Differentiation among Spanish sheep breeds using microsatellites

Genetic variability at 18 microsatellites was analysed on the basis of individual genotypes in five Spanish breeds of sheep – Churra, Latxa, Castellana, Rasa-Aragonesa and Merino -, with Awassi also being studied as a reference breed. The degree of population subdivision calculated between Spanish breeds from F_ST diversity indices was around 7% of total variability. A high degree of reliability was obtained for individual-breed assignment from the 18 loci by using different approaches among which the Bayesian method provided to be the most efficient, with an accuracy for nine microsatellites of over 99%. Analysis of the Bayesian assignment criterion illustrated the divergence between any one breed and the others, which was highest for Awassi sheep, while no great differences were evident among the Spanish breeds. Relationships between individuals were analysed from the proportion of shared alleles. The resulting dendrogram showed a remarkable breed structure, with the highest level of clustering among members of the Spanish breeds in Latxa and the lowest in Merino sheep, the latter breed exhibiting a peculiar pattern of clustering, with animals grouped into several closely set nodes. Analysis of individual genotypes provided valuable information for understanding intra- and inter-population genetic differences and allowed for a discussion with previously reported results using populations as taxonomic units.     

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K_m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Effects of dietary polyunsaturated n-3 fatty acids on dyslipidemia and insulin resistance in rodents and humans. A review

For many years, clinical and animal studies on polyunsaturated n-3 fatty acids (PUFAs), especially those from marine oil, eicosapentaenoic acid (20:5,n-3) and docosahexaenoic acid (22:6,n-3), have reported the impact of their beneficial effects on both health and diseases. Among other things, they regulate lipid levels, cardiovascular and immune functions as well as insulin action. Polyunsaturated fatty acids are vital components of the phospholipids of membrane cells and serve as important mediators of the nuclear events governing the specific gene expression involved in lipid and glucose metabolism and adipogenesis. Besides, dietary n-3 PUFAs seem to play an important protecting role against the adverse symptoms of the Plurimetabolic syndrome. This review highlights some recent advances in the understanding of metabolic and molecular mechanisms concerning the effect of dietary PUFAs (fish oil) and focuses on the prevention and/or improvement of dyslipidemia, insulin resistance, impaired glucose homeostasis, diabetes and obesity in experimental animal models, with some extension to humans.     

Alteration of copper physiology in mice overexpressing the human Menkes protein ATP7A

The Menkes protein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physiological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken beta-actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human protein. Immunohistochemical analysis revealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebellum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 males (18%), and T25#5 females (23%), as well as in the brains of 60-day-old T22#2 females and males (23% and 30%, respectively).     

Use of the SSLP-based method for detection of rare apomictic events in a sexual AtSERK1 transgenic Arabidopsis population

Here we present a screening method to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of single sequence length polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1) gene expressed under the control of the AtLTP1 promoter in sexual Arabidopsis plants. In one of the three tested F2 transgenic populations expressing the AtLTP1::AtSERK1 construct we observed two plants with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for a number of heterozygous loci, it was concluded that these two putative apomictic plants either lost their clonal ability in the next generation or resulted from incidental recombination events displaying the genotype of the parent. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Synthesis of alpha-glucans in fission yeast spores is carried out by three alpha-glucan synthase paralogues, Mok12p, Mok13p and Mok14p

Fission yeast possesses a family of (1,3)-alpha-glucan synthase-related genes; one of them, mok1+/ags1+, plays an essential function in morphogenesis during vegetative growth. Here we show that three mok1+ paralogues -mok12+, mok13+ and mok14+- are required for sporulation to succeed, acting at different stages of the spore wall maturation process. Mutation of mok12+ affected the efficiency of spore formation and spore viability. Deletion of mok13+ does not affect spore viability but the spores showed reduced resistance to stress conditions. mok14Delta mutant spores failed to accumulate the amylose-like spore wall-specific polymer. mok12+, mok13+ and mok14+ expression was restricted to sporulating cells and the proteins localized to the spore envelope but with different timing. mok11+ was also induced during the sporulation process although its deletion did not show apparently a sporulation defect. In vegetative cells, beta-glucans are more abundant than alpha-glucans (55% versus 28%). In spores, the situation was the opposite, alpha-glucans accounted for 46% while beta-glucans were approximately 38% of the total polysaccharides. We found at least two types of alpha-glucan polymers, Mok12p and Mok13p, were involved in the synthesis of the greater part of alpha-glucan in the spores envelope, a polymer that is mainly digested with alpha-1,3 glucanase, while Mok14p, homologous to starch synthases, was required for the synthesis of the iodine-reactive polymer that is made of alpha-1,4 glucose residues.     

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack.     

