Retinol-binding Protein Gene Research Articles

Reactive astrocytes targeting with oral vitamin A: Efficient neuronal regeneration for Parkinson's disease treatment and reversal of associated liver fibrosis.

A recent approach to cure neurodegenerative diseases is to reprogram fibroblasts into functioning neurons using multiple exogenous transcription factors (TFs) and micro-RNAs. Administering agents that can endogenously induce these TFs may bypass the limitations of this approach. Astrocytes may represent a part of the extrahepatic-stellate system involved in vitamin-A (VA ) homeostasis. Activated-stellate cells lose their VA -storage capacity, and this was previously applied for hepatic-stellate cells (HSCs) targeting to treat liver fibrosis. Accordingly, it is hypothesized that Parkinson's disease (PD) may be coupled with retinoid depletion that may extract VA from VA -rich-HSCs triggering liver fibrosis. Thus, VA administration may selectively target VA -deficient reactive astrocytes and HSCs. Besides, VA has the regenerative capability and may induce endogenous-TFs generation. Fluorescently labeled VA -coupled liposomes (FLV) were traced using confocal laser microscope in rats with induced PD for detecting brain accumulation and uptake into fluorescently labeled astrocytes. Liver fibrosis associated with PD was assessed biochemically and histopathologically, while VA deficiency was confirmed by assessing retinol-binding protein gene expression in the brain and liver. Multiple VA doses were tested for reversing PD-associated liver fibrosis, generating TFs (involved in reprograming astrocytes/fibroblasts into different neuronal types) and capability of dopaminergic-neurons regeneration. Fluorescently labeled VA -coupled liposomes revealed selective brain accumulation and uptake into astrocytes. PD was associated with significant liver fibrosis and VA deficiency in the brain and liver. Furthermore, VA -medium dose (VAMD) was the optimum one for reversing PD-associated liver fibrosis, generating multiple astrocytes/fibroblasts reprogramming TFs, regenerating dopaminergic neurons, and improving PD. VA -medium dose pursued brain targeting in PD with the potential capability of regenerating neurons and restoring dopaminergic transmission. This may place this therapy as an essential treatment in PD management protocol.

Differential retention of duplicated retinoid-binding protein (crabp &rbp) genes in the rainbow trout genome after two whole genome duplications and their responses to dietary canola oil

The complexity of retinoid intracellular signaling by lipid-binding proteins is further confounded in salmonids, owing to a whole genome duplication (WGD), followed by a second salmonid-specific WGD. As such, many more cellular retinoic acid-binding protein (crabp)/retinol-binding protein (rbp) genes may exist in salmonids, compared to other vertebrate species. In this study, genomic database searches and phylogenetic analyses identified seven crabp and eight rbp genes in the rainbow trout. A parsimonious explanation for the number of crabp/rbp genes in the rainbow trout genome is duplicated genes were lost from the genome by nonfunctionalization following the salmonid-WGD, and the extant duplicate crabp/rbp genes of the much earlier teleost-WGD have been retained by either sub- or neofunctionalization of tissue-specific regulatory mechanisms. Nutritional regulation of retinoid-binding protein genes in the liver was also determined in the fish fed diets containing various amounts of canola oil (CO) for 12 weeks along with hepatic fatty acid profile, Vitamin A (VitA) level, and growth parameters. The crabp/rbp transcripts were modulated markedly by experimental diets except for rbp2a.1/rbp2a.2 at the end of the trial. This result suggests that rbp2a.1/rbp2a.2 don't exhibit obvious divergence of transcriptional regulatory mechanisms that control their nutritional regulation by dietary CO. Growth parameters didn't differ markedly among dietary treatments. An understanding of VitA homeostasis, especially the regulatory effects of retinoids on transcriptional control of target genes, may have considerable importance to basic fish development, physiology, and aquaculture and this study may lead to future research on molecular VitA metabolism especially in salmonids experienced salmonid-WGD.

