Effects of dam breed and dietary source of n-3 polyunsaturated fatty acids on the growth and carcass characteristics of lambs sourced from hill sheep flocks

The objectives of this study were to investigate the effect of dietary lipid source on the growth and carcass characteristics of lambs sourced from a range of crossbred hill ewes. Over a 2-year period, 466 lambs representing the progeny of Scottish Blackface (BF × BF), Swaledale (SW) × BF, North Country Cheviot (CH) × BF, Lleyn (LL) × BF and Texel (T) × BF ewes were sourced from six commercial hill flocks and finished on one of four diets: grass pellets (GP), cereal-based concentrate (CC), CC enriched with oilseed rape (CR) and CC enriched with fish oil (CF). Dry matter intake (DMI) was highest (P < 0.001) in lambs offered GP; however, carcass weight gain (CWG) and feed conversion efficiency were higher (P < 0.001) in lambs fed concentrate-based diets. For lambs offered concentrate-based diets, DMI and live weight gain were lower (P < 0.001) for CF than CC or CR. Lambs with T × BF dams achieved a higher (P < 0.05) daily CWG and CWG/kg DMI than BF × BF, SW × BF or LL × BF dams. When lambs were slaughtered at fat score 3, CH × BF, LL × BF and T × BF dams increased carcass weight by 0.8 to 1.4 kg (P < 0.001) and conformation score (CS) by 0.2 to 0.4 units (P < 0.001) compared with BF × BF or SW × BF dams. However, breed effects on carcass conformation were reduced by 50% when lambs were slaughtered at a constant carcass weight. Diets CC and CR increased carcass weight by 0.8 to 1.6 kg (P < 0.001) and CS by 0.1 to 0.3 units (P < 0.001) compared with GP and CF. Both, dam breed and dietary effects on carcass conformation were associated with an increase (P < 0.001) in shoulder width of the lambs. Lambs fed CF and slaughtered at a constant carcass weight had more subcutaneous fat over the Longissumus dorsi (P < 0.05), Iliocostalis thoracis (P < 0.001) and Obliquus internus abdominis (P < 0.001) compared with those fed CC. However, these effects were removed when lambs were slaughtered at a constant fat score. At both endpoints, lambs from T × BF dams contained less (P < 0.05) perinephric and retroperitoneal fat than SW × BF or LL × BF dams fed GP or CC, respectively. The results from this study show that using crossbred ewes sired by CH, LL or T sires will increase carcass weight and improve carcass conformation of lambs sourced from hill flocks. Inclusion of oilseed rape in lamb finishing diets had only minor effects on performance compared with a standard CC but feeding fish oil or GP impacted negatively on lamb growth and carcass quality.     

Ubiquitination of the prototype foamy virus envelope glycoprotein leader peptide regulates subviral particle release

Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28LP and gp38LP are ubiquitinated variants of the major gp18LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two- to threefold increase in single-cycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP.     

A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.     

Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases and therefore effects of AGEs on cells are the objective of numerous investigations. Since AGEs used in biochemical studies are usually not chemically characterized, comparison of data is difficult if not impossible. To find a suitable characterization protocol, human serum albumin was reacted with different concentrations of glucose, methyl glyoxal, and glyoxylic acid. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine), the carboxymethyl lysine and carbonyl content, and the fibrillar state. Additionally, their fluorescence and absorbance characteristics were extensively studied. Although we found significant differences in the degree of modification and in AGE-specific fluorescence when using different modifiers, the results provide important information and allow comparing AGEs derived from different modifier concentrations. The results also suggest strong conformational changes within the modified proteins. In the present paper we propose a set of parameters that is sufficient to partially characterize AGEs used for biochemical studies.     

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.     

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

Background  

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD⁺ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions.     

Physical delimitation of the pepper Bs3 resistance gene specifying recognition of the AvrBs3 protein from Xanthomonas campestris pv. vesicatoria

The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three YAC clones. Genetic mapping of the corresponding YACends revealed however, that these YACs do not cover Bs3 and subsequent screens with newly developed YACend markers failed to identify new YAC clones. Marker saturation at the Bs3 locus was carried out by amplified fragment length polymorphism (AFLP). The analysis of 1,024 primer combinations resulted in the identification of 47 new Bs3-linked AFLPs. High-resolution linkage mapping of Bs3 was accomplished by inspecting more than 4,000 F₂ segregants resulting in a genetic resolution of 0.01 cM. Using tightly Bs3-linked YACend- and AFLP-derived markers we established a Bs3-spanning BAC contig and physically delimited the target gene within one BAC clone. The analysis of the Bs3-containing genomic region revealed substantial local variation in the correlation of genetic and physical distances.     

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.