Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC10643153 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundMaternal embryonic leucine zipper kinase (MELK) is an atypical member of the snf1/AMPK family of serine-threonine kinases, involved in diverse physiological and pathological processes, including cell proliferation, apoptosis, embryogenesis, cancer treatment resistance, and RNA processing. MELK is highly expressed in human cancers and is associated with more aggressive forms of astrocytoma, glioblastoma, breast cancer, and melanoma to date, no information about porcine MELK (pMELK) has been reported.MethodsIn this study, the pMELK coding sequence was cloned from swine spleen and characterized. We also quantitatively determined the expression of MELK in 11 tissues isolated from a piglet and determined its subcellular localization when expressed in swine umbilical vein endothelial cells (SUVEC) as a fusion protein. Moreover, we report the functional characterization of pMELK protein concerning its role in apoptosis.ResultsSequencing analysis showed that full-length of pMELK is 2,072 bp with 17 exons, encoding 655 amino acids, including an S-TKc conserved domain. Comparison of pMELK with ten other mammalian species of their orthologous sequences showed >91% homology and an evolutionary distance <0.05, demonstrating that MELK is highly conserved in evolution. Relative quantification of MELK expression in 11 tissue samples isolated from 30-day-old piglets showed MELK expression in all tested organs and the highest expression in the superficial inguinal lymph node. Constructed a plasmid named pEGFP-MELK, and the fusion protein GFP-MELK was successfully expressed in SUVECs. Fluorescence microscopy revealed the subcellular distribution of the fusion protein GFP-MELK was limited to the cytoplasm. About function, Flow cytometry analysis showed that overexpression of GFP-pMELK in SUVEC cells enhances staurosporine (STS)-induced apoptosis, but not significantly different. The pMELK protein also was found to interact with porcine BCL-G and transient transfection of the recombinant plasmid pCMV-HA-pMELK into SUVEC cells stably expressing GFP-pBCL-G protein inhibited pBCL-G -induced apoptosis significantly.ConclusionsThe present study provided useful information on pMELK basic details and function in apoptosis offer a potential new molecular model for disease interventions and disease related to human MELK and BCL-G.
| S-EPMC7154462 | biostudies-literature
Project description:Adipose tissue is a key regulator of energy balance playing an active role in lipid storage as well as in synthesizing several hormones directly involved in the pathogenesis of obesity. Obesity represents a peculiar risk factor for a growing list of cancers and is frequently associated to poor clinical outcome. The mechanism linking obesity and cancer is not completely understood, but, amongst the major players, there are both chronic low-grade inflammation and deregulation of adipokines secretion. In obesity, the adipose tissue is pervaded by an abnormal number of immune cells that create an inflammatory environment supporting tumor cell proliferation and invasion. Adiponectin (APN), the most abundant adipokine, shows anti-inflammatory, anti-proliferative and pro-apoptotic properties. Circulating levels of APN are drastically decreased in obesity, suggesting that APN may represent the link factor between obesity and cancer risk. The present review describes the recent advances on the involvement of APN and its receptors in the etiology of different types of cancer.
| S-EPMC6412253 | biostudies-literature
Project description:BackgroundIn mammals, L-threonine is an indispensable amino acid. The conversion of L-threonine to glycine occurs through a two-step biochemical pathway involving the enzymes L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase. The L-threonine 3-dehydrogenase enzyme has been purified and characterised, but the L-threonine 3-dehydrogenase gene has not previously been identified in mammals.ResultsTranscripts for L-threonine 3-dehydrogenase from both the mouse and pig are reported. The ORFs of both L-threonine dehydrogenase cDNAs encode proteins of 373 residues (41.5 kDa) and they share 80% identity. The mouse gene is located on chromosome 14, band C. The amino-terminal regions of these proteins have characteristics of a mitochondrial targeting sequence and are related to the UDP-galactose 4-epimerases, with both enzyme families having an amino-terminal NAD+ binding domain. That these cDNAs encode threonine dehydrogenases was shown, previously, by tiling 13 tryptic peptide sequences, obtained from purified L-threonine dehydrogenase isolated from porcine liver mitochondria, on to the pig ORF. These eukaryotic L-threonine dehydrogenases also have significant similarity with the prokaryote L-threonine dehydrogenase amino-terminus peptide sequence of the bacterium, Clostridium sticklandii. In murine tissues, the expression of both L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase mRNAs were highest in the liver and were also present in brain, heart, kidney, liver, lung, skeletal muscle, spleen and testis.ConclusionsThe first cloning of transcripts for L-threonine dehydrogenase from eukaryotic organisms are reported. However, they do not have any significant sequence homology to the well-characterised Escherichia coli L-threonine dehydrogenase.