Novel Functions of the Fatty Acid and Retinol Binding Protein (FAR) Gene Family Revealed by Fungus-Mediated RNAi in the Parasitic Nematode, Aphelenchoides besseyi

RNA interference (RNAi) is a powerful tool for the analysis of gene function in nematodes. Fatty acid and retinol binding protein (FAR) is a protein that only exists in nematodes and plays an important role in their life activities. The rice white-tip nematode (RWTN), Aphelenchoides besseyi, is a migratory endoparasitic plant nematode that causes serious damage in agricultural production. In this study, the expression levels of eight RWTN genes were effectively decreased when RWTN was fed Ab-far-n (n: 1–8) hairpin RNA transgenic Botrytis cinerea (ARTBn). These functions of the far gene family were identified to be consistent and diverse through phenotypic changes after any gene was silenced. Such consistency indicates that the body lengths of the females were significantly shortened after silencing any of the eight Ab-far genes. The diversities were mainly manifested as follows: (1) Reproduction of nematodes was clearly inhibited after Ab-far-1 to Ab-far-4 were silenced. In addition, silencing Ab-far-2 could inhibit the pathogenicity of nematodes to Arabidopsis; (2) gonad length of female nematodes was significantly shortened after Ab-far-2 and Ab-far-4 were silenced; (3) proportion of male nematodes significantly increased in the adult population after Ab-far-1, Ab-far-3, and Ab-far-5 were silenced, whereas the proportion of adult nematodes significantly decreased in the nematode population after Ab-far-4 were silenced. (4) Fat storage of nematodes significantly decreased after Ab-far-3, Ab-far-4, and Ab-far-7 were silenced. To our knowledge, this is the first study to demonstrate that Ab-far genes affect sex formation and lipid metabolism in nematodes, which provides valuable data for further study and control of RWTNs.

Molecular cloning, characterization, and steady-state levels of retinol-binding protein (rbp) genes in response to dietary soybean oil in brown trout (Salmo trutta)

The cDNA of rbp1a, rbp2a, rbp2b, rbp5, and rbp7b genes was cloned and characterized in brown trout, and their mRNA expressions in response to different levels of dietary soybean oil (SBO) were determined along with their growth parameters, muscle fatty acid (FA) profile, and hepatic retinol level. Dietary SBO did not affect the growth parameters, survival, and hepatosomatic index. The FA composition and retinol content of fish reflected those of experimental diets. The retinol-binding proteins (Rbps; except Rbp5) contained lipocalin domains. The phylogenetic analysis showed that brown trout rbps are orthologs of the rbp/RBPs from other vertebrates; this was also supported by identity/similarity analysis. The rbp genes were exhibited to have conserved synteny with zebrafish and human rbps/RBPs. The tissue-specific transcription of duplicated rbp2a and rbp2b genes varied among all the tested tissues. Moreover, the diet including 100% SBO affected the rbp2a and rbp2b mRNA transcriptions differently, and this was explained by the divergence of the transcriptional regulation site of these genes due to the teleost-specific whole genome duplication. rbp1b and rbp7a were lost in the brown trout genome most likely because of nonfunctionalization. The mRNA expression of all the rbp genes studied was affected by dietary SBO. Therefore, their mRNA expressions could be modulated by dietary FAs and retinol content.

A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi

ABSTRACT RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (Ab-far-1, Ab-far-4, and combinatorial Ab-far-1 and Ab-far-4) of the rice white tip nematode (RWTN), Aphelenchoides besseyi, were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTBn) were generated using Agrobacterium-mediated transformation technology. After RWTN feeding on ARTBn, the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs. Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi); ARTB1 (Ab-far-1 hpRNA transgenic Botrytis cinerea); ARTB4 (Ab-far-4 hpRNA transgenic Botrytis cinerea); ARTB1/4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea); EVTB (Empty vector transgenic B. cinerea); GRTB (eGFP hpRNA transgenic B. cinerea); WTB (Wild-type B. cinerea).

Cloning and functional study of lipocalin: retinol-binding protein-like gene family of the ridgetail white prawn, Exopalaemon carinicauda.

