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Id Subject Object Predicate Lexical cue
T160 0-104 Sentence denotes BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1
T1 0-104 Sentence denotes BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1
T2 107-115 Sentence denotes Abstract
T161 116-126 Sentence denotes Background
T3 116-126 Sentence denotes Background
T162 127-272 Sentence denotes Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and are secreted proteins with pleiotropic roles in many different cell types.
T4 127-272 Sentence denotes Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and are secreted proteins with pleiotropic roles in many different cell types.
T163 273-393 Sentence denotes A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases.
T5 273-393 Sentence denotes A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases.
T164 394-487 Sentence denotes In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells.
T6 394-487 Sentence denotes In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells.
T165 489-496 Sentence denotes Results
T7 489-496 Sentence denotes Results
T166 497-619 Sentence denotes The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8.
T8 497-619 Sentence denotes The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8.
T167 620-760 Sentence denotes Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected.
T9 620-760 Sentence denotes Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected.
T168 761-961 Sentence denotes Furthermore, we found that BMP-6 had an antiproliferative effect both in naïve (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L.
T10 761-961 Sentence denotes Furthermore, we found that BMP-6 had an antiproliferative effect both in naïve (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L.
T169 962-1029 Sentence denotes Additionally, BMP-6 induced cell death in activated memory B cells.
T11 962-1029 Sentence denotes Additionally, BMP-6 induced cell death in activated memory B cells.
T170 1030-1153 Sentence denotes Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin.
T12 1030-1153 Sentence denotes Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin.
T171 1154-1249 Sentence denotes Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM.
T13 1154-1249 Sentence denotes Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM.
T172 1251-1261 Sentence denotes Conclusion
T14 1251-1261 Sentence denotes Conclusion
T173 1262-1400 Sentence denotes In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.
T15 1262-1400 Sentence denotes In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.
T16 1403-1413 Sentence denotes Background
T1034 1414-1581 Sentence denotes Members of the transforming growth factor β (TGF-β) superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis [1].
T17 1414-1581 Sentence denotes Members of the transforming growth factor β (TGF-β) superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis [1].
T1035 1582-1759 Sentence denotes These cytokines can be divided into three subgroups: TGF-β, the activins/inhibins, and the bone morphogenetic proteins (BMPs), of which the latter constitute the largest family.
T18 1582-1759 Sentence denotes These cytokines can be divided into three subgroups: TGF-β, the activins/inhibins, and the bone morphogenetic proteins (BMPs), of which the latter constitute the largest family.
T1036 1760-1903 Sentence denotes BMPs are 30–38 kDa hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation [2,3].
T19 1760-1903 Sentence denotes BMPs are 30–38 kDa hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation [2,3].
T1037 1904-2041 Sentence denotes Several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues [1].
T20 1904-2041 Sentence denotes Several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues [1].
T1038 2042-2202 Sentence denotes TGF-β has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date. [4].
T21 2042-2202 Sentence denotes TGF-β has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date. [4].
T1039 2203-2289 Sentence denotes In contrast, the effect of BMPs in the immune system has not been widely investigated.
T22 2203-2289 Sentence denotes In contrast, the effect of BMPs in the immune system has not been widely investigated.
T1040 2290-2435 Sentence denotes In that respect, BMP- 2, -4 and -7 have been found to control differentiation of hematopoietic stem cells [5] and early T cell development [6,7].
T23 2290-2435 Sentence denotes In that respect, BMP- 2, -4 and -7 have been found to control differentiation of hematopoietic stem cells [5] and early T cell development [6,7].
T1041 2436-2556 Sentence denotes BMP-6 has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells [8].
T24 2436-2556 Sentence denotes BMP-6 has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells [8].
T1042 2557-2674 Sentence denotes Furthermore, BMP-2, -4, 6 and -7 had an antiproliferative and a proapoptotic effect on multiple myeloma cells [9-11].
