Bovine Receptor Research Articles

Modulation of Bovine Endometrial Cell Receptors and Signaling Pathways as a Nanotherapeutic Exploration against Dairy Cow Postpartum Endometritis.

Simple SummaryThe provision of updated information on the molecular pathogenesis of bovine endometritis with host-pathogen interactions and the possibility of exploring the cellular sensors mechanism in a nanotechnology-based drug delivery system against persistent endometritis were reported in this review. The mechanism of Gram-negative bacteria and their ligands has been vividly explored, with the paucity of research detail on Gram-positive bacteria in bovine endometritis. The function of cell receptors, biomolecules proteins, and sensors were reportedly essential in transferring signals into cell signaling pathways to induce immuno-inflammatory responses by elevating pro-inflammatory cytokines. Therefore, understanding endometrial cellular components and signaling mechanisms across pathogenesis are essential for nanotherapeutic exploration against bovine endometritis. The nanotherapeutic discovery that could inhibit infectious signals at the various cell receptors and signal transduction levels, interfering with transcription factors activation and pro-inflammatory cytokines and gene expression, significantly halts endometritis.In order to control and prevent bovine endometritis, there is a need to understand the molecular pathogenesis of the infectious disease. Bovine endometrium is usually invaded by a massive mobilization of microorganisms, especially bacteria, during postpartum dairy cows. Several reports have implicated the Gram-negative bacteria in the pathogenesis of bovine endometritis, with information dearth on the potentials of Gram-positive bacteria and their endotoxins. The invasive bacteria and their ligands pass through cellular receptors such as TLRs, NLRs, and biomolecular proteins of cells activate the specific receptors, which spontaneously stimulates cellular signaling pathways like MAPK, NF-kB and sequentially triggers upregulation of pro-inflammatory cytokines. The cascade of inflammatory induction involves a dual signaling pathway; the transcription factor NF-κB is released from its inhibitory molecule and can bind to various inflammatory genes promoter. The MAPK pathways are concomitantly activated, leading to specific phosphorylation of the NF-κB. The provision of detailed information on the molecular pathomechanism of bovine endometritis with the interaction between host endometrial cells and invasive bacteria in this review would widen the gap of exploring the potential of receptors and signal transduction pathways in nanotechnology-based drug delivery system. The nanotherapeutic discovery of endometrial cell receptors, signal transduction pathway, and cell biomolecules inhibitors could be developed for strategic inhibition of infectious signals at the various cell receptors and signal transduction levels, interfering on transcription factors activation and pro-inflammatory cytokines and genes expression, which may significantly protect endometrium against postpartum microbial invasion.

Single nucleotide polymorphisms (SNPs) in bovine follicle stimulating hormone receptor (bFSHR) gene in Pasundan cattle

Single nucleotide polymorphisms (SNPs) in bovine follicle stimulating hormone receptor (bFSHR) gene in Pasundan cattle

Polymorphism in the Exon 11 of Bovine Luteinizing Hormone Receptor Gene and its Association with Service per Conception in Pasundan Cows

Polymorphism in the Exon 11 of Bovine Luteinizing Hormone Receptor Gene and its Association with Service per Conception in Pasundan Cows

The interplay between non-esterified fatty acids and bovine peroxisome proliferator-activated receptors: results of an in vitro hybrid approach

BackgroundIn dairy cows circulating non-esterified fatty acids (NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors (PPAR). PPAR are activated by fatty acids (FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows.MethodsThe dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300e digital dispenser and luciferase reporter in several bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase (LPL), and palmitic acid (C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors.ResultsNEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum (R2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation.ConclusionOur results support dose-dependent activation of PPAR by circulating NEFA in bovine, specifically δ and γ isotypes. Data also support the possibility of increasing PPAR activation by dietary FA; however, this nutrigenomics approach maybe only effective in pre-partum but not post-partum cows.

