Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.     

Apoptotic process of porcine intestinal M cells

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer’s patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18⁺/PCNA⁺ cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18⁺/alkaline phosphatase⁺ cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex. This investigation was supported by a Grant-in-Aid for Scientific Research (16658107) from the Ministry of Education, Culture, Sports, Science and Technology, by two grants (Prion Project and Secure and Healthy Livestock Farming Project) from the Ministry of Agriculture, Forestry and Fisheries, and by a grant from the Naito Foundation.     

Kinetic mechanism of porcine testicular 17ß-hydroxysteroid dehydrogenase

A study on product inhibition of 17ß-hydroxysteroid dehydrogenase from porcine testes was carried out by measuring the initial velocities of NADPH formation using testosterone as the substrate steroid. Type of inhibition by NADPH against NADP⁺ was competitive in both saturated and unsaturated concentrations of testosterone. In the saturated concentration of NADP⁺, activity of the enzyme was not inhibited by NADPH against testosterone. In the unsaturated concentrations of NADP⁺, however, NADPH brought mixed type inhibition against testosterone. The similar modes of inhibition by the product steroid, androstenedione were observed in the saturated and unsaturated concentrations of NADP⁺ and testosterone.The fluorescence of NADPH was increased in the presence of the enzyme, and fluorometric titration indicated that 1 mol of NADPH was bound to 1 mol of the 17ß-hydroxysteroid dehydrogenase. Addition of testosterone to enzyme-NADPH complex reduced the intensity of fluorescence of NADPH, suggesting formation of testosterone-enzyme-NADPH complex as a ternary dead end complex.From the analyses of product inhibition and spectral changes of NADPH, the kinetic mechanism of the enzyme was revealed as rapid equilibrium random system with two dead end complexes which consisted of the two reduced reactants bound to the enzyme and the two oxidized ones bound to it.     

Sequencing and characterization of the porcine α-galactosidase A gene: towards the generation of a porcine model for Fabry disease

Fabry disease is an inherited lysosomal disorder caused by a deficiency of alpha-galactosidase A (α-gal A). The systemic accumulation of substrate, mainly globotriaosylceramide (Gb3), results in organ failure. Although Gb3 accumulation has been observed in an α-gal A-deficient mouse model, important clinical manifestations were not seen. The pursuit of effective treatment for Fabry disease through gene therapy, for example, has been hampered by the lack of a relevant large animal model to assess the efficacy and safety of novel therapies. Towards assembling the tools to generate an alternative animal model, we have sequenced and characterized the porcine ortholog of the α-gal A gene. When compared to the human α-gal A, the porcine α-gal A showed a high level of homology in the coding regions and located at chromosome Xq22. Cell lysate and supernatants from Fabry patient-derived fibroblasts transduced with a lentiviral vector (LV) carrying the porcine α-gal A cDNA (LV/porcine α-gal A), showed high levels of α-gal A activity and its enzymological stability was similar to that of human α-gal A. Uptake of secreted porcine α-gal A was observed into non-transduced cells and was partially inhibited by soluble mannose-6-phosphate. Furthermore, Gb3 accumulation was reduced in Fabry patient-derived fibroblasts transduced with the LV/porcine α-gal A. In conclusion, we elucidated and characterized the porcine α-gal A gene and enzyme. Similarity in enzymatic profile and chromosomal location between α-gal A of porcine and human origins may be of great advantage for the development of a large animal model for Fabry disease.     

Bioinformatic and expression analysis of novel porcine β-defensins

β-Defensins are a major group of mammalian antimicrobial peptides. Although more than 30 β-defensins have been identified in humans, only one porcine β-defensin has been reported. In this article we report the identification and initial characterization of 11 novel porcine β-defensins (pBD). Using bioinformatic approaches, we screened 287,821 porcine expressed sequence tags for similarity of their predicted peptides to known human β-defensins and identified full-length or partial sequences for the 11 novel pBDs. Similar to the previously identified pBD1, all of these peptides have a consensus β-defensin motif. A differential expression pattern for these newly identified genes was found. For example, unlike most β-defensins, pBD2 and pBD3 were expressed in bone marrow and in other lymphoid tissues including thymus, spleen, lymph nodes, duodenum, and liver. Including pBD2 and pBD3, six porcine β-defensins were expressed in lung and skin. Several newly identified porcine β-defensins, including pBD123, pBD125, and pBD129, were expressed in male reproductive tissues, including lobuli testis and some segments of the epididymis. Phylogenetic analysis indicates that in most cases the evolutionary relationship between individual porcine β-defensins and their human orthologs is closer than the relationship among β-defensins in the same species. These findings establish the existence of multiple porcine β-defensins and suggest that the pig may be an ideal model for the characterization of β-defensin diversity and function. The nucleotide sequence data reported in this article have been submitted to GenBank.     

