Coumarin-Ser-Asp-Lys-Pro-OH, a fluorescent substrate for determination of angiotensin-converting enzyme activity via high-performance liquid chromatography

N-Acetyl-Ser-Asp-Lys-Pro-OH (AcSDKP-OH), a negative regulator of hematopoietic stem cell proliferation, is shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), a zinc-dipeptidyl carboxypeptidase, involved in cardiovascular homeostasis. Recently, a study carried out on captopril-treated volunteers revealed that the kinetics of [3H]AcSDKP-OH hydrolysis in vitro in the plasma of donors correlates closely to the plasmatic ratio angiotensin II/angiotensin I, which characterized the conversion activity of ACE. This prompted us to design a fluorescent substrate, 2-[7-(dimethylamino)-2-oxo-2H-chromen-4-yl]acetyl-SDKP-OH, or coumarin-SDKP-OH, which could be an alternative to the radiolabeled analogue used in that study, allowing an easier and more rapid determination of enzyme activity. We report here the synthesis and the determination of the kinetics constants of this fluorescent derivative compared with those of [3H]AcSDKP-OH with human plasma ACE (133 and 125 microM, respectively), which are in the same range as those of the physiological substrate angiotensin I. Furthermore, the hydrolysis of the fluorescent substrate shows the same sensitivity toward chloride concentration as the natural substrate, demonstrating its specificity for N-domain hydrolysis. This fluorescent derivative was used to develop a sensitive assay for the determination of ACE activity in human plasma.     

Genetics of frost hardiness in <Emphasis Type="Italic">Juglans regia</Emphasis> L. and relationship with growth and phenology

The growing interest in broadleaf timber plantations in the Mediterranean area has promoted several studies focusing on the identification and characterization of variability sources in main timber-producing species. J. regia is one of these species, well-adapted to this area, but with freezing, damages registrations. Breeding focused on productive traits should include knowledge of adaptation, required to obtain a good selection capable of producing a suitable turnover in timber plantations. In this study, the features evaluated were autumn and winter frost hardiness and some vegetative traits on 22 half-sib J. regia progenies. Budsticks were exposed to sub-zero temperatures in a controlled chamber and using measurements of relative electrolyte content, the LT50 values (°C) were calculated by each individual. The study was carried out on seven-year-old progenies. The familiar heritability of autumn frost hardiness was 0.68, and on winter, it was 0.77. The autumn frost behaviour correlated genetically with the length of the growing season (0.574 ± 0.351), and both autumn and winter frost hardiness correlated inversely with secondary annual growth measured at breast height (?0.654 ± 0.259 and ?0.740 ± 0.227, respectively). These results pointed that growth could therefore be improved without increasing the frost vulnerability. This should be important for growers, particularly under climate change conditions.     

Basal body positioning is controlled by flagellum formation in Trypanosoma brucei

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

Peptidomic analysis of skin secretions from Rana heckscheri and Rana okaloosae provides insight into phylogenetic relationships among frogs of the Aquarana species group

The members of the Aquarana (or Rana catesbeiana species group) form a monophyletic group comprising seven species: R. catesbeiana, Rana clamitans, Rana grylio, Rana virgatipes, Rana septentrionalis, Rana heckscheri and Rana okaloosae. Previous work has led to structural characterization of the antimicrobial peptides present in electrically-stimulated skin secretions from the first five species listed and this study presents the primary structures of orthologs from the river frog R. heckscheri and the Florida bog frog R. okaloosae. Peptidomic analysis of R. heckscheri and R. okaloosae skin secretions led to the identification of peptides with antimicrobial activity belonging to the ranalexin, ranatuerin-2, and temporin families. In addition, a peptide (GFLDIIKDTGKDFAVKILNNLKCKLAGGCPR) was isolated from R. okaloosae whose primary structure identified it as a member of the palustrin-2 family. Consistent with previous data based upon morphological analysis and comparisons of the nucleotide sequences of mitochondrial and ribosomal genes, cladistic analysis based upon a comparison of the amino acid sequences of antimicrobial peptides indicates a sister-group relationship between R. heckscheri and R. grylio and a close, but less well defined, phylogenetic relationship between R. okaloosae and R. clamitans.     

Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.     

Lipoprotein lipase inhibits hepatitis C virus (HCV) infection by blocking virus cell entry

A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.     

The timing of leaf fall affects cold acclimation by interactions with air temperature through water and carbohydrate contents

Three parameters (i.e. the water content, soluble sugar content and minimal air temperature) can be used to predict the cold acclimation process of walnut trees. In order to test this assumption, two-year-old walnuts were defoliated at two different dates, i.e. mechanical defoliation in early October (early leaf fall, EF) or natural defoliation in early November (natural leaf fall, NF) and conditioned in either outdoor freeze-deprived or cold-deprived (T_min > 13 °C) greenhouses over winter. Even if early defoliation date could have affected short day signal perception (SDSP), water balance and carbohydrate metabolism were more altered. EF treatment, by stopping transpiration, significantly increased tree's water content and at warm temperature high root activity stopped normal winter dehydration. Starch content decreased in all treatments, but there was only a significant increase in soluble sugar content when water content had sufficiently decreased. Thus, depending on date of defoliation, cold-deprived trees were or were not able to acclimate to frost (minimal frost hardiness = −21.8 °C vs. −22.1 °C in controls (freeze-deprived) for NF and −13.7 °C vs. −25.3 °C in controls for EF). Different treatments showed the relationship between minimal water content observed during winter and maximal soluble sugars synthesized. Thus, the cold acclimation process appeared dependent on these physiological parameters (water and soluble sugar contents) through the interaction between air temperature and timing of leaf fall.