Gonadotropine Releasing Hormone (GnRH) Receptor Profile from Cow’s Hypothalamus

Gonadotropine Releasing Hormone (GnRH) is one of the recommended hormones to overcome ovulation problems and it can increase pregnancy rate so that it is used in government programs to increase cattle population in Indonesia, although the results are not yet optimal. The purpose of this study is to compare the composition of bovine GnRH receptor nucleotides with the GenBank reference so that the level of genetic diversity of bovine receptors in Indonesia is known. PCR product sequencing using Promoter F and Exon 1 R primers were further aligned with the reference sequences of Bos Taurus GnRHR mRNA GenBank using the MEGA X program. The results of the analysis found the presence of Single Nucleotide Polymorphism (SNP) in the coding region of 1st cow position 38(A> T), 261(C > T), 342(C >T), 411(C >T) and 495(C > T) and 2nd cow positions 261(C >T). Changes in amino acids were also detected in 1st cow position 13 (H> L). The presence of SNP was found to indicate genomic variation between individuals at cattle receptors in Indonesia.

Detection of polymorphisms in the bovine leptin receptor gene affects fat deposition in two Chinese beef cattle breeds.

Leptin receptor (LEPR) gene play a pivotal role in the regulation of fat deposition and energy homeostasis. This study investigated the presence and frequency of polymorphisms of bovine LEPR gene and determine whether the polymorphisms are associated with the fat deposition in two Chinese beef cattle breeds. Quantitative real-time polymerase chain reactions identified that the expression of LEPR gene was highest in the liver and subcutaneous fat. Four single nucleotide polymorphisms (SNPs) were identified including g.24169C>T, g.24256T>A, g.24267 G>C and g.24413T>A. A greater backfat thickness was associated with the AA genotype of g.24256T>A compared to the TT genotype. A greater intramuscular fat content was associated with the GG genotype of g.24267 G>C compared to the CC genotype. Both g.24169C>T and g.24413T>A were not correlated with fat deposition. These results indicated that the SNP g.24256T>A and g.24267 G>C of LEPR gene may be useful markers for genetic improvement of fat deposition in Chinese beef cattle breeds.

MiR-145 is involved in the proliferation of bovine mammary epithelial cells and regulates bovine insulin receptor substrate 1

MicroRNAs (miRNAs) are short noncoding RNAs that can regulate target gene expression at the posttranscriptional level. It has been reported that bta-miR-145 shows differential expression between the different lactation stages of bovine mammary glands, but the function of bta-miR-145 in normal mammary glands remains unknown. The overexpression of bta-miR-145 significantly decreased the proliferation of mammary epithelial cells (p < .05), and this effect was reversed upon bta-miR-145 inhibition. Using several miRNA prediction programmes, insulin receptor substrate 1 (IRS1) was predicted to be a potential target of miR-145. Using the DAVID database, many predicted targets of bta-miR-145 were found to be involved in the MAPK signalling pathway associated with cell proliferation, including IRS1. Bovine IRS1 3’RACE sequencing and reference comparison revealed that possible binding sites for bta-miR-145 still exist in IRS1. The IRS1 mRNA level was not affected by the transfection of primary bovine mammary epithelial cells with 150 pmol of a bta-miR-145 mimic or 300 pmol of a bta-miR-145 inhibitor for 24 h (pBMEC, p > .05), whereas the IRS1 protein level was changed significantly after bta-miR-145 mimic or inhibitor transfection for 72 h (p < .05). Taken together, these results suggest that bta-miR-145 is involved in the proliferation of bovine mammary epithelial cells by targeting IRS1, which is related to the MAPK signalling pathway. Highlights Bta-miR-145 affects bovine mammary epithelial cell vitality in primary bovine mammary epithelial cells. IRS1 was verified as target gene of bta-miR-145. IRS1 belongs to MAPK signaling pathway, which regulate cell proliferation.