T25 2557-2674 Sentence denotes Furthermore, BMP-2, -4, 6 and -7 had an antiproliferative and a proapoptotic effect on multiple myeloma cells [9-11].
T1043 2675-2887 Sentence denotes In addition, by gene expression profiling, BMP-6 significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large B cell lymphomas (DLBCL) [12].
T26 2675-2887 Sentence denotes In addition, by gene expression profiling, BMP-6 significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large B cell lymphomas (DLBCL) [12].
T1044 2888-3144 Sentence denotes BMP-6, like the other BMP members, signals through ligation and heterodimerzation of BMP type I [activin-like-kinase (ALK)] and type II serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating Smad proteins.
T27 2888-3144 Sentence denotes BMP-6, like the other BMP members, signals through ligation and heterodimerzation of BMP type I [activin-like-kinase (ALK)] and type II serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating Smad proteins.
T1045 3145-3380 Sentence denotes BMP-6 can signal through the ligation of the type I receptors Act-RIA, BMP-RIA, and BMP-RIB and the type II receptors BMP-RII, Act-RIIA and Act-RIIB, which lead to the phosphorylation of the receptor Smads (Smad-1, Smad-5, and Smad-8).
T28 3145-3380 Sentence denotes BMP-6 can signal through the ligation of the type I receptors Act-RIA, BMP-RIA, and BMP-RIB and the type II receptors BMP-RII, Act-RIIA and Act-RIIB, which lead to the phosphorylation of the receptor Smads (Smad-1, Smad-5, and Smad-8).
T1046 3381-3517 Sentence denotes The R- Smads then form complexes with the co-Smad (Smad4) and are translocated into the nucleus where they exert gene regulation [1,13].
T29 3381-3517 Sentence denotes The R- Smads then form complexes with the co-Smad (Smad4) and are translocated into the nucleus where they exert gene regulation [1,13].
T1047 3518-3675 Sentence denotes Given the reported role of BMP-6 in B-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human B cells.
T30 3518-3675 Sentence denotes Given the reported role of BMP-6 in B-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human B cells.
T1048 3676-3773 Sentence denotes We studied the effects of BMP-6 on proliferation and apoptosis on resting and stimulated B cells.
T31 3676-3773 Sentence denotes We studied the effects of BMP-6 on proliferation and apoptosis on resting and stimulated B cells.
T1049 3774-3949 Sentence denotes Furthermore, the expression of BMP receptors and BMP-6 induced activation of the Smad signalling pathway with subsequent regulation of the target genes Id1–Id4, were resolved.
T32 3774-3949 Sentence denotes Furthermore, the expression of BMP receptors and BMP-6 induced activation of the Smad signalling pathway with subsequent regulation of the target genes Id1–Id4, were resolved.
T1050 3950-4028 Sentence denotes Finally, we investigated whether B cells also were capable of producing BMP-6.
T33 3950-4028 Sentence denotes Finally, we investigated whether B cells also were capable of producing BMP-6.
T34 4030-4037 Sentence denotes Results
T2626 4039-4101 Sentence denotes BMP-6 inhibits anti-IgM induced proliferation of human B cells
T35 4039-4101 Sentence denotes BMP-6 inhibits anti-IgM induced proliferation of human B cells
T2627 4102-4240 Sentence denotes The effects of BMP-6 on normal and neoplastic hematopoietic cells prompted us to investigate the effects of BMP-6 on normal human B cells.
T36 4102-4240 Sentence denotes The effects of BMP-6 on normal and neoplastic hematopoietic cells prompted us to investigate the effects of BMP-6 on normal human B cells.
T2628 4241-4392 Sentence denotes All experiments in this study were performed under serum-free conditions as FCS has been shown to interfere with BMP-signalling [14](own observations).
T37 4241-4392 Sentence denotes All experiments in this study were performed under serum-free conditions as FCS has been shown to interfere with BMP-signalling [14](own observations).
T2629 4393-4566 Sentence denotes To study the effect of BMP-6 on proliferation, B-cells from healthy volunteers were stimulated with anti-IgM and/or CD40L in the presence or absence of BMP-6 for three days.
