Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum

Key message In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. Abstract The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Colocalization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50 % of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species

Inter- and intrachromosomal asynchrony of cell division cycle events in root meristem cells of Allium cepa: possible connection with gradient of cyclin B-like proteins

Alternate treatments of Allium cepa root meristems with hydroxyurea (HU) and caffeine give rise to extremely large and highly elongated cells with atypical images of mitotic divisions, including internuclear asynchrony and an unknown type of interchromosomal asynchrony observed during metaphase-to-anaphase transition. Another type of asynchrony that cannot depend solely on the increased length of cells was observed following long-term incubation of roots with HU. This kind of treatment revealed both cell nuclei entering premature mitosis and, for the first time, an uncommon form of mitotic abnormality manifested in a gradual condensation of chromatin (spanning from interphase to prometaphase). Immunocytochemical study of polykaryotic cells using anti-β tubulin antibodies revealed severe perturbations in the microtubular organization of preprophase bands. Quantitative immunofluorescence measurements of the control cells indicate that the level of cyclin B-like proteins reaches the maximum at the G2 to metaphase transition and then becomes reduced during later stages of mitosis. After long-term incubation with low doses of HU, the amount of cyclin B-like proteins considerably increases, and a significant number of elongated cells show gradients of these proteins spread along successive regions of the perinuclear cytoplasm. It is suggested that there may be a direct link between the effects of HU-mediated deceleration of S- and G2-phases and an enhanced concentration of cyclin B-like proteins. In consequence, the activation of cyclin B-CDK complexes gives rise to an abnormal pattern of premature mitotic chromosome condensation with biphasic nuclear structures having one part of chromatin decondensed, and the other part condensed

Influence of the light factor on the course of the cell cycle in the successive generations of the antheridial filaments of Chara vulgaris L.

The exposure to continuous light causes a rise of the mitotic index in the successive generations of the antheridial filaments of Chara vulgaris, whereas culture in darkness leads to a considerable depression of mitosis, and if prolonged, to its complete cessation. Light reverses the effect of the depression of mitosis caused by darkness: in the 16-, and 32-cell generations mitotic activity reappeares as early as after 3 h, but in the 2-, 4-, and 8-cell generations only after 18 h. The size of the cells in the antheridial filaments of plants exposed to continuous illumination is larger as compared with the control material. After being kept in darkness the cells become smaller. The analysis of the size of the latter suggests the inhibition of growth in initial and final periods of interphase. In all generations continuous light reduces the duration of the cell cycle while darkness protracts it. The duration of S phase is similar in all generations of antheridial filaments (ca. 16 h) and is not modified by the light conditions. Thus, the light factor seems to interfere mainly with G2 phase, and its role in the regulation of the cell cycle is correlated with the duration of this phase. The role of light in the kinetics of the cell cycle of the particular generations of antheridial filaments in Chara is discussed in the aspect of the nutritional requirements of cells, hormonal regulation and phytochrome

IAA and BAP affect protein phosphorylation-dependent processes during sucrose-mediated G1 to S and G2 to M transitions in root meristem cells of Vicia faba

In carbohydrate-starved root meristems of Vicia faba subsp. minor, the expression of two Principal Control Points located at the final stages of the G1 (PCP1) and G2 (PCP2) phases has been found to be correlated with a marked decrease of protein phosphorylation within cell nuclei, nucleoli and cytoplasm. Adopting the same experimental model in our present studies, monoclonal FITC conjugated antibodies that recognize phosphorylated form of threonine (αTPab-FITC) were used to obtain an insight about how the indole-3-acetic acid (IAA), benzyl-6-aminopurine (BAP), and the mixture of both phytohormones influence the time-course changes in an overall protein phosphorylation during sucrose-mediated PCP1→S and PCP2→M transitions. Unsuspectedly, neither IAA, BAP, nor the mixture of both phytohormones supplied in combination with sucrose did up-regulate protein phosphorylation. However using the block-and-release method, it was shown that root meristems of Vicia provided with sucrose alone indicated higher levels of αTPab-FITC. Contrarily, phytohormones supplied in combination with sucrose induced apparent decline in phosphorylation of cell proteins, which - when compared with the influence of sucrose alone - became increasingly evident in time. Thus, it seems probable, that a general decline in the amount of αTPab-FITC labeled epitopes may overlay specific phosphorylations and dephosphorylations governed by the main cell cycle kinases and phosphatases

