scholarly journals The Molecular Forms of GDF9 In A Range of Mammalian Species

2021 ◽  
Author(s):  
◽  
Abdulaziz Alhussini
Keyword(s):  
Litter Size ◽  
Western Blotting ◽  
Red Deer ◽  
Transforming Growth Factor ◽  
Mammalian Species ◽  
Molecular Size ◽  
Biologically Active ◽  
Molecular Forms ◽  
Western Blots ◽  
Mature Form

<p>Growth Differentiation Factor (GDF) 9 is a member of the transforming growth factor β (TGFβ) superfamily that is exclusively expressed within and secreted from, the oocyte. This protein has generated much interest as it has been found to play a major role in follicular growth and maturation in mammals, and may be involved in determining litter size. Like most TGFβ family members, it is synthesised as a pre-pro-mature protein and is cleaved at various stages to allow the biologically active mature form to bind its Type II receptor. The aim of this study was to improve our understanding of the different molecular forms of GDF9 that are present within ovarian follicles of a range of mammalian species that differ in litter size. To achieve this aim, Western blotting experiments were performed to illustrate the molecular forms that were present within, and secreted from, the oocytes of rats, pigs, sheep and red deer. The detection of bands that represented the different molecular forms of GDF9 was undertaken using a monoclonal antibody that targeted a conserved region in the mature form of ovine GDF9.  The predominant forms of GDF9 found within and secreted from the oocyte across the species were the promature and cleaved mature forms of GDF9. Densitometry analysis of the Western blots revealed that pig, sheep, and red deer had significantly more of the promature, than the mature, form within the oocyte. Conversely, there were no significant differences between the levels of promature and mature forms of GDF9 in the secreted media. Moreover, the levels of the specific molecular forms of GDF9 were not different between pigs, sheep and red deer. The levels of GDF9 in rat samples were low which may be due to a lower affinity of the monoclonal GDF9 antibody due to sequence differences between rat and ovine GDF9.  Interestingly, applying a crosslinking reagent to the oocyte lysate and conditioned media samples revealed the appearance of a high molecular size band. The appearance of this band, which was more prominent in the rat and pig, was concomitant with the disappearance of the mature GDF9 band. The differential levels of these presumptive GDF9 multimers in these two species that have large litters may suggest that rat and pig mature GDF9 binds other oocyte secreted factors more readily than GDF9 from mono-ovulatory species. Importantly, no homo- or hetero- mature dimers of GDF9 were detected in any of the species studied.  In summary, GDF9 was predominantly present as promature and cleaved mature forms both within the oocyte and in the secretions from the oocyte. Overall, these results indicated that the promature form was present in higher levels than the cleaved mature form. With the exception of the rat, there were no detectable species differences in the levels of the GDF9 forms within or secreted from the oocyte using Western blotting methodologies.</p>

scholarly journals The Molecular Forms of GDF9 In A Range of Mammalian Species

2021 ◽  
Author(s):  
◽  
Abdulaziz Alhussini
Keyword(s):  
Litter Size ◽  
Western Blotting ◽  
Red Deer ◽  
Transforming Growth Factor ◽  
Mammalian Species ◽  
Molecular Size ◽  
Biologically Active ◽  
Molecular Forms ◽  
Western Blots ◽  
Mature Form

<p>Growth Differentiation Factor (GDF) 9 is a member of the transforming growth factor β (TGFβ) superfamily that is exclusively expressed within and secreted from, the oocyte. This protein has generated much interest as it has been found to play a major role in follicular growth and maturation in mammals, and may be involved in determining litter size. Like most TGFβ family members, it is synthesised as a pre-pro-mature protein and is cleaved at various stages to allow the biologically active mature form to bind its Type II receptor. The aim of this study was to improve our understanding of the different molecular forms of GDF9 that are present within ovarian follicles of a range of mammalian species that differ in litter size. To achieve this aim, Western blotting experiments were performed to illustrate the molecular forms that were present within, and secreted from, the oocytes of rats, pigs, sheep and red deer. The detection of bands that represented the different molecular forms of GDF9 was undertaken using a monoclonal antibody that targeted a conserved region in the mature form of ovine GDF9.  The predominant forms of GDF9 found within and secreted from the oocyte across the species were the promature and cleaved mature forms of GDF9. Densitometry analysis of the Western blots revealed that pig, sheep, and red deer had significantly more of the promature, than the mature, form within the oocyte. Conversely, there were no significant differences between the levels of promature and mature forms of GDF9 in the secreted media. Moreover, the levels of the specific molecular forms of GDF9 were not different between pigs, sheep and red deer. The levels of GDF9 in rat samples were low which may be due to a lower affinity of the monoclonal GDF9 antibody due to sequence differences between rat and ovine GDF9.  Interestingly, applying a crosslinking reagent to the oocyte lysate and conditioned media samples revealed the appearance of a high molecular size band. The appearance of this band, which was more prominent in the rat and pig, was concomitant with the disappearance of the mature GDF9 band. The differential levels of these presumptive GDF9 multimers in these two species that have large litters may suggest that rat and pig mature GDF9 binds other oocyte secreted factors more readily than GDF9 from mono-ovulatory species. Importantly, no homo- or hetero- mature dimers of GDF9 were detected in any of the species studied.  In summary, GDF9 was predominantly present as promature and cleaved mature forms both within the oocyte and in the secretions from the oocyte. Overall, these results indicated that the promature form was present in higher levels than the cleaved mature form. With the exception of the rat, there were no detectable species differences in the levels of the GDF9 forms within or secreted from the oocyte using Western blotting methodologies.</p>