| S-EPMC117216 | biostudies-literature
Project description:Transforming growth factor alpha (TGF alpha) was originally identified as a product of tumour tissues and transformed cells in culture. Although it is now clear that expression of this factor is not restricted to neoplastic cells, there remains relatively little information about the sites of expression of TGF alpha in normal tissues. Therefore, an amplified DNA fragment encoding the pig TGF alpha precursor was cloned by reverse transcription-PCR (RT-PCR) using RNA isolated from normal skin tissue as the template. Nucleotide sequence analysis predicts a 160-residue transmembrane polypeptide that differs from the rat, mouse and human TGF alpha precursors at 14, 15 and six sites respectively. The distribution of TGF alpha mRNA in a wide variety of pig tissues was analysed by RT-PCR, using oligonucleotide primers based on the pig TGF alpha cDNA sequence. TGF alpha transcripts were detected in RNA isolated from 17 of the 22 tissues analysed, including four previously unreported sites. Using an antibody raised against a synthetic TGF alpha peptide, we have immunolocalized TGF alpha protein to cells within the red pulp of the spleen and to the distal convoluted tubules of the kidney.
| S-EPMC1137770 | biostudies-other
Project description:Distant hybridization leads to obvious changes in genotypes and phenotypes, giving rise to species with novel capabilities. However, the fusion of distinct genomes also polymerizes the DNA or gene variations that occur during the course of evolution. Knowledge of the early stages of post-hybridization evolution is particularly important. Here, we investigated the full-length (FL) transcriptomes and the sequences resulting from the genome resequencing of the red crucian carp-like homodiploid fish (RCC-L) and goldfish-like homodiploid fish (GF-L) derived from the interspecific hybridization of koi carp (KOC) and blunt snout bream (BSB) to provide molecular evidence for the hybrid origin of the goldfish (GF). We compared the orthologous genes in the transcriptomes of RCC-L and GF-L with those of KOC and BSB. We also mapped the orthologous genes to the common carp (CC) and BSB genomes and classified them into eight gene patterns in three categories (chimaera, mutant, and biparental origin genes). The results showed that 48.20% and 46.50% of the genes were chimaera and that 3.70% and 8.30% of the genes were mutations of orthologous genes in RCC-L and GF-L, respectively. In RCC-L and GF-L, 63.70% and 68.20% of the genetic materials were from KOC, and 12.30% and 11.90% of the genetic materials were from BSB. The sequences from the genome resequencing of RCC-L and GF-L were mapped to the genome sequences of CC and BSB, revealing that the similarities of both RCC-L and GF-L to the CC genome (92.52%, 90.18%) were obviously higher than to the BSB genome (50.33%, 49.18%), supporting the suggestion that the genomes of both RCC-L and GF-L were mainly inherited from KOC but had some DNA fragments from BSB. Overall, our results provide molecular biological evidence for the hybrid origin of red crucian carp (RCC) and GF.
| S-EPMC7063666 | biostudies-literature
Project description:Chicken is an important economic animal that encounter various stressors including high temperature, high stocking density, bacterial infections and transportation, etc. affecting the poultry production with serious economic loss. To be in response to the varied stimulus, the hypothalamic-pituitary-adrenal (HPA) axis is activated through the controlling of the synthesis and secretion of glucocorticoids (GCs). Present study characterized a novel gene C5H11ORF96, that demonstrated significant upregulation after the DEX injection in chicken, which simulates the stress stimulus. Our results showed that: (1) cC5H11ORF96 cDNA encodes a 120 amino acids protein, which shares high sequence identity with that of birds, mammals, reptiles, frogs and fish; (2) cC5H11ORF96 has a fully conserved RFKTQP motif and high proportion of serine, indicating its multiple potential phosphorylation sites; (3) cC5H11ORF96 is widely expressed in various chicken tissues, with high expression levels in the parathyroid gland, adrenal gland, and pituitary; (4) glucocorticoids (GCs) and stress significantly upregulate C5H11ORF96 mRNA and protein expression in the chicken pituitary and hypothalamus, suggesting its involvement in regulating stress response by influencing the negative feedback of GCs on the HPA axis in chickens. The characterization of the C5H11ORF96 gene in the chicken stress response provides potential targets for stress adaptability and poultry production. Meanwhile, our finding provides essential insights into the physiological functions of C11ORF96 gene in vertebrates.
