Expression and regulation of ccBAX by miR-124 in the caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

Bcl2 family proteins play a critical role in cell death or survival. BAX, the death-promoting protein of bcl2 family, mediated mitochondrial pathway inducing cells' apoptosis in mammal. MiRNAs have been implicated as negative regulators down-regulating genes' expression after post-transcriptional level. At present, little is known about the regulatory mechanism of miRNA on the Bcl2 family proteins during CyHV-2 infection in silver crucian carp (Carassius auratus gibelio). In this study, the ccBAX (silver crucian carp BAX) gene was cloned and expressed, and polyclonal antibodies were raised in mouse against the purified ccBAX-GST fusion protein. The structure analysis indicated that ccBAX protein included four conserve domains (BH1, BH2, BH3 and transmembrane domains) and the expression of ccBAX protein occurred throughout the cells. Furthermore, two miRNAs (miR-124 and miRNA-29b) were identified to negatively regulate ccBAX gene expression in GiCF cell. miR-124 was found to suppress the expression of WT-ccBAX (wild type), but not the MT-ccBAX (mutant). Overall, the results demonstrated that the expression of the ccBAX gene was significantly down-regulated by miR-124 in silver crucian carp (Carassius auratus gibelio) during CyHV-2 infection.

A Novel Multi-Epitope Vaccine Based on Urate Transporter 1 Alleviates Streptozotocin-Induced Diabetes by Producing Anti-URAT1 Antibody and an Immunomodulatory Effect in C57BL/6J Mice.

Hyperuricemia (HUA) is related to diabetes. Uric acid-induced inflammation and oxidative stress are risk factors for diabetes and its complications. Human urate transporter 1 (URAT1) regulates the renal tubular reabsorption of uric acid. IA-2(5)-P2-1, a potent immunogenic carrier designed by our laboratory, can induce high-titer specific antibodies when it carries a B cell epitope, such as B cell epitopes of DPP4 (Dipeptidyl peptidase-4), xanthine oxidase. In this report, we describe a novel multi-epitope vaccine composing a peptide of URAT1, an anti-diabetic B epitope of insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in streptozotocin-induced diabetes C57BL/6J mice successfully induced specific anti-URAT1 antibody, which inhibited URAT1 action and uric acid reabsorption, and increased pancreatic insulin level with a lower insulitis incidence. Vaccination with U-IA-2(5)-P2-1 (UIP-1) significantly reduced blood glucose and uric acid level, increased Th2 cytokines interleukin (IL)-10 and IL-4, and regulated immune reactions through a balanced Th1/Th2 ratio. These results demonstrate that the URAT1-based multi-epitope peptide vaccine may be a suitable therapeutic approach for diabetes and its complications.

Immunoreactivity of urate transporters, GLUT9 and URAT1, is located in epithelial cells of the choroid plexus of human brains.

It has been suggested that urate plays a protective role in neurons, while hyperuricemia is correlated with atherosclerosis and cardiovascular disease. However, whether there is a system that directly transports urate into the brain remains to be clarified. In this study, the localization of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), which are known to be representative reabsorptive urate transporters, was immunohistochemically examined in autopsied human brains. Immunoreactivity of GLUT9 was observed on the apical side of the cytoplasm of epithelial cells in the choroid plexus and in the cilia of ependymal cells of the human brain. Immunoreactivity of URAT1 was observed on the basolateral side of the cytoplasm of epithelial cells in the choroid plexus. In addition, immunoreactivity of GLUT9 and URAT1 was not observed in microvessels of the human brains. The choroid plexus and renal proximal tubule were similar in having a polarized distribution of these two transporters with the two transporters on opposite membranes, but the two transporters' distribution differs between the choroid plexus and the kidney in terms of which membrane (apical/basal) expresses which transporter. These findings support the hypothesis of the direct transport of intravascular urate into the central nervous system through the choroid plexus.

Prostaglandin Transporter (PGT/SLCO2A1) Protects the Lung from Bleomycin-Induced Fibrosis.

Prostaglandin (PG) E2 exhibits an anti-fibrotic effect in the lung in response to inflammatory reactions and is a high-affinity substrate of PG transporter (SLCO2A1). The present study aimed to evaluate the pathophysiological relevance of SLCO2A1 to bleomycin (BLM)-induced pulmonary fibrosis in mice. Immunohistochemical analysis indicated that Slco2a1 protein was expressed in airway and alveolar type I (ATI) and II (ATII) epithelial cells, and electron-microscopic immunohistochemistry further demonstrated cell surface expression of Slco2a1 in ATI cells in wild type (WT) C57BL/6 mice. PGE2 uptake activity was abrogated in ATI-like cells from Slco2a1-deficient (Slco2a1-/-) mice, which was clearly observed in the cells from WT mice. Furthermore, the PGE2 concentrations in lung tissues were lower in Slco2a1-/- than in WT mice. The pathological relevance of SLCO2A1 was further studied in mouse BLM-induced pulmonary fibrosis models. BLM (1 mg/kg) or vehicle (phosphate buffered saline) was intratracheally injected into WT and Slco2a1-/- mice, and BLM-induced fibrosis was evaluated on day 14. BLM induced more severe fibrosis in Slco2a1-/- than in WT mice, as indicated by thickened interstitial connective tissue and enhanced collagen deposition. PGE2 levels were higher in bronchoalveolar lavage fluid, but lower in lung tissues of Slco2a1-/- mice. Transcriptional upregulation of TGF-ß1 was associated with enhanced gene transcriptions of downstream targets including plasminogen activator inhitor-1. Furthermore, Western blot analysis demonstrated a significant activation of protein kinase C (PKC) δ along with a modest activation of Smad3 in lung from Slco2a1-/- mice, suggesting a role of PKCδ associated with TGF-ß signaling in aggravated fibrosis in BLM-treated Slco2a1-/- mice. In conclusion, pulmonary PGE2 disposition is largely regulated by SLCO2A1, demonstrating that SLCO2A1 plays a critical role in protecting the lung from BLM-induced fibrosis.