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

2019 ◽  
Vol 14 (04) ◽  
Author(s):  
N. S. Dangar ◽  
G. M. Pandya ◽  
U. V. Ramani ◽  
Y. D. Padheriya ◽  
T. Sangma ◽  
...  
Keyword(s):  
Litter Size ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
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.

scholarly journals JNK and p38 Inhibitors Prevent Transforming Growth Factor-β1-Induced Myofibroblast Transdifferentiation in Human Graves’ Orbital Fibroblasts

2021 ◽  
Vol 22 (6) ◽  
pp. 2952
Author(s):  
Tzu-Yu Hou ◽  
Shi-Bei Wu ◽  
Hui-Chuan Kau ◽  
Chieh-Chih Tsai
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Mapk Pathway ◽  
Transforming Growth Factor Β1 ◽  
Tissue Growth ◽  
Mitogen Activated Protein Kinase ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Western Blots ◽  
Orbital Fibroblasts ◽  
Tgf Β1

Transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation from orbital fibroblasts is known to dominate tissue remodeling and fibrosis in Graves’ ophthalmopathy (GO). However, the signaling pathways through which TGF-β1 activates Graves’ orbital fibroblasts remain unclear. This study investigated the role of the mitogen-activated protein kinase (MAPK) pathway in TGF-β1-induced myofibroblast transdifferentiation in human Graves’ orbital fibroblasts. The MAPK pathway was assessed by measuring the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) by Western blots. The expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and fibronectin representing fibrogenesis was estimated. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for extracellular matrix (ECM) metabolism were analyzed. Specific pharmacologic kinase inhibitors were used to confirm the involvement of the MAPK pathway. After treatment with TGF-β1, the phosphorylation levels of p38 and JNK, but not ERK, were increased. CTGF, α-SMA, and fibronectin, as well as TIMP-1 and TIMP-3, were upregulated, whereas the activities of MMP-2/-9 were inhibited. The effects of TGF-β1 on the expression of these factors were eliminated by p38 and JNK inhibitors. The results suggested that TGF-β1 could induce myofibroblast transdifferentiation in human Graves’ orbital fibroblasts through the p38 and JNK pathways.

scholarly journals Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shauna Kehoe ◽  
Katarina Jewgenow ◽  
Paul R. Johnston ◽  
Susan Mbedi ◽  
Beate C. Braun
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Ovarian Follicle ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Ovarian Follicles ◽  
Domestic Cat ◽  
Transcriptional Analysis ◽  
Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

scholarly journals Long non-coding (lnc)RNA profiling and the role of a key regulator lnc-PNRC2-1 in the transforming growth factor-β1-induced epithelial–mesenchymal transition of CNE1 nasopharyngeal carcinoma cells

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199651
Author(s):  
Jie Yang ◽  
Enzi Feng ◽  
Yanxin Ren ◽  
Shun Qiu ◽  
Liufang Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Nasopharyngeal Carcinoma ◽  
Rna Sequencing ◽  
Western Blotting ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Carcinoma Cells ◽  
Mesenchymal Transition ◽  
Emt Markers ◽  
Tgf Β1

