Molecular characterization and expression analysis of the gene coding for the porcine beta(3) integrin subunit (CD61).

Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(3) integrin subfamily are composed of a beta(3) subunit (CD61) non-covalently associated with two alpha subunits, alpha(IIb) (CD41) and alpha(v) (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine beta(3) integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides insight into the physiological function of the porcine beta(3) integrins and should be of importance in understanding the role of this integrin family in biological processes.

Molecular cloning, chromosomal location, and expression analysis of porcine CD14.

CD14 is a membrane-associated glycosylphosphatidylinositol (GPI)-anchored protein that binds lipopolysaccharide (LPS) of Gram-negative bacteria and enables LPS-dependent responses in a variety of cells. In this study a cDNA containing the porcine CD14 coding sequence has been cloned and its complete sequence determined. The amino acid sequence deduced from pig CD14 cDNA encodes a 373 amino acid polypeptide that exhibits 75%, 72%, 69%, 66%, 57% and 56% similarity to CD14 from cow, horse, human, rabbit, mouse and rat, respectively. Structural analysis showed that the porcine CD14 is a membrane glycoprotein with a GPI-anchor site and an extracellular domain containing 11 leucine-rich repeats. In addition, the LPS-binding regions identified in the human CD14 are highly conserved in the N-terminal domain of the porcine sequence. Fluorescence in situ hybridization was used to locate the CD14 gene on the pig chromosome 2, band q28. Expression analysis revealed that porcine CD14 transcripts were detected in all tissues and cells examined, suggesting that the expression of porcine CD14 gene is not restricted to myeloid cell lineage. Finally, we report that LPS stimulation significantly up-regulated CD14 gene expression in porcine alveolar macrophages.

Localization of porcine CD29 transcripts and protein in pig cells and tissues by RT-PCR and immunohistochemistry.

Integrins are heterodimeric cell adhesion proteins with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(1) integrin subfamily are composed of a beta(1) subunit (CD29) non-covalently associated with different alpha subunits to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. Here, we have studied the CD29 expression in porcine tissues and cells at two different levels: expression of the CD29 mRNA by RT-PCR and localization of the protein by immunohistochemistry. CD29 transcripts were detected in a variety of tissues and cells: platelets, PBMC, granulocytes, alveolar macrophages, smooth muscle, intestine, lung, liver, spleen, lymph node, skin, testis, heart, kidney and bone marrow. Our results suggest that CD29 gene transcription occurs in all organs examined, although with different intensities. The precise localization of CD29 protein in paraffin-embedded tissues was detected by using a specific polyclonal antibody indicating that its expression is limited to smooth muscle, epithelium cells, endothelium of blood vessels and myeloid cells and is no detectable in cells of the lymphoid lineage. The distribution of the CD29 in normal tissues provide insight into the physiological function of the porcine beta(1) integrins and should be of importance in understanding the role of this integrin family in pathological processes.

Molecular cloning and structural analysis of the porcine homologue to CD97 antigen.

CD97 is a member of a novel subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seven-span transmembrane region, known as EGF-TM7. We here report the cloning of cDNA encoding the pig homologue of CD97. A pig CD97 specific probe was generated by PCR amplification of pig leukocyte cDNA, using primers based on consensus regions among the known sequences of mouse and human CD97. Screening of a pig aorta smooth muscle cDNA library identified one clone containing an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 141 amino acid sequence consisting of three EGF domains, a mucin-like spacer region of 276 amino acid, containing a G-protein coupling motif of 52 amino acids, followed by a 250 amino acid region containing seven membrane spanning domains and a 47 amino acid cytoplasmic tail. The amino acid sequence of the clone was 75, 67 and 59% homologous to cattle, human and mouse CD97 antigen, respectively. Therefore, it was termed pig CD97. Pig CD97 antigen shares many structural features with human, cattle and mouse CD97. RT-PCR analysis of cDNA from different pig cells and tissues showed that CD97 was highly expressed in leukocytes and lymph node cells. This is the first report describing the identification of a member of the EGF-TM7 family in the pig.

Monoclonal antibody that recognizes a domain on heterogeneous nuclear ribonucleoprotein K and PTB-associated splicing factor.

hnRNP K protein is a member of the heterogeneous nuclear protein (hnRNP) complex that, besides its function as a translational regulator of human mRNA, is also considered to be a transcription factor involved in tumorigenesis. PSF is a protein part of the human spliceosome and essential in RNA splicing. Here we report the generation of one monoclonal antibody GG6H9.1C3 that recognized both hnRNP K and PSF proteins using Western blot analysis, flow cytometry, and immunocytochemistry.

Comparison of three monoclonal and three polyclonal antibodies in the immunohistochemical diagnosis of canine autoimmune skin diseases.

The aim of this study was to evaluate the utility of three monoclonal antibodies (mAbs), and two anticanine IgG and one anticanine IgM polyclonal antibodies (pAbs) for the immunohistochemical diagnosis of canine autoimmune skin diseases. Skin biopsies from 11 cases of pemphigus (7 foliaceus, 3 vulgaris and 1 erythematosus), 12 cases of discoid lupus erythematosus (DLE) and 12 cases of chronic hyperplasic dermatitis were used. The CA4E7 mAb (IgG1 + IgG2) showed similar sensitivity, but higher specificity and lower background than the two anti-IgG pAbs for the immunohistochemical diagnosis of pemphigus and DLE. The CA4F1 mAb (IgG2) and CA3H1 mAb (IgG2) showed moderate and low interepithelial reactivity, respectively, in autoimmune skin diseases, but strong staining of the cytoplasm of plasma cells of the inflammatory infiltrates. These results suggest that the CA4E7 mAb may be valuable in the immunohistochemical diagnosis of such disorders.