Pig Parotid Research Articles

238 PRODUCTION OF CLEAVAGE-RESISTANT PHYTASE TRANSGENIC PIGS BY HANDMADE CLONING

Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions. Large quantities and high concentrations of manure waste that contains phytate phosphorus are generated. The use of phytase can effectively solve the problem of high phosphorus pollution in the fecal material of monogastric animals. Enviropigs, producing phytase in the salivary glands and secreting the enzyme in the saliva, were first generated at the University of Guelph (Guelph, ON, Canada) in 1999. However, phytase is easily inactivated in digestive processing. To address this problem, we improved the transgene construct and successfully generated phytase transgenic pigs by handmade cloning. The Escherichia coli periplasmic phosphoanhydride phosphohydrolase (appA) gene was subcloned. Using fragment substitution method, we designed a phytase gene that was insensitive to cleavage by pepsin and trypsin and had a higher affinity for the substrate. After codon optimization, the designed phytase gene was named Cafp and subcloned downstream of the pig parotid secretory protein (PSP) gene promoter. The tissue-specific vector p-PSP-Intron-Cafp was constructed and transferred into Landrace fetal fibroblasts by electroporation. The cell lines carrying Cafp were used as nuclear donors in handmade cloning. Cloned embryos were cultured in vitro to blastocysts and transferred to recipient sows. The presence of Cafp was tested by PCR and sequencing of cloned pigs. Phytase activity in saliva, feed, and feces was detected by the ammonium molybdate method with a slight modification. Immunohistochemistry (IHC) was used to determine tissue-specific expression. Three cell lines carrying Cafp were obtained. We generated 1027 blastocysts; 712 were of good quality and transferred to 6 recipients. Fourteen piglets were born, of which 6 survived. The PCR and sequencing results showed that 7 (3 live and 4 dead) of the 14 piglets carried Cafp. Phytase activity in the saliva of the 6 live cloned pigs was tested at 4 months of age and only 1 pig had 0.155 FTU mL–1 enzyme activity. The enzyme in the other 2 pigs may be inactivated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, which represents a 25.4% decrease in fecal emissions compared with the average of controls. The IHC results on the 3 later dead, Cafp-positive pigs showed that the transgene was expressed only in parotids, confirming tissue-specific gene expression. In summary, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to manage phosphorus nutrition and environmental pollution in animal husbandry.

Transgenic mice expressing yeast CUP1 exhibit increased copper utilization from feeds.

Copper is required for structural and catalytic properties of a variety of enzymes participating in many vital biological processes for growth and development. Feeds provide most of the copper as an essential micronutrient consumed by animals, but inorganic copper could not be utilized effectively. In the present study, we aimed to develop transgenic mouse models to test if copper utilization will be increased by providing the animals with an exogenous gene for generation of copper chelatin in saliva. Considering that the S. cerevisiae CUP1 gene encodes a Cys-rich protein that can bind copper as specifically as copper chelatin in yeast, we therefore constructed a transgene plasmid containing the CUP1 gene regulated for specific expression in the salivary glands by a promoter of gene coding pig parotid secretory protein. Transgenic CUP1 was highly expressed in the parotid and submandibular salivary glands and secreted in saliva as a 9-kDa copper-chelating protein. Expression of salivary copper-chelating proteins reduced fecal copper contents by 21.61% and increased body-weight by 12.97%, suggesting that chelating proteins improve the utilization and absorbed efficacy of copper. No negative effects on the health of the transgenic mice were found by blood biochemistry and histology analysis. These results demonstrate that the introduction of the salivary CUP1 transgene into animals offers a possible approach to increase the utilization efficiency of copper and decrease the fecal copper contents.

β-Glucanase specific expression in the parotid gland of transgenic mice

The feasibility of using the pig parotid secretory protein promoter to drive the β-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the β-glucanase gene and the β-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The β-glucanase activity in the saliva was found to be 0.18U/mL. After feeding a diet containing 2% β-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign β-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.

Structural and functional studies on a proline-rich peptide isolated from swine saliva endowed with antifungal activity towards Cryptococcus neoformans

A proline-rich peptide of 2733Da, isolated from pig parotid granule preparations was tested against different pathogenic fungi. It showed interesting antifungal activity towards a clinical isolate of Cryptococcus neoformans, with an EC50 of 2.2μM. Neither cytotoxic nor haemolytic effects were observed towards mammalian cells. Circular dichroism and infrared spectroscopic studies showed that the peptide adopted a combination of polyproline type-II, β-turn and unordered conformations at physiological temperatures. Temperature dependent experiments evidenced a tendency to adopt a polyproline-II helix conformation. From experiments with lipid vesicles, Neutral Red Uptake (NRU), haemolytic assays, and confocal microscopy studies, it could be hypothesized that the peptide may exert its antifungal effect by interacting with an intracellular target rather than through membrane damage.