Objectives To identify key long non-coding (lnc)RNAs responsible for the epithelial–mesenchymal transition (EMT) of CNE1 nasopharyngeal carcinoma cells and to investigate possible regulatory mechanisms in EMT. Methods CNE1 cells were divided into transforming growth factor (TGF)-β1-induced EMT and control groups. The mRNA and protein expression of EMT markers was determined by real-time quantitative PCR and western blotting. Differentially expressed genes (DEGs) between the two groups were identified by RNA sequencing analysis, and DEG functions were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. EMT marker expression was re-evaluated by western blotting after knockdown of a selected lncRNA. Results TGF-β1-induced EMT was characterized by decreased E-cadherin and increased vimentin, N-cadherin, and Twist expression at both mRNA and protein levels. Sixty lncRNA genes were clustered in a heatmap, and mRNA expression of 14 dysregulated lncRNAs was consistent with RNA sequencing. Knockdown of lnc-PNRC2-1 increased expression of its antisense gene MYOM3 and reduced expression of EMT markers, resembling treatment with the TGF-β1 receptor inhibitor LY2109761. Conclusion Various lncRNAs participated indirectly in the TGF-β1-induced EMT of CNE1 cells. Lnc-PNRC2-1 may be a key regulator of this and is a potential target to alleviate CNE1 cell EMT.

scholarly journals Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

1988 ◽  
Vol 8 (3) ◽  
pp. 1247-1252 ◽  
Author(s):  
E Lazar ◽  
S Watanabe ◽  
S Dalton ◽  
M B Sporn
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Amino Acid ◽  
Growth Factor ◽  
Aspartic Acid ◽  
Transforming Growth Factor ◽  
Biologically Active ◽  
Single Amino Acid ◽  
Transforming Growth Factor Alpha ◽  
Factor Alpha

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

scholarly journals Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

2015 ◽  
Vol 82 (4) ◽  
pp. 1286-1294 ◽  
Author(s):  
Evelyn Durmaz ◽  
Yan Hu ◽  
Raffi V. Aroian ◽  
Todd R. Klaenhammer
Keyword(s):  
Bacillus Thuringiensis ◽  
Lactococcus Lactis ◽  
Copy Number ◽  
Oral Delivery ◽  
Membrane Integrity ◽  
Biologically Active ◽  
High Copy Number ◽  
Cry Protein ◽  
Western Blots ◽  
Content Type

ABSTRACTTheBacillus thuringiensiscrystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) inLactococcus lactisfor potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production,cry5Bwas cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes inLactococcuslysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strainL. lactisKP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates fromL. lactiscultures expressing both Cry5B and tCry5B,in vivochallenges ofCaenorhabditis elegansworms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly fromL. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.

Intracellular distribution of α-galactosidase in leaves of Cucurbita pepo

1979 ◽  
Vol 57 (18) ◽  
pp. 1904-1911 ◽  
Author(s):  
Brian Thomas ◽  
John A. Webb
Keyword(s):  
Cucurbita Pepo ◽  
Developmental Stages ◽  
Molecular Size ◽  
Total Activity ◽  
Intracellular Distribution ◽  
Molecular Forms ◽  
Enzyme Release ◽  
Enzyme Preparations ◽  
Isolated Protoplasts

The intracellular distribution of α-galactosidase in leaves of Cucurbita pepo was studied at different developmental stages using tissue strips, homogenates, and isolated protoplasts. About 85% of the total activity was found in the 500 g supernatant after tissues were homogenized either in water, in buffer at pH 5.6 or at pH 7.0, or in buffer containing 0.8 M KCl. Isolated protoplasts contained less than 10% of the total activity which was confined to the 20 000 g supernatant after lysis. p-Nitrophenyl-α-D-galactoside was readily hydrolysed when incubated with leaf strips but less than 3% of α-galactosidase could be leached from strips held for 4 h in 100 mM phosophate buffer or in buffer containing either 0.8 M KCl, 1 mM EDTA, or 1 mM dithioerythritol. It is concluded that at all stages of leaf development a high proportion of α-galactosidase is located in the exocellular region, not strongly bound either to the outer surface of the plasmalemma or to the cell wall but prevented from diffusing through the wall matrix by some physical attribute such as molecular size. Enzyme release occurred only following breakage or enzymatic digestion of the wall. The in vivo properties of the exocellular enzyme in leaf strips were compared with those of three molecular forms of α-galactosidase (LI, LII, and LIII) which were partially purified from mature leaves. The exocellular enzyme was active over a broad pH range with optima at pH 3.0 and pH 6.0; this resembles a combination of pH optima for LI and LIII. Inhibition by Cu2+ and p-chloromercuribenzoate resembled that for LIII and LII, respectively. Galactose and galactinol at a 5 mM concentration were 25–30% inhibitory for all enzyme preparations; melibiose, raffinose, and stachyose were very weakly inhibitory. The function of an exocellular α-galactosidase and its bearing on the transport of galactosylsucrose oligosaccharides to and from the minor veins of C. pepo are discussed.