Inhibition of porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages with the 3′UTR-targeted amiRNA北大核心CSCD

［Objective］ To study the inhibitory effect of 3′ untranslated region （UTR）-targeted artificial microRNA （amiRNA） against porcine reproductive and respiratory syndrome virus （PRRSV） replication in porcine alveolar macrophages （PAM）. ［Methods］ Recombinant adenovirus （rAd） expressing the 3′ UTR-targeted or control amiRNA and green fluorescent protein （GFP） reporter gene were generated by transfecting AAV-293 cells with the transfer vector. The expression of sequence-specific amiRNA was detected by quantitative RT-PCR. The anti-PRRSV effect of amiRNA was detected by quantitative RT-PCR， Western blotting and viral titration assay. ［Results］ Two rAds，namely rAd-amiR3UTR-GFP and rAd-amiRcon-GFP， were generated. Both primary PAM and 3D4/163 cells could be transduced by rAd with different transduction efficiencies. The amiR3UTR was expressed in dose-and timedependent manners in rAd-transduced PAM cells. The amiR3UTR， but not amiRcon， had significant and stable inhibitory effects against replication of three different PRRSV strains in a dose-dependent manner. ［Conclusion］ The rAd-delivered amiR3UTR had strong anti-PRRSV effect against different PRRSV strains and rAd-amiR3UTR-GFP could be explored further as the alternative strategy against PRRS.     

Are hypothalamic neurons transsynaptically connected to porcine adipose tissue?

Specific anatomical sites and pathways responsible for mediating metabolic and neuroendocrine effects of leptin are still poorly understood. Therefore, we examined distribution of leptin receptor-containing neurons transsynaptically connected with the porcine fat tissue by means of combined viral transneuronal tracing and immunohistochemical staining method. Pseudorabies virus (PRV) was injected into the perirenal fat tissue in pigs, and after survival periods of 3, 5, 7, 9, and 11 days, hypothalami were processed immunohistochemically with primary antisera against PRV and leptin receptor (OBR). PRV labeled neurons were found in paraventricular nucleus (PVN), ventromedial nucleus (VMN), anterior hypothalamic area (AHA), preoptic area (PA), arcuate nucleus (ARC), and supraoptic nucleus (SON) by nine days after injection of the virus. Double-labeling immunofluorescence demonstrated that OBR were co-localized in nearly all virus-infected neurons. The present results provide the first morphological data demonstrating a multisynaptic circuit of neurons of CNS origin which innervates porcine fat tissue.     

Absence of 5′-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess $\text{p-}\text{nitrophenyl}$ phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed.     

Immunocytochemical localization of 17β-hydroxysteroid dehydrogenase in porcine testis

Summary The immunocytochemical localization of 17-hydroxysteroid dehydrogenase (17-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.Supported by grants from the Ministry of Education, Science, and Culture, Japan     

Does seminal plasma affect angiogenesis in the porcine oviduct?

In the present study we examined the effect of seminal plasma (SP) on angiogenesis in the porcine oviduct. Gene expressions of vascular endothelial growth factor (VEGF) and its two receptors (Flt-1: fms-like tyrosine kinase and Flk-1/KDR: fetal liver kinase-1/kinase insert domain-containing receptor) as well as fibroblast growth factors (FGF-1 and 2) and von Willenbrand factor (VWF) were determined in the oviduct of SP-treated and control (PBS-treated) gilts. Moreover, vascular density (VD) indicated by endothelial cell area immunolocalized by VWF staining, was assessed in the oviducts. Real-time PCR revealed significantly higher expression of FGF-2 and VWF on day 1 (p < 0.05) after SP administration in comparison to control animals. In contrast, Flt-1 mRNA level on day 1 was lower in SP-treated gilts compared to controls (p < 0.05). In the examined oviductal sections, VD did not differ between control and SP-treated animals. However, in SP-treated animals VD was higher on day 5 than on day 1 (p < 0.05) or 3 (p < 0.01). SP had no significant effect on VEGF, Flk-1/KDR and FGF-1 mRNA expression. In conclusion, our results suggest that SP affects the vascular network by changing the expression of factors contributing to angiogenesis.     