Stimulating neuronal processes that benefit patients and our profession

Stimulating neuronal processes that benefit patients and our profession

Structural characterization of a new statherin from pig parotid granules

This study describes the identification and structural characterization of Sus scrofa statherin. HPLC-electrospray ionization mass spectrometry analysis on pig parotid secretory granule extracts evidenced a peptide with a molecular mass value of 5381.1 +/- 0.6 Da and its truncated form, devoid of the C-terminal Ala residue, with a molecular mass value of 5310.1 +/- 0.6 Da. The complete sequence of pig statherin gene was determined by sequencing the full-length cDNA obtained by rapid amplification of cDNA ends. The gene is 549 base pairs long and contains an open reading frame of 185 nucleotides, encoding a 42-amino acid secretory polypeptide with a signal peptide of 19 residues. This sequence presents some typical features of the four statherins characterized till now, showing the highest degree of amino acid identity with bovine (57%) and human statherin (39%). Pig statherin is mono-phoshorylated on Ser-3, while primate statherins already characterized are di-phosphorylated on Ser-2 and Ser-3. This difference, probably connected to the Asp-4 --> Glu substitution, suggests the involvement of the Golgi-casein kinase, which strictly recognizes the SX(E/pS) consensus sequence.

Effect of Same‐dose Single or Dual Field Irradiation on Damage to Miniature Pig Parotid Glands

AimTo evaluate the effect of single or dual field irradiation (IR) with the same dose on damage to miniature pig parotid glands.MethodologySixteen miniature pigs were divided into two IR groups (n=6) and a control group (n=4). The irradiation groups were subjected to 20 Gy X-radiation to one parotid gland using single-field or dual-field modality by linear accelerator. The dose-volume distributions between two IR groups were compared. Saliva from parotid glands and blood were collected at 0, 4, 8 and 16 weeks after irradiation. Parotid glands were removed at 16 weeks to evaluate tissue morphology.ResultsThe irradiation dose volume distributions were significantly different between single and dual field irradiation groups (t=4.177, P=0.002), although dose volume histogramin (DVH) indicated the equal maximal dose in parotid glands. Saliva flow rates from IR side decreased dramatically at all time points in IR groups, especially in dual field irradiation group. The radiation caused changes of white blood cell count in blood, lactate dehydrogenase and amylase in serum, calcium, potassium and amylase in saliva. Morphologically, more severe radiation damage was found in irradiated parotid glands from dual field irradiation group than that from single field irradiation group.ConclusionData from this large animal model demonstrated that the radiation damage from the dual field irradiation was more severe than that of the single field irradiation at the same dose, suggesting that dose-volume distribution is an important factor in evaluation of the radiobiology of parotid glands.

Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control1,2

This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastric animals offers a promising biological approach to relieve the requirement for dietary phosphate supplements and to reduce phosphorus pollution from animal agriculture.

Guanylin and uroguanylin in the parotid and submandibular glands: potential intrinsic regulators of electrolyte secretion in salivary glands.

The intestinal peptides guanylin and uroguanylin regulate the electrolyte/water transport in the gastrointestinal epithelium via activation of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis gene product. Because a major but incompletely understood function of the salivary glands is the CFTR-mediated secretion of an electrolyte-rich fluid, we investigated the rat and guinea pig parotid and submandibular glands for expression, cellular distribution, and subcellular localization of guanylin and uroguanylin. RT-PCR analyses with guanylin and uroguanylin-specific primers revealed that both peptides are highly expressed in the parotid and submandibular glands. At the translational level, western blotting analyses with peptide-specific guanylin and uroguanylin antibodies identified the expected 12.5-kDa immunoreactive peptides in these organs. At the cellular level, guanylin and uroguanylin were exclusively confined to epithelial cells of the intralobular and interlobular ducts. At the subcellular level, the immunoreactivities were localized by preembedding immunoelectron microscopy to small vesicles which were concentrated at the apical part of the secretory epithelial cells. The expression and cell-specific localization of guanylin and uroguanylin in the salivary glands indicate that these peptides may be specifically involved in the regulation of CFTR-mediated electrolyte/water secretion in the salivary gland ductal system.