T38 4393-4566 Sentence denotes To study the effect of BMP-6 on proliferation, B-cells from healthy volunteers were stimulated with anti-IgM and/or CD40L in the presence or absence of BMP-6 for three days.
T2630 4567-4681 Sentence denotes We found that BMP-6 led to a 35% mean reduction of anti-IgM- induced DNA synthesis (n = 8; p ≤ 0.0002, Figure 1A).
T39 4567-4681 Sentence denotes We found that BMP-6 led to a 35% mean reduction of anti-IgM- induced DNA synthesis (n = 8; p ≤ 0.0002, Figure 1A).
T2631 4682-4795 Sentence denotes Similar results were obtained for B cells treated with anti-IgM and CD40L (26% mean reduction, n = 6; p ≤ 0.023).
T40 4682-4795 Sentence denotes Similar results were obtained for B cells treated with anti-IgM and CD40L (26% mean reduction, n = 6; p ≤ 0.023).
T2632 4796-4985 Sentence denotes The BMP-6-induced inhibition of proliferation was dose-dependent in both peripheral B cells (Figure 1B) and the Burkitt lymphoma cell line Ramos (40% reduction of DNA synthesis, Figure 1C).
T41 4796-4985 Sentence denotes The BMP-6-induced inhibition of proliferation was dose-dependent in both peripheral B cells (Figure 1B) and the Burkitt lymphoma cell line Ramos (40% reduction of DNA synthesis, Figure 1C).
T2633 4986-5084 Sentence denotes The BMP-6 effects could be reversed by addition of the extracellular inhibitor Noggin (Figure 1D).
T42 4986-5084 Sentence denotes The BMP-6 effects could be reversed by addition of the extracellular inhibitor Noggin (Figure 1D).
T2634 5085-5220 Sentence denotes Similarly, a combination of the soluble BMP receptors BMP-RIB-Fc and BMP-RII-Fc also neutralized the effects of BMP-6 (data not shown).
T43 5085-5220 Sentence denotes Similarly, a combination of the soluble BMP receptors BMP-RIB-Fc and BMP-RII-Fc also neutralized the effects of BMP-6 (data not shown).
T2635 5221-5308 Sentence denotes Next, we wanted to test whether BMP-6 had different effect on naïve and memory B cells.
T44 5221-5308 Sentence denotes Next, we wanted to test whether BMP-6 had different effect on naïve and memory B cells.
T2636 5309-5525 Sentence denotes Naïve (CD19+CD27-) and memory (CD19+CD27+) B cells were isolated from peripheral blood by cell sorting of immunobead-isolated CD19+ B cells [15], and tested for their capacity to proliferate in the presence of BMP-6.
T45 5309-5525 Sentence denotes Naïve (CD19+CD27-) and memory (CD19+CD27+) B cells were isolated from peripheral blood by cell sorting of immunobead-isolated CD19+ B cells [15], and tested for their capacity to proliferate in the presence of BMP-6.
T2637 5526-5770 Sentence denotes However, BMP-6 inhibited anti-IgM induced DNA synthesis in the two subpopulations to a similar extent, with a mean reduction of DNA-synthesis of 45% (n = 5; p ≤ 0,004) for naïve B cells and 48% (n = 5; p ≤ 0,001) for memory B cells (Figure 1E).
T46 5526-5770 Sentence denotes However, BMP-6 inhibited anti-IgM induced DNA synthesis in the two subpopulations to a similar extent, with a mean reduction of DNA-synthesis of 45% (n = 5; p ≤ 0,004) for naïve B cells and 48% (n = 5; p ≤ 0,001) for memory B cells (Figure 1E).
T3578 5772-5836 Sentence denotes BMP-6 induces cell death in human memory B cells and Ramos cells
T47 5772-5836 Sentence denotes BMP-6 induces cell death in human memory B cells and Ramos cells
T3579 5837-5930 Sentence denotes Next, we wanted to establish whether BMP-6 also could affect the viability of normal B cells.