Lipocalin is a large family with complex functions including retinol-binding protein (RBP), crustacyanin (CRCN), apolipoprotein D, etc. In shrimps, it is well known that CRCN is related to body color. Recently, retinoic acid/retinol-binding protein was found in shrimp. However, little is known about the function of RBP and relationships among the gene members of lipocalin in shrimps. Based on the transcriptome sequences responding to starvation stress, three genes of the lipocalin-retinol-binding protein-like gene family (lipocalin-1, lipocalin-2, and lipocalin-3) were cloned by RACE from the ridgetail white prawn, Exopalaemon carinicauda. Homology analysis showed that these three genes had high similarity with the known insect apolipoprotein D gene and vertebrate retinol-binding protein gene, and they are of the same type in terms of evolution. Fluorescence quantitative PCR showed that the above three genes were mainly expressed in the ventral nerve cord of E. carinicauda. The expression characteristics of the three genes at different developmental stages showed that they were more highly expressed at the larval stage, which suggests that they might be related to embryonic and larval development. The RNA interference tests showed that after silencing lipocalin-1 and lipocalin-3, the body color of individual shrimps turned slightly red and the blue pigment in the epidermis largely disappeared, but no significant change took place in the appearance of individuals after silencing lipocalin-2. In addition, on the 6th and 16thdays of interference, dead shrimps appeared in the lipocalin-1 and lipocalin-3 interference groups. The dead shrimps had hard crusts and remained in a molting posture. Totally, this study showed that the retinol-binding protein-like gene obtained in this study had certain biological functions in the growth and development and body color formation as CRCN; in addition, it also plays a role in nerve system and molting of E. carinicauda.

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode.

Silencing of murine transthyretin and retinol binding protein genes has distinct and shared behavioral and neuropathologic effects

The murine genes encoding transthyretin (TTR) and retinol binding protein (RBP) were independently silenced by targeted disruption more than 10years ago. Studies of both strains showed surprisingly little impact on either thyroid function or retinoid metabolism. Silencing TTR led to a relatively mild behavioral phenotype. In order to gain insight into the behavioral effect and determine if it was related to TTR’s function as the carrier of RBP we carried out simultaneous studies with homozygous Rbp4−/− and Ttr−/− animals 4–7months of age. Both strains showed behavioral differences relative to Ttr and Rbp4 wild-type animals and each other. The patterns were discrete for each knockout although there was some overlap. Neuropathologic examination of the cortex and hippocampus revealed cortical and hippocampal (CA3) neuronal loss in both and some degree of gliosis, more pronounced in the Rbp4−/− mice. There also appeared to be a major reduction in proliferating neuroblasts in the subventricular zone in both strains, which was also more severe in the Rbp4−/− mice. This is the first description of behavioral abnormalities in Rbp4−/−mice. The data also indicate that it is unlikely that the behaviors seen in Ttr−/− mice are related to its function as an RBP carrier.

Characterization of the venom allergen—like protein (vap-1) and the fatty acid and retinol binding protein (far-1) genes in Meloidogyne hispanica

The root-knot nematode (RKN) Meloidogyne hispanica has been found in all continents associated with a wide host range, including economically, important plants and can be considered a species of emerging importance. Considerable progress has been made to identify nematode effector genes as they are important targets for the development of novel control strategies. The effector genes, venom allergen-like protein (vap-1) and fatty acid and retinol binding protein (far-1), were identified, isolated and sequenced in M. hispanica (Mhi-vap-1 and Mhi-far-1) using the genome information available for the RKNs M. incognita and M. hapla. These genes are differentially expressed during M. hispanica development and their amplification products were observed from cDNA of the eggs, second-stage juveniles (J2) and adult females. However, Mhi-vap-1 showed the highest level of expression in J2. In situ hybridization analysis revealed that the Mhi-vap-1 and Mhi-far-1 transcripts are accumulated within the J2 subventral oesophageal glands. The specific expression in the subventral oesophagel glands and presence of the secretion signal peptide for both genes suggests that these proteins are secreted by the J2 and may play a role in the early parasitic stage of the infection process. These genes were also isolated and sequenced in M. arenaria, M. incognita and M. javanica; and phylogenetic analysis revealed that the predicted protein sequences belonging to M. hispanica and several other species of plant-parasitic nematodes have a high degree of conservation.