Abstract 269: Miofibroblast Tgf-beta Signaling in a Proteotoxic Mouse Model

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Bidur Bhandary ◽  
Qinghang Meng ◽  
Hanna Osinska ◽  
Kritton Shay-Winkler ◽  
James Gulick ◽  
...  
Keyword(s):  
Heart Disease ◽  
Mouse Model ◽  
Cardiac Function ◽  
Transforming Growth Factor Beta ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Prolonged Survival ◽  
Tgfβ Signaling ◽  
Specific Expression ◽  
Western Blots

Introduction: Transforming Growth Factor Beta (TGFβ) is an important cytokine in mediating the fibrogenic response and, in particular, cardiac fibrosis. Extensive fibrosis accompanies the cardiac remodeling that occurs during development of the protein conformation-based disease caused by cardiomyocyte-specific expression of a mutant, small, heat shock-like protein and chaperone, aB crystallin (CryABR120G). During the onset of fibrosis, fibroblasts are activated to the so-called “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that TGFβ signaling processes that result in significant cardiac fibrosis in a mouse model of proteotoxic heart disease are mediated by cardiac fibroblasts, rather than cardiomyocytes. Here, we have partially ablated TGFβ signaling only in cardiac myofibroblasts to observe if cardiac fibrosis is reduced. Aims and Methods: The objective of this study was to understand the contributions of fibroblast-derived TGFβ signaling to the development of cardiac fibrosis in a proteotoxic mouse model that results in significant cardiac fibrosis. To test the hypothesis we partially deleted the myofibroblast specific canonical and non-canonical signaling by crossing CryAB R120G mice with Tgfbr1 or Tgfbr2 floxed mice. The double transgene containing mice were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter. Echocardiography, Masson’s Trichome staining, PCR arrays, IHC and western blots were performed to characterize the fibrotic progression in CryAB R120G transgenic mice. Results: We observed that myofibroblast-targeted partial knockdown of Tgf βr1 signaling prolonged survival, modestly reducing fibrosis and improving cardiac function . Similarly, Tgf βr2 partial knockdown prolonged survival, modestly reducing fibrosis without improving cardiac function during fibrosis development in CryAB R120G mice. Conclusion: These findings suggest that, in a model of proteotoxic heart disease, myofibroblast based TGFβ signaling in the heart may contribute to cardiac hypertrophy/dysfunction but cannot account entirely for the fibrotic response.

scholarly journals Ferulic Acid Attenuates TGF-β1-Induced Renal Cellular Fibrosis in NRK-52E Cells by Inhibiting Smad/ILK/Snail Pathway

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Ming-gang Wei ◽  
Wei Sun ◽  
Wei-ming He ◽  
Li Ni ◽  
Yan-yu Yang
Keyword(s):  
Ferulic Acid ◽  
Western Blotting ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Underlying Mechanism ◽  
Tubular Epithelial Cell ◽  
Epithelial Cell Line ◽  
Proximal Tubular ◽  
Mesenchymal Transformation ◽  
E Cadherin

Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. Central to this process is epithelial-mesenchymal transformation (EMT) of proximal tubular epithelial cells driven by transforming growth factor-β1 (TGF-β1) signaling. The present study aimed to investigate the effect of Ferulic acid (FA) on EMT of renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-β1 and to elucidate its underlying mechanism against EMT related to TGF-β1/Smads pathway. The NRK-52E cells were treated for 48 h with TGF-β1 (5 ng/mL) in different concentrations of FA (0 to 200 µM). Fibronectin, a mesenchymal marker, was assessed by western blotting. Western blotting was also used to examine the EMT markers (E-cadherin, andα-smooth muscle actin (α-SMA)), signal transducer (p-Smad2/3), and EMT initiator (Snail). ILK was also assayed by western blotting. The results showed that TGF-β1 induced spindle-like morphological transition in NRK-52E cells. Smad2/3 signaling pathway activation, increased fibronectin,α-SMA, ILK, and Snail expression, and decreased E-cadherin expression in TGF-β1-treated NRK-52E cells. FA efficiently blocked P-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. These findings suggest that FA may serve as a potential fibrosis antagonist for renal proximal tubule cells by inhibiting EMT process.

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