Effect of prolactin on CD 2T cell activation pathway

To evaluate the effect of irradiation on microvascular endothelial cells in miniature pig parotid glands.A single 25-Gy dose of irradiation (IR) was delivered to parotid glands of 6 miniature pigs. Three other animals served as non-IR controls. Local blood flow rate in glands was measured pre- and post-IR with an ultrasonic Doppler analyzer. Samples of parotid gland tissue were taken at 4 h, 24 h, 1 week, and 2 weeks after IR for microvascular density (MVD) analysis and sphingomyelinase (SMase) assay. Histopathology and immunohistochemical staining (anti-CD31 and anti-AQP1) were used to assess morphological changes. MVD was determined by calculating the number of CD31- or AQP1-stained cells per field. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay was used to detect apoptotic cells. The activity of acid and neutral Mg2+-dependent SMase (ASMase and NSMase, respectively) was also assayed.Local parotid gland blood flow rate decreased rapidly at 4 h post-IR and remained below control levels throughout the 14-day observation period. Parotid MVD also declined from 4 to 24 hours and remained below control levels thereafter. The activity levels of ASMase and NSMase in parotid glands increased rapidly from 4 to 24 h post-IR and then declined gradually. The frequency of detecting apoptotic nuclei in the glands followed similar kinetics.Single-dose IR led to a significant reduction of MVD and local blood flow rate, indicating marked damage to microvascular endothelial cells in miniature pig parotid glands. The significant and rapid increases of ASMase and NSMase activity levels may be important in this IR-induced damage.

The role of protein kinase C in carbachol-induced and of cAMP-dependent protein kinase in isoproterenol-induced secretion in primary cultured guinea pig parotid acinar cells.

Stimulation of secretion by muscarinic agonists in guinea pig parotid or pancreatic acini is accompanied by a translocation of protein kinase C (PKC) from the cytosol to the particulate fraction [Machado-De Domenech and Söling (1987) Biochem. J. 242, 749-754] and by a PKC-mediated phosphorylation of the ribosomal protein S6 [Padel and Söling (1985) Eur. J. Biochem. 151, 1-10]. In order to decide whether PKC is directly involved in the secretory process, the effect of down regulation of PKC by phorbol 12-myristate 13-acetate (PMA) was studied in primary cultured guinea pig parotid acinar cells. These cells secrete in response to carbachol and isoproterenol. Only the carbachol response is associated with an increase in cytosolic calcium. Carbachol plus isoproterenol lead to an over-additive stimulation of secretion, an effect which depends completely on the presence of external calcium. Down regulation of PKC by about 90% did not significantly affect carbachol-induced exocytosis, whereas isoproterenol-stimulated secretion was almost doubled. The secretory response to permeable cAMP analogues was also enhanced in PKC-down-regulated acini, indicating a post-receptor effect. The increased response to isoproterenol was also observed in the absence of external calcium. The isoproterenol effect was significantly inhibited by the relatively specific cAMP-dependent protein kinase inhibitor H-89, which had only a minor effect on carbachol-induced exocytosis. Although down regulation of total PKC by up to 90% did not significantly affect the secretory response to carbachol, RO 31-8220, a relatively specific inhibitor of PKC, abolished carbachol-induced secretion in normal as well as in PMA-down-regulated cells. This indicates that a PKC isoform resistant to down regulation by PMA is involved in carbachol- but not in cAMP-mediated secretion.

CAMP and Ca(2+)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton.

The potential involvement of actin and fodrin (brain spectrin) in secretory events has been assessed in primary cultured guinea pig parotid acinar cells, using as a tool affinity purified anti-alpha-fodrin antibody, phalloidin, and immunofluorescence techniques. In resting parotid acinar cells fodrin and actin appeared as a continuous ring under the plasma membrane of most of the cells. Upon stimulation with secretagogues fodrin and actin labeling at the level of the plasma membrane disappeared almost completely. To establish a correlation between secretion and cytoskeletal changes at the individual cell level, anti-alpha-amylase-antibodies were used to label secreted amylase exposed at the surface of secreting cells. The number of cells expressing alpha-amylase on their surface followed bulk secretion of alpha-amylase. A strict correlation between secretion and alteration of the actin-fodrin labeling was observed at the individual cell level. The cytoskeletal changes occurred in parallel with secretion independently of the secretagogue used (carbamoylcholine in the presence of Ca2+, isoproterenol in presence or absence of Ca2+, forskolin, or dibutyryl-cyclic-AMP). The changes were reversible upon removal of the secretagogue. Since Ca2+, as well as cAMP-mediated secretion, was associated with the same kind of cytoskeletal changes, a reorganization of the cytoskeleton may play an essential part in regulated secretion.

Early effects of beta-adrenergic and muscarinic secretagogues on lipid and phospholipid metabolism in guinea pig parotid acinar cells. Stimulation of 2,3-sn-diacylglycerol formation by isoproterenol.

