Development and identification of candidate aptamers for growth differentiation factor 9

Tertiary Structure ◽

Affinity Maturation ◽

Detection Methods ◽

Target Molecule ◽

Differentiation Factor ◽

Growth Differentiation Factor 9

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted growth factors that are essential for fertility and ovarian development in many mammalian species. It is hypothesised that the ratios of these growth factors within ovarian follicles are unique to each species and determine litter size in mammals. The current detection method for these proteins is by Western blotting with monoclonal antibodies, however this methodology generates semi-quantitative results at best. Systematic evolution via exponential enrichment (SELEX) is a method involving iterative rounds of affinity maturation. It has been used for generating synthetic DNA- and RNA-based sequences (aptamers) that are capable of recognising a target molecule with high sensitivity. To understand more about the biological functions of BMP15 and GDF9, reliable detection methods need to be developed so these proteins can be measured in biological samples. The generation of aptamers that recognise BMP15 and GDF9 of multiple species could enable a novel detection system to be developed to further explore the role of BMP15 and GDF9 in determining litter size in mammals. In this study, recombinant BMP15 and GDF9 proteins from species that vary in litter size were produced and purified by IMAC and HPLC for aptamer validation. Membrane-SELEX was used to generate candidate aptamers against recombinant human GDF9. After ten rounds of SELEX, seven candidate aptamers were identified and sequenced. Alignment of the sequences revealed a conserved region of 29bp in four of the seven candidate aptamers. These sequences also showed a high (85-90%) guanine/thymine content consistent with G-quadruplex forming aptamers, a tertiary structure that increases specificity to the target molecule. These four sequences also showed homology with that of a previously published aptamer for BMP15. This is interesting as BMP15 and GDF9 are both members of the transforming growth factor beta superfamily and as such, share a high peptide homology suggesting convergent evolution occurred in two unrelated SELEX experiments against related proteins. Whilst time constraints did not allow for characterisation of the resultant GDF9 aptamer candidates, nor their validation using recombinant ovine and mouse GDF9 produced herein, this study showed that GDF9 aptamer candidates were generated by SELEX that exhibited sequence similarities to an aptamer generated against a structurally-related protein.</p>

Development and identification of candidate aptamers for growth differentiation factor 9

10.26686/wgtn.17135972.v1 ◽

2021 ◽

Author(s):

◽

Orin Robb

Keyword(s):

Growth Factors ◽

Litter Size ◽

Tertiary Structure ◽

Affinity Maturation ◽

Detection Methods ◽

Target Molecule ◽

Differentiation Factor ◽

Growth Differentiation Factor 9

Association Study of Fecundity Gene BMPR1B with Prolificacy in Surti Goats under Farm and Field Condition

THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY ◽

10.21887/ijvsbt.14.4.5 ◽

2019 ◽

Vol 14 (04) ◽

Author(s):

N. S. Dangar ◽

G. M. Pandya ◽

U. V. Ramani ◽

Y. D. Padheriya ◽

T. Sangma ◽

...

Keyword(s):

Litter Size ◽

Pcr Primers ◽

Dual Purpose ◽

Protein Receptor ◽

Morphogenetic Protein ◽

Pcr Rflp ◽

Base Size ◽

Mutation Information

The Surti is a dual purpose goat breed of Gujarat. The bone morphogenetic protein receptor type 1B (BMPR1B) gene of transforming growth factor beta (TGF-β) superfamily ligands is playing a role in ovulation as well as litter size. Mutation in Exon-6 region of BMPR1B gene with base size 190 bp reported increasing litter size. Based on the known mutation information in goat and sheep, PCR primers were designed to screen polymorphism in total 100 Surti goats, 50 Surti goats from University Farm, Navsari and 50 Surti goats from field units of Southern part of Gujarat. During PCR-RFLP study no polymorphic sites were found for Exon-6 region of BMPR1B on Surti goats. Moreover, the twinning rate was 10% in first parity and higher in subsequent second (62.5%) and third (76.8%) parties.

Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽

10.3390/biomedicines9060679 ◽

2021 ◽

Vol 9 (6) ◽

pp. 679

Author(s):

Benedict-Uy Fabia ◽

Joshua Bingwa ◽

Jiyeon Park ◽

Nguyen-Mihn Hieu ◽

Jung-Hoon Ahn

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Pseudomonas Fluorescens ◽

Abc Transporter ◽

Deletion Mutant ◽

Gene Knockout ◽

Type I ◽

Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

Abstract 165: Amp-activated Protein Kinase (ampk) α1 in Macrophages Promotes Collateral Remodeling and Arteriogenesis in Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.165 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Huaiping Zhu

Keyword(s):