T48 5837-5930 Sentence denotes Next, we wanted to establish whether BMP-6 also could affect the viability of normal B cells.
T3580 5931-6044 Sentence denotes Cell viability was determined by propidium iodide (PI) staining after culture with or without BMP-6 for 48 hours.
T49 5931-6044 Sentence denotes Cell viability was determined by propidium iodide (PI) staining after culture with or without BMP-6 for 48 hours.
T3581 6045-6205 Sentence denotes Interestingly, BMP-6 showed a small, but reproducible mean increase of cell death from 17 to 23% (n = 5; p ≤ 0,003) in anti-IgM stimulated CD27+ memory B cells.
T50 6045-6205 Sentence denotes Interestingly, BMP-6 showed a small, but reproducible mean increase of cell death from 17 to 23% (n = 5; p ≤ 0,003) in anti-IgM stimulated CD27+ memory B cells.
T3582 6206-6334 Sentence denotes Furthermore, Ramos cells showed a mean increase in cell death from 20 to 50% (n = 3, p < 0,001, figure 3) after BMP-6 treatment.
T51 6206-6334 Sentence denotes Furthermore, Ramos cells showed a mean increase in cell death from 20 to 50% (n = 3, p < 0,001, figure 3) after BMP-6 treatment.
T3583 6335-6500 Sentence denotes In contrast, cell death of total CD19+ cells (n = 6; p ≤ 0,32; data not shown) or CD27-IgG- naïve B cells was not significantly affected (n = 5; p ≤ 0,65, figure 2).
T52 6335-6500 Sentence denotes In contrast, cell death of total CD19+ cells (n = 6; p ≤ 0,32; data not shown) or CD27-IgG- naïve B cells was not significantly affected (n = 5; p ≤ 0,65, figure 2).
T4052 6502-6539 Sentence denotes Human B cells express BMP-6 receptors
T53 6502-6539 Sentence denotes Human B cells express BMP-6 receptors
T4053 6540-6645 Sentence denotes Detailed knowledge regarding expression of different BMP receptors in B cells is currently not available.
T54 6540-6645 Sentence denotes Detailed knowledge regarding expression of different BMP receptors in B cells is currently not available.
T4054 6646-6774 Sentence denotes To further elucidate the role of BMPs in human B cells, we performed western blot analysis for type I and type II BMP receptors.
T55 6646-6774 Sentence denotes To further elucidate the role of BMPs in human B cells, we performed western blot analysis for type I and type II BMP receptors.
T4055 6775-6938 Sentence denotes This analysis revealed that the type I receptors Act-RIA, BMP-RIB and the type II receptors BMP-RII and Act-RIIb are expressed on resting human B-cells (Figure 4).
T56 6775-6938 Sentence denotes This analysis revealed that the type I receptors Act-RIA, BMP-RIB and the type II receptors BMP-RII and Act-RIIb are expressed on resting human B-cells (Figure 4).
T4056 6939-7087 Sentence denotes Ramos cells expressed the type I receptors Act-RIA, weakly BMP-RIB and the type II receptor BMP-RII, but more weakly than normal B cells (Figure 4).
T57 6939-7087 Sentence denotes Ramos cells expressed the type I receptors Act-RIA, weakly BMP-RIB and the type II receptor BMP-RII, but more weakly than normal B cells (Figure 4).
T4057 7088-7165 Sentence denotes HL60 cells were used for comparison and weakly expressed Act-RIA and BMP-RII.
T58 7088-7165 Sentence denotes HL60 cells were used for comparison and weakly expressed Act-RIA and BMP-RII.
T4058 7166-7308 Sentence denotes Taken together, these data show that normal human B cells and Ramos cells express a set of BMP receptors, previously shown to bind BMP-6 [16].
T59 7166-7308 Sentence denotes Taken together, these data show that normal human B cells and Ramos cells express a set of BMP receptors, previously shown to bind BMP-6 [16].
