scholarly journals Oocyte-derived forms of ruminant BMP15 and GDF9 and a theoretical model to explain their synergistic response

2021 ◽  
Author(s):  
◽  
Derek Heath
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Synergistic Effects ◽  
Follicular Development ◽  
Molecular Forms ◽  
Type I ◽  
Type Ii ◽  
Synergistic Response

<p>Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact.  Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions.  The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9.  Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using promature porcine TGFB1 and human BMP9 as templates. These models, together with the previously determined forms of GDF9 and BMP15 produced by the ruminant oocyte, were used to visualise their potential interactions, both with each other and with their receptors. This report describes a model showing the possible interactions involved in a synergistic response. In this model, the mature domain is presented to the type II receptor by the proregion and heterodimers form at the level of the receptor. Differences, following heterodimerisation in the conformation and orientation between GDF9 and its type I receptor, as well as between type I and type II receptors, relative to that in homodimers, could explain how heterodimerisation leads to increased Smad3 phosphorylation and subsequent down-stream somatic cell responses.</p>

scholarly journals Oocyte-derived forms of ruminant BMP15 and GDF9 and a theoretical model to explain their synergistic response

2021 ◽  
Author(s):  
◽  
Derek Heath
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Synergistic Effects ◽  
Follicular Development ◽  
Molecular Forms ◽  
Type I ◽  
Type Ii ◽  
Synergistic Response

<p>Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact.  Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions.  The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9.  Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using promature porcine TGFB1 and human BMP9 as templates. These models, together with the previously determined forms of GDF9 and BMP15 produced by the ruminant oocyte, were used to visualise their potential interactions, both with each other and with their receptors. This report describes a model showing the possible interactions involved in a synergistic response. In this model, the mature domain is presented to the type II receptor by the proregion and heterodimers form at the level of the receptor. Differences, following heterodimerisation in the conformation and orientation between GDF9 and its type I receptor, as well as between type I and type II receptors, relative to that in homodimers, could explain how heterodimerisation leads to increased Smad3 phosphorylation and subsequent down-stream somatic cell responses.</p>

scholarly journals In vivo and In vitro Regulation of Type I IFN Synthesis by Synergistic Effects of CD40 and Type II IFN

2006 ◽  
Vol 176 (10) ◽  
pp. 5995-6003 ◽  
Author(s):  
Jennifer A. Greene ◽  
Jennifer L. DeVecchio ◽  
Meetha P. Gould ◽  
Jeffery J. Auletta ◽  
Frederick P. Heinzel
Keyword(s):  
Synergistic Effects ◽  
Type I ◽  
Type Ii ◽  
Type I Ifn

scholarly journals Molecular forms of ruminant BMP15 and GDF9 and putative interactions with receptors

Reproduction ◽  
2017 ◽  
Vol 154 (4) ◽  
pp. 521-534 ◽  
Author(s):  
Derek A Heath ◽  
Janet L Pitman ◽  
Kenneth P McNatty
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Follicular Development ◽  
Molecular Forms ◽  
Published Data ◽  
Type I ◽  
Mutant Form ◽  
Surface Receptors

Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted factors with demonstrable effects on ovarian follicular development and ovulation rate. However, the molecular forms of BMP15 and GDF9 produced by oocytes remain unclear. The aims herein, using Western blotting (WB) procedures with specific monoclonal antibodies (mabs), were to identify the molecular forms of BMP15 and GDF9 synthesised and secreted by isolated ovine (o) and bovine (b) oocytes in vitro. The mabs were known to recognise the biological forms of BMP15 or GDF9 since they had previously been shown to inhibit their bioactivities in vitro and in vivo. Using recombinant variants of oBMP15 and oGDF9, including a cysteine mutant form of oBMP15 (S356C) and a human (h) BMP15:GDF9 heterodimer (cumulin), it was established that the mabs were able to identify monomeric, dimeric, promature and higher-molecular-weight forms of BMP15 and GDF9 and cumulin (GDF9 mab only). After using non-reducing, reducing and reducing + cross-linking conditions, the major oocyte-secreted forms of o and b BMP15 and GDF9 were the cleaved and uncleaved monomeric forms of the promature proteins. There was no evidence for dimeric or heterodimeric forms of either mature BMP15 or GDF9. From in silico modelling studies using transforming growth factor beta (TGFB), activin or BMP crystal templates, and both present and previously published data, a model is proposed to illustrate how the monomeric forms of BMP15 and GDF9 may interact with their type II and type I cell-surface receptors to initiate the synergistic actions of these growth factors.

