Effect of cadmium stress on antioxidative enzymes during the germination of Serbian spruce [Picea omorika (Pan..) Purkynĕ]

When considering the effect of heavy metals on trees generally and on forest ecosystems especially,  importance is given to their influence on seed germination in metal polluted soil. There is insufficient data on  this subject, especially when conifers are concerned. In this work, the influence of high cadmium  concentrations on percentage germination, specific activities and isoenzyme patterns of catalase (CAT),  superoxide dismutase (SOD), and peroxidase (POD) during the germination of Serbian spruce [Picea omorika (Pan..) Purkynĕ] was studied. Cadmium chloride concentrations of up to 0.1 mM did not cause an inhibition of germination, while 1 mM concentration inhibited germination and the activities of catalase, superoxide  dismutase and peroxidase. The isoenzyme profile of catalase and superoxide dismutase did not change at high cadmium concentrations, while peroxidase expression of basic peroxidase (B5) with pI value of 9.1 increased. This isoform of POD can play an important role in the early development of Serbian spruce and its defense mechanism against heavy metals.Key words: Seed germination, catalase, peroxidase, superoxide dismutase, heavy metals

Oxalate oxidase and non-enzymatic compounds of the antioxidative system in young Serbian spruce plants exposed to cadmium stress

We studied changes in the concentrations of ascorbate and glutathione, composition of soluble phenolics, and activity of oxalate oxidase in 75-day-old Serbian spruce plants after exposure to 5 mu M and 50 mu M cadmium for 6-48 h. The presence of OxOx activity in a conifer species is here demonstrated for the first time. Both Cd concentrations induced a decrease of OxOx activity in treated plants in comparison with the control at all sampling dates. The concentrations of reduced glutathione, its oxidized form, and reduced ascorbate in the plants decreased during 48-h treatment with cadmium. Among simple phenolics, only catechin increased significantly during Cd treatment

Cell Death Parameters as Revealed by Whole-Cell Patch-Clamp and Interval Weighted Spectra Averaging: Changes in Membrane Properties and Current Frequency of Cultured Mouse Microglial Cells Induced by Glutaraldehyde

The physiological and biochemical factors that lead to cell death have not been recognized completely. To our knowledge, there are no data on the bioelectric parameters that characterize early period of cell death, as well as on the appearance of related membrane current frequencies. We studied early parameters of glutaraldehyde (GA)-induced cell death, by examining the membrane properties of mouse microglia using the whole-cell patch-clamp technique. In addition, we investigated the GA-induced changes in the membrane current frequency, to see if characteristic frequencies would appear in dying cell. For data analysis, we applied a new approach, an improved multiple moving window length analysis and interval weighted spectra averaging (IWSA). We chose GA for its ability to induce almost instantaneous cell death. The 0.6 \% GA did not induce changes in the bioelectric membrane properties of microglia. However, the 3 \% GA caused significant decrease of membrane capacitance and resistance accompanied by the prominent increase in the membrane currents and nearly ohmic current response of microglial cells. These data indicate that 3 \% GA caused complete loss of the membrane function consequently inducing instantaneous cell death. The membrane function loss was characterized by appearance of the 1.26-4.62 Hz frequency peak in the IWSA spectra, while no significant increase of amplitudes could be observed for cells treated with 0.6 \% GA. To our knowledge, this is the first record of a frequency associated with complete loss of the membrane function and thus can be considered as an early indicator of cell death.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}III45012, III41014, OI 173022

Multiple forms of superoxide dismutase in the apoplast and whole-needle extract of Serbian spruce [Picea omorika (Panc.) Purkyně]

Activity and isoenzyme composition of superoxide dismutase in the apoplast and whole-needle extract of Picea omorika (Panč.) Purkyně, was studied. Total SOD activity of the soluble fraction of the needle extract exceeded markedly that of the apoplastic SOD. Several acidic and two slow-migrating basic isoforms were found in the whole extract. Extracellular SOD had an extremely acidic isoform. Using specific inhibitors, we identified Cu/Zn- and MnSOD forms in the total extract, but only MnSOD in the apoplast. The Feisoform was not present in a detectable amount.Proučavana je aktivnost i izoenzimski sastav superoksid dismutaze u apoplastu i ekstraktu celih četina omorike [Picea omorika (Panč.) Purkyně]. Ukupna aktivnost superoksid dismutaze u solubilnoj frakciji ekstrakta četina je bila značajno veća od aktivnosti superoksid dismutaze u apoplastu. U ekstraktu celih četina nađene su nekoliko kiselih i dve sporo-migrirajuće bazne izoforme. Vanćelijska superoksid dismutaza sadrži samo jednu ekstremno kiselu izoformu. Koristeći specifične inhibitore, Cu/Zn- i Mn- superoksid dismutazne forme su identifikovane u ekstraktu celih četina, dok je u apoplastu nađena samo Mn- superoksid dismutaza. Feizoforma nije prisutna u merljivoj koncentraciji

