Involvement of calcium signalling in dormancy release of grape buds

Artificial induction of grape bud dormancy release by hydrogen cyanamide (HC) serves as a reliable model system to explore the events occurring shortly after the induction of dormancy release. Recently, a group of genes with remarkable differences in expression level between HC-treated and control buds was identified. The identification of several calcium signalling-related genes within that group raised the hypothesis of the involvement of Ca(2+) signalling in grape bud dormancy release. Therefore, the effects of HC treatment on the expression profiles of several calcium sensors, the effect of the plasma membrane calcium channel blocker LaCl(3) and the calcium chelator EGTA on HC-induced and chilling-induced bud-break, and the effect of HC application on calcium-dependent protein phosphorylation activities in the bud tissue were studied. Here the HC-induced expression of Ca(2+)-ATPase is described, indicating that this treatment might evoke an increase in [Ca(2+)]cyt. Similar induction was confirmed for calmodulin, calmodulin-binding protein, and calcium-dependent protein kinase (CDPK). Both LaCl(3) and EGTA blocked the inducing effect of HC on bud-break, and their inhibitory effects were removed by supplying exogenous Ca(2+). Calcium-dependent histone phosphorylation was up to 70% higher in HC-treated buds. Endogenous protein phosphorylation assays detected four proteins exhibiting increased phosphorylation following HC treatment, of which two were phosphorylated in a calcium-dependent manner. One of these, a 47 kDa protein, presented strong and Ca(2+)-dependent phosphorylation only in HC-treated buds. The potential role of CDPK in the phosphorylation of this protein was supported by an immunoprecipitation assay. The data suggest, for the first time, that calcium signalling is involved in the mechanism of bud dormancy release.     

Impact of using multiple causes of death codes to compute site-specific, death certificate-based cancer mortality statistics in the United States

Background: Cancer mortality statistics, an important indicator for monitoring cancer burden, are traditionally restricted to instances when cancer is determined to be the underlying cause of death (UCD) based on information recorded on standard certificates of death. This study's objective was to determine the impact of using multiple causes of death codes to compute site-specific cancer mortality statistics. Methods: The state cancer registries of California, Colorado and Idaho provided linked cancer registry and death certificate data for individuals who died between 2002 and 2004, had at least one cancer listed on their death certificate and were diagnosed with cancer between 1993 and 2004. These linked data were used to calculate the site-specific proportion of cancers not selected as the UCD (non-UCD) among all cancer-related deaths (any mention on the death certificate). In addition, the retrospective concordance between the death certificate and the population-based cancer registry, measured as confirmations rates, was calculated for deaths with cancer as the UCD, as a non-UCD, and for any mention. Results: Overall, non-UCD deaths comprised 9.5 percent of total deaths; 11 of the 79 cancer sites had proportions greater than 3 standard deviations from 9.5 percent. The confirmation rates for UCD and for any mention did not differ significantly for any of the cancer sites. Conclusion and impact: The site-specific variation in proportions and rates suggests that for a few cancer sites, death rates might be computed for both UCD and any mention of the cancer site on the death certificate. Nevertheless, this study provides evidence that, in general, restricting to UCD deaths will not under report cancer mortality statistics.     

Mutagenesis at two distinct phosphate-binding sites unravels their differential roles in regulation of Rubisco activation and catalysis

Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.     

Relationship between unicellular cyanobacteria established by indirect immunofluorescence of intact cells

Antisera prepared against intact, viable cells were used to show the applicability of a serological approach to detect relationships between unicellular cyanobacteria. Antisera were raised against eight unicellular cyanobacteria and two chlorophycean unicellular organisms. The staining reactivity of each antiserum was tested by the fixed indirect immunofluorescence assay against the different organisms, and each organism was tested for its reactivity with all of the different antisera. Absorption of antisera with the appropriate heterologous antigens was used to further characterize the relationship betweenAnacystis nidulans andSynechococcus cedrorum, and also the relationship between two subcultures of an isolate distinguished by morphological features. Absorption of antiserum was also used for the removal of antibodies to contaminating bacteria. The approaches used are suggested as a useful tool for determining relationships between unicellular cyanobacteria.     

