflex, when corrected for reduced muscle size or reduced CMAP, was not affected by the toxin treatment, suggestive of the lack of the toxin’s direct effect on monosynaptic reflex. The enzymatic activity of the toxin, examined by cleaved synaptosomal-associated protein 25 (cSNAP-25) immunohistochemistry, was still present in neuromuscular junctions and spinal cord. The central occurrence of the cSNAP- 25, present in second order spinal cord cholinergic neurons, depended on the toxin’s central transcytosis. Long term motor effects of BoNT-A both on normal motor performance (day 1-62 ), as well as the spastic paralysis (days 62-78), are influenced by the toxin’s ongoing central action mediated by retrograde transport and transcytosis. These data suggest that clinically relevant beneficial effect of BoNT-A result from toxin’s combined peripheral and central effects.

Introduction: Due to presumable action on local neuromuscular terminals, botulinum toxin type A (BoNT-A) is widely used in different muscle hyperactivity disorders. However, clinical observations suggest possible central effects. Herein, we examined the contribution of BoNT A long term central effects on normal muscle function and recovery from paralysis, as well as tetanus neurotoxin (TeNT) evoked spasticity. Materials and methods: BoNT-A was bilaterally injected into rat gastrocnemius muscle (2 U/kg) or sciatic nerve (5 U/kg). To prevent spinal transcytosis, BoNT-A-neutralizing antitoxin was administered into the lumbar intrathecal (i.t.) spine. To examine late antispastic action on disinhibited muscle tone and stretch reflex, 1.5 ng TeNT was injected unilaterally into gastrocnemius muscle on day 62 post BoNT-A. BoNT-A enzymatic activity was examined in injected muscle and spinal cord by cleaved synaptosomal-associated protein 25 (cSNAP-25) immunohistochemistry. Results: I.t. antitoxin significantly accelerated the flaccid paralysis and motor performance recovery in different motor tests (gait ability score, digit abduction score, rota-rod, beam walking and swimming performance). Dependently on its central effects, BoNT-A reduced the TeNT-evoked increased muscle tone, however, did not affect the disinhibited stretch reflex. In contrast to cSNAP-25 continuous presence in injected muscle, BoNT-A action in second order spinal cord cholinergic neurons depended on the toxin’s central transcytosis. Conclusion: Normal motor performance (day 1-62), as well as the spastic paralysis (days 62- 78), are influenced by BoNT-A ongoing central action, suggesting a clinically relevant benefit resulting from combined peripheral and central toxin effects.

Introduction: Botulinum toxin type A (BoNT-A) is a potent neurotoxin with anticholinergic effect. It is a standard therapy in various movement disorders, presumably due to action on local neuromuscular terminals. However, clinical findings and recent experimental data, points to the possible central effects. Moreover, BoNT-A non- local actions have been reported after BoNT-A treatments as effects on distant muscles located far from treated neuromuscular junctions or muscle spindle zone. Herein, after toxin's peripheral injection, the aim was to examine the contribution of the transcytosis-dependent central toxin action, on normal tone muscle function and recovery, as well as tetanus neurotoxin (TeNT) evoked spasticity Methods: Rats were bilaterally injected with BoNT- A into the gastrocnemius muscle (2 U/kg) or sciatic nerve (5 U/kg). To stop the toxin central transcytosis, BoNT-A-neutralizing antitoxin was intrathecally (i.t.) administered after 24 hours. After recovery from flaccid paralysis, TeNT was intramuscularly (i.m.) injected to animals on day 62, and animals were followed for next 14 days, until they recovered from evoked spastic paralysis. Results: In different motor tests (gait ability score, digit abduction score, rota-rod, beam walking, catwalk), i.t. antitoxin significantly accelerated the flaccid paralysis and motor performance recovery. TeNT-evoked increase in muscle tone was reduced by BoNT-A dependently on its central effect. However, the H-reflex, when corrected for reduced muscle size or reduced compound muscle action potential (CMAP), was not affected by the toxin treatment, suggestive of the lack of the toxin’s direct effect on monosynaptic reflex. The toxin enzymatic activity examined by cleaved synaptosomal associated protein 25 (cSNAP-25) immunohistochemistry, was still present in neuromuscular junctions and spinal cord. cSNAP-25, presence in second order spinal cord cholinergic neurons, depended on the toxin’s central transcytosis. Conclusion: Long term motor effects of BoNT-A both on normal motor performance (day 1-62), as well as the spastic paralysis (days 62-78), are influenced by the toxin’s ongoing central action mediated by retrograde transport and transcytosis. These data suggest that clinically relevant beneficial effect of BoNT-A result from toxin’s combined peripheral and central effects.