T4599 7310-7352 Sentence denotes BMP-6 induces phosphorylation of Smad1/5/8
T60 7310-7352 Sentence denotes BMP-6 induces phosphorylation of Smad1/5/8
T4600 7353-7449 Sentence denotes Upon ligand binding, the type II receptor transphosphorylates and activates the type I receptor.
T61 7353-7449 Sentence denotes Upon ligand binding, the type II receptor transphosphorylates and activates the type I receptor.
T4601 7450-7499 Sentence denotes Type I receptors can signal via several pathways.
T62 7450-7499 Sentence denotes Type I receptors can signal via several pathways.
T4602 7500-7644 Sentence denotes We examined the effect of BMP-6 on Smad phosphorylation, as the activation of Smad is considered to be a major signalling pathway for BMPs [17].
T63 7500-7644 Sentence denotes We examined the effect of BMP-6 on Smad phosphorylation, as the activation of Smad is considered to be a major signalling pathway for BMPs [17].
T4603 7645-7750 Sentence denotes B cells were cultured in serum-free media over night and then treated with BMP-6 for various time points.
T64 7645-7750 Sentence denotes B cells were cultured in serum-free media over night and then treated with BMP-6 for various time points.
T4604 7751-7886 Sentence denotes Total protein lysates were prepared, and the amounts of the phosphorylated forms of Smad1/5/8 were determined by western blot analysis.
T65 7751-7886 Sentence denotes Total protein lysates were prepared, and the amounts of the phosphorylated forms of Smad1/5/8 were determined by western blot analysis.
T66 7887-7965 Sentence denotes Interestingly, treatment with 500 ng/ml BMP-6 induced phosphorylation of Smad.
T4605 7887-8108 Sentence denotes Interestingly, treatment with 500 ng/ml BMP-6 induced phosphorylation of Smad. The BMP-6 induced phosphorylation was high at the earliest time point tested (15 minutes), and remained high for at least 48 hours (Figure 5).
T67 7966-8108 Sentence denotes The BMP-6 induced phosphorylation was high at the earliest time point tested (15 minutes), and remained high for at least 48 hours (Figure 5).
T4606 8109-8197 Sentence denotes A similar phosphorylation was observed in Ramos cells, but not in HL60 cells (Figure 6).
T68 8109-8197 Sentence denotes A similar phosphorylation was observed in Ramos cells, but not in HL60 cells (Figure 6).
T4607 8198-8331 Sentence denotes Furthermore, we also tested whether other known downstream signalling pathways of BMP-6 could be triggered by BMP-6 in human B cells.
T69 8198-8331 Sentence denotes Furthermore, we also tested whether other known downstream signalling pathways of BMP-6 could be triggered by BMP-6 in human B cells.
T4608 8332-8478 Sentence denotes However, we did not observe any significant changes in the level of phospho-STAT3 or phospho-p38 upon BMP-6 treatment of B cells (data not shown).
T70 8332-8478 Sentence denotes However, we did not observe any significant changes in the level of phospho-STAT3 or phospho-p38 upon BMP-6 treatment of B cells (data not shown).
T5443 8480-8513 Sentence denotes BMP-6 induces upregulation of Id1
T71 8480-8513 Sentence denotes BMP-6 induces upregulation of Id1
T5444 8514-8655 Sentence denotes Next, we wanted to explore whether the BMP-6 induced phosphorylation of Smad 1/5/8 also could induce transcriptional changes of target genes.
T72 8514-8655 Sentence denotes Next, we wanted to explore whether the BMP-6 induced phosphorylation of Smad 1/5/8 also could induce transcriptional changes of target genes.
T5445 8656-8794 Sentence denotes In this regard, the inhibitors of DNA binding proteins (Ids) are considered to be some of the major target genes for Smad-signalling [17].
T73 8656-8794 Sentence denotes In this regard, the inhibitors of DNA binding proteins (Ids) are considered to be some of the major target genes for Smad-signalling [17].