Genomic organization and transcription of the medaka and zebrafish cellular retinol-binding protein (rbp) genes

In this study, we examined the evolutionary trajectories and the common ancestor of medaka rbp genes by comparing them to the well-studied rbp/RBP genes from zebrafish and other vertebrates. We describe here gene structure, sequence identity, phylogenetic analysis and conserved gene synteny of medaka rbp genes and their putative proteins as well as the tissue-specific distribution of rbp transcripts in adult medaka and zebrafish. Medaka rbp genes consist of four exons separated by three introns that encode putative polypeptides of 134–138 amino acids, a genomic organization characteristic of rbp genes. Medaka Rbp sequences share highest sequence identity and similarity with their orthologs in vertebrates, and form a distinct clade with them in phylogenetic analysis. Conserved gene synteny was evident among medaka, zebrafish and human rbp/RBP genes, which provides compelling evidence that the medaka rbp1, rbp2a, rbp2b, rbp5, rbp7a and rbp7b genes arose from a common ancestor of vertebrates. Moreover, the duplicated rbp2 and rbp7 genes most likely exist owing to a whole-genome duplication (WGD) event specific to the teleost fish lineage. Selection pressure and the nonparametric relative rate test of the medaka and zebrafish duplicated rbp2 and rbp7 genes suggest that these duplicated genes are subjected to purifying selection and one paralog might have evolved at an accelerated rate compared to its sister duplicate since the WGD. The steady-state levels of medaka and zebrafish rbp1, rbp2a, rbp2b and rbp5 transcripts in various tissues suggest that medaka rbp1, rbp2a and rbp2b genes have retained the regulatory elements of an ancestral RBP1 and RBP2 genes, and the medaka rbp5 gene has acquired new function. Furthermore, the tissue-specific regulations of rbp7a and rbp7b genes have diverged markedly in medaka and zebrafish since the teleost-specific WGD.

Characterization of the Expression Profile and Genetic Polymorphism of the Cellular Retinol-Binding Protein (CRBP IV) Gene in Erlang Mountainous Chickens.

In this study, we cloned the coding sequence of chicken CRBP IV, quantified the mRNA expression in Erlang Mountainous Chickens, and investigated a polymorphism in this gene and its association with egg production traits among 349 individuals. The cloned fragment contained a 384 bp open reading frame, which encoded a predicted protein of 127 amino acids and was highly conserved among species. Expression of CRBP IV mRNA was detected in all eight tissues (small intestine, heart, liver, kidney, oviduct, ovary, pituitary, and hypothalamus) at different ages (12, 24, 32 and 45 w). High expression was found in small intestine, pituitary, kidney and liver, whereas it was low in the heart (p < 0.05). The CRBP IV mRNA levels changed with age in the various tissues, and were highly expressed in all tissues at 32 w, except for the heart. We identified one nucleotide substitution (c. 826T>C) in the second exon, which caused an amino acid change (p. S49L). Genotypes (TT, TC and CC) had significant effects on the age at first egg (AFE), total eggs for 300 days (TE300) and highest continuous laying days (HCLD). The CC genotype would be genetically advantageous to improve egg production traits due to earlier AFE, more TE300, and longer HCLD.

Bovine hepatic and adipose retinol-binding protein gene expression and relationship with tumor necrosis factor-α

