Porcineβ-lactoglobulin A and C

Summary The occurrence of the dominant ‘whey’ protein in samples of milk from 1180 sows is examined. It exhibits genetic polymorphism with some unusual features. Although immunologically different from bovine β-lactoglobulin, it is shown by chemical studies of the isolated protein to be a β-lactoglobulin. Two homozygous genetic variants, designated porcine β-lactoglobulin A and C, are isolated and their amino acid compositions and peptide maps compared. It is shown that the C variant has +1 His, −1 Gln, and +1 Asp, −1 Glu, with respect to the A variant. These variants, containingca. 162 residues per molecule, are considered in relationship to porcine β-lactoglobulins isolated by other workers. The sequence of the first 50 residues is determined and compared with sequence of the bovine protein. The sequences ofca. 70% of the remaining residues is proposed on the basis of the composition of tryptic peptides and assumed homology.     

Expression and Identification of Porcine β-Defensin 1 in Escherichia coli and Up-Regulation by Streptococcus Infection in Porcine Tongue In Vivo

Defensins are members of a major family of antimicrobial peptides that play an important role in the innate immune response, which are of interest as potential novel pharmaceutical agents. We successfully constructed expression system of porcine β-defensin 1 (pBD-1) with his-tag in Escherichia coli Rossetta (DE3) cells and investigated the effect of Streptococcus ATCC 19714 infection on the mRNA expression of pBD-1 in porcine tongue in vivo. The results showed that active pBD-1 not to be affected by the presence of his-tag was obtained and displayed a high antimicrobial activity against Streptococcus ATCC 19714 at a concentration of 42 ± 5.2 μg/ml. The mRNA level of pBD-1 after infection in the tongue mucosa was initially changed with significant up-regulation at 3 h and reached the highest level at 6 h (about tenfold higher than 0 h), thereafter reduced to normal level at 12 h. The results indicated that pBD-1 is shown as a potent antimicrobial activity, and the expression level of pBD-1 against Streptococcus ATCC 19714 is up-regulation in the porcine tongue.     

Porcine α1,3-galactosyltransferase: tissue-specific and regulated expression of splicing isoforms

Expression of the Galα_1,3 Gal epitope on membrane glycolipids and glycoproteins is known to vary widely from one tissue to another. In the course of studying the mechanisms underlying this variability, we have isolated from pig cDNA four sequences corresponding to four isoforms of α_1,3-galactosyltransferase (α_1,3GT), the Golgi enzyme that links galactose in α_1,3 on the galactose residue of N-acetyllactosamine. The isoforms differ from each other in the alternative presence of two nucleotide stretches of 36 and 63 base pairs in a segment encoding the stem region of the protein. Stable expression experiments show that all four isoenzymes can confer α-galactosyltransferase activity to HeLa cells, and that they are all located within the Golgi compartment, indicating that variations in length in the stem region do not affect enzyme activity or cellular localization. Analysis of RNA from different pig organs and cells shows quantitative differences between tissues in levels of α_1,3GT, as well as qualitative differences, the four isoforms being unequally represented in different tissues.     

Cloning of Porcine Platelet Glycoprotein Ibα and Comparison with the Human Homolog

