Pig enamel revisited – Incremental markings in enamel of wild boars and domestic pigs

The nature and periodicity of incremental markings in pig enamel is currently debated. To broaden the basis for a correct interpretation of growth marks in pig enamel, we analyzed their periodicity in teeth of wild boars and domestic pigs. For that, the numbers of enamel incremental markings were recorded in ground sections and compared with crown formation times for the respective teeth derived from literature data on tooth development and eruption in Sus scrofa. Our study revealed that laminations with a daily periodicity are the dominant incremental feature of pig enamel. In wild boar M₃s, daily enamel secretion (apposition) rates ranged between a minimum of 6.1?µm in the inner and a maximum of 30.6?µm in the outer enamel.Long-period (supra-daily) incremental markings were present as perikymata at the outer enamel surface (OES). Contrary to the situation in primate enamel, in pig enamel the long-period incremental lines terminating in perikyma grooves were mostly structurally indistinguishable from the daily laminations. Typically, five sub-daily increments were present between successive laminations. The incremental pattern in pig enamel can be misinterpreted if the laminations are mistaken for long-period markings (striae of Retzius) and the sub-daily growth marks for daily prism cross-striations. The findings of the present study demonstrate the critical importance of correctly characterizing the incremental markings and their periodicity in enamel, and caution against an uncritical transfer of the interpretation of the nature of incremental markings in primate enamel to other mammalian taxa.     

Epitope Recognition in the Human–Pig Comparison Model on Fixed and Embedded Material

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig—the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.     

Expression of Human Ecto 5′ Nucleotidase in Pig Endothelial Cells and Its Implication for Adenosine Production and Xenotransplantation

Human endothelial activity of ecto-5′-nucleotidase (E5′N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and anti-aggregatory adenosine. We have shown in this study that human E5′N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5′N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.     

Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract

BackgroundThe expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (G_αgust) and α-transducin (G_αtran) G protein subunits. This study tested whether G_αtran and G_αgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract.ResultIn the stomach, G_αgust and G_αtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, G_αgust and G_αtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of G_αtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of G_αgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included G_αtran / 5HT-IR and G_αtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as G_αgust /5-HT-IR or G_αgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in G_αtran-IR cells in the duodenal crypts and a significant increase of G_αgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of G_αtran-G_αgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of G_αtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01).ConclusionThis study showed an upregulation of selected subpopulations of G_αgust / G_αtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.     

Purification and Some Properties of a Neutral α-Glucosidase from Pig Serum

A neutral α-glucosidase was purified from pig serum by precipitation with ammonium sulfate, chromatographies on DEAE-cellulose and -Sephadex A–50, and gel filtration on Bio-Gel P–300 and Sephadex G–200. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient (s_20,w) was calculated to be 10.7 S, and the isoelectric point, 4.0. The molecular weight was estimated to be approximately 2.7 × 10⁵ by thin-layer gel filtration and SDS-disc electrophoresis.

The enzyme exhibited also glucoamylase activity. The optimal pH was found to be in the pH range of 6.0 to 7.0 for maltose and soluble starch. The ratio of velocity of hydrolysis for maltose (Km, 0.72 mg/ml), soluble starch (Km, 9.8 mg/ml) and shellfish glycogen (Km, 55.6 mg/ml) was calculated to be 100: 110: 5.15 in this order.     

Identification of the Critical Therapeutic Entity in Secreted Hsp90α That Promotes Wound Healing in Newly Re-Standardized Healthy and Diabetic Pig Models

Chronic and non-healing skin wounds represent a significant clinical, economic and social problem worldwide. Currently, there are few effective treatments. Lack of well-defined animal models to investigate wound healing mechanisms and furthermore to identify new and more effective therapeutic agents still remains a major challenge. Pig skin wound healing is close to humans. However, standardized pig wound healing models with demonstrated validity for testing new wound healing candidates are unavailable. Here we report a systematic evaluation and establishment of both acute and diabetic wound healing models in pigs, including wound-creating pattern for drug treatment versus control, measurements of diabetic parameters and the time for detecting delayed wound healing. We find that treatment and control wounds should be on the opposite and corresponding sides of a pig. We demonstrate a strong correlation between duration of diabetic conditions and the length of delay in wound closure. Using these new models, we narrow down the minimum therapeutic entity of secreted Hsp90α to a 27-amino acid peptide, called fragment-8 (F-8). In addition, results of histochemistry and immunohistochemistry analyses reveal more organized epidermis and dermis in Hsp90α-healed wounds than the control. Finally, Hsp90α uses a similar signaling mechanism to promote migration of isolated pig and human keratinocytes and dermal fibroblasts. This is the first report that shows standardized pig models for acute and diabetic wound healing studies and proves its usefulness with both an approved drug and a new therapeutic agent.     

Investigation of Testosterone,Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.     

Prokaryotic Expression and In Vitro Functional Analysis of IL-1β and MCP-1 from Guinea Pig

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.     