Growth Factors ◽

Hind Limb ◽

Arterial Occlusion ◽

Arterial Stenosis ◽

Surrounding Tissue ◽

Chronic Coronary Occlusion ◽

Collateral Arteries ◽

Collateral Vessels

Efficient arteriogenesis is vital for recovery after cardiovascular events (such as chronic coronary occlusion, myocardial infarction). Identifying the biological factors that affect arteriogenesis will help design new treatments for patients with chronic arterial stenosis and occlusions. Circulating monocytes and macrophages recruited within the surrounding tissue of collateral vessels following arterial occlusion have been reported to be essential to arteriogenesis in collateral arteries/arterioles because they promote the proliferation of vascular smooth muscle cells (VSMCs) and endothelial cells through secreted cytokines. Although a growing number of putative arteriogenic factors have been identified, the exact mechanisms that regulate collateral remodeling have remained largely unknown. Here we report that AMPK, an energy and redox sensor, is required for monocyte-mediated collateral remodeling. Collateral arteriogenesis was monitored in WT, global AMPKα1 knockout (KO), or macrophage-specific AMPKα1 KO mice with or without hind limb ligation. Compared to WT mice with ligation, global AMPKα1 KO mice displayed significant reduction in blood flow recovery and impaired remodeling of collateral arterioles. Similar impairments were observed in macrophage-specific AMPK α1 KO mice following hind limb ligation. Mechanistically, we found that AMPKα1 promotes the production of growth factors, such as transforming growth factor beta, by directly phosphorylating the inhibitor of nuclear factor kappa B (NF-κB) kinase alpha, resulting in an NF-κB-dependent production of growth factors. Collectively, our findings suggest a novel role for macrophage AMPKα1 in arteriogenesis and collateral remodeling and indicate that AMPKα1 activation might be a therapeutic target for treating occlusive vascular disorders.

Regulation of mesenchymal extracellular matrix protein synthesis by transforming growth factor-beta and glucocorticoids in tumor stroma

Journal of Cell Science ◽

10.1242/jcs.108.6.2153 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2153-2162 ◽

Cited By ~ 3

Author(s):

J.F. Talts ◽

A. Weller ◽

R. Timpl ◽

M. Ekblom ◽

P. Ekblom

Keyword(s):

Extracellular Matrix ◽

Growth Factors ◽

Growth Factor ◽

Mrna Expression ◽

Tenascin C ◽

Extracellular Matrix Components ◽

Growth Factor Beta ◽

Matrix Components

We have here studied the composition and regulation of stromal extracellular matrix components in an experimental tumor model. Nude mice were inoculated with WCCS-1 cells, a human Wilms' tumor cell line. In the formed tumors the stroma was found to contain mesenchymal extracellular matrix proteins such as tenascin-C, fibulins-1 and 2 and fibronectin, but no nidogen. Nidogen was confined to basement membranes of tumor blood vessels. Since glucocorticoids have been shown to downregulate tenascin-C expression in vitro, we tested whether dexamethasone can influence biosynthesis of extracellular matrix components during tumor formation in vivo. A downregulation of tenascin-C mRNA and an upregulation of fibronectin mRNA expression by dexamethasone was noted. Transforming growth factor-beta 1 mRNA levels were unaffected by the dexamethasone treatment. Glucocorticoids can thus downregulate tenascin-C synthesis although local stimulatory growth factors are present. The competition between a negative and a positive extrinsic factor on synthesis of stromal extracellular matrix components was studied in a fibroblast/preadipocyte cell line. Transforming growth factor-beta 1 stimulated tenascin-C synthesis but did not affect fibronectin or fibulin-2 synthesis. Dexamethasone at high concentrations could completely suppress the effect of transforming growth factor-beta 1 on tenascin-C mRNA expression. Transforming growth factor-beta 1 could in turn overcome the downregulation of tenascin-C mRNA expression caused by a lower concentration of dexamethasone. We therefore suggest that the limited expression of tenascin-C in part is due to a continuous suppression by physiological levels of glucocorticoids, which can be overcome by local stimulatory growth factors when present in sufficient amounts.

Growth factors and metanephrogenesis

AJP Renal Physiology ◽

10.1152/ajprenal.1992.262.4.f523 ◽

1992 ◽

Vol 262 (4) ◽

pp. F523-F532 ◽

Cited By ~ 3

Author(s):

M. R. Hammerman ◽

S. A. Rogers ◽

G. Ryan

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Kidney Development ◽

Polypeptide Growth Factors ◽

Metanephric Kidney ◽

Epidermal Growth ◽

Factor Alpha ◽

Editorial Review

The formation of all organs during embryogenesis, including kidney, is dependent on the timed and sequential expression of a number of polypeptide growth factors. Synthesis and actions of one or more members of the insulin-like growth factor, epidermal growth factor/transforming growth factor-alpha, transforming growth factor-beta, platelet-derived growth factor, fibroblast growth factor, and nerve growth factor families have been characterized in the developing metanephric kidney. Studies originating from a number of laboratories have defined the localization of growth factor mRNAs, receptors and peptides, have delineated patterns of growth factor synthesis, and have established the growth factor dependency of embryonic kidney development. The results of these investigations will be summarized in this editorial review and integrated within the broader context of growth factor cellular physiology and growth factor expression in nonrenal systems.

Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells

Revista Mvz Córdoba ◽

10.21897/rmvz.1367 ◽

2018 ◽

pp. 6778-6787 ◽

Cited By ~ 1

Author(s):

Pablo S Reineri ◽

María S. Coria ◽

María G. Barrionuevo ◽

Olegario Hernández ◽

Santiago Callejas ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Growth Factor Receptor ◽

Follicular Development ◽

Fibroblast Growth ◽

Rt Pcr ◽

Follicular Cells

Introduction. Growth and follicular maturation involve transformations of various components of the follicle, such as the oocyte, granulosa and techa cells. Several growth factors, including differentiation growth factor 9 (GDF9), bone morphogenic protein 15 (BMP15) and basic fibroblast growth factor (FGF2) are important for follicular development and oocyte maturation, by its ability to increase the proliferation of granulosa, techa cells and the ovarian stroma. Objetive. Evaluate mRNA expression of GDF9, BMP15, FGF2 and their main receptors, transforming growth factor beta receptor 1 (TGFβ-R1), bone morphogenetic protein receptor, type IB (BMPR-IB) and fibroblast growth factor receptor 2 (FGFR2) in bovine follicular cells. Materials and methods. Total RNA was isolated from pooled samples of oocytes (OOs), cumulus cells (CCs) of cumulus oocyte complexes (COCs) and follicular cell pellets (PCs) of 70 ovaries obtained from 96 beef heifers, collected at a local abattoir. The expression pattern of growth factors and their receptors in follicular bovine cells was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Results. The mRNA transcripts encoding GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB and FGFR2 genes were detected, by RT-PCR, in all studied cells. This is the first time that the expression of TGFβ-R1 and BMPR-IB receptors is reported in bovine oocytes. Conclusions. The presence of growth factors and receptor transcripts in the studied cells indicate that these factors could act as paracrine and autocrine regulators of folliculogenesis.

Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor

Methods in Molecular Biology - Cytokine Protocols ◽

10.1007/978-1-61779-439-1_8 ◽

2011 ◽

pp. 117-132 ◽

Cited By ~ 4

Author(s):

Paulette M. Robinson ◽

Timothy D. Blalock ◽

Rong Yuan ◽

Alfred S. Lewin ◽

Gregory S. Schultz

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Connective Tissue ◽

Connective Tissue Growth Factor ◽

Hammerhead Ribozyme ◽

Tissue Growth ◽

Growth Factor Beta

Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2669-2677.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2669-2677

Author(s):

G E Panganiban ◽

K E Rashka ◽

M D Neitzel ◽

F M Hoffmann

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Cell Protein ◽

Stable Complex ◽

Tgf Beta ◽

Amino Terminal ◽

Growth Factor Beta ◽

Carboxy Terminal

The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.

Basic FGF and TGF-beta 1 influence commitment to melanogenesis in neural crest-derived cells of avian embryos

Development ◽

10.1242/dev.111.2.635 ◽

1991 ◽

Vol 111 (2) ◽

pp. 635-645 ◽

Cited By ~ 5

Author(s):

K.M. Stocker ◽

L. Sherman ◽

S. Rees ◽

G. Ciment

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Neural Crest ◽

Cell Fate ◽

Culture Medium ◽

Neutralizing Antibody ◽

Pigment Cells ◽

Tgf Beta

In previous studies, we showed that neural crest (NC)-derived cells from embryonic quail dorsal root ganglia (DRG) and peripheral nerve (PN), which do not normally give rise to melanocytes, become committed to melanogenesis following treatment in culture with the phorbol ester drug 12-O-tetradecanoyl phorbol-13-acetate (TPA). These and other observations support the notion that melanocytes and Schwann cells are derived from a common bipotent intermediate in the neural crest lineage—the melanocyte/Schwann cell progenitor. In this study, we test the possibility that peptide growth factors found in the embryonic environment might act similarly to TPA to influence the fates of these cells. DRG and PN explants were cultured in medium supplemented with a variety of growth factors, and then the cultures were examined for the presence of pigment cells. We found that basic fibroblast growth factor (bFGF), but not various other growth factors, induced pigmentation in about 20% of these cultures. When low concentrations of TPA were included in the culture medium, bFGF augmented the TPA-induced pigmentation, significantly increasing the proportion of pigmented cultures. These effects of bFGF were age-dependent, and could be blocked by addition of a bFGF-neutralizing antibody to the culture medium. In contrast to these stimulatory effects of bFGF, transforming growth factor-beta 1 (TGF-beta 1) was found to inhibit the TPA- or bFGF-induced pigmentation of DRG cultures. These data suggest, therefore, that at least some NC-derived cells are responsive to bFGF and TGF-beta 1, and that these growth factors may play an important role in the control of NC cell fate.