Assembly and sorting of the tonoplast potassium channel AtTPK1 and its turnover by internalization into the vacuole

The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.     

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.     

Endotoxemia reduces skeletal muscle protein synthesis in neonates

Protein synthesis in skeletal muscle is reduced by as much as 50% as early as 4 h after a septic challenge in adults. However, the effect of sepsis on muscle protein synthesis has not been determined in neonates, a highly anabolic population whose muscle protein synthesis rates are elevated and uniquely sensitive to insulin and amino acid stimulation. Neonatal piglets (n = 10/group) were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 microg. kg(-1). h(-1)]. Plasma amino acid and glucose concentrations were kept at the fed level by infusion of dextrose and a balanced amino acid mixture. Fractional protein synthesis rates were determined by use of a flooding dose of [(3)H]phenylalanine. LPS infusion produced a septic-like state, as indicated by an early and sustained elevation in body temperature, heart rate, and plasma tumor necrosis factor-alpha, interleukin-1, cortisol, and lactate concentrations. Plasma levels of insulin increased, whereas glucose and amino acids decreased, suggesting the absence of insulin resistance. LPS significantly reduced protein synthesis in longissimus dorsi muscle by only 11% and in gastrocnemius by only 15%, but it had no significant effect in masseter and cardiac muscles. LPS increased protein synthesis in the liver (22%), spleen (28%), kidney (53%), jejunum (19%), diaphragm (21%), lung (50%), and skin (13%), but not in the stomach, pancreas, or brain. These findings suggest that, when substrate supply is maintained, skeletal muscle protein synthesis in neonates compared with adults is relatively resistant to the catabolic effects of sepsis.     

Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53-Eps8 complex, supporting the existence of a Cdc42-IRSp53-Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53-Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.     

Follicular fluid adrenomedullin concentrations in spontaneous and stimulated cycles: relationship to ovarian function and endothelin-1 and nitric oxide

The objective of this study was to determine concentration of adrenomedullin (AM) in follicular fluid and whether a correlation exists between AM and nitric oxide (NO) or endothelin-1 (ET-1) levels in follicular fluid, serum 17beta-estradiol or other parameters of ovarian function in spontaneous and gonadotrophin stimulated ovarian cycles. Follicular fluid samples were obtained at oocyte retrieval from 50 women who underwent an in vitro fertilization (IVF) program: 40 undergoing ovarian hyperstimulation with recombinant FSH and 10 had spontaneous ovarian cycles. AM, ET-1, and NO were detected in all of the follicular fluid samples and their concentrations were similar in spontaneous and stimulated cycles. In patients undergoing ovarian stimulation, follicular fluid AM levels correlated with serum 17beta-estradiol concentration. No correlation was found between follicular AM concentration and parameters of ovarian function. Similarly, no relationship was observed between ET-1, NO, and AM follicular fluid concentrations in either spontaneous or stimulated cycles.This study suggests a possible regulatory effect of the sexual hormones on AM production by the ovary during the ovulatory process. The site of AM secretion and its function (if any), however, remain to be established.     

Mapping and characterization of polymorphism in mtDNA of Cryphonectria parasitica: evidence of the presence of an optional intron

The mitochondrial DNA (mtDNA) of the filamentous ascomycete Cryphonectria parasitica is large and polymorphic so, to better understand the nature of the polymorphisms within populations, a small collection of Italian strains of the fungus was examined. Known mtDNA polymorphisms were mapped and found to cluster in four regions of the mtDNA molecule, particularly in the RFLP region 2 where five different mtDNA haplotypes out of 13 strains were identified. This region included an area of 8.4kbp which was entirely sequenced in strain Ep155 showing the presence of two introns. An internal 3.2kbp portion was sequenced also in six additional strains. Sequence comparison of the C. parasitica mitochondrial intronic ORFs revealed similarities to known endonucleases such as those of Podospora anserina and Neurospora crassa. DNA sequence analysis showed that three polymorphisms of this mtDNA region within this population of 12 strains were due to the optional presence in the ND5 gene of an intron and of an intervening sequence within the intron. Evidence was also found within this population of mixed mitochondrial types within a single strain.     

A structure–activity study to identify novel and efficient substrates of the human semicarbazide-sensitive amine oxidase/VAP-1 enzyme

Kinetic studies were performed with various alkanamines as “substrate probes” of the properties of the active site of the human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). We found that the enzyme–substrate recognition step is mainly controlled by apolar interactions and that a “good” substrate has a molecular structure containing a long aliphatic chain and a second positive charge at a distance greater than 12 ? from the reactive amino group. In this context, we identified a novel substrate for the human SSAO/VAP-1, 1,12-diaminododecane (DIADO), which is characterised by the highest catalytic efficiency reported to date in comparison to the prototypic substrate benzylamine. Computational docking studies revealed the structural basis of this behaviour, highlighting the key role played by Lys393 in hindering substrate docking.     

Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake

Purpose

The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron.

Methods and Results

Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of ⁵⁷Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of ⁵⁷FeSO₄ or of ⁵⁷Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice.

Conclusions

The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis.     

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments.     

Metabolomic patterns associated to QTc interval in shiftworkers: an explorative analysis

Objectives: ¹H NMR-metabolomic approach was used to investigate QTc interval correlation with plasma metabolic profiles in shiftworkers.

Methods: Socio-demographic data, electrocardiographic QTc interval and plasma metabolic profiles from 32 male shiftworkers, were correlated by multivariate regression analysis.

Results: We found a positive correlation between QTc interval values, body mass index, glycemia and lactate level and a negative correlation between QTc interval and both pyroglutamate and 3-hydroxybutyrate plasma level.

Conclusions: Our analysis provides evidence of the association between clinical, metabolic profiles and QTc interval values. This could be used to identify markers of early effects and/or susceptibility in shiftworkers.