T5446 8795-8971 Sentence denotes Thus, B cells were pre-incubated over night in X-VIVO 15, and then cultured in medium alone or in the presence of BMP-6 for various time points before preparation of total RNA.
T74 8795-8971 Sentence denotes Thus, B cells were pre-incubated over night in X-VIVO 15, and then cultured in medium alone or in the presence of BMP-6 for various time points before preparation of total RNA.
T5447 8972-9034 Sentence denotes The amount of Id1–Id4 mRNA was quantified by real-time RT-PCR.
T75 8972-9034 Sentence denotes The amount of Id1–Id4 mRNA was quantified by real-time RT-PCR.
T5448 9035-9144 Sentence denotes Interestingly, we observed a specific four-fold upregulation of Id1 mRNA in BMP-6-treated B cells (Figure 7).
T76 9035-9144 Sentence denotes Interestingly, we observed a specific four-fold upregulation of Id1 mRNA in BMP-6-treated B cells (Figure 7).
T5449 9145-9326 Sentence denotes The up-regulation of Id1 mRNA was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of BMP-6 and returned to baseline after 24 hours.
T77 9145-9326 Sentence denotes The up-regulation of Id1 mRNA was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of BMP-6 and returned to baseline after 24 hours.
T5450 9327-9470 Sentence denotes In contrast, no significant changes were observed for Id2 and Id3 mRNA, whereas Id4-transcripts were not detectable (Figure 7, data not shown).
T78 9327-9470 Sentence denotes In contrast, no significant changes were observed for Id2 and Id3 mRNA, whereas Id4-transcripts were not detectable (Figure 7, data not shown).
T5451 9471-9611 Sentence denotes Western blot analysis revealed that the BMP-6-induced upregulation of Id1 mRNA also was correlated with upregulation of Id1 protein as well.
T79 9471-9611 Sentence denotes Western blot analysis revealed that the BMP-6-induced upregulation of Id1 mRNA also was correlated with upregulation of Id1 protein as well.
T5452 9612-9811 Sentence denotes The increase in Id1 protein level was detectable after one hour and increased until 24 hours after BMP-6 addition, showing a 16-fold upregulation compared with t0 (p ≤ 0.020, n = 4) (Figure 8 and 9).
T80 9612-9811 Sentence denotes The increase in Id1 protein level was detectable after one hour and increased until 24 hours after BMP-6 addition, showing a 16-fold upregulation compared with t0 (p ≤ 0.020, n = 4) (Figure 8 and 9).
T5453 9812-9934 Sentence denotes In line with the mRNA data, no consistent change in the amounts of Id2 and Id3 protein could be observed (Figure 8 and 9).
T81 9812-9934 Sentence denotes In line with the mRNA data, no consistent change in the amounts of Id2 and Id3 protein could be observed (Figure 8 and 9).
T5454 9935-10020 Sentence denotes We were able to block the Id1 specific band with a blocking peptide (data not shown).
T82 9935-10020 Sentence denotes We were able to block the Id1 specific band with a blocking peptide (data not shown).
T5455 10021-10194 Sentence denotes Taken together, these data suggest that Id1 could be a possible target gene for mediating the effects of BMP-6 in human B cells, whereas Id2 and Id3 not seem to be involved.
T83 10021-10194 Sentence denotes Taken together, these data suggest that Id1 could be a possible target gene for mediating the effects of BMP-6 in human B cells, whereas Id2 and Id3 not seem to be involved.
T6481 10196-10223 Sentence denotes BMP-6 production in B cells
T84 10196-10223 Sentence denotes BMP-6 production in B cells
T6482 10224-10428 Sentence denotes The fact that BMP-6 has been reported to act as an autocrine stimulator in chondrocytes [18] and ovarium [19], prompted us to investigate whether normal human B cells could produce BMP-6 upon stimulation.
T85 10224-10428 Sentence denotes The fact that BMP-6 has been reported to act as an autocrine stimulator in chondrocytes [18] and ovarium [19], prompted us to investigate whether normal human B cells could produce BMP-6 upon stimulation.
T6483 10429-10592 Sentence denotes Ramos cells, which have been described to express BMP-6 mRNA endogenously [20], and the T cell line Jurkat, served as positive and negative controls, respectively.
T86 10429-10592 Sentence denotes Ramos cells, which have been described to express BMP-6 mRNA endogenously [20], and the T cell line Jurkat, served as positive and negative controls, respectively.
T6484 10593-10734 Sentence denotes Endogenous BMP-6 mRNA levels in normal B cells were quantified by real-time RT-PCR after stimulation with anti-IgM for different time points.
T87 10593-10734 Sentence denotes Endogenous BMP-6 mRNA levels in normal B cells were quantified by real-time RT-PCR after stimulation with anti-IgM for different time points.
T6485 10735-10912 Sentence denotes Interestingly, the up-regulation of BMP-6 mRNA was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-IgM.
T88 10735-10912 Sentence denotes Interestingly, the up-regulation of BMP-6 mRNA was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-IgM.
T6486 10913-11002 Sentence denotes The level of BMP-6 mRNA was back to baseline after 24 hours upon stimulation (Figure 10).
T89 10913-11002 Sentence denotes The level of BMP-6 mRNA was back to baseline after 24 hours upon stimulation (Figure 10).
T6487 11003-11103 Sentence denotes Furthermore, both FCS and human AB-serum induced significant upregulation of BMP-6 mRNA (Figure 11).
T90 11003-11103 Sentence denotes Furthermore, both FCS and human AB-serum induced significant upregulation of BMP-6 mRNA (Figure 11).
T6488 11104-11267 Sentence denotes Interestingly, in a separate study we have found that normal human T cells do not express BMP-6 mRNA after activation (Sivertsen et al, manuscript in preparation).
T91 11104-11267 Sentence denotes Interestingly, in a separate study we have found that normal human T cells do not express BMP-6 mRNA after activation (Sivertsen et al, manuscript in preparation).
T6489 11268-11379 Sentence denotes Next, we wanted to detect BMP-6 protein in normal B-cells and tested various commercially available antibodies.
T92 11268-11379 Sentence denotes Next, we wanted to detect BMP-6 protein in normal B-cells and tested various commercially available antibodies.
T6490 11380-11506 Sentence denotes However, in our hands these anti-BMP-6 antibodies did only recognize the recombinant BMP-6 protein and not the native protein.
T93 11380-11506 Sentence denotes However, in our hands these anti-BMP-6 antibodies did only recognize the recombinant BMP-6 protein and not the native protein.
T94 11508-11518 Sentence denotes Discussion
T7807 11519-11762 Sentence denotes Recent studies have demonstrated an important role for BMP superfamily members in hematopoietic stem cells, early thymocytes [6,7] and B-cell malignancies [8,11,12], but a role for BMPs in normal human B cells has previously not been reported.
T95 11519-11762 Sentence denotes Recent studies have demonstrated an important role for BMP superfamily members in hematopoietic stem cells, early thymocytes [6,7] and B-cell malignancies [8,11,12], but a role for BMPs in normal human B cells has previously not been reported.
T7808 11763-11876 Sentence denotes The present study demonstrated a significant antiproliferative effect of BMP-6 in peripheral blood CD19+ B cells.
T96 11763-11876 Sentence denotes The present study demonstrated a significant antiproliferative effect of BMP-6 in peripheral blood CD19+ B cells.
T7809 11877-11991 Sentence denotes Additionally, BMP-6 induced cell death in CD27+ memory B cells as well as in a Burkitt lymphoma cell line (Ramos).
T97 11877-11991 Sentence denotes Additionally, BMP-6 induced cell death in CD27+ memory B cells as well as in a Burkitt lymphoma cell line (Ramos).
T7810 11992-12077 Sentence denotes Importantly, BMP-6 induced a rapid and marked increase in Smad-1/5/8 phosphorylation.