Badanie poziomów dioksyn w surowym mleku krów i kóz w Polsce

Dioxins due to their toxic properties may pose health risks. These compounds comprise seven congeners of polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 12 dl-PCBs. Because the main source of human exposure to dioxins is food of animal origin, the goal of the studies was to assess the levels of 29 congeners of dioxins in cow and goat’s milk from Poland. High resolution gas chromatography coupled with high resolution mass spectrometry (HRGC-HRMS) were used to analyze more than 120 samples in the period of 2006 to 2011. In 94 samples of raw cow's milk an average concentration of PCDD/PCDFs was 0.84 ± 0.60 pg WHO-TEQ/g fat whereas the sum of 29 congeners was 1.35 ± 0.89 pg WHO-TEQ/g fat. The concentrations of dioxins and dl-PCBs were low (30% of limits for whole milk) and the samples met the requirements of the national and European legislation. Within the period of examinations two samples of cow's milk demonstrated the concentrations of PCDD/PCDFs at the action level (2 pg WHO-TEQ/g fat) whereas only one sample revealed the concentration exceeding the permissible content of PCDD/PCDFs (3 pg WHO-TEQ/g fat). Raw goat's milk contains generally higher concentrations than those found in cow’s samples tested (PCDD/PCDFs and dl-PCBs) and the average concentration of PCDD/PCDFs was 1.49 ± 0.97 pg WHO-TEQ/g fat. Of the 33 examined goat’s milk samples, 6 demonstrated the concentrations of dioxins at the “action level” whereas in 1 sample the content of dioxins exceeded the acceptable limits. The obtained results allow to conclude that cow and goat’s milk contains generally low levels of dioxins. High concentrations of dioxins found could be a result of improper feeding of food producing animals.Dioksyny ze względu na właściwości toksyczne mogą stanowić zagrożenie dla zdrowia. Do grupy tej należy 7 kongenerów polichlorowanych dibenzo-p-dioksyn (PCDD), 10 polichlorowanych dibenzofuranów (PCDF) oraz 12 dl-PCB. Ponieważ głównym źródłem narażenia ludzi na dioksyny jest żywność pochodzenia zwierzęcego, celem pracy była ocena poziomów 29 kongenerów dioksyn w mleku krowim i kozim pochodzącym z terenu kraju. Metodą wysokorozdzielczej chromatografii gazowej sprzężonej z wysokorozdzielczą spektrometrią mas (HRGC-HRMS) analizowano w latach 2006-2011 ponad 120 próbek. W 94 próbkach surowego mleka krowiego średnie stężenie PCDD/PCDF wynosiło 0,84 ± 0,60 pg WHO-TEQ/g tł., zaś suma 29 kongenerów 1,35 ± 0,89 pg WHO-TEQ/g tł. Zawartość dioksyn i dl-PCB była niska (do 30% dopuszczalnych limitów dla mleka pełnego) i próbki spełniały wymagania przepisów krajowych i unijnych. W badanym okresie jedynie w dwóch próbkach mleka krowiego stwierdzono stężenie na poziomie progu podejmowania działań (2 pg WHO-TEQ/g tłuszczu) oraz w 1 próbce stężenie przekraczające dopuszczalną zawartość PCDD/PCDF (3 pg WHO-TEQ/g tłuszczu). Surowe mleko kozie generalnie zawierało wyższe niż krowie stężenia badanych związków (zarówno PCDD/PCDF, jak i dl-PCB) i średnia dla PCDD/PCDF wynosiła 1,49 ± 0,97 pg WHO-TEQ/g tłuszczu. W 6 spośród 33 badanych próbek mleka koziego stężenia dioksyn znajdowały się na poziomie „action level”, zaś w jednej próbce zawartość dioksyn przekroczyła dopuszczalne limity. Uzyskane wyniki pozwalają na stwierdzenie, że mleko zawiera niskie poziomy dioksyn, jakkolwiek zdarzały się incydenty wynikające najprawdopodobniej z błędów żywieniowych zwierząt hodowlanych (karma zawierająca dioksyny)