Botulinum toxin type A (BoNT-A) is a potent neurotoxin with anticholinergic effect. It is a stand ard therapy in various movement disorders, presumably due to action on local neuromuscular terminals. However, observations in clinics and recent experimental data, points to the possible central effects. The aim was to examine the contribution of the transcytosis-dependent central toxin action on the long term muscular function recovery in rats, as well as tetanus neurotoxin (TeNT) evoked spastic paralysis after peripheral application. Rats were bilaterally injected with BoNT-A into the gastrocnemius muscle (2 U/kg) or sciatic nerve (5 U/kg). To stop the toxin central transcytosis, BoNT-A-neutralizing antitoxin was intrathecally (i.t.) administered after 24 hours. After recovery from flaccid paralysis, TeNT was intramuscularly (i.m.) injected to animals on day 62. In different motor tests (gait ability score, digit abduction score, rota-rod, beam walking and swimming performance), i.t. antitoxin significantly accelerated the flaccid paralysis and motor performance recovery. TeNT-evoked increase in muscle tone was reduced by BoNT-A dependent ly on its central effect. However, the H-reflex, when corrected for reduced muscle size or reduced compound muscle action potential (CMAP), was not affected by the toxin treatment, suggestive of the lack of the toxin’s direct effect on monosynaptic reflex. The toxin enzymatic activity exam ined by cleaved synaptosomal-associated protein 25 (cSNAP-25) immunohistochemistry, was still present in neuromuscular junctions and spinal cord. cSNAP-25, presence in second order spinal cord cholinergic neurons, depended on the toxin’s central transcytosis. Conclusion:Long term motor effects of BoNT-A both on normal motor performance (day 1-62), as well as the spas tic paralysis (days 62-78), are influenced by the toxin’s ongoing central action mediated by retro grade transport and transcytosis. These data suggest that clinically relevant beneficial effect of BoNT-A result from toxin’s combined peripheral and central effects.

Selenium (Se) deficiency in human and animal nutrition is primarily due to low levels of Se in soils. It can be prevented by enriching crops, such as wheat, with Se by genetic and agronomic biofortification. Although Se is not essential for plants, it shows a double effect on their metabolism. This study aimed to elucidate the impact of five different concentrations (0.4, 4, 20, 40 and 400 mg kg1) of selenate and selenite on the oxidative status and detoxifying systems of wheat (Triticum aestivum L., cv. Kraljica) shoots and roots and to determine biochemical and molecular tissue-specific responses. According to morpho-physiological analyses, selenite was found to have a lower toxicity threshold than selenate. Measurement of oxidative stress biomarkers showed that Se did not cause oxidative damage to wheat seedlings due to activation of detoxification mechanisms at the biochemical and molecular level, which depended on the type of tissue, concentration and form of applied Se. These results contribute to a better understanding of wheat seedlings' physiological, biochemical, and molecular response to Se and the development of more effective biofortification strategies.

An increased cadmium content in agricultural soils causes changes in the physiological and biochemical processes in plants, affecting their growth and yield. Different plant species, but also the different cultivars of the same species, widely vary in the terms of cadmium accumulation and tolerance. In this respect, the production of soybean (Glycine max L.) as a leading oil and protein culture is endangered by a low cadmium tolerance. Therefore, the aim of this study is to determine the differences in the biochemical mechanisms of cadmium tolerance in the leaves of three Croatian soybean cultivars (Korana, Ika, and Lucija), which differ in the capacity of cadmium accumulation. The soybean was hydroponically cultivated under the controlled conditions and exposed to cadmium (100 μmol L-1 ) for seven days. As the indicator of oxidative stress, the content of H2O2 and lipid peroxidation levels, expressed in terms of the thiobarbituric acid’s reactive substances, was determined, while an antioxidative status was determined by the activities of the antioxidative enzymes, such as the catalase, ascorbate peroxidase, glutathione peroxidase and guaiacol peroxidase. A cadmium treatment increased the lipid peroxidation levels in the leaves of all three soybean cultivars, indicating the presence of an oxidative stress. Also, the activity of glutathione peroxidase was increased in all cadmium‐treated plants, while other enzymes demonstrated different activity patterns in the investigated cultivars. The cultivar Korana, which manifested the highest level of cadmium accumulation, also manifested the highest level of tolerance with regard to two other cultivars. The most sensitive cultivar, Ika, with a moderate accumulation capacity, manifested the lowest level of an antioxidative response. The biochemical mechanisms of tolerance to a cadmium stress differed in the three investigated soybean cultivars, and a tolerance to cadmium was partly achieved by an increased antioxidative response.