Immunohistochemical and Functional Analysis of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) and Ecto-5′-Nucleotidase (CD73) in Pig Aortic Valves

Extracellular nucleotides control mechanisms such as thrombosis or inflammation that are important in several pathologies, including heart valve disease and calcification. Ectonucleoside triphosphate diphosphohydrolase 1 (eNTPD1, CD39) and ecto-5′-nucleotidase (e5NT, CD73) are ectoenzymes that convert adenosine triphosphate to adenosine diphosphate, adenosine monophosphate and finally to adenosine. Changes in activities of these enzymes influence extracellular nucleotide concentrations and therefore could be involved in valve pathology. This study aimed to analyze type of cells, specific area, level of expression and biochemical function of CD39 and CD73 in pig aortic valves. Samples were collected from aortic valves of domestic pigs. Histological sections were cut from paraffin embedded tissue blocks. Following incubation with primary antibody against CD39 or CD73, washing and secondary goat anti-rabbit secondary antibodies, slides were viewed with NanoZoomer scanner. Substantial expression CD39 and CD73 was observed in two main types of valve cells: endothelial and valve interstitial cells. Subsequently, biochemical function of CD39 and CD73 was evaluated in cells cultured from pig aortic valve. Breakdown of extracellular nucleotides added to cell medium was analyzed with high performance liquid chromatography. In the interstitial cells, the CD73 products formation was much faster than in endothelium, while for the CD39 activity this relation was opposite. Expression and high concentration of CD39 and CD73 products in endothelium are expected, but presence of CD73 in valve interstitial cells is a surprise. We conclude that CD39 and CD73 and their enzymatic activities that convert extracellular nucleotides are highly expressed and could have special function in the valve.     

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5′-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 μM ATP or 50 μM ADP or 50 μM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2 ± 1.1 and 24.5 ± 3.4 μM respectively while it remained below 1 μM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7 ± 0.1 and 5.7 ± 2.2 μM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.     

Pig renal glutaminase—Another view

Olsen et al. (1973) interpret their observations of pig renal glutaminase in terms of a conformationsl change in the dimers making up a polymer. It is argued here that the same data can be used to explain the same polymer structure without involving conformational changes, and a model is proposed to support this view.     

Recombinant Bivalent Vaccine against Foot-and-Mouth Disease VirusSerotype O／A Infection in Guinea Pig

Foot-and-mouth disease (FMD) is a highly contagiousdisease of cloven-hoofed animals such as cattle and pig.The disease causes explosive epidemics and heavyeconomic losses in the agriculture worldwide [1]. FMDvirus (FMDV) shows a high genetic and antigenicvariability, and has seven serotypes: O, A, C, AsiaI, SAT1,SAT2 and SAT3 [2]. The FMDV control is mainly imple-mented using chemically inactivated virus vaccines, whichmay contain residual living virus and pose a risk of virusreleas…     

Frequency Dependence of Electrical Coupling in Deiters″ Cells of the Guinea Pig Cochlea

Immunolabeling with antibodies against connexins 26 and 30 showed that, in the guinea pig cochlea, supporting Deiters″ cells are massively interconnected and form an orderly network within the organ of Corti. In paired patch-clamp recordings the coupling ratio (CR) of adjacent Deiters″ cells at the apex of the cochlea (～0.31) was 3-fold smaller than in isolated cell pairs due to shunting afforded by multicellular connectivity. With sinusoidal current stimuli the delay in signal propagation between adjacent cells increased with increasing frequency whereas the amplitude did not change significantly up to 200 Hz (corner frequency F_c ～220 Hz). Depolarizing voltage commands applied to an outer hair cell (OHC) elicited outward potassium currents in the OHC and inward currents in the abutting Deiters″ cells, supplying direct evidence for potassium buffering in the organ of Corti. Computational analysis indicates that electrical signals injected into a Deiters″ cell are transmitted across a network segment spanning 8 cell diameters. Thus electrical coupling in the organ of Corti is unlikely to influence the selectivity of frequency filtering performed mechanically by the mammalian cochlea.     

Favipiravir (T-705) Inhibits Junín Virus Infection and Reduces Mortality in a Guinea Pig Model of Argentine Hemorrhagic Fever

Background

Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses.

Methodology/Principal Findings

To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1–2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33–40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection.

Conclusions/Significance

The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses.     

Does the Order of Invasive Species Removal Matter? The Case of the Eagle and the Pig

Background

Invasive species are recognized as a primary driver of native species endangerment and their removal is often a key component of a conservation strategy. Removing invasive species is not always a straightforward task, however, especially when they interact with other species in complex ways to negatively influence native species. Because unintended consequences may arise if all invasive species cannot be removed simultaneously, the order of their removal is of paramount importance to ecological restoration. In the mid-1990s, three subspecies of the island fox Urocyon littoralis were driven to near extinction on the northern California Channel Islands owing to heightened predation by golden eagles Aquila chrysaetos. Eagles were lured to the islands by an abundant supply of feral pigs Sus scrofa and through the process of apparent competition pigs indirectly facilitated the decline in foxes. As a consequence, both pigs and eagles had to be removed to recover the critically endangered fox. Complete removal of pigs was problematic: removing pigs first could force eagles to concentrate on the remaining foxes, increasing their probability of extinction. Removing eagles first was difficult: eagles are not easily captured and lethal removal was politically distasteful.

Methodology/Principal Findings

Using prey remains collected from eagle nests both before and after the eradication of pigs, we show that one pair of eagles that eluded capture did indeed focus more on foxes. These results support the premise that if the threat of eagle predation had not been mitigated prior to pig removal, fox extinction would have been a more likely outcome.

Conclusions/Significance

If complete eradication of all interacting invasive species is not possible, the order in which they are removed requires careful consideration. If overlooked, unexpected consequences may result that could impede restoration.