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In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.

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We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.

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A 24-year-old patient presented with flaccid quadriplegia due to severe hypokaliemia, initially presumed to have been induced by glycyrrhizin. Persistence of low potassium levels and hypertension led to the diagnosis of primary hyperaldosteronism related to an adrenal cortical tumor. After surgery, the patient recovered from hypertension and hypokaliemia.

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A rapid, sensitive, and specific liquid chromatography method for the simultaneous determination of four protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir) in human plasma is described. After a liquid-liquid extraction with terbutyl methyl ether and a sequential washing of the reconstituted sample with hexane, protease inhibitors are separated on a phenyl column using a simple binary mobile phase of ammonium acetate buffer:acetonitrile (48:52) (pH = 7.5) with an ultraviolet detection at 260 nm. The standard curves are linear in the range 0.025-1 microg/mL for saquinavir, 0.1-4 microg/mL for indinavir and nelfinavir, and 0.25-10 microg/mL for ritonavir, with an average recovery ranging from 79% to 99%, and with both low interday and intraday coefficients of variation (<15%). This assay is simple, rapid (15-minute interval between runs) , and useful for therapeutic monitoring of the protease inhibitors on a routine basis.

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The cytological location of ion channel antagonist-binding sites was studied in sunflower protoplasts using the fluorescent probes DM-Bodipy-PAA and DM-Bodipy-DHP. The binding specificity of the probes was established by competition experiments with Bepridil, phenylalkylamine (Verapamil) and dihydropyridine (Nifedipine) which are known as calcium and potassium channel antagonists. Quantitative image analysis of the fluorescence emitted by the protoplasts showed the existence of interactions between PAA- and DHP-binding sites. Moreover, studies on the cytolocalization of the PAA receptors by confocal imaging showed that in freshly isolated protoplasts, DM-Bodipy-PAA binds exclusively at sites located in the cortical region of the cell.

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Sunflower protoplasts were cultured in liquid medium under high atmospheric pressure (0.2 to 0.6 MPa) and the plating efficiency, cell wall synthesis and microtubule organization were assessed. In 7-day-old cultures under a pressure of 0.4 MPa and above, the division rate was strongly reduced by more than 60% as compared to the control. Although most of the protoplasts had begun to regenerate a new cell wall they were unable to complete this process. Pressure also had an inhibitory effect on microtubule synthesis. The percentage of protoplasts showing a disassembled cortical network of microtubules was significantly increased up to 60% of the population. These effects were reversible: when protoplasts were transferred to normal pressure most of them rapidly recovered their capacity to divide and afterwards developed normally. Culturing protoplasts under a pressurized atmosphere revealed to be a good model system for studying cortical microtubule dynamics.

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Leukocytoconcentration is an easy, fast and inexpensive technique for the diagnosis of leishmaniasis from peripheral blood. The technique involves concentration of blood parasites on a small surface of a microscope slide while the red blood cells are removed by lysis. The results, compared with those of other methods (examination of cultures of blood samples and bone marrow smears), were very good and accurate. All but one of our cases of leishmaniasis were patients with HIV co-infection. Leukocytoconcentration facilitates follow-up of cases and fast detection of any relapse.

PIP: The biological diagnosis of an infectious disease is ideally based upon isolating and identifying the pathogenic agent in the host tissue and establishing cultures after direct examination under a microscope. That procedure allows both an accurate diagnosis and an epidemiological survey of the disease. When leishmaniasis occurs in an AIDS patient or any other immunocompromised patient, however, the procedure is often unsatisfactory for the following reasons: the samples are difficult to collect, there may be few parasites, and their growth is slow or impeded by other pathogenic agents. The clinical features, when they are not specific, may be attributed to an etiology other than leishmaniasis. This paper describes an easy, fast, and inexpensive technique for diagnosing leishmaniasis from peripheral blood. Leukocytoconcentration involves concentrating blood parasites on a small surface of a microscope slide while the red blood cells are removed by lysis. The results, compared with those derived from examining cultures of blood samples and bone marrow smears, were very good and accurate. All but one of the cases of leishmaniasis studied were patients co-infected with HIV. Leukocytoconcentration facilitates the follow-up of cases and fast detection of any relapse.

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Antigenic diversity in five stocks of the tick-borne rickettsia Cowdria ruminantium, the causal agent of heartwater disease of ruminants, was studied by cross-immunity trials in goats and sheep. Complete absence of cross-protection was found only between the Kümm and Kwanyanga stocks, and in all other combinations there were various degrees of cross-immunity. Immunological strain differences were more pronounced in goats than in sheep.

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The Goodwin and Trainor model of pattern generation in calcium-regulated strain fields is studied in the case where calcium input and calcium output processes are involved. It is shown that the properties of the original model may still remain provided that the input-output processes are not unstable. In this last case, despite the eventual stabilizing effect of the calcium exchange term, perturbations of the generalized system can grow and lead to inhomogeneous solutions. Applications to cell differentiation and cell growth are discussed.

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In Bryales protonema , elongation rate plays an essential role in the determination of cell size. It regulates the intermitotic increase of apical cells and could act on nucleus movements. In constant growth conditions, the duration of the mitotic cycle of the apical cell is strongly correlated to the growth rate. However, the relationship between elongation rate and the rhythm of cellular division is not linear. In the apical cell, the distance of the nucleus from the apex is also correlated to the growth activity. This could determine the position of the daughter apical nucleus after mitosis.

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