Porcine Protein Research Articles

Molecular cloning of the gene encoding pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3) in pigs

The pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3) are members of the nuclear receptor superfamily that act as transcription factors. They regulate the expression of several important genes that encode enzymes responsible for the metabolism of endogenous hormones and exogenous substances. We determined the nucleotide sequences of porcine CARand PXR genes bp using cDNA clones derived from porcine cDNA libraries. The porcine CAR gene has a 1407 bp mRNA, which contains a 216 bp 5'-untranslated region (UTR), a 144 bp 3'-UTR, and a 1047 bp coding region that encodes 348 amino acids. The mRNA sequence of porcine PXR is composed of 2567 bp that contains a 273 bp 5'-UTR, a 1266 bp putative coding region encoding 421 amino acids, and a 1028 bp 3'-UTR. The porcine CAR and porcine PXR proteins showed a high degree of sequence identity in their DNA-binding domain (DBD) and ligand-binding domain (LBD) with 80% - 90% amino acid identity in comparison with those of humans. However, the AB domain of the porcine CAR and PXR proteins is considerably different in comparison with that of humans. Eighty six percentage and 43 % sequence identity is found between porcine and human CAR proteins and porcine and human PXR proteins, respectively. The mRNA expression of both porcine CAR and PXR was detected in liver, small intestine, and kidney bp using reverse-transcription (RT) -PCR.

208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation.

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals. The objective of the present study was to clarify the cAMP/PK-A pathway during the capacitation of porcine spermatozoa and to evaluate this impact on the p32 sperm tyrosine phosphoprotein appearance. The presence of p32 was assessed after incubating fresh pig sperm with IBMX/db-cAMP, H-89, a PK-A inhibitor or bistyrphostin, a tyrosine kinase inhibitor, in capacitating (CM) or non-capacitating conditions (NCM) by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. When pig spermatozoa were incubated in CM supplemented with H-89 (50 microM) or bistyrphostin (1.2 microM), capacitation decreased significantly (P < 0.001). The p32 sperm tyrosine phosphoprotein, previously shown to be associated with capacitation of porcine sperm though not necessarily an end point of this phenomenon, was not modulated by IBMX/db-cAMP (100 microM/1 mM), H-89 (50 microM) nor bistyrphostin (1.2 microM). Our results indicate, therefore, that pig sperm are regulated somewhat differently than as described for other mammals, because although the cAMP/PK-A and tyrosine kinase pathways are involved in capacitation, they do not influence the appearance of p32.

Structural and expression analysis of the porcine FUS2 gene

The putative porcine tumor suppressor gene FUS2 or N-acetyltransferase (Nat6) assigned to SSC13q21 spans 864-basepairs (bp) of genomic DNA, consisting of a single exon encoding a protein of 288 amino acids (aa), with 73% identity to the human and 74% to the mouse protein. Similar to man and mouse, the gene possesses an N-acetyltransferase domain, but the cell attachment motif arginine–glycine–aspartate (RGD) is exclusively found in the pig gene. Expression studies of the gene in several organs by RT-amplification and by quantitative polymerase chain reaction (Q-PCR) showed that FUS2 is widely expressed in porcine tissues. A point mutation was detected at position 836 of the coding sequence (G to A) leading to an amino acid substitution from cystein (C) to tyrosine (Y) at position 278 of the protein. Genes of the tumor suppressor gene (TSG) cluster act together to suppress tumor growth through their functional activation of tumor suppressing pathways. Studies in humans have proven that mutations in N-acetyltransferase genes are associated with some kind of cancers. Knowledge of structure and function of the respective porcine genes and proteins is important. Pigs—in particular minipigs—will be the non-rodent biomodels for human oncology and cancer therapy in the future.