Retinol-binding protein (RBP) is the main transport system for retinol in circulation, is a relatively small protein with one binding site for retinol in the all-trans form, and is bound to transthyretin. The objectives of this study were to characterize the temporal pattern of bovine hepatic mRNA expression of RBP during the periparturient period and to determine if a relationship exists between the expression of RBP and that of tumor necrosis factor (TNF)-α in dairy cows. In experiment 1, we assessed hepatic mRNA expression of RBP during the periparturient period. Liver tissues were sampled from periparturient dairy cows (n=9) at −21, −4, +1, +7, and +21 relative to parturition and frozen in liquid N2. Total RNA was extracted from each tissue sample and cDNA was generated. Gene expressions of RBP and β-actin (as a housekeeping gene) were measured as relative quantity using reverse transcription-PCR. Data were analyzed using cycle threshold values, adjusted to β-actin, and significance was determined at P<0.05. Serum samples (−21, −4, +1, +7, and +21 relative to parturition) were analyzed for retinol concentration using a standard HPLC-based method. Cows had variable expression of hepatic RBP and serum retinol over the transition period, with a decline near parturition and a rebound toward prepartum levels later in lactation. In experiment 2, liver and visceral (intestinal) adipose tissues were sampled from dairy cows (n=28) at slaughter. Expression of RBP and TNF-α was detected in all samples and variations among cows were highly significant for both genes. Across tissues, expression of RBP was positively correlated with that of TNF-α (r=0.60). Within adipose tissue, expression of RBP and TNF-α was weakly correlated (r=0.23), whereas in hepatic tissue, expression was strongly correlated (r=0.62). In experiment 3, late-lactation dairy Holstein cows were blocked by parity and feed intake, and randomly assigned to control, recombinant bovine (rb)TNF challenge, or pair-fed control treatment (n=5/treatment). Cows were injected with either rbTNF (subcutaneous injection of 2µg/kg of body weight in saline) or sterile saline (control and pair-fed control animals) once daily for 7d. Liver biopsy was performed on d 7 and samples were processed for expression of RBP and TNF-α. Although TNF challenge caused an upregulation of hepatic TNF-α expression, as expected, it did not alter hepatic RBP expression. Overall, the temporal pattern of hepatic RBP gene expression during the periparturient period followed, to a great extent, that of plasma retinol. Although a strong positive correlation was previously detected between bovine hepatic RBP and TNF-α transcripts, rbTNF challenge did not cause alter RBP expression. These observations collectively imply that regulation of RBP at the transcription level is influenced by physiological state but may be independent from that of transthyretin, which is altered by proinflammatory stimuli (such as TNF-α) via induction of transcription factor nuclear factor-interleukin 6.

Alterations in retinoid status after long-term exposure to PBDEs in zebrafish (Danio rerio)

This study examined the disruptive effect of exposure to polybrominated diphenyl ethers (PBDEs) on retinoid content in zebrafish (Danio rerio). Adult zebrafish were exposed to an environmentally relevant concentration (0.45μg/L) and a higher concentration (9.6μg/L) of DE-71 for 60 days. Retinoid content and gene transcription levels were examined in female zebrafish. PBDE exposure caused a significant decrease of retinyl ester content in the intestine and a downregulation of intestinal cellular retinol binding protein gene transcription (CRBP1a). In the liver, retinyl ester content was significantly decreased, while retinol content was increased. An upregulation of liver CRBP2a and retinol binding protein (RBP) gene transcription and an increased level of RBP protein were observed. In the eyes, both the retinal and retinyl ester content were increased and CRBP1a gene transcription was upregulated. However, the gene encoding for retinal dehydrogenase (RALDH2), responsible for retinoic acid synthesis, was downregulated in the eyes. CYP26a, the gene responsible for retinoic acid degradation, was upregulated, which indicated an increased level of retinoic acid. In the ovaries, the increased deposition of retinoids was also observed, while gene transcription levels of both CRBPs (CRBP1a and CRBP1b) were upregulated. An increased deposition of retinal was measured in the eggs. Overall, this study demonstrated that long-term exposure of zebrafish to environmentally relevant concentrations of DE-71 disrupted the transport, storage and metabolism of retinoid in various tissues. This study also indicated that retinoid levels in zebrafish are sensitive to PBDE exposure and highlighted the importance of liver storage, which appears to support important functions in reproduction and vision.

Uptake of Dietary Retinoids at the Maternal-Fetal Barrier: IN VIVO EVIDENCE FOR THE ROLE OF LIPOPROTEIN LIPASE AND ALTERNATIVE PATHWAYS

Dietary retinoids (vitamin A and its derivatives) contribute to normal embryonic development. However, the mechanism(s) involved in the transfer of recently ingested vitamin A from mother to embryo is not fully understood. We investigated in vivo whether lipoprotein lipase (LPL) facilitates the placental uptake of dietary retinyl ester incorporated in chylomicrons and their remnants and its transfer to the embryo. We examined the effects of both genetic ablation (MCK-L0 mice) and pharmacological inhibition (P-407) of LPL by maintaining wild type and MCK-L0 mice on diets with different vitamin A content or administering them an oral gavage dose of [(3)H]retinol with or without P-407 treatment. We showed that LPL expressed in placenta facilitates uptake of retinoids by this organ and their transfer to the embryo, mainly through its catalytic activity. In addition, through its "bridging function," LPL can mediate the acquisition of nascent chylomicrons by the placenta, although less efficiently. Quantitative real-time PCR and Western blot analysis showed that placental LPL acts in concert with LDL receptor and LRP1. Finally, by knocking out the retinol-binding protein (RBP) gene in the MCK-L0 background (MCK-L0-RBP(-/-) mice) we demonstrated that the placenta acquires dietary retinoids also via the maternal circulating RBP-retinol complex. RBP expressed in the placenta facilitate the transfer of postprandial retinoids across the placental layers toward the embryo.