The Interplay Between Non-Esterified Fatty Acids and Bovine Peroxisome Proliferator-Activated Receptors: Results of an In Vivo-In Vitro Hybrid Approach

Abstract Objectives Metabolic stressors and energy deficiency related to parturition induce lipolysis in dairy cows with a subsequent increase in circulating non-esterified fatty acids (NEFA). We hypothesized that circulating NEFA directly activate the Peroxisome Proliferator-Activated Receptors (PPAR), a transcription factor with known nutrigenomics response to fatty acids, in a dose-dependent manner. Methods Whole-blood samples were collected from five cows at different times between −40 and +10 days relative to parturition to isolate serum. NEFA were extracted from said samples through solid-phase extraction. A bioluminescent reported, bound to a PPAR response element, was used to assess PPAR activation in response to the treatments. To estimate individual PPAR isotype activation, PPAR isotype-specific antagonists were used, along with the treatments. Results Isolated NEFA activate PPAR linearly up to 400 μM, with a decrease in their effect when albumin is introduced. A hybrid approach, treating cells with bovine serum from different stages of parturition, revealed that much of the PPAR activation can be explained by the amount of NEFA in the serum, and that its activation follows a quadratic tendency (calculated maximum activation at 1.47 mM NEFA of 4.8-fold vs. DMEM control, R2 = 0.91). Further analysis of the effect of serum NEFA on PPAR revealed that they mostly activate PPARδ and PPARγ, but not PPARα. However, addition of palmitic acid to the media activate PPARδ and PPARα, but not PPARγ, and only when circulating NEFA are low (0.16 mM, prepartum), while additional palmitate did not increase PPAR activation in serum with high NEFA (0.71 mM, postpartum). Finally, the addition of lipoprotein lipase to the serum, in order to mimic release of NEFA from VLDL, increased PPAR activation, recapitulating the role of dietary lipids in the regulation of PPAR. Conclusions Taken together, our results support the activation of PPAR by NEFA, helping to explain prior observations on PPAR activation in peripartum cows. Additionally, we outlined the role of dietary fatty acids in activating PPAR and their interaction with serum NEFA. Our results aid in setting the foundation for nutrigenomics approaches in ruminant nutrition. Funding Sources This study was funded by the Oregon Beef Council.

In silico prediction of the functional and structural consequences of the non-synonymous single nucleotide polymorphism A122V in bovine CXC chemokine receptor type 1.

The current study aimed to assess whether the A122V causal polymorphism promotes alterations in the functional and structural proprieties of the CXC chemokine receptor type 1 protein (CXCR1) of cattle Bos taurus by in silico analyses. Two amino acid sequences of bovine CXCR1 was selected from database UniProtKB/Swiss-Prot: a) non-polymorphic sequence (A7KWG0) with alanine (A) at position 122, and b) polymorphic sequence harboring the A122V polymorphism, substituting alanine by valine (V) at same position. CXCR1 sequences were submitted as input to different Bioinformatics' tools to examine the effects of this polymorphism on functional and structural stabilities, to predict eventual alterations in the 3-D structural modeling, and to estimate the quality and accuracy of the predictive models. The A122V polymorphism exerted tolerable and non-deleterious effects on the polymorphic CXCR1, and the predictive structural model for polymorphic CXCR1 revealed an alpha helix spatial structure typical of a receptor transmembrane polypeptide. Although higher variations in the distances between pairs of amino acid residues at target-positions are detected in the polymorphic CXCR1 protein, more than 97% of the amino acid residues in both models were located in favored and allowed conformational regions in Ramachandran plots. Evidences has supported that the A122V polymorphism in the CXCR1 protein is associated with increased clinical mastitis incidence in dairy cows. Thus, the findings described herein prove that the replacement of the alanine by valine amino acids provokes local conformational changes in the A122V-harboring CXCR1 protein, which could directly affect its post-translational folding mechanisms and biological functionality.

Bovine dopamine type-2 receptor SNP has no effect on growth, semen characteristics and prolactin concentrations in beef bulls treated with a dopamine agonist

