Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA

The AKT2 K(+) channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA (beta-glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K(+) transport and membrane polarization during stress situations.     

Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.     

Ghrelin, macronutrient intake and dietary preferences in long-evans rats

Ghrelin is a recently discovered peptide that is primarily produced by the stomach. As a ligand of the growth hormone (GH) secretagogue (GHS) receptor, it stimulates GH secretion but it also stimulates feeding and has adipogenic effects in rodents. Although its circulating levels are modulated by fasting and refeeding, its relationship with diet composition is not known. In the present paper, we measured plasma ghrelin as well as two important hormones (leptin and insulin) in Long-Evans rats placed in two different feeding situations, e.g., either with imposed diets or with food choice. In the first case, the rats were fed unbalanced diets (either high-carbohydrate (HC) or high-fat (HF) diets) for 14 weeks, whereas in the second situation, they had the choice between these 2 diets for 2 weeks and were selected for their fat or carbohydrate preference. The intake of the HF diet for 14 weeks was associated with lower levels of ghrelin (-30% vs control diet; P < 0.01). These levels increased when the percentage of carbohydrate in the diet increased (+26 to +42% vs control diet; P < 0.01 or less). Ghrelin was inversely correlated with plasma leptin (r = -0.55; P < 0.003) and blood glucose (r = -0.58; P < 0.001) as well as with body weight (r = -0.63; P < 0.0001) and body fat content estimated by the sampling of specific fat pads (r = -0.62; P < 0.0001). In the food choice experiment, fat-preferring rats had plasma ghrelin levels lower than the carbohydrate-preferring rats (-33%; P < 0.0002). Ghrelin secretion was therefore very sensitive to the diet composition. Its down-regulation by fat ingestion might serve as a counterregulatory mechanism to limit the development of dietary-induced adiposity. Ghrelin may signal when a high calorie diet is ingested.     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

Effect of tyramine, a dietary amine, on glycerol and lactate release by isolated adipocytes from old rats

Amine degradation by adipocyte amine oxidases leads to the production of metabolites that interact with lipid and glucose metabolisms and their hormonal regulations. To further investigate these interactions, we determined the effect of a dietary amine, tyramine (TYR), on glycerol and lactate releases, respectively taken as indices of lipolytic and glycolytic activities of isolated adipocytes. Old male Wistar rats were used to prepare adipocytes by collagenase dissociation of retroperitoneal fat pads. The two tested doses of tyramine (10 microM and 1 mM) had no effect on basal glycerol release. On the other hand, TYR, at the highest dose tested (1 mM), weakly but significantly increased basal lactate release, which was elevated in adipocytes from old rats. Norepinephrine (NE), highly stimulated adipocyte lipolysis with a submaximal effect at 1 microM which was slightly but significantly inhibited by TYR 1 mM. Insulin 1 nM (INS) also poorly inhibited the NE-stimulated lipolysis in adipocytes isolated from old rats. TYR was able to potentiate the poor antilipolytic efficiency of INS. Under similar conditions, a high dose of NE greatly reduced lactate production and TYR (1 mM) reversed this inhibition of lactate release. INS was also able to totally reverse the inhibitory effect of NE on lactate release, but there was no potentiation between insulin and tyramine effects. It can be concluded that high doses of TYR interact with norepinephrine and insulin, at least on the control of glycerol and lactate release, by counteracting catecholamine effects and by mimicking insulin actions.     

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.     

Ancient introgression of Lepus timidus mtDNA into L. granatensis and L. europaeus in the Iberian Peninsula

A 587 bp fragment of cytochrome b sequences from 90 individuals of 15 hare (Lepus) species and two outgroups were phylogenetically analysed and compared to an analysis derived from 474 bp sequences of the nuclear transferrin gene. Mountain hare (Lepus timidus) type mtDNA was observed in L. granatensis and L. europaeus from the Iberian Peninsula, far away from the extant distributional range of L. timidus. In addition to these two hare species, other hare species may also contain mtDNA from L. timidus. This species may have introgressed with other species of Lepus that occur within its present range, or where fossils indicate its historical presence during glacial periods. L. timidus mtDNA is common in the northern part of the L. granatensis range. Finally, we reassessed the phylogenetic relationships of the five European hare species based on both mitochondrial and nuclear DNA sequences.     

Effects of deletion of the prolactin receptor on ovarian gene expression

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, and is mainly considered as a regulator of reproduction and cell growth. Null mutation of the PRL receptor (R) gene leads to female sterility due to a complete failure of embryo implantation. Pre-implantatory egg development, implantation and decidualization in the mouse appear to be dependent on ovarian rather than uterine PRLR expression, since progesterone replacement permits the rescue of normal implantation and early pregnancy. To better understand PRL receptor deficiency, we analyzed in detail ovarian and corpora lutea development of PRLR-/- females. The present study demonstrates that the ovulation rate is not different between PRLR+/+ and PRLR-/- mice. The corpus luteum is formed but an elevated level of apoptosis and extensive inhibition of angiogenesis occur during the luteal transition in the absence of prolactin signaling. These modifications lead to the decrease of LH receptor expression and consequently to a loss of the enzymatic cascades necessary to produce adequate levels of progesterone which are required for the maintenance of pregnancy.