TWO EXTREME X-RAY MUTATIONS OF MORPHOLOGICAL INTEREST

Retinol-binding proteins (RBPs) are a kind of circulating carrier proteins for serum and cellular retinol and retinol acid, which are lipid-soluble vitamins, and are members of hydrophobic binding protein family. Serum RBPs were synthesized primarily in liver, then was released into blood streams, and then to various tissues. Under the interaction with substances such as retinol, pre-albumin and the receptors of cellular surface, they play important roles in storage, metabolism of VitA and transport of VitA to the target cells. Cellular RBPs play the similar function as serum RBPs in intracell. This review introduces action mechanism, tissue localization and developmental expression of retinol-binding proteins. This review also introduces the structure, chromosome mapping and their relationships with reproductive performance of retinol-binding protein genes.

The effects of environmental salinity on trunk kidney proteome of juvenile ayu (Plecoglossus altivelis)

As the life cycle of ayu spans river, brackish and seawater environments, it would be a suitable fish model for studying the responses to salinity changes in aquatic animals. We investigated the effect of salinity on trunk kidney proteome in ayu (Plecoglossus altivelis) using two-dimensional gel electrophoresis and mass spectrometry. The proteins involved in the process of energy metabolism, biosynthesis, DNA methylation and cell differentiation were mainly affected, and 10 significantly changed proteins were identified. Our result showed that isocitrate dehydrogenase (ICD), pyruvate dehydrogenase (E1), O-glycosyl hydrolase, mitochondrial precursor of ATP synthase subunit beta, mitochondrial ferrtin (MtF), retinol binding protein (RBP) were down-regulated, whereas aldehyde dehydrogenase, cytokeratin 1, S-adenosylhomocysteine hydrolase, Cys-Met metabolism PLP-dependent enzyme were up-regulated when ayu transferred from freshwater to brackish water. Partial coding sequences of E1, ICD, MtF and RBP genes were determined, and the effects of salinity on their mRNA expression in ayu trunk kidney were tested by real-time PCR subsequently. Their possible direct or indirect roles in the adaptation of ayu to salinity are discussed.

Transcriptional activity of the murine retinol-binding protein gene is regulated by a multiprotein complex containing HMGA1, p54nrb/NonO, protein-associated splicing factor (PSF) and steroidogenic factor 1 (SF1)/liver receptor homologue 1 (LRH-1)

Retinol-binding protein (RBP4) transports retinol in the circulation from hepatic stores to peripheral tissues. Since little is known regarding the regulation of this gene, we analysed the cis-regulatory sequences of the mouse RBP4 gene. Our data show that transcription of the gene is regulated through a bipartite promoter: a proximal region necessary for basal expression and a distal segment responsible for cAMP-induction. This latter region contains several binding sites for the structural HMGA1 proteins, which are important to promoter regulation. We further demonstrate that HMGA1s play a key role in basal and cAMP-induction of Rbp4 transcription and the RBP4 and HMGA1 genes are coordinately regulated in vitro and in vivo. HMGA1 acts to recruit transcription factors to the RBP4 promoter and we specifically identified p54nrb/NonO and protein-associated splicing factor (PSF) as components that interact with this complex. Steroidogenic factor 1 (SF1) or the related liver receptor homologue 1 (LRH-1) are also associated with this complex upon cAMP-induction. Depletion of SF1/LRH-1 by RNA interference resulted in a dramatic loss of cAMP-induction.Collectively, our results demonstrate that basal and cAMP-induced Rbp4 transcription is regulated by a multiprotein complex that is similar to ones that modulate expression of genes of steroid hormone biosynthesis. Since genes related to glucose metabolism are regulated in a similar fashion, this suggests that Rbp4 expression may be regulated as part of a network of pathways relevant to the onset of type 2 diabetes.