A dopamine type-2 receptor (DRD2) SNP, previously found to be correlated with serum prolactin (PRL) concentrations in cattle, was evaluated for impact on growth traits, serum prolactin concentration, and semen quality. Over a four-year period, yearling beef bulls were allowed diets containing or lacking ergot alkaloids (EA). Every 21 or 28 d semen was collected for semen motility and morphology assessment and blood samples were collected to measure serum PRL concentrations. In addition, body condition score and scrotal circumference were evaluated. Serum PRL concentrations were assessed using a radioimmunoassay. In the first year, all bulls were sacrificed at the end of a 126-day study. Testicles and epididymis were collected at the end of the study or 60 days after removal from treatment. Immunohistochemistry was performed on testis, epididymis, and sperm cells, incubated with or without a primary antibody for DRD2 and counterstained with DAPI. Isolation of DNA was performed on sperm pellets using DNAzol (Thermo Fisher Scientific, Waltham, MA, USA) methods. Polymerase chain reaction was performed to amplify the region of the DRD2 gene containing the SNP of interest. The products were subjected to restriction fragment length polymorphism analysis. Further, all samples were subjected to genotyping using a custom Taqman genotyping assay (Applied Biosystems, Foster city, CA, USA). The presence of DRD2 was detected in the testis, epididymis, and sperm cells. The DRD2 genotype was not associated with semen quality, serum PRL, or growth traits. Consumption of EA resulted in lesser PRL serum concentrations but had no effect on values for other variable examined.

Polymorphisms in the Bovine Tumour Necrosis Factor Receptor Type Two Gene (TNF-RII) and Cell Subpopulations Naturally Infected with Bovine Leukaemia Virus.

IntroductionNumerous mutations in the bovine tumour necrosis factor receptor type two (TNF-RII) gene have been identified, but their biological consequences remain poorly understood. The aim of this study was to determine whether polymorphism in the analysed loci of the bovine TNF-RII gene is linked with the size of cell subpopulations naturally infected with bovine leukaemia virus (BLV) which serve important immune functions in the host.Material and MethodsSamples originated from 78 cows. Polymorphisms in the studied gene were determined by PCR-RFLP and DNA sequencing by capillary electrophoresis. BLV infection was diagnosed by the immunofluorescence (IMF) technique and nested PCR. Cell subpopulations were immunophenotyped with IMF.ResultsSimilar and non-significant differences in the average percentages of TNFα±, IgM+TNFα±, and CD11b+TNFα±cells infected with BLV were noted in individuals with various genotypes in the polymorphic sites g.-1646T > G and g. 16534T > C of the TNF-RII gene, and significant differences in the percentages of these subpopulations were observed between selected microsatellite genotypes (g.16512CA(n)).ConclusionSTR polymorphism and the number of CA dinucleotide repeats in intron 1 of the TNF-RII gene influence the frequency of TNF+, CD11b+TNF+, and IgM+TNF+ subpopulations naturally infected with BLV. Polymorphism in the gene’s other two sites do not affect the size of these cell subpopulations.

DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy

BackgroundRefractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α.ResultsBoth TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig’s anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1β and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α.ConclusionsCollectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application.

Docosahexaenoic acid and TUG-891 activate free fatty acid-4 receptor in bovine neutrophils

Fatty acids are well known metabolic intermediaries but also have a role in the immune response. Long-chain fatty acids such as omega-6 and -9 activate neutrophil function through free fatty acid (FFA)-1 receptor in bovines. Although omega-3 has also been suggested to influence neutrophil function, the details remain unclear. The goal of this study was to determine the presence of the bovine FFA4 receptor and its effect on neutrophil responses. We treated bovine neutrophils with the natural and synthetic agonists of FFA4 receptor docosahexaenoic acid (DHA) and TUG-891, respectively, and assessed oxidative and no oxidative response. We detected protein and mRNA FFA4 receptor expression through immunofluorescence, immunoblot, and RT-PCR analysis. DHA and TUG-891 both increased intracellular calcium mobilisation in bovine neutrophils, with 50% effective concentrations of 99 μM and 73 μM, respectively, which was partially reduced after treatment with the FFA4 antagonist AH7614. Furthermore, DHA and TUG-891 increased matrix metalloproteinase (MMP)-9 granules release and superoxide production. AH7614 and the intracellular calcium chelator BAPTA-AM decreased the superoxide production induced by TUG-891 and by both DHA and TUG-891, respectively, suggesting a key role of intracellular calcium in FFA4 agonists-induced superoxide production. These results highlight an important mechanism of bovine neutrophil responses mediated via FFA4 receptor, which can further inform the development of new formulations for DHA-enriched feed supplements to enhance innate immunity in dairy cattle.