Optimal methods of antigen retrieval for organic anion transporters in cryosections of the rat kidney.

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.

Development of high-specificity antibodies against renal urate transporters using genetic immunization.

Recently three proteins, playing central roles in the bidirectional transport of urate in renal proximal tubules, were identified: two members of the organic anion transporter (OAT) family, OAT1 and OAT3, and a protein that designated renal urate-anion exchanger (URAT1). Antibodies against these transporters are very important for investigating their expressions and functions. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages including high specificity and high recognition to the native protein compared with current methods. We fused high antigenicity fragments of the three transporters to the plasmids pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin, respectively. Gene gun immunization with these recombinant plasmids and two other adjuvant plasmids, which express granulocyte/ macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, induced high level immunoglobulin G antibodies, respectively. The native corresponding proteins of URAT1, OAT1 and OAT3, in human kidney can be recognized by their specific antibodies, respectively, with Western blot analysis and immunohistochemistry. Besides, URAT1 expression in Xenopus oocytes can also be recognized by its corresponding antibody with immuno-fluorescence. The successful production of the antibodies has provided an important tool for the study of UA transporters.

Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.

In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

[Preparation and identification of the rabbit antibody against human sialin].

AIM: To prepare the rabbit antibody against human sialin and identify its properties. METHODS: Recombinant expression vector pGEX-5X-1-sialin was constructed, in which the sialin cDNA encoding the 1-38 aa was fused to the C-terminal of the gene encoding the GST protein. The GST-sialin (N1-38) fusion protein was expressed in E. coli JM109 at 37 degrees C in the presence of IPTG at 0.1 mmol/L for induction for 3 hours, purified by GSTrap FF, and then used as the immunogen to prepare the rabbit polyclonal antibody. The properties of antiserum against human sialin were identified by ELISA, Western blot and immunocytochemistry. RESULTS: The recombinant expression plasmid pGEX-5X-1-sialin was constructed. The GST-sialin (N1-38) fusion protein was highly expressed with a molecular weight of 30 kDa, and the yield of the fusion protein was about 20% to 30% in total E. coli protein. The titre of antiserum against human sialin was 1:32,000. Western blot analysis proved the rabbit polyclonal antibody could identify both GST-sialin (N1-38) fusion protein and GST. Besides, it specially recognized a 55 kDa band expressed in the human submandibular gland (HSG) cell line. The antigen recognized by the antibody was located in the cytoplasm and nucleus of HSG cell. CONCLUSION: The successful preparation of the polyclonal antibody against human sialin will provide efficient affinity reagent for further functional study of sialin expressed in human salivary glands.

The role of ABC-transporter gene polymorphisms in chemotherapy induced immunosuppression, a retrospective study in childhood acute lymphoblastic leukaemia.

We examined the association of functional ABCB1 (MDR1) and ABCG2 (BCRP) polymorphisms with acute side effects of chemotherapy. Analyses were performed on clinical data from 138 patients treated with the ALL-BFM-95 protocol implying several substrates of these transporters. ABCB1 3435T>C, 2677G>T/A 1236C>T and ABCG2 421C>A genotypes were determined. A higher proportion of ABCB1 3435TT patients suffered excessive infectious complications than those harbouring at least one C allele (OR=2.5, p=0.03) during the whole half-year-long intensive phase of chemotherapy. Weaker associations were calculated when ABCB1 1236T-2677T-3435T haplotype homozygotes were tested against the remaining part of the population (OR=2.3, p=0.09). During the reinduction phase of therapy, the occurrence of severe leukocytopenia was similar among ABCB1 genotype groups. The frequency of any toxicities were not shown to differ according to the ABCG2 421C>A genotype. Our data suggest that the ABCB1 3435T>C genotype is associated with the infectious complications of the applied chemotherapy regimen.

Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification.

BACKGROUND: Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney. METHODS: Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody's generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry. RESULTS: The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58 kD hURAT1 and 64 kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody. CONCLUSION: Genetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.

Identification of the gonad-specific anion transporter SLCO6A1 as a cancer/testis (CT) antigen expressed in human lung cancer.

Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of many of the antigens recognized by the immune system of cancer patients, which are collectively referred to as the cancer immunome. We used SEREX to screen a testicular cDNA expression library with sera obtained from non-small cell lung cancer patients and isolated cDNA clones for 82 antigens. These included a total of 31 antigens previously identified by SEREX, and 51 that did not match entries in the Cancer Immunome Database and were considered newly identified antigens. Overall, the antigens comprised 62 known proteins and 20 uncharacterized gene products. Six antigens (NY-TLU-6, -37, -39, -57, -70, -75) were identified as putative cell surface proteins that are potential targets for monoclonal antibody-based immunotherapy. Of these, the gonad-specific anion transport protein SLCO6A1 (NY-TLU-57) was shown to be tissue-restricted. RT-PCR showed it to be expressed strongly only in normal testis, and weakly in spleen, brain, fetal brain, and placenta. In addition, NY-TLU-57 mRNA was found in lung tumor samples (5/10) and lung cancer cell lines (6/11), as well as bladder (5/12) and esophageal (5/12) tumor samples. These data suggest that SLCO6A1 is a putative cancer/testis (CT) cell surface antigen of potential utility as a target for antibody-based therapy for a variety of tumor types. The analysis also permits us to estimate the eventual size of the SEREX-defined cancer immunome at around 4000 genes. This emphasizes the importance of continued SEREX screening to define the cancer immunome.

Subcellular immunolocalisation of fatty acid translocase (FAT)/CD36 in human type-1 and type-2 skeletal muscle fibres.

Limited information exists about the putative role and expression in human skeletal muscle cells of the 88-kDa integral membrane protein fatty acid translocase (FAT), highly homologous to the human leucocyte differentiation factor CD36. Therefore, we investigated in healthy male individuals the muscle (m. vastus lateralis) fibre type specific expression and subcellular localisation of FAT/CD36. For this purpose four different monoclonal antibodies raised against human and mouse FAT/CD36 were used. Acetone or methanol/acetone fixation were tested. Serial cryosections were cut at -20 degrees C, thaw-mounted on uncoated glass slides and air-dried before processing indirect immunofluorescence assays. Images were examined in a Nikon ER800 microscope, digitally captured, processed and analysed by LUCIA laboratory software. Three antibodies showed that FAT/CD36 was: (1) most abundantly expressed in capillary endothelium, (2) colocalised with caveolin-3, which indicates that FAT/CD36 is in the sarcolemma, or its close vicinity, and (3) abundantly expressed in (or in the close vicinity) of the sarcolemma and intracellular structures of type-1 muscle fibres, and much less abundantly in the sarcolemma of type-2 muscle fibres. One of the antibodies raised against mouse CD36 also detected myosin heavy chain 1, which makes it unsuitable in skeletal muscle research. The fixation (acetone or methanol/acetone) was found to be highly important for the result.

The CD36 protein functions as an immunogenic domain of the RT8 alloantigen.

Completely concordant distributions of Cd36 and Rt8 deletion/null and wild-type alleles among inbred and congenic strains, together with Western blot analysis of RT8/CD36 proteins, indicated that the CD36 protein functions as an immunogenic domain of the RT8 alloantigen.

CD36/fatty acid translocase in rats: distribution, isolation from hepatocytes, and comparison with the scavenger receptor SR-B1.

The new mAb UA009 recognizes an antigen expressed by microvascular endothelium, by lymphatic endothelium, and by some epithelia in a number of organs, including the small intestine, lactating mammary gland, kidney, lung, sebaceous glands, and circumvallate papillae of the tongue. This antigen is also expressed abundantly in the splenic red pulp and marginal zone and by monocytes, macrophages, and erythrocytes (but not by platelets). Among tissues that store or metabolize fatty acids, the antigen is expressed by adipocytes, cardiomyocytes, and red skeletal muscle. Importantly, it is expressed by steroidogenic cells in the adrenal gland, testis, and ovary, whereas in the liver it is expressed by hepatocytes in a pattern that is dependent on gender and genetic background. mAb UA009 immunoprecipitated a mol wt 85-kDa surface protein from detergent extracts of hepatocytes from Dark Agouti female rats. The N-terminal amino acid sequence of this protein was identical to fatty acid translocase (FAT), the rat cluster of differentiation 36 (CD36) ortholog. The mAb also reacted with COS-7 cells transfected with cDNA encoding FAT. cDNAs encoding a CD36/FAT-like polypeptide were prepared from both liver and heart RNA by RT-PCR. The nucleotide sequences obtained from these cDNAs (Dark Agouti rats) revealed identity and 99% similarity, respectively, with the published sequences of Cd36/Fat in rats of the Wistar and Sprague-Dawley strains. The absence of the UA009 antigen in CD36/FAT-deficient SHR/N rats confirmed the identity of the UA009 antigen and CD36/FAT. We suggest that CD36/FAT might function in the liver as a sex-regulated accessory molecule, either in reverse cholesterol transport and/or in fatty acid uptake.