Immobilization of chemically modified horse radish peroxidase within activated alginate beads

Imobilizacija peroksidaze iz rena unutar alginatnih kuglica je poboljšana hemijskom modifikacijom enzima i polisaharidnih lanaca. Peroksidaza i alginat su oksidovani perjodatom i naknadno modifikovani etilendiaminom. Najveća specifična aktivnost od 0,43 U/ml gela i 81% vezane aktivnosti je dobijeno korišćenjem aminovane peroksidaze i alginata oksidovanog perjodatom. Imobilizovani enzim je zadržao 75% originalne aktivnosti nakon 2 dana inkubacije u 80% (v/v) dioksanu i imao je povećanu aktivnost pri baznim pH vrednostima u poređenju sa nativnim enzimom. Tokom višestruke upotrebe u šaržnom reaktoru za oksidaciju pirogalola imobilizovana peroksidaza je zadržala 75% početne aktivnosti.Immobilization of horseradish peroxidase (HRP) within alginate beads was enabled by chemical modification of the enzyme and polysaccharide chains. HRP and alginate were oxidized by periodate and subsequently modified with ethylenediamine. Highest specific activity of 0.43 U/ml of gel and 81% of bound enzyme activity was obtained using aminated HRP and alginate oxidized by periodate. Immobilized enzyme retained 75% of its original activity after 2 days of incubation in 80% (v/v) dioxane and had increased activity in basic solutions compared to native enzyme. During repeated use in batch reactor for pyrogallol oxidation immobilized peroxidase retained 75% of its original activity

Mitochondrial activity detected by cantilever based sensor

Our team recently demonstrated that cantilever based devices can detect signature of life in a chemistry independent manner. In this technique, the organism of interest is attached onto a classical AFM cantilever. If alive, it induces nanometre scale oscillations of the cantilever that disappear once the organism is killed. The technique was successfully used on bacteria, yeast, vegetal and mammalian cells. In this work we demonstrate that the method can also be applied to sub-cellular organelles, such as mitochondria. Mitochondria are involved in cellular energy production and are present in most eukaryotic cells. Nowadays, it is believed that mitochondria were originally prokaryotes that colonized eukaryotic cells and that live in an endosymbiotic way ever since. Here we present that mitochondria are also animated by nanometre scale oscillations that depend on their metabolic state and that stop once they are inhibited. This observation opens novel avenues to investigate the numerous mitochondria-related diseases in humans

Antioxidant volatiles of the freshwater bryozoan <i>Hyalinella punctata</i>

<div><p>Two volatile samples of the bryozoan <i>Hyalinella punctata</i> (seasonally collected from the same locality) were isolated (hydrodistillation, Clevenger apparatus), identified (GC and GC–MS) and screened <i>in vitro</i> (EPR) for anti-hydroxyl radical activity. The main components of sample 1 (2-ethyl-1-hexanol 37.00%, dodecanol 21.40% and hexanal 8.40%) and sample 2 (2-ethyl-1-hexanol 30.50%, 7-tridecanol 24.60% and 1-hexadecanol 11.80%) were relatively similar. However, more components were present in the sample 2 (17 and 25, respectively). EPR measurements indicated significant anti-hydroxyl radical activity of the both samples (75.00 ± 6.00% and 87.00 ± 8.00%, respectively) whereas the generation of other types of free radicals in reaction with hydroxyl radicals was not observed. According to the best of our knowledge, this is the first report of 31 organic compounds from the phylum Bryozoa. Alcohols, aldehydes, ketones, esters and ethers of lower molecular mass appear to be characteristic for the volatiles of these organisms commonly known as moss animals.</p></div