On the relative efficiency of two- vs. one-stage production of astaxanthin by the green alga Haematococcus pluvialis

Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.     

Immunological evaluation of patients with hematological malignancies receiving ambulatory cytokine-mediated immunotherapy with recombinant human interferon-α2a and interleukin-2

Summary Immunological parameters were evaluated in patients treated with cytokine-mediated immunotherapy (CMI) consisting of low doses of recombinant human interferon 2a (rIFN) and recombinant human interleukin-2 (rIL-2) administered either concomitantly or sequentially by subcutaneous self-injections in an outpatient setting. Twenty-six patients with hematological malignancies and 2 metastatic melanoma patients in a progressive stage were enrolled in this clinical trial. Of the 26 patients, 24 were at a stage of minimal residual disease, including 14 patients who had received autologous bone marrow transplantation (ABMT) 2–5 months previously, 7 chronic myelogenous leukemia (CML) and 3 acute myeloid leukemia (AML) patients. Two patients (1 CML and 1 mult. myeloma) were treated at a stage of progressive disease. Non-MHC-restricted cytotoxicity directed against natural-killer(NK)-resistant (Daudi) and NK-sensitive (K562) target cells was assessed before, during and after CMI, either in fresh peripheral blood samples (spontaneous activity) or after in vitro rIL-2 activation (induced activity). Spontaneous killing activity was low prior to treatment, but increased upon termination of treatment in 10/15 evaluated cycels. rIL-2-activated cytotoxicity in vitro was markedly elevated in 8/12 and 6/8 patients after one and two cycles, respectively, of sequential treatment, as well as in 3/8 CML and 5/6 patients after one and two cycles, respectively, of concomitant treatment Activation of the T cell mitogenic response was demonstrated in 6/9 patients after concomitant CMI, while no such effect was observed throughout a sequential treatment in lymphoma and leukemia patients after ABMT. Although a direct correlation between immune stimulation and the in vivo antitumor response cannot yet be determined, our clinical observations support a beneficial therapeutic effect in a substantial number of patients. These results indicated that the ambulatory CMI protocol of rIL-2 and rIFN could stimulate the host defense immune system and may be helpful in mediating the in vivo antitumor response in patients with minimal residual disease.     

Patterns of carbohydrate and fatty acid changes under nitrogen starvation in the microalgae Haematococcus pluvialis and Nannochloropsis sp.

The aim of this research was to study the impact of nitrogen starvation on the production of two major secondary metabolites, fatty acids and carbohydrates, in two microalgae: Nannochloropsis sp. and Haematococcus pluvialis. The major response to nitrogen starvation in both algae occurred within the first 2 days, accompanied by a sharp reduction in chlorophyll content. However, the pattern of the response differed between the two microalgae. In H. pluvialis, the first response to nitrogen starvation was intensive production of carbohydrates, accumulating to up to 63% of dry weight by day 1; on day 2, the total carbohydrate content decreased and was partially degraded, possibly to support fatty acid synthesis. Under these conditions, H. pluvialis accumulated up to 35% total fatty acids in the biomass. In Nannochloropsis sp., the immediate and major response, which was maintained throughout the entire period of exposure to stress, was production of fatty acids, accumulating up to 50% of dry weight, while carbohydrate content in the biomass remained stable at 18%. In addition, we tested the effect of the lipid-synthesis inhibitor sesamol, known to inhibit malic enzyme, on the balance between total fatty acid and carbohydrate contents in H. pluvialis and Nannochloropsis sp. In both cultures, sesamol inhibited fatty acid accumulation, but the carbohydrate content was reduced as well, albeit to a lesser extent. These findings demonstrate the complexity of the stress-response and the potential link between fatty acid and carbohydrate synthesis.     

Isolation of hyperactive mutants of the MAPK p38/Hog1 that are independent of MAPK kinase activation

Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis. The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation. Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation. Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1. Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38alpha, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38. It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions.