Eukaryotic expression, Co-IP and MS identify BMPR-1B protein-protein interaction network

BACKGROUND: BMPR-1B is part of the transforming growth factor ß super family and plays a pivotal role in ewe litter size. Functional loss of exon-8 mutations in the BMPR-1B gene (namely the FecB gene) can increase both the ewe ovulation rate and litter size. RESULTS: This study constructed a eukaryotic expression system, prepared a monoclonal antibody, and characterized BMPR-1B/FecB protein-protein interactions (PPIs). Using Co-immunoprecipitation coupled to mass spectrometry (Co-IP/MS), 23 proteins were identified that specifically interact with FecB in ovary extracts of ewes. Bioinformatics analysis of selected PPIs demonstrated that FecB associated with several other BMPs, primarily via signal transduction in the ovary. FecB and its associated interaction proteins enriched the reproduction process via BMP2 and BMP4 pathways. Signal transduction was identified via Smads proteins and TGF-beta signaling pathway by analyzing the biological processes and pathways. Moreover, other target proteins (GDF5, GDF9, RhoD, and HSP 10) that interact with FecB and that are related to ovulation and litter size in ewes were identified. CONCLUSIONS: In summary, this research identified a novel pathway and insight to explore the PPi network of BMPR-1B.

Polymorphism of bone morphogenetic protein receptor-IB [BMPR-IB] gene with litter size and kids growth of some goat breeds in Egypt

Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms [PCR-RFLP] and Single Nucleotide polymorphism [SNP] techniques were used to study the association between bone morphogenetic protein receptor IB [BMPR IB] gene polymorphism with litter size trait and kids growth. Forty four Female goats were precisely selected according to their litter size and kids growth. PCR amplification of 190 bp of the BMPR-IB gene was genotyped in all goats and sequenced only in those produced the highest and lowest litter size and kids growth. Restriction analysis of PCR-RFLP using Ava II and Hind III of the BMPRIB gene [190-bp] do not produce restriction fragments. By DNA sequencing, eight single nucleotide polymorphisms [SNP's] at seven different positions were obtained. Furthermore, with translation of SNPs to corresponding amino acids, change of six amino acids in three female goats were obtained as the following, Baladi goat with high litter size, glutamic acid [E] changed to aspartic acid [P] and isoleucine [I] changed to valine [V]. In high litter size, Zaraibi goat, valine [V] changed to leucine acid [L] and glutamine [Q] changed to histidine [H] and threonine [T] changed to proline [P]. These findings can be used in a marker-assisted selection [MAS] for selection for high litter size trait in goats. There are negative relationships in most goats between SNPs in BMPR IB gene and relative growth gain [RGG]

Expression of bone morphogenetic protein receptor IA in rats after contusive spinal cord injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the expression pattern of bone morphogenetic protein receptor IA (BMPR IA) in rats after contusive spinal cord injury.</p><p><b>METHODS</b>The expressions of BMPR IA, IB, and II were detected by immunochemistry in the spinal cord of normal adult rats, and the expression of BMPR IA was detected in the infinite horizons impactor model at 1, 3, 7, 14, 30, and 60 days after spinal cord injury.</p><p><b>RESULTS</b>In the spinal cord of normal adult rats, BMPR IA and II were expressed predominantly in the oligodentrocytes and neurons in the grey matter, and also in some astrocytes and numerous microglia cells. Only a low level of BMPR IB expression was detected in the neurons of the grey matter. After spinal cord injury, the expression of BMP IA markedly increased with sustained strong expression in the astrocytes till one month after the injury; its expression was also increased obviously in the microglia cells activated by the injury.</p><p><b>CONCLUSION</b>The expression of BMPR IA increases significantly in the astrocytes and activated microglia cells in rats after contusive spinal cord injury, suggesting the involvement of BMP signaling pathway in the physiological and pathological role of glia cells.</p>

Experiment on induction of fibroblasts on 3-D cell-foam structures to express osteoblastic phenotype and its mechanism / 中华外科杂志

<p><b>OBJECTIVE</b>To study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.</p><p><b>METHODS</b>The fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.</p><p><b>RESULTS</b>Fibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.</p><p><b>CONCLUSIONS</b>Fibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.</p>

Preliminary study of ALK3 downstream genes related to ventricular septum defect / 生物工程学报

To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.

Expression of core-binding factor a1 by human skin fibroblasts induced in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.</p><p><b>METHODS</b>The fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.</p><p><b>RESULTS</b>TNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.</p><p><b>CONCLUSION</b>Human skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.</p>