Molecular characterization of porcine hyaluronidase genes 1, 2, and 3 clustered on SSC13q21

Hyaluronidase genes (HYAL) encode hyaluronidase enzymes required for hyaluronan degradation. Both in humans and in mouse, clustered hyaluronidase genes have been identified. Here, the porcine hyaluronidase cluster consisting of genes HYAL1, HYAL2 and HYAL3 was characterized. The porcine cDNA sequences and proteins share homologies to human orthologs of 85 and 81% for HYAL1, 87 and 89% for HYAL2 and 86 and 83% for HYAL3, respectively. The porcine hyaluronidase proteins approximately share a 40% homology with each other. Furthermore, genes FUS1 and FUS2 were found within this cluster, which was assigned to SSC13q21. A total of seven SNPs were detected in the genes (four in HYAL1, two in HYAL2 and one in HYAL3). Three of the four SNPs in HYAL1 led to amino acid exchanges (C622G → Asp²⁴ to Glu; C633T → Pro²⁸ to Leu, and G1298T → Ala²⁵⁰ to Ser). The amino acid replacements induce putative changes in the extended strand at Asp²⁴, in the extended strand and the random coil at Pro²⁸, and finally in the random coil and the alpha helix at Ala²⁵⁰. Frequency estimations for four SNPs located in genes HYAL1 and HYAL3 using animals (n = 295) of nine European and six Chinese pig breeds indicated several significant deviations. For example, there were no significant differences in allele frequencies between pigs representing breeds Hampshire and Jiangquhai at SNP C633T (HYAL1), but between Hampshire respectively Jiangquhai animals and Rongchang pigs. Analysis of the same breeds at SNP C588T (HYAL3) indicates significant differences between Hampshire and Jiangquhai respectively Rongchang, but not between Jiangquhai and Rongchang. The breed Göttingen Minipig displayed significant differences concerning two SNPs with respect to the other European pig breeds tested. For all three hyaluronidase genes, N-glycosylation sites are typical. For HYAL2 the lysosomal character was proven. The catalytic site responsible for HAase activity is conserved in the three enzymes. Expression of hyaluronidases was determined by RT-PCR and quantitative PCR. Broad gene expression was observed in different tissues for the three genes, respectively.

A novel monoclonal antibody inhibits the immune response of human cells against porcine cells: identification of a porcine antigen homologous to CD58.

Human CD58 is an adhesion molecule that interacts with CD2 on lymphocytes. We describe here an antibody that blocks responses of human peripheral blood mononuclear cells (PBMCs) to porcine cells and reacts with a porcine protein with homology to CD58. Antibodies were isolated with a screen for inhibition of the human antiporcine response. One of these antibodies was used for immunoaffinity purification of a protein that was identified by molecular weight determination, endoglycosidase sensitivity, and microsequencing analysis as a porcine homologue of CD58. The antigen recognized by this antibody was a cell surface protein of relative molecular mass (Mr)=45,000 containing N-linked carbohydrate chains. Immunoaffinity purification of this protein and microsequencing revealed homology to sheep CD58 as well as sequences that were common to this protein and both sheep and human CD58. The protein was widely distributed on porcine cells, including lymphocytes, endothelial cells, muscle cells, and neuronal cells. This antibody efficiently inhibited lysis of porcine targets by human PBMCs in addition to preventing proliferation of the human PBMCs in response to the porcine cells. The CD2 interaction with porcine cells is important for the efficient recognition of porcine tissue, and inhibition of the human antiporcine immune response with the antibody is likely to be caused by the disruption of the human CD2 interaction with this porcine homologue of CD58. The antibody may prove to be useful for the blocking of this interaction without interfering with other functions of T cells.

EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins

Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins.

Functional Properties of Protein Fractions Isolated from Porcine Blood

ABSTRACT: Red cell concentrate, serum, albumin, and immunoglobulin fractions were separated from porcine blood collected at a HACCP‐implemented (Hazard Analysis and Critical Control Points system‐implemented) slaughterhouse in Hermosillo, México. Functional and microbiological properties were examined. Red cell concentrate and serum isolates were high in protein (92 and 85%, respectively) but limited in methionine and isoleucine. Porcine serum and albumin fractions showed emulsifying properties comparable to bovine sources. Porcine immunoglobulins were 96% soluble. All fractions were of excellent microbiological quality. These results indicate the optimal sanitary conditions during slaughter and processing procedures, as well as the potential functionality of porcine protein fractions.