scholarly journals Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei

2006 ◽  
Vol 189 (3) ◽  
pp. 807-817 ◽  
Author(s):  
Narisara Chantratita ◽  
Vanaporn Wuthiekanun ◽  
Khaemaporn Boonbumrung ◽  
Rachaneeporn Tiyawisutsri ◽  
Mongkol Vesaratchavest ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Vaccine Development ◽  
Colony Morphology ◽  
Phenotypic Switching ◽  
Type I ◽  
Type Ii ◽  
Altered Expression ◽  
Replication Fitness

ABSTRACT Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

scholarly journals LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

2019 ◽  
Vol 47 (12) ◽  
pp. 6369-6385
Author(s):  
Jia-Yi Fan ◽  
Qian Huang ◽  
Quan-Quan Ji ◽  
En-Duo Wang
Keyword(s):  
Tertiary Structure ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Type I ◽  
Type Ii ◽  
Specific Domain ◽  
Recognition Mechanism ◽  
Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

scholarly journals ACT001, a novel PAI-1 inhibitor, exerts synergistic effects in combination with cisplatin by inhibiting PI3K/AKT pathway in glioma

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Xiaonan Xi ◽  
Ning Liu ◽  
Qianqian Wang ◽  
Yahui Chu ◽  
Zheng Yin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Synergistic Effect ◽  
Glioma Cell ◽  
Synergistic Effects ◽  
Akt Pathway ◽  
Phase I Clinical Trials ◽  
Glioma Cell Proliferation ◽  
Pai 1

Abstract PAI-1 plays significant roles in cancer occurrence, relapse and multidrug resistance and is highly expressed in tumours. ACT001, which is currently in phase I clinical trials for the treatment of glioblastoma (GBM). However, the detailed molecular mechanism of ACT001 is still unclear. In this study, we investigated the effects of ACT001 on glioma cell proliferation and clarified its mechanism. We discovered that PAI-1 was the direct target of ACT001 by a cellular thermal shift assay. Then, the interaction between ACT001 and PAI-1 was verified by Biacore assays, thermal stability assays and ACT001 probe assays. Furthermore, from the proteomic analysis, we found that ACT001 directly binds PAI-1 to inhibit the PI3K/AKT pathway, which induces the inhibition of glioma cell proliferation, invasion and migration. Moreover, the combination of ACT001 and cisplatin showed a synergistic effect on the inhibition of glioma in vitro and in vivo. In conclusion, our findings demonstrate that PAI-1 is a new target of ACT001, the inhibition of PAI-1 induces glioma inhibition, and ACT001 has a synergistic effect with cisplatin through the inhibition of the PAI-1/PI3K/AKT pathway.

scholarly journals MicroRNA-224 Is Involved in Transforming Growth Factor-β-Mediated Mouse Granulosa Cell Proliferation and Granulosa Cell Function by Targeting Smad4

2010 ◽  
Vol 24 (3) ◽  
pp. 540-551 ◽  
Author(s):  
Guidong Yao ◽  
Mianmian Yin ◽  
Jie Lian ◽  
Hui Tian ◽  
Lin Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Transforming Growth Factor ◽  
Cell Function ◽  
Ectopic Expression ◽  
Follicular Development ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Mrna Levels ◽  
Tgf Β1

Abstract Many members of the TGF-β superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-β1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-β-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-β1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFβ superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-β1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-β1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-β1. In addition, both miR-224 and TGF-β1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-β1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.