Application of screening method for dioxin determination in crisis situations

Dioksyny i dioksynopodobne PCB były źródłem wielu różnych awarii i katastrof ekologicznych oraz skażeń żywności i pasz na świecie. W Europie do najbardziej znanych i brzemiennych w skutkach należy katastrofa ekologiczna w 1976 roku w Seveso (Włochy). Innym przykładem awarii było skażenie mleka, jaj i tłuszczu zwierząt wypasanych na farmach w pobliżu spalarni komunalnych emitujących do środowiska duże ilości tych związków, skażenie mleka krów w wyniku pobierania dioksyn wraz z zanieczyszczoną pulpą cytrusową oraz największa katastrofa, która wystąpiła w Belgii i Europie (1999 rok) w wyniku dodania do pasz 200 litrów oleju skażonego PCB i dioksynami. Powyższe wydarzenia, jak również podjęta przez Wspólnotę Europejską strategia ochrony zdrowia mieszkańców przed szkodliwym działaniem dioksyn zawartych w żywności (ponad 90% dioksyn pobieranych jest wraz z żywnością), ujawniły potrzebę posiadania szybkich metod skryningowych do monitorowania na szeroką skalę żywności i pasz. Dla określenia aktywności biologicznej obecnych w paszy PCDD, PCDF i dl-PCB w opracowanych metodach przesiewowych wykorzystano ekspresję genu reporterowego, aktywność enzymów mikrosomalnych, wiązanie ligandu do receptora i przeciwciał. Obecnie biotest CALUX (ekspresja genu reporterowego) uznawany jest za najlepszą metodę przesiewowego oznaczania dioksyn i dl-PCB w żywności i paszach oraz jest jedynym testem stosowanym w rutynowych badaniach monitoringowych oraz sytuacjach kryzysowych. W pracy przedstawiono rezultaty stosowania metody przesiewowej, bazującej na genetycznie modyfikowanych komórkach hepatomy mysiej, wrażliwych na dioksyny. Podczas incydentów dioksynowych w naszym kraju z metody tej korzystano w badaniach żywności pochodzenia zwierzęcego i roślinnego (mięso i jego przetwory, mleko i produkty mleczne, pasze i składniki paszowe). Zastosowanie metody przesiewowej oznaczania dioksyn umożliwiło szybką selekcję próbek dodatnich i zwiększenie przepustowości laboratorium, co jest istotne w sytuacjach kryzysowych dla skrócenia czasu oczekiwania na wynik. W sytuacjach kryzysowych dotyczących skażenia dioksynami potwierdzono, że szybka metoda przesiewowa jest ważnym narzędziem w ocenie i zarządzaniu ryzykiem oraz bezpieczeństwem żywności.Dioxins and dioxin-like compounds have caused numerous ecological disasters and contamination of food and feed. In Europe, the most famous and fateful environmental disasters was the Seveso accident (Italy) in 1976. Another example was the contamination of milk, eggs and fat of animals grazing on farms near municipal waste incinerators emitting into the environment large amounts of these compounds, contamination of the milk of cows as a result of feeding citrus pulp contaminated with dioxin, and the biggest catastrophe that has occurred in Belgium and in Europe (1999) by adding to feed 200 dm3 of PCB and dioxin contaminated oil. These events, as well as taken by the European Community strategy for health protection of inhabitants from the harmful effects of dioxins in food (more than 90% of dioxin comes from the food), revealed a need for a rapid screening methods for monitoring food and feedstuffs at a large scale. To determine the biological activity of PCDD, PCDF and dl-PCBs present in the feed, developed screening methods use the expression of reporter gene, activity of microsomal enzymes, the binding of ligand to the receptor and to antibody. At present, bioassay CALUX (reporter gene expression) is considered to be the best screening test for determination of dioxins and dl-PCBs in food and feed, and it is the only test used in routine monitoring and in the crisis situations. The paper presents the results of a screening method application based on genetically engineered murine hepatomy cell line, sensitive to dioxins. This method was used to determine dioxins in food of animal and plant origin (meat and its products, milk and milk products, feedstuffs and feedstuff components) during dioxin crises. Application of the screening test allowed rapid selection of positive samples, increase laboratory throughput, and shorten waiting time for results. Shortening the waiting time for results is important in crisis situations. Rapid screening method is an important tool in assessing and managing risk and food safety in emergency situations on the dioxin contamination