Selenium (Se) is an important microelement with a dual effect ; at lower concentrations it is an essential element for animals, humans and some lower plants, but at higher concentrations, it can become toxic. The aim of this study was to investigate the influence of different concentrations of two selenium forms, selenate and selenite, on the oxidative and antioxidative status of wheat shoots and roots. Wheat grains were exposed to five different concentrations(0.1 mg L-1, 1 mg L-1, 5 mg L-1, 10 mg L-1, 100 mg L-1) of Se in a form of selenite and selenate forseven days. As biomarkers of the oxidative status in wheat, lipid peroxidation (LPO) levels and total H2O2 content were determined. The antioxidative status was determined by total reduced glutathione levels (tGSH), as well as by catalase (CAT) and ascorbate peroxidase (APX) activities. Both Se forms did not cause any significant changes in LPO levels and H2O2 content in shoots, while in roots selenite and selenite induced oxidative stress. tGSH levels in roots and shoot were reduced after the exposure to various concentrations of selenite and selenate relative to their controls. The results showed that both forms of Se decreased CAT activity in roots, while in shoots CAT activity did not change. Furthermore, only the highest concentrations of selenite and selenite increased the APX activity in shoots and roots. In conclusion, the plant response to Se depends on its chemical form, applied concentration and the analyzed part of the plant. The obtained results can serve as a basis for further studies of molecular and biochemical effects of Se on plants.

Anthropogenic activities increased cadmium content in agriculture soils leading to its entry into the food chain, thus causing numerous pathological consequences for the consumers. In addition, cadmium causes changes in physiological and biochemical processes in plants affecting their growth and yield. Different plant species, but also different cultivars of the same species, vary widely in the terms of cadmium accumulation and tolerance. Considering it, production of soybean (Glycine max L.) as a leading oil and protein culture is endangered by low cadmium tolerance. Therefore, the aim of this study is to determine differences in the biochemical mechanisms of cadmium tolerance in leaves of three Croatian soybean cultivars (Korana, Ika and Lucija), which differ in the capacity of cadmium accumulation. As the indicators of oxidative stress, content of H 2 O 2 and lipid peroxidation levels expressed in terms of thiobarbituric acid reactive substances were determined, while antioxidative status was determined by the activities of antioxidative enzymes such as catalase, ascorbate peroxidase, glutathione peroxidase and guaiacol peroxidase. Cadmium treatment increased lipid peroxidation levels in leaves of all three soybean cultivars, indicating presence of oxidative stress. Also, the activity of glutathione peroxidase was increased in all cadmium treated plants, while other enzymes showed different activity patterns in investigated cultivars. Cultivar Korana, that showed the highest level of cadmium accumulation, also demonstrated the highest level of tolerance in regard to other two cultivars. The most sensitive cultivar Ika with moderate accumulation capacity showed the lowest level of antioxidative response. Biochemical mechanisms of tolerance to cadmium stress differed in three investigated soybean cultivars, and tolerance to cadmium was partly achieved by increased antioxidative response.

Agronomic bio-fortification of wheat with selenium is a successful approach in improving its content in wheat grains. Due to its chemical similarity to sulphur, selenium is metabolized via sulphur assimilation pathways in plants. Therefore, in addition to oxidative stress induction, the application of selenium could also disturb protein structure and consequently influence glutathione metabolism in plants. Glutathione is involved in different processes such as biosynthetic pathways, detoxification of xenobiotics, antioxidant defence and maintenance of redox homeostasis. The aim of this study was to determine the impact of different concentrations of selenate and selenite on the glutathione metabolism in wheat seedlings (Triticum aestivum L.). For this purpose, contents of total, reduced and oxidised glutathione were determined, as well as the activities of the enzymes included in the glutathione metabolism such as glutathione reductase, glutathione peroxidase and glutathione S-transferase. Results showed that both forms of selenium affected total, reduced and oxidised glutathione content and activities of different enzymes in wheat seedlings. The glutathione content and enzyme activities depended on applied concentration of selenate and selenite. Both forms of selenium had more significant impact on the measured parameters in the root compared to wheat shoots, suggesting tissue-specific response. Obtained results can serve as a basis for further research on selenium toxicity and detoxification mechanisms in wheat. New insights into the selenium impact on glutathione metabolism could contribute to the further development of bio-fortification strategies.

Selenium (Se) deficiency in human and animal nutrition could lead to development of different pathophysiological conditions. Accordingly, biofortification of wheat, as one of the most important food crop, with Se is sustainable strategy for overcome this problem. Although not essential for plants, Se shows dual impact on their metabolism, at low levels it has a beneficial effect, while it is toxic at higher levels. The aim of this study was to elucidate the impact of increased concentrations of selenate and selenite on the oxidative stress and antioxidative response in wheat seedlings (Triticum aestivum L.). As an indicators of oxidative stress, content of H2O2 and lipid peroxidation levels expressed in terms of thiobarbituric acid reactive substances were determined, while antioxidative status was determined by the content of total glutathione and by the activities of antioxidative enzymes such as catalase and ascorbate peroxidase. Results showed that the effect of Se on oxidative stress and antioxidative response in wheat seedlings depended on its chemical form and applied concentration. Both forms of selenium had more significant impact on the measured parameters in the root compared to wheat shoots, suggesting tissue-specific response. Obtained results can serve as a basis for further research on the Se impact on oxidative and antioxidative status in order to get better insight into the wheat detoxification mechanisms.