Effect of gamma-irradiation on the physicochemical properties of porcine and bovine blood plasma proteins

To elucidate the effect of γ-irradiation on the physicochemical properties of blood plasma proteins, bovine and porcine blood, released from a slaughterhouse, was collected and plasma proteins were prepared. Physicochemical properties of blood plasma protein powders and solutions, such as molecular weight distribution, secondary structure, solubility, and viscosity, were examined after γ-irradiation at 1, 5, 7, and 10 kGy. Oxygen radicals, such as hydroxyl and superoxide anion radicals, generated by γ-irradiation, affected the physicochemical properties of the blood plasma protein solutions, but not the plasma protein powders. Circular dichroism and sodium dodecyl sulphate–polyacrylamide gel electrophoresis studies showed that an increase of γ-irradiation decreased the ordered structure of plasma protein solutions and caused initial fragmentation of the polypeptide chains and subsequent aggregation. However, solubility and viscosity of the irradiated plasma protein powders, as well as secondary structure and molecular weight profile, were not changed significantly with radiation dose.

Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein.

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron-sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus-human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron-sulphur flavoprotein.

Development of a baculovirus expression system for soluble porcine tumor necrosis factor receptor type I and soluble porcine tumor necrosis factor receptor type I-IgG fusion protein

Tumor necrosis factor-α (TNF-α) is a key mediator of inflammatory responses and gram-negative bacterial sepsis, but the role that it plays during Salmonella enterica species bacterial infections in swine has not yet been elucidated. To facilitate studies on the role of TNF-α on the pathology associated with Salmonella infections in pigs, recombinant soluble porcine TNF receptor type I (rspTNF-RI) and soluble TNF receptor type I fused to the Fc region of porcine IgG1 (rspTNF-RI-IgG) were expressed in insect cells using a baculovirus expression system. The proteins were secreted into the cell culture media and purified by anti-soluble porcine TNF-RI antibody and protein G affinity chromatography, respectively. The yield of protein using this method was approximately 1.5 mg rspTNF-RI and 4 mg rspTNF-RI-IgG/L of cell culture medium. In in vitro assays, rspTNF-RI-IgG was approximately 10-fold (0.97 vs. 10.00 pmol/ml) more effective than rspTNF-RI at completely inhibiting the cytotoxic activity of 500 U of recombinant porcine TNF-α on 3×10 4 WEHI 164 murine fibrosarcoma, clone 13, cells. Compared to previously described methods, this method yields significantly more biologically active rspTNF-RI.

Novel method for detecting bovine immunoglobulin G in dried porcine plasma as an indicator of bovine plasma contamination.

Current U.S. Food and Drug Administration regulations prohibit feeding of protein derived from mammalian tissue, excluding blood and blood products and any product that consists entirely of porcine or equine protein. A novel lateral flow immunoassay device has been developed that can quickly and qualitatively determine the presence of bovine immunoglobulin G (IgG), a major component in blood products, at very low levels (0.01% v/v). The device can be used to test for bovine IgG commingling in spray-dried porcine plasma used in the feed industry. Producers and consumers alike could use this device to verify product content at threshold levels.

Isolation of GIF from porcine brain and studies of its zinc transfer kinetics with apo-carbonic anhydrase.

Neuronal growth inhibitory factor (GIF) of porcine brain, was isolated and purified by a similar procedure which was used on the isolation of human and bovine GIF. The native porcine protein with stoichiometry of 4Cu+, 3Zn2+ was obtained for the first time. The kinetics of zinc transfer from Cu4Zn3MT-3 to apo-carbonic anhydrase were studied, and zinc transfer rate constants and thermodynamic parameters were obtained. It is found that like other MTs, porcine Cu4Zn3MT-3 can also transfer its zinc atom to apoCA, even much easier than other MTs. A possible association mechanism has been proposed, the formation of Cu4Zn3MT3-apoCA complex may be the rate-determining step. The obtained data indicate besides its neuronal growth inhibitory function, GIF might play a role in cellular Zn homeostasis in brain.