miR-125a inhibits porcine preadipocytes differentiation by targeting ERRα

MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor which plays an important role in adipocyte differentiation. Our previous Solexa sequencing results indicated a high expression of miR-125a in adult pig backfat. In this study, we predicated and experimentally validated ERRα as a target of miR-125a. To explore the role of miR-125a in porcine preadipocytes differentiation, miRNA agomir and antagomir were used to perform miR-125a overexpression or knockdown, respectively. Our results showed that overexpression of miR-125a could dramatically reduce the mRNA expression of adipogenic markers PPARγ, LPL, and aP2, as well as its target gene ERRα. Western blotting showed the protein level of aP2 and ERRα was also significantly down-regulated. The overexpression of miR-125a also led to a notable reduction in lipid accumulation which was detected by Oil Red O staining. In contrast, we observed promoted differentiation of porcine preadipocytes upon miR-125a inhibition. In conclusion, we verified miR-125a inhibits porcine preadipocytes differentiation through targeting ERRα for the first time, which may provide new insights in pork quality improvement and obesity control.     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

Do porcine Sertoli cells represent an opportunity for Duchenne muscular dystrophy?

Sertoli cells (SeC) are responsible for the immunoprivileged status of the testis thanks to which allogeneic or xenogeneic engraftments can survive without pharmacological immune suppression if co‐injected with SeC. This peculiar ability of SeC is dependent on secretion of a plethora of factors including maturation factors, hormones, growth factors, cytokines and immunomodulatory factors. The anti‐inflammatory and trophic properties of SeC have been largely exploited in several experimental models of diseases, diabetes being the most studied. Duchenne muscular dystrophy (DMD) is a lethal X‐linked recessive pathology in which lack of functional dystrophin leads to progressive muscle degeneration culminating in loss of locomotion and premature death. Despite a huge effort to find a cure, DMD patients are currently treated with anti‐inflammatory steroids. Recently, encapsulated porcine SeC (MC‐SeC) have been injected ip in the absence of immunosuppression in an animal model of DMD resulting in reduction of muscle inflammation and amelioration of muscle morphology and functionality, thus opening an additional avenue in the treatment of DMD. The novel protocol is endowed with the advantage of being potentially applicable to all the cohort of DMD patients regardless of the mutation. This mini‐review addresses several issues linked to the possible use of MC‐SeC injected ip in dystrophic people.     

Fragments of human γG immunoglobulins produced by a porcine pancreatic esterase

The effect of a porcine pancreatic esteroproteolytic enzyme on human IgG has been described. A sequential breakdown of the molecule occurs. The first cleavage results in the formation of an F′c fragment together with an F(ab)₂ fragment. A subsequent proteolysis of the F(ab)₂ fragment liberates two Fab fragments. Each fragment has been characterized by its antigenic properties, molecular weight, and sulfhydryl content.     

Complete nucleotide sequence of a CDNA encoding porcine tumor necrosis factor‐alpha

Abstract

Tumor necrosis factor‐alpha (TNF‐α), produced by immune cells, is a cytokine with a central role in the mediation of inflammatory responses to infection and injury. We report the nucleotide and corresponding amino acid sequence of a full length porcine TNF‐α cDNA. The complete cDNA nucleotide sequence is 1650 bp in length (not including the poly A tail) and has an open reading frame of 696 bp encoding a 232 amino acid protein. The porcine TNF cDNA sequence shows homologies of 86, 77, and 82% to human, murine, and lapine TNF cDNA sequences in the coding regions, respectively. The 5’ untranslated region of the cDNAs shows little sequence similarity. However, the 3´ untranslated region contains highly conserved sequences among all species, especially the TTATTTAT motif characteristic of cytokine messages.