Topoclimatic conditions in summer seasons in the Kaffioyra region (NW Spitsbergen) in the years 2005-2009

W artykule przedstawiono zróżnicowanie temperatury i wilgotności względnej powietrza oraz kierunku i prędkości wiatru w rejonie Kaffioyry (NW Spitsbergen) w sezonach letnich 2005-2009. Na podstawie pomiarów w 8 punktach stwierdzono znaczne różnice topoklimatyczne uwarunkowane rodzajem podłoża, wyso-kością nad poziom morza, odległością od morza, ekspozycją oraz lokalną cyrkulacją atmosferyczną. W rejonie Kaffioyry często występują sytuacje inwersyjne, związane nie tylko ze stratyfikacją termiczno-wilgotnościową napływających mas powietrza, ale również oddziaływaniem czynników lokalnych. Zróżnicowanie topoklima-tyczne zmienia się w zależności od stopnia zachmurzenia i pory doby oraz w czasie formowania się wiatrów lokalnych (wiatry lodowcowe i fenowe).The paper presents the spatial differentiation of the meteorological conditions in the summer seasons in the Kaffiřyra in the period 2005-2009. The meteorological measurement points (4 automatic weather stations and 4 electronic devices measuring temperature and humidity, 2 m a.g.l.) were located on the Kaffiřyra Plain (KH) on the Waldemar Glacier area (ATA, LW1, LW2) and on the mountains: Kuven (KU), Grĺfjellet (GF) and Prins Heinrichfjella (PH1, PH2). The analysed five seasons had changeable weather conditions dependent on types of synoptic situations. The highest air temperatures were recorded on the coast (KH 5.8°C) and on the marginal zone of the Waldemar Glacier (ATA 5.1°C). On the glaciated area air temperature is decreasing with the altitude (LW2 2.9°C). The largest temperature lapse-rate is recorded at the transitional area between the glacier and its marginal zone. Growing altitude lowers air temperature on the mountain ridges (GF 4.0°C, PH2 3.6°C), but temperature inversions are recorded quite frequently in the region. Relative air humidity is high due to low temperature and large frequency of occurrence of maritime air masses. The highest mean relative air humidity was recorded on the coast (KH 88%) and on the firn field of the Waldemar Glacier (LW2 84%) as well as on the mountain ridges (PH2 92%). The course of the relative humidity is significantly influenced by foehn winds. Wind directions and velocity in the study area are strongly dependent on the synoptic situation and influence of local factors, mainly orography (foehn winds). Wind regime in the Waldemar Glacier significantly differs from that observed in the Kaffiřyra (here the tunnel effect is observed as a consequence of the narrow Forlandsundet, presences to the abovementioned plain), mainly due to katabatic winds occurrence