External PS in Sickle RBC: Relationships among Aminophospholipid Translocase (APLT), Phospholipid Scramblase (PLSCR), Cell Maturity, and Cell Density.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 148-148
Author(s):  
Latorya E. Arnold ◽  
Mary B. Palascak ◽  
Clinton H. Joiner ◽  
Robert S. Franco
Keyword(s):  
Annexin V ◽  
Type I ◽  
Type Ii ◽  
Color Flow ◽  
Low Level ◽  
Aminophospholipid Translocase ◽  
Phospholipid Scramblase ◽  
High Level

Abstract External phosphatidylserine (PS) is present on some sickle RBC and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Phospholipid scramblase (PLSCR) disrupts phospholipid (PL) asymmetry by causing nonspecific PL equilibration across the membrane. Aminophospholipid translocase (APLT) maintains PL asymmetry by returning externalized PS to the inner membrane leaflet. It has been proposed that both APLT inhibition and PLSCR activation are required for PS externalization. Sickle RBC with low level external PS (Type I PS+) are present in cells of all densities and include some reticulocytes. Sickle RBC with high external PS (Type II PS+) are primarily found in the dense fraction. Type II cells are thought to be more important because: the high level of external PS should have greater consequence; high level external PS occurs primarily in pathologically dehydrated sickle RBC; and low level external PS appears to be physiological in immature RBC. We have previously shown that dense, dehydrated sickle RBC, including the small number of dense transferrin receptor positive (TfR+) reticulocytes, have markedly inhibited APLT. In the current studies, we examined the relationships among external PS, APLT, PLSCR, and density in mature RBC and TfR+ reticulocytes using 3-color flow cytometry. APLT and PLSCR activities were assayed using fluorescent PL analogues (NBD-PS and NBD-PC, respectively), and expressed as the fraction of probe internalized. External PS was measured with Annexin V-PE and TfR+ reticulocytes were identified with anti-TfR-PE/Cy5. PS+ cells had lower APLT activity compared to PS- cells that did not reach significance for n=3 (NBD-PS internalization fraction for PS-: 0.586±0.053; Type I PS+: 0.517±0.158, Type II PS+: 0.523±0.033). PS- sickle RBC had a uniformly low PLSCR activity similar to normal RBC (NBD-PC internalization fractions ∼ 0.1). In mature sickle RBC, PLSCR was more active in PS+ cells (PS-: 0.097±0.096; Type I PS+: 0.163±0.070, Type II PS+: 0.248±0.043; n=3; PS- vs Type I PS+: p=0.06; PS- vs Type II PS+: p=0.04; Type I versus Type II: p=0.03). TfR+ reticulocytes had increased APLT and PLSCR activity compared to mature sickle RBC, but there was no apparent relationship between PLSCR and external PS. Since dense sickle RBC had markedly inhibited APLT, we evaluated the relationship between dehydration and APLT activity. Dehydration of AA RBC from an MCHC of 35.6±2.2 to 49.2±2.0 g/dL inhibited APLT (from 0.484±0.068 to 0.301±0.076; n=7, p= 0.01). Dehydration of SS RBC from an MCHC of 34.8±3.5 to 50.1±3.9 g/dL also inhibited APLT (from 0.460±0.060 to 0.361±0.047; n=3, p=0.006), but not as low as in SS RBC dehydrated in vivo (0.222±0.036 at 44.7±5.6 g/dL; n=4, p=0.007 vs. SS RBC dehydrated in vitro). Rehydration of AA and SS RBC that had been dehydrated in vitro reversed APLT inhibition. However, APLT activity was not reversed upon rehydration of sickle RBC dehydrated in vivo. In summary, our data show that: many dense sickle RBC with significantly inhibited APLT are PS-, indicating that APLT inhibition alone does not result in PS externalization; dehydration contributes to, but is not entirely responsible for, the APLT inhibition seen in dense sickle RBC; and PS+ sickle RBC have increased PLSCR activity.

Oocyte-secreted factros regulate granulosa cell steroidogenesis

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 317-321 ◽  
Author(s):  
Barbara C. Vanderhyden
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Inhibitory Factor ◽  
Preovulatory Follicles ◽  
Loss Of Contact

Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellaset al., 1991; Huanget al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhydenet al., 1990, 1992) and cumulus expansion (Buccioneet al., 1990; Salustriet al., 1990; Vanderhydenet al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinisein vitro(Channinget al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisationin vitro(Ledwitz-Rigbyet al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.

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