Glycoprotein Ib–IX–V (GPIb–IX–V) is a platelet adhesion receptor complex that initiates platelet aggregation. Glycoprotein Ibα (GPIbα) is the central component of the GPIb–IX–V complex, anchoring the complex to the cytoskeleton and harboring the binding site for von Willebrand factor (vWF). Previous studies suggest that the coagulation function in pigs differs from that in humans, especially with respect to the interaction between vWF and platelets. However, we have little knowledge about the function of porcine platelets, which is important with regard to studies of cardiovascular disease, clotting, and surgery that use pigs as animal models. To extend this information, we cloned and analyzed the porcine GPIbα sequence. Porcine GPIbα contains 1891 nucleotides and includes an open reading frame that encodes 627 amino acids. The nucleotide sequence showed 67% identity with human GPIbα, whereas the deduced amino acid sequences were 59% identical. The vWF binding domain shares the highest identity among different species, whereas the PEST domain shows variations. Evaluation of platelet function by using ristocetin-induced platelet aggregation revealed remarkably lower levels of aggregation in porcine than human platelets. According to the sequence analysis and platelet aggregation tests, we propose that the function of GPIbα, especially regarding the ristocetin–vWF–GPIbα interaction, differs between pigs and humans. This characterization of porcine GPIbα will enhance our knowledge of the porcine coagulation system.Abbreviations: GPIbα, glycoprotein Ibα, vWF, von Willebrand factorGlycoprotein (GP) Ib–IX–V is one of the major adhesive receptors expressed on the surface of circulating platelets and is essential for platelet adhesion and clot formation at sites of vascular injury.² Platelet adhesion in high-shear areas is initiated by GPIbα, a subunit of the GPIb–IX–V complex, via binding to von Willebrand factor (vWF), a multimeric adhesive protein associated with collagen in the vessel wall.^3,13,27 After GPIbα-dependent adhesion to vWF, platelets become activated and undergo cytoskeletal rearrangements associated with shape changes, spreading, and the secretion of platelet agonists that amplify the platelet aggregation and activation mediated by platelet integrin α_IIbβ₃.¹The GPIb–IX–V complex consists of 4 transmembrane subunits—GPIbα, GPIbβ, GP IX, and GP V—which are present at a ratio of 2:2:2:1.²⁶ The entire ligand-binding capacity of the GPIb–IX–V complex is situated in the N-terminal globular region (amino acids 1 through 282) of GPIbα.²⁸ Mutations in GPIbα lead to Bernard–Soulier syndrome and pseudo-von Willebrand disease.^15,24 Thrombi that cause complications in arterial thrombosis are associated with GPIb–IX–V, especially GPIbα.²¹ Because the interactions between GPIbα and its ligand are critical to the vascular processes of thrombosis and inflammation, the complex is under intense scrutiny as a potential therapeutic target.²⁹Pigs share many physiologic and anatomic similarities with humans and offer several breeding and handling advantages relative to nonhuman primates, making the pig an optimal species for preclinical experimentation. During the last several years, porcine animal models have gained a great deal of importance^23,30 in cardiovascular diseases,^6,33 ischemia–reperfusion injury,¹⁰ transplant surgery, and many other areas of biomedical research.¹⁷ In particular, the pig has been identified as an ideal cell, tissue, and organ donor for xenotransplantation. Because differences exist between species, it is necessary to take the physiologic differences between pigs and humans into account when developing animal models and when analyzing the results obtained by using these models.Our early studies revealed differences in the process of coagulation between pigs and humans.⁵ Currently we know little about which functions of platelets are conserved between species or about porcine GPIb–IX–V and its differences from the human complex. In the current study, we cloned the coding sequences of porcine GPIbα and compared its nucleotide sequence, deduced protein sequence, and 3D structure model with those of human GPIbα, focusing on important functional domains and vWF interaction sites. We also investigated the ability of porcine platelets to be agglutinated or activated when treated with ristocetin. This work represents a step toward understanding the value and limitations of the pig as a preclinical model for coagulation-related studies.     

The Interrelationship between Polyunsaturated Fatty Acids, α-Tocopherol and Glutathione Peroxidase in Chicken and Porcine Skeletal Muscles

Two species of chrysomelid leaf beetles found in Brazil, Diabrotica speciosa and Cerotoma arcuata, are strongly attracted to the root of Ceratosanthes hilariana (Cucurbitaceae). Root extracts stimulate a compulsive feeding response. The major feeding stimulants isolated from these extracts were cucurbitacin B and its 23,24-dihydro derivative.     

Carbohydrate and Protein-based Inhibitors of Porcine Pancreatic α-Amylase: Structure Analysis and Comparison of Their Binding Characteristics

The crystal structures of porcine pancreatic α-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 Å, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface.V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder.This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments.A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.     

Chemical Modification of Sulfhydryl Groups in Porcine Pancreatic α-Amylase,and Purification and Properties of the Modified Amylase

The behavior of SH groups of porcine pancreatic α-amylase, called PPA II, was studied by chemical modification with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). Only two SH groups in PPA II reacted, in a pseudo-first-order reaction, and the modification was accompanied with the inactivation of the amylase. The reactivity of SH groups with DTNB was influenced by the ionic strength of the medium. The SH groups were protected against modification by the addition of some substrate analogs; maltopentaitol, maltotetraitol, maltotriitol and cyclomaltohexaose were effective analogs, whereas maltitol, d-glucitol and methyl α-d-glucoside did not protect these groups. The modified enzymes (M₁ and M₂), in which one and two SH groups reacted with DTNB, respectively, were purified in an electrophoretically homogeneous state by chromatography on Bio-Gel P-2 and TSK-Gel DEAE-Toyopearl 650S. The optimum pH of the modified enzyme (M₂) was 6.9~7.0, which was the same as that of the native PPA II. The isoelectric points of M₁ and M₂ were estimated to be 5.8 and 5.2, respectively, by the method of Catsimpoolas. The CD spectrum of PPA II was altered partially by the modification of SH groups with DTNB. Moreover, a precipitin line with a spur was observed in a double immunodiffusion test of PPA II and M₂ to rabbit antiserum of PPA II. It is concluded that the free SH group(s) in PPA II, located near the substrate binding site, don’t participate directly in its catalytic activity, but that the SH group(s) are involved in the antigenicity of PPA II.     

Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 production at both the mRNA and protein levels in porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and monocyte-derived dendritic cells (DCs). We subsequently demonstrated that the NF-κB signaling pathway was essential for PRRSV infection-induced IL-15 production. First, addition of an NF-κB inhibitor drastically reduced PRRSV infection-induced IL-15 production. We then found that NF-κB was indeed activated upon PRRSV infection, as evidenced by IκB phosphorylation and degradation. Moreover, we revealed an NF-κB binding motif in the cloned porcine IL-15 (pIL-15) promoter, deletion of which abrogated the pIL-15 promoter activity in PRRSV-infected alveolar macrophages. In addition, we demonstrated that PRRSV nucleocapsid (N) protein had the ability to induce IL-15 production in porcine alveolar macrophage cell line CRL2843 by transient transfection, which was mediated by its multiple motifs, and it also activated NF-κB. These data indicated that PRRSV infection-induced IL-15 production was likely through PRRSV N protein-mediated NF-κB activation. Our findings provide new insights into the molecular mechanisms underling the IL-15 production induced by PRRSV infection.     

Porcine Epidemic Diarrhea Virus RNA Present in Commercial Spray-Dried Porcine Plasma Is Not Infectious to Na?ve Pigs

Porcine epidemic diarrhea virus emerged in North America in April 2013 and has since been identified in 30 U.S. States, Canada and Mexico. The rapid spread of PEDV has raised concerns about the role of feed and particularly pork-by-product components such as spray-dried porcine plasma (SDPP) in PEDV transmission. The aim of this study was to determine the infectivity of PEDV RNA present in commercial SDPP. Specifically, 40 3-week-old PEDV naïve pigs were randomly assigned to one of five treatment groups. At day post inoculation (dpi) 0, NEG-CONTROL pigs were sham-inoculated, PEDV-CONTROL pigs received cell culture propagated PEDV, and SDPP-CONTROL pigs were switched to a diet with 5% SDPP containing 5.1±0.1 log₁₀ PEDV RNA copies/g. To evaluate a potential positive effect of anti-PEDV antibodies in SDPP on PEDV challenge, four days prior to PEDV challenge the pigs in the SDPP-PEDV group were switched to and remained on a 5% SDPP diet through dpi 28. Another group, EGG-PEDV, was orally administered a commercial egg-derived liquid PEDV globulin product from dpi -4 through 6. All PEDV-CONTROL pigs began shedding PEDV in feces by dpi 3 and seroconverted between dpi 7 and 14, whereas pigs in NEG-CONTROL and SDPP-CONTROL groups remained PEDV RNA negative and did not seroconvert to PEDV for the study duration. This indicates no evidence of infectivity of the PEDV RNA in the SDPP lot utilized. Furthermore, under the study conditions SDPP or egg-derived liquid PEDV globulin addition did not significantly alter PEDV-shedding or overall disease course after experimental challenge.     

Porcine FcγRIIb mediates enhancement of porcine reproductive and respiratory syndrome virus (PRRSV) infection

Antibody-dependent enhancement (ADE) of virus infection caused by the uptake of virus-antibody complexes by FcγRs is a significant obstacle to the development of effective vaccines to control certain human and animal viral diseases. The activation FcγRs, including FcγRI and FcγRIIa have been shown to mediate ADE infection of virus. In the present paper, we showed that pocine FcγRIIb, an inhibitory FcγR, mediates ADE of PRRSV infection. Stable Marc-145 cell lines expressing poFcγRIIb (Marc-poFcγRII) were established. The relative yield of progeny virus was significantly increased in the presence of sub-neutralization anti-PRRSV antibody. The Fab fragment and normal porcine sera had no effect. Anti-poFcγRII antibody inhibited the enhancement of infection when cells were infected in the presence of anti-PRRSV antibody, but not when cells were infected in the absence of antibody. These results indicate that enhancement of infection in these cells by anti-PRRSV virus antibody is FcγRII-mediated. Identification of the inhibitory FcγR mediating ADE infection should expand our understanding of the mechanisms of pathogenesis for a broad range of infectious diseases and may open many approaches for improvements to the treatment and prevention of such diseases.     

The C/EBPβ-Dependent Induction of TFDP2 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Proliferation

Virologica Sinica - Porcine reproductive and respiratory syndrome (PRRS) is an important infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), leading to...     

Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse

Authentic or na?ve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop na?ve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to na?ve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.