The crystal and molecular structures of diferric porcine and rabbit serum transferrins at resolutions of 2.15 and 2.60 A, respectively.

The serum transferrins are monomeric proteins with a molecular mass of around 80 kDa and are responsible for the transport of iron in vertebrates. The three-dimensional structures of diferric porcine and rabbit serum transferrin have been refined against X-ray diffraction data extending to 2.15 and 2.60 A, respectively. Data for both proteins were collected using synchrotron radiation at temperatures of 277 K. The porcine protein crystallizes in the space group C2, with unit-cell parameters a = 223.8, b = 44.9, c = 78.9 A, beta = 105.4 degrees with one molecule in the asymmetric unit. The structure was solved by molecular-replacement methods using rabbit serum transferrin as the search model. The structure was refined using REFMAC, with a final residual of 13.8% (R(free) = 18.2% for a 5% data sample) for all data to 2.15 A. The final model comprises 5254 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, one N-acetyl glucosamine moiety and 494 water molecules. The rabbit protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = 127.2, c = 144.9 A and one molecule per asymmetric unit. The structure was solved using the method of multiple isomorphous replacement and refined using REFMAC to give a final residual of 18.6% (R(free) = 22.2% for a 5% data sample) for all data to 2.60 A. The final model comprises 5216 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, a Cl(-) anion and 206 solvent molecules; there is no clear indication of the carbohydrate moiety attached to Asn490 (rabbit serum numbering). Both molecules adopt a bilobal structure typical for members of the transferrin family. Each of the structurally homologous lobes contains two dissimilar domains with a single iron-binding site buried within the interdomain cleft. The porcine serum protein lacks an interdomain disulfide bridge close to the connecting peptide between the lobes, but this seems to have little effect on the overall orientation of the lobes. The N-lobes of both proteins possess lysine residues, one from each of the two domains, that lie in close proximity to one another to form the so-called dilysine trigger. The more acid-labile release of iron from serum transferrins than from lactoferrins is discussed.

Porcine Fas-ligand gene: genomic sequence analysis and comparison with human gene

Thymic Fas-ligand ( FasL) cDNA and hepatic FasL genomic sequences were obtained from a 2-month-old LW pig. From these nucleotide sequences, amino acid sequence was deduced and compared with FasL sequences obtained from various animals. This comparison reveals that porcine FasL is closer to that of human, macaca and cat, and differs more from mouse and rat. The extracelluar domains of porcine and human FasL proteins appear to be functionally compatible. The complete genomic DNA sequence of porcine FasL was also compared with its human counterpart. Exons showed 80–89% nucleotide homology between pig and human, while introns showed 64–69% nucleotide homology. Sequence comparison by Harr plot analysis revealed many stretches within introns having identical sequences, suggesting that the sites may have unidentified common functions. One potential extra exon between exons 2 and 3 was located within porcine intron 2. This potential exon has no counterpart in human FasL intron 2. Whether or not this extra exon can be expressed and could cause additional immunological responses remains to be investigated. For future xenotransplantation, it is important to compare porcine and human genomic sequences, and to investigate their system compatibilities.

Definition of antigen specificity for antimitochondrial proteins detected by Western blotting using native mitochondrial proteins in primary biliary cirrhosis