| S-EPMC11625329 | biostudies-literature
Project description:BackgroundRecent clinical studies suggest that the prophylactic use of recombinant factor VIIa (rFVIIa) markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Given the short biological half-life of rFVIIa, it is unclear how rFVIIa could be effective in prophylactic treatment.ObjectivesTo examine the extravascular distribution of pharmacologically administered rFVIIa to obtain clues on how rFVIIa could work in prophylaxis.MethodsRecombinant mouse FVIIa tagged with AF488 fluorophore (AF488-FVIIa) was administered into mice via the tail vein. At different time intervals following the administration, mice were exsanguinated and various tissues were collected. The tissue sections were processed for immunohistochemistry to evaluate distribution of rFVIIa.ResultsrFVIIa, immediately following the administration, associated with the endothelium lining of large blood vessels. Within 1 h, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. In the liver, rFVIIa was localized to sinusoidal capillaries and accumulated in hepatocytes. In bone, rFVIIa was accumulated in the zone of calcified cartilage and some of it was retained there for a week. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to regions that contain TF expressing cells.ConclusionsThe present study demonstrates that pharmacologically administered rFVIIa readily associates with the vascular endothelium and subsequently enters into extravascular spaces where it is likely to bind to TF and is retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa.
| S-EPMC2849270 | biostudies-literature
Project description:Summary Objective Fibroblast growth factor 18 (FGF18) is involved in chondrogenesis and articular cartilage repair. We investigated tissue distribution and pharmacokinetics of radioactive [3H]sprifermin, a recombinant human FGF18, in rats after a single intravenous (i.v.) or intra-articular (i.a.) injection. Design In two studies (48–96-h [n = 23] and 28-day [n = 12]), 35 male albino (Sprague Dawley) rats received single i.v. or i.a. dose [3H]sprifermin (0.24 mg/kg). Radioactivity was measured in blood, serum, and (in animals receiving i.a. administration) in the knee joint by liquid scintillation counting. Radioactivity in organs, tissues, and distribution in the whole body were measured with whole-body autoradiography. Results After i.v. injection, radioactivity peaked in serum and whole blood after 4 and 24 h, respectively, with greater total radioactivity in serum. After i.a. injection, radioactivity peaked in serum and whole blood after 24 and 48 h, respectively; intact [3H]sprifermin was not detected in vena caval serum and systemic exposure was low, approximately 20% of that with i.v. injection. Following i.v. injection, radioactivity was mainly found in the liver, adrenal glands, kidney, and spleen; following i.a. injection, radioactivity was preferentially concentrated in articular cartilage after initial distribution in the joint capsule, and still evident in the joint after 28 days. Conclusions After i.a. injection of [3H]sprifermin in rats, radioactivity was concentrated in the knee joint, particularly articular cartilage, with low levels in other investigated tissues. Systemic exposure to sprifermin was greater with i.v. than i.a. injection. Subsequent clinical investigation in patients with osteoarthritis has reported consistent results.
| S-EPMC9718268 | biostudies-literature
Project description:DC-SIGN, a human C-type lectin, is involved in the transmission of many enveloped viruses. Here we report the cloning and characterization of the cDNA and gene encoding porcine DC-SIGN (pDC-SIGN). The full-length pDC-SIGN cDNA encodes a type II transmembrane protein of 240 amino acids. Phylogenetic analysis revealed that pDC-SIGN, together with bovine, canis and equine DC-SIGN, are more closely related to mouse SIGNR7 and SIGNR8 than to human DC-SIGN. pDC-SIGN has the same gene structure as bovine, canis DC-SIGN and mouse SIGNR8 with eight exons. pDC-SIGN mRNA expression was detected in pig spleen, thymus, lymph node, lung, bone marrow and muscles. pDC-SIGN protein was found to express on the surface of monocyte-derived macrophages and dendritic cells, alveolar macrophages, lymph node sinusoidal macrophage-like, dendritic-like and endothelial cells but not of monocytes, peripheral blood lymphocytes or lymph node lymphocytes. A BHK cell line stably expressing pDC-SIGN binds to human ICAM-3 and ICAM-2 immunoadhesins in a calcium-dependent manner, and enhances the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) to target cells in trans. The results will help better understand the biological role(s) of DC-SIGN family in innate immunity during the evolutionary process.
| S-EPMC7103218 | biostudies-literature
Project description:Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (approximately 320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (approximately 840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors. In situ hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate.
| S-EPMC21752 | biostudies-literature