Bovine scavenger receptor class A (SR-A) exhibit specific patterns of regulation in the endometrium during the oestrous cycle and early pregnancy.

In mammals, tight regulation of maternal endometrial function is critical for pregnancy success. In bovine species, endometrial expression of members of the scavenger receptor class A (SR-A) has been listed in high-throughput analyses, but very little is known about the involvement of these immune factors during implantation in mammals. To provide first insights into the contribution of SR-A to endometrial physiology, we analysed the expression and regulation of all members of SR-A (SR-A1, SR-A3-SR-A6) during the oestrous cycle and early pregnancy in cattle. Levels of SR-A1 were increased on Day 20 of pregnancy, whereas SR-A3 levels were increased on Day 13 of the oestrous cycle and of the pregnancy. Although SR-A4 levels were reduced on Day 20 of the oestrous cycle, they remained high in pregnant animals. SR-A5 levels increased by Day 13 of the oestrous cycle and decreased on Day 20, but remained high in pregnant animals. Interferon-τ does not affect SR-A gene expression, whereas progesterone regulates the expression of the SR-A3 and SR-A5 transcripts. Endometrial SR-A3 appeared significantly higher in cows carrying invitro-produced embryos than in AI cows. Our data suggest that members of the SR-A family are involved in endometrial remodelling and regulation of endometrial gland physiology, both processes being critical for implantation in mammals.

Polymorphisms of TLR1, TLR4 and SLC11A1 Genes in Some Cattle Breeds Reared in Turkey

The objective of this study was to examine the allelic and genotypic profiles of four SNPs of bovine Toll-Like receptor 1 (TLR1), Toll-Like Receptor 4 (TLR4) and Solute Linkete Carier 11A1 (SLC11A1) genes in Holstein, two Turkish native breeds and their crossbreds in Turkey. For this purpose, a total of 1023 cattle from Holstein (HL, n= 410), Anatolian Black (AB, n= 106), East Anatolian Red (EAR, n= 84), Anatolian Black Crossbreed (ABC, n= 124) and East Anatolian Red crossbreed (EARC, n= 299) breeds were examined for four SNPs. Samples were genotyped using by the PCR-RFLP method. According to the TLR1 (+1380) SNP, EAR was in Hardy-Weinberg equilibrium (HWE) while the AB, HL and native crossbreeds were deviated from HWE. All cattle breeds were in HWE for the TLR1 (+1596) SNP. In terms of TLR4 (+10) SNP, the CC genotype had the highest frequency in HL and native crossbreeds whereas the AB and EAR breeds were monomorphic and only CC genotype was found these breeds. According to the SLC11A1 (+1066) SNP, the CC genotype had the highest while the GG genotype frequency the lowest in all breeds. In addition EAR breed was in HWE while the AB, HL and native crossbreeds deviated from HWE.

Production and validation of a polyclonal serum against bovine FSH receptor

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.

Depot-specific and GH-dependent regulation of IGF binding protein-4, pregnancy-associated plasma protein-A, and stanniocalcin-2 in murine adipose tissue

IntroductionPregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF)-I action through proteolytic cleavage of IGF binding protein-4 (IGFBP-4). Recently, stanniocalcin-2 (STC2) was discovered as an inhibitor of PAPP-A. Most members of the IGF system are expressed in adipose tissue (AT), but there is a relative paucity of information on the distribution of IGFBP-4, PAPP-A, and STC2 in different AT depots. Since IGF-I expression in AT is highly GH-dependent, we used bovine GH transgenic (bGH) and GH receptor knockout (GHR−/−) mice to investigate AT depot-specific expression patterns of IGFBP-4, PAPP-A, and STC2, and whether the regulation is GH-dependent. MethodsSeven-month-old male bGH, GHR−/− and wild type (WT) control mice were used. Body composition was determined, and subcutaneous, epididymal, retroperitoneal, mesenteric and brown adipose tissue (BAT) depots were collected. RNA expression of Igfbp4, Pappa, and Stc2 was assessed by reverse transcription quantitative PCR and IGFBP-4 protein by Western blotting. ResultsIgfbp4, Pappa, and Stc2 RNA levels were differentially expressed in an AT depot-dependent manner in WT mice. Igfbp4 RNA levels were significantly higher in all white AT depots than in BAT. Pappa was most highly expressed in the mesenteric depot: levels were 7.5-fold higher in mesenteric than in subcutaneous AT (p < .001). Although intraabdominal in origin, epididymal and retroperitoneal Pappa expression levels were 69% and 68% lower, respectively, as compared to mesenteric levels (p < .001). Stc2 RNA expression was significantly higher in all intraabdominal white AT as compared to subcutaneous AT and BAT; levels in epididymal, retroperitoneal, and mesenteric were all more than three-fold higher than in subcutaneous AT (p < .001) and 12-fold higher than in BAT (p < .001). Gene expression patterns in bGH and GHR−/− mice mimicked those in WT mice, suggesting that GH does not affect the transcription of the STC2-PAPP-A-IGFBP-4-axis in AT. However, proteins levels of intact IGFBP-4 were significantly increased in bGH mice and decreased in GHR−/− mice, whereas the PAPP-A-generated IGFBP-4 fragment level was unaltered. ConclusionExpression of Igfbp4, Pappa, and Stc2 differ between AT depots and is generally higher in white AT than in BAT. The transcription appears to occur in a GH-independent manner, whereas IGFBP-4 protein levels are highly influenced by altered GH activity.

Predicting the effects of the single nucleotide polymorphism A122V on CXC chemokine receptor type 1 of Bos taurus (Artiodactyla: Bovidae) cattle by in silico analyses

Este estudo objetivou predizer bioquimicamente as estruturas primárias e secundárias da proteína receptora de quimiocina CXCR1 bovina não polimórfica e polimórfica carreando o polimorfismo A122V por análises in silico. Duas sequências da proteína CXCR1 de bovino Bos taurus foram selecionadas a partir da base de dados de sequências de proteínas UniProtKB/Swiss-Prot: a) uma sequência não polimórfica (A7KWG0), contendo o aminoácido alanina (A) na posição 122, e b) uma sequência polimórfica, apresentando o aminoácido valina (V) na mesma posição. As estruturas primárias e secundárias da proteína foram preditas empregando os programas ProtParam e Chou &amp; Fasman Protein Secondary Structure Prediction CFSSP. Diferenças nos parâmetros físicos e químicos não foram previstas a partir da estrutura primária de ambas as proteínas analisadas. A presença de um domínio helicoidal, situado nas posições 100 e 150, foi exclusivamente encontrada na proteína CXCR1 não polimórfica. Resíduos de aminoácidos de propriedades bioquímicas variáveis foram detectados na posição 122 na proteína CXCR1 dos ruminantes e humana, sugerindo que esse peptídeo é altamente polimórfico em vertebrados. Os resultados aqui descritos predizem diferenças no padrão da estrutura secundária da proteína CXCR1 bovina não polimórfica e polimórfica A122V empregando ferramentas de Bioinformática.

Solubilization of the serotonin1A receptor monitored utilizing membrane dipole potential

Solubilization of membrane proteins by amphiphilic detergents represents a crucial step in studies of membrane proteins in which proteins and lipids in natural membranes are dissociated giving rise to mixed clusters of proteins, lipids and detergents in the aqueous dispersion. Although solubilization is a popular method, physicochemical principles underlying solubilization are not well understood. In this work, we monitored solubilization of the bovine hippocampal serotonin1A receptor, a representative member of the GPCR family, using membrane dipole potential measured by a dual fluorescence ratiometric approach with a potential-sensitive fluorophore. Our results show that membrane dipole potential is a good indicator of solubilization and reflects the change in dipolar environment upon solubilization due to dipolar reorganization associated with solubilization. To the best of our knowledge, these results constitute the first report linking membrane dipole potential with solubilization. We envision that these results are potentially useful in providing a molecular mechanism for membrane protein solubilization.