The early effects (0-120 s) of the beta-adrenergic secretagogue isoproterenol (2.10(-5) M) and the muscarinic secretagogue carbamoylcholine (2.10(-6) M) on various parameters of lipid and phospholipid metabolism were studied in isolated guinea pig parotid acinar cells. Both agonists enhanced within 10-20 s the incorporation of radioactive palmitate into the diacylglycerol, the triglyceride, and the phosphatidylinositol fractions but significantly diminished radioactive palmitate recovered in the acyl-CoA fraction. Carbamoylcholine decreased and isoproterenol increased the recovery of radioactive palmitate in the free fatty acid fraction. All changes had returned almost to control levels after 120 s. In cells prelabeled with [3H]arachidonate, carbamoylcholine exerted similar effects, whereas isoproterenol was almost ineffective. Both agonists stimulated the incorporation of radioactive glycerol into diacylglycerols 2-3-fold, while only carbamoylcholine stimulated the incorporation of [32P]phosphate into phosphatidylinositol and phosphatidate. Both agonists induced an increase in total diacylglycerols, carbamoylcholine being about twice as effective as isoproterenol. A lower concentration of carbamoylcholine (6.5.10(-7) M) had the same quantitative effect as 2.10(-5) M isoproterenol on the increase of total diacylglycerols. Even under these conditions carbamoylcholine, but not isoproterenol led to a significant translocation of protein kinase C from the soluble to the particulate fraction. Isoproterenol remained ineffective in this respect also when intracellular free calcium was increased with a calcium ionophore. This is explained by the finding that isoproterenol stimulates preferentially the formation of 2,3-sn-diacylglycerol, and carbamoylcholine preferentially stimulates the formation of 1,2-sn-diacylglycerol. The results indicate that in the guinea pig parotid acinar cell the two agonists do not only lead to activation of a triglyceride lipase (isoproterenol) or phosphoinositide-specific phospholipase(s) (carbamoylcholine), but also to a rapid change of flux through a number of other enzyme-catalyzed reactions involved in diacylglycerol turnover.

Phosphorylation of the ribosomal protein S6 during agonist‐induced exocytosis in exocrine glands is catalyzed by calcium‐phospholipid‐dependent protein kinase (protein kinase C)

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulation of exocytosis by cAMP-dependent or calcium-dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & Söling (1983) FEBS Lett. 159, 112-118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin-dependent, phospholipid-dependent, and cAMP-dependent protein kinases. Only the phospholipid-dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP-dependent enzyme phosphorylated only peptides A and C, and the calmodulin-dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid-dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanoylphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid-dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium-dependent and cAMP-dependent secretagogues.

Histology and ultrastructure of the pig parotid gland.

AbstractParotid glands of adult pigs were studied by light and electron microscopy. The parenchyma consists of acini, intercalated ducts, striated ducts, and excretory ducts.Acini had little affinity for periodic acid‐Schiff and were alcian blue‐negative at pH 2.6 or 0.5. These results indicate a paucity of neutral mucins and an absence of sialo‐ and sulfomucins. Histologically, acinar cells had vacuoles which corresponded ultrastructurally to large electron‐lucent secretory granules. The latter contained electron‐dense bodies and lipid droplets. Acinar cells differed histochemically and ultrastructurally from typical serous cells and were classified as special serous.Intercalated duct cells near acini contained electron‐dense secretory granules and numerous microfilaments. Cells in distal segments lacked secretory granules.Striated ducts were lined by two types of columnar epithelial cells, light cells and dark cells. Light cells were characterized by numerous infoldings of the basal plasma membrane, mitochondria between the infoldings, and electron‐lucent vesicles in the apical cytoplasm. The mitochondria contained tubular cristae. Dark cells were characterized by an abundance of microfilaments and numerous infranuclear processes which extended to the basement membrane.Excretory ducts, in addition to light and dark cells, also contained basal cells and goblet cells. Mitochondria in the light cells had flattened rather than tubular cristae.The pig parotid is a unique salivary gland and the most atypical mammalian parotid gland studied thus far. Mitochondria with tubular cristae and vacuolated special serous cells with lipid in the secretory granules are hallmarks of the pig parotid.

REgeneration of the guinea pig parotid gland after 4-hydroxyaminoquinoline-1-oxide-induced necrosis.

SummaryA single iv injection of 4-hy-droxyaminoquinoline-1-oxide (4-HAQO) in a dose of 22 mg/kg body weight induced marked necrosis of parotid acinar tissue in guinea pigs within 48 hr. After the necrotic phase, marked regenerative activity of the acinar cells was noted. [3H]Thymidine auto-radiographic studies revealed a labeling index of 15% cells in the parotid gland at 72 hr after 4-HAQO injection. A significant increase in the mitotic activity of the parotid acinar cells was also evident at 72 and 78 hr after 4-HAQO administration. The acinar cell repair, as well as formation of the new acini, were complete at 108 hr. It is clear from the studies that 4-HAQO, a pancreati-cotoxic agent and a potent carcinogen, also causes necrosis of the parotid acinar tissue, which is followed by regeneration and restitution.