Mapping, Tissue Distribution and Polymorphism of Porcine Retinol Binding Protein Genes (RBP5 and RBP7)

The retinoids (vitamin A and its derivatives) play a critical role in vision, growth, reproduction, cell differentiation and embryonic development. Using the IMpRH panel, porcine cellular retinol binding protein genes 5 and 7 (RBP5 and RBP7) were assigned to porcine chromosomes 5 and 6, respectively. The complete coding sequences (CDS) of the RBP5 and RBP7 genes were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) method, and the deduced amino acid sequences of both genes were compared to human corresponding proteins. The mRNA distributions of the two genes in adult Wuzhishan pig tissues (lung, skeletal muscle, spleen, heart, stomach, large intestine, lymph node, small intestine, liver, brain, kidney and fat) were examined. A total of nine single nucleotide polymorphisms (SNPs) were identified in two genes. Three of these SNPs were analyzed using the polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) method in Laiwu, Wuzhishan, Guizhou, Bama, Tongcheng, Yorkshire and Landrace pig breeds. Association analysis of genotypes of these SNP loci with economic traits was done in our experimental populations. Significant associations of different genotypes of RBPS-A/G 63 , RBPS-A/G 517 and RPBS-T/C intron1-90 loci with traits including maximum carcass length (LM), minimum carcass length (LN), marbling score (MS), back fat thickness at shoulder (SBF), meat color score (MCS) and hematocrit (HCT) were detected. These SNPs may be useful as genetic markers in genetic improvement for porcine production.

Mapping, tissue distribution and polymorphism of porcine genes encoding retinol binding proteins (RBP1 and RBP4)

AbstractUsing the INRA – University of Minnesota porcine 7000 Rad Radiation Hybrid panel (IMpRH) containing 118 clones, porcine cellular retinol binding protein gene 1 (RBP1) and plasma retinol binding protein gene 4 (RBP4) were assigned to porcine chromosomes 13 and 14, respectively. The mRNA distributions of the two genes in adult Wuzhishan pig tissues (lung, skeletal muscle, spleen, heart, stomach, large intestine, lymph node, small intestine, liver, brain, kidney and fat) were examined. One single nucleotide polymorphism (SNP) was identified in Laiwu, Wuzhishan, Guizhou, Bama pig breeds and a Tongcheng experimental population and analysed using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Significant associations were found between different genotypes of A/G156 and maximum carcass length (LM), minimum carcass length (LN), haemoglobin (HGB), mean corpuscular volume haemoglobin (MCH), and day to slaughter weight (age) traits in the Tongcheng experimental population. This SNP locus might be used as a genetic marker in pig breeding and production.

A Trend of Central Versus Peripheral Structuring in Mitochondrial and Nuclear Gene Sequences of the Japanese Wood Mouse, Apodemus speciosus

A phylogeographic analysis was performed on Japanese endemic wood mice (Apodemus speciosus) using nuclear interphotoreceptor retinol binding protein (IRBP) gene sequences (1,152 bp), together with previously published mitochondrial cytochrome b (cyt b) data. In the IRBP analysis, 40 haplotypes were recovered from 84 individuals by statistical and subcloning methods. Substantial sequence variation was determined from the IRBP data (pi=0.0047), and no significant evidence of recombination was detected. From the phylogenetic analysis, the 40 haplotypes fell into two major groups with geographic associations, irrespective of the karyotype groups (2n=46 and 2n=48), yielding a trend of central (Hokkaido, Honshu, Shikoku, Kyushu, and Sado) and peripheral (Izu, Oki, Tsushima, and Satsunan Is.) groupings. This geographic pattern is similar to that observed in the cyt b data, with a different insular grouping of Sado, Hokkaido, Izu, and Satsunan islands, and also to that of morphological features. In both gene data sets, nested clade analyses revealed allopatric fragmentation in the "peripheral island clades" and range expansion in the "central island clades." A mismatch analysis using cyt b data also suggested expansion of the central islands clade. Thus, the trend of central vs. peripheral structuring may be attributable to past demographic dynamics in the two distinct haplotype clades, such as range expansion of one clade in the central area of the Japanese Islands, leaving the other clade in the periphery.