Design of a Potent d-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance

The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific d-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. d-Peptides (peptides composed of d-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first d-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.The HIV envelope protein (Env) mediates viral entry into cells (11). Env is cleaved into surface (gp120) and transmembrane (gp41) subunits that remain noncovalently associated to form trimeric spikes on the virion surface (16). gp120 recognizes target cells by interacting with cellular receptors, while gp41 mediates membrane fusion. Peptides derived from heptad repeats near the N and C termini of the gp41 ectodomain (N and C peptides) interact in solution to form a six-helix bundle, representing the postfusion structure (3, 55, 56). In this structure, N peptides form a central trimeric coiled coil (N trimer), creating grooves into which C peptides bind. This structure, in conjunction with the dominant-negative inhibitory properties of exogenous N and C peptides, suggests a mechanism for Env-mediated entry (10, 22, 58-60).During entry, gp41 forms an extended prehairpin intermediate that leaves the exposed N-trimer region vulnerable to inhibition for several minutes (18, 35). This intermediate ultimately collapses as the C-peptide regions bind to the N-trimer grooves to form a trimer of hairpins (six-helix bundle), juxtaposing viral and cellular membranes and inducing fusion. Enfuvirtide (Fuzeon), the only clinically approved HIV fusion inhibitor, is a C peptide that binds to part of the N-trimer groove and prevents six-helix bundle formation in a dominant-negative manner (61). Enfuvirtide is active in patients with multidrug resistance to other classes of inhibitors and is a life-prolonging option for these patients (30, 31). However, enfuvirtide use is restricted to salvage therapy due to several limitations, including (i) high dosing requirements (90 mg, twice-daily injections), (ii) high cost (∼$30,000/year/patient in the United States), and (iii) the rapid emergence of resistant strains (21, 47).A deep hydrophobic pocket at the base of the N-trimer groove is an especially attractive inhibitory target because of its high degree of conservation (3, 12, 48), poor tolerance to substitution (4, 34), and critical role in membrane fusion (2). Indeed, this region is conserved at both the amino acid level (for gp41 function in membrane fusion) and the nucleotide level (for the structured RNA region of the Rev-responsive element). Enfuvirtide binds to the N-trimer groove just N terminal to the pocket and is significantly more susceptible to resistance mutations than 2nd-generation C-peptide inhibitors, such as T-1249, that also bind to the pocket (8, 13, 29, 44, 46, 47, 58).Peptide design, molecular modeling, and small-molecule screening have produced a diverse set of compounds that interact with the gp41 pocket and inhibit HIV-1 entry with modest potency, but often with significant cytotoxicity (7, 14, 15, 17, 23, 24, 26, 34, 51, 54). The first direct evidence that pocket-specific binders are sufficient to inhibit HIV entry came with the discovery of protease-resistant d-peptides identified using mirror-image phage display (12). In this technique, a phage library is screened against a mirror-image version of the target protein (synthesized using d-amino acids) (50). By symmetry, mirror images (d-peptides) of the discovered sequences will bind to the natural l-peptide target. As the mirror images of naturally occurring l-peptides, d-peptides cannot be digested by natural proteases. Protease resistance provides d-peptides theoretical treatment advantages of extended survival in the body and possible oral bioavailability (41, 42, 49).These 1st-generation d-peptide entry inhibitors possess potency against a laboratory-adapted isolate (HXB2) at low to mid-μM concentrations (12). We previously reported an affinity-matured 2nd-generation d-peptide called PIE7, pocket-specific inhibitor of entry 7 (57). A trimeric version of PIE7 is the first high-affinity pocket-specific HIV-1 inhibitor and has potency against X4-tropic (HXB2) and R5-tropic (BaL) strains at sub-nM concentrations. However, significant further optimization is required to create a robust clinical candidate for two reasons. First, this d-peptide is much less potent (requiring high nM concentrations) against JRFL, a primary R5-tropic strain. Therefore, improved PIE potency is necessary to combat diverse primary strains. Second, by improving the affinity of our inhibitors for the pocket target, we hope to provide a reserve of binding energy that will delay the emergence of drug resistance, as described below.We and others have reported a potency plateau for some gp41-based fusion inhibitors that is likely imposed by the transient exposure of the prehairpin intermediate (9, 27, 53, 57). For very high-affinity inhibitors, association kinetics (rather than affinity) limits potency so that two inhibitors with significantly different affinities for the prehairpin intermediate can have similar antiviral potencies. We proposed that overengineering our d-peptides with substantial affinity beyond this potency plateau would provide a reserve of binding energy that would combat affinity-disrupting resistance mutations (57). Such a resistance capacitor should also prevent the stepwise accumulation of subtle resistance mutations in Env by eliminating the selective advantage that such mutants would otherwise confer.Here, we report on the design and characterization of a 3rd-generation pocket-specific d-peptide, PIE12-trimer, with ∼100,000-fold improved target binding compared to that of the best previous d-peptide, significantly broadened inhibitory potency, and an enhanced resistance capacitor that provides a strong barrier to viral resistance. We achieved this increased potency via structure-guided phage display and crosslinker optimization. PIE12-trimer has a dramatically improved resistance profile compared to the profiles of earlier d-peptides, as well as those of enfuvirtide and T-1249. These results validate the resistance capacitor hypothesis and establish PIE12-trimer as a leading anti-HIV therapeutic candidate.