The major autoantigens to anti-mitochondrial antibody (AMA) in primary biliary cirrhosis (PBC) have previously been identified to be PDC-E2, BCOADC-E2, and OGDC-E2. However, analysis of these autoantigens to AMA cannot be examined using the two routine assays; immmunofluorescence and ELISA. Moreover, there are some problems in specificity and sensitivity in these routine assays. So, analysis with Western blotting using native mitochondrial protein as the antigen is required; it allows the identification of the molecular weights for the proteins which react with AMA in patients' sera. However, since the antigen-proteins used are not unified, molecular weights of AMA corresponding proteins vary among laboratories. In the present study, as the first step to help address this issue, we investigated the antigen specificity of protein bands detected by Western blotting using our in-house bovine and porcine heart mitochondrial proteins. Three major recombinant mitochondrial proteins were prepared. The antigen specificity was examined by the absorption tests preincubated with the three recombinant mitochondrial proteins. The molecular weights of developing our bovine and porcine heart mitochondrial proteins using SDS-PAGE were multiple protein bands including 74, 52, 50, and 43 kDa protein bands. Of them, the 74, 50, and 43 kDa protein bands were absorbed with preincubations of recombinant PDC-E2, BCOADC-E2, and OGDC-E2 protein, respectively. AMA specificity of these three major proteins with our Western blotting was confirmed.

Dimerization, stability and electrostatic properties of porcine beta-lactoglobulin.

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico-chemical characterization of porcine beta-lactoglobulin, purified from pooled milk: size-exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine beta-lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer-dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess alpha helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine beta-lactoglobulin in the crystal structure. Interaction studies revealed that porcine beta-lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.

Cloning, expression and localization of human BM88 shows that it maps to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis.

Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in human brain whereas immunohistochemistry on human brain sections demonstrated that the expression of BM88 is confined to neurons. The initial mapping assignment of human BM88 to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis, was retrieved from the UniGene database maintained at the National Centre for Biotechnology Information (NCBI, Bethesda, MD, U.S.A.). We confirmed this localization by performing fluorescence in situ hybridization on BM88-positive cosmid clones isolated from a human genomic library. These results suggest that BM88 may be a candidate gene for genetic disorders associated with alterations at 11p15.5.

Bacterially induced activation of interleukin-18 in porcine intestinal mucosa

Interleukin (IL)-18 is a cytokine with structural and functional properties similar to IL-1β and IL-12, respectively. It is activated by caspase-1 cleavage, like IL-1β, and induces interferon (IFN)-γ, like IL-12. In order to study the role of IL-18 in the immune response to infectious diseases of mucosal surfaces we cloned and expressed porcine IL-18 and developed antibodies to the protein. Porcine IL-18 retains the caspase-1 cleavage site present in other mammalian IL-18 proteins, but has two potential N-linked glycosylation sites not found in those proteins. Porcine interleukin-18 mRNA and protein are expressed in immune tissues including lymph nodes and gut associated lymphoid tissues. Specific cell types containing IL-18 include lung and splenic macrophages, nonadherent spleen cells and intestinal epithelial cells. Although IL-18 transcription is moderately induced by lipopolysaccharide, the magnitude and total expression level are small compared to those of interleukin-1β. In vivo and ex vivo infection of intestinal mucosa with Salmonella choleraesuis resulted in a decrease in size of IL-18, consistent with cleavage of the preprotein by caspase-1. Thus, IL-18 is present in mucosal tissues where it could play a role in the immune response to invading pathogens.

Heat-Induced Gelation of Charcoal-Treated Serum Protein.

The gel strength of charcoal-treated bovine serum and porcine serum proteins at a concentration of 8% was determined with different concentrations of NaCl after heating at 80°C for 15 min at pH 7.0. The charcoal treatment considerably enhanced the gel strength of porcine serum protein, and also improved gel strength of bovine serum protein at NaCl concentrations between 75 mM and 150 mM. The gel strength of commercial PSA and BSA and that of fractionated bovine and porcine proteins by ion-exchange chromatography were also determined at a constant NaCl concentration. With the charcoal treatment, the gel strength of fractions containing albumin increased, while that containing globulin decreased. The gel strength of charcoal-treated bovine serum and porcine serum remarkably increased by the addition of the egg white, ovalbumin and glutathione. The promotion of gel formation with glutathione and ovalbumin seemed to result from the interaction between sulfhydryl groups in glutathione and ovalbumin and intramolecular disulfide bonds in the serum protein, thereby resulting in the highly organized matrix.