T98 11992-12077 Sentence denotes Importantly, BMP-6 induced a rapid and marked increase in Smad-1/5/8 phosphorylation.
T7811 12078-12210 Sentence denotes Furthermore, the BMP-6 induced Smad phosphorylation was followed by a selective upregulation of Id1 mRNA and subsequent Id1 protein.
T99 12078-12210 Sentence denotes Furthermore, the BMP-6 induced Smad phosphorylation was followed by a selective upregulation of Id1 mRNA and subsequent Id1 protein.
T7812 12211-12347 Sentence denotes In the present study, the demonstrated antiproliferative effect of BMP-6 in anti-IgM treated B cells was significant and dose-dependent.
T100 12211-12347 Sentence denotes In the present study, the demonstrated antiproliferative effect of BMP-6 in anti-IgM treated B cells was significant and dose-dependent.
T101 12348-12474 Sentence denotes Importantly, the anti-proliferative effect of BMP-6 could be completely neutralized by the use of a natural inhibitor, Noggin.
T7813 12348-12567 Sentence denotes Importantly, the anti-proliferative effect of BMP-6 could be completely neutralized by the use of a natural inhibitor, Noggin. This is in line with others, showing that Noggin can function as a BMP-6 antagonist [21,22].
T102 12475-12567 Sentence denotes This is in line with others, showing that Noggin can function as a BMP-6 antagonist [21,22].
T7814 12568-12723 Sentence denotes In addition, the combination of soluble BMP-RIB-Fc and BMP-RII-Fc fusion proteins also neutralized the anti-proliferative effect of BMP-6 in human B cells.
T103 12568-12723 Sentence denotes In addition, the combination of soluble BMP-RIB-Fc and BMP-RII-Fc fusion proteins also neutralized the anti-proliferative effect of BMP-6 in human B cells.
T7815 12724-12830 Sentence denotes Interestingly, as for other TGF family members, bifunctional effects have also been demonstrated for BMPs.
T104 12724-12830 Sentence denotes Interestingly, as for other TGF family members, bifunctional effects have also been demonstrated for BMPs.
T7816 12831-13084 Sentence denotes Whereas several of the BMPs have been shown to promote proliferation in various cell types including condrocytes [23], liver [24] and granulosa cells [25], antiproliferative effects and induction of apoptosis has been reported for B and T lineage cells.
T105 12831-13084 Sentence denotes Whereas several of the BMPs have been shown to promote proliferation in various cell types including condrocytes [23], liver [24] and granulosa cells [25], antiproliferative effects and induction of apoptosis has been reported for B and T lineage cells.
T7817 13085-13237 Sentence denotes Similar effects as demonstrated for BMP-6 on human B cells in the present study, were demonstrated for BMP-2, 4, 6 and -7 in human myeloma cells [9-11].
T106 13085-13237 Sentence denotes Similar effects as demonstrated for BMP-6 on human B cells in the present study, were demonstrated for BMP-2, 4, 6 and -7 in human myeloma cells [9-11].
T7818 13238-13355 Sentence denotes Other members of the BMP-family have also been reported to induce apoptosis, including in mouse B lineage cells [26].
T107 13238-13355 Sentence denotes Other members of the BMP-family have also been reported to induce apoptosis, including in mouse B lineage cells [26].
T7819 13356-13413 Sentence denotes Additionally, BMP-4 inhibits thymocyte proliferation [6].
T108 13356-13413 Sentence denotes Additionally, BMP-4 inhibits thymocyte proliferation [6].
T7820 13414-13550 Sentence denotes Taken together, these data suggest that the role of BMPs in the regulation of proliferation and apoptosis is highly cell type dependent.
T109 13414-13550 Sentence denotes Taken together, these data suggest that the role of BMPs in the regulation of proliferation and apoptosis is highly cell type dependent.
T7821 13551-13675 Sentence denotes To examine how BMP-6 exerts its functional effects in B cells, we analysed BMP receptor expression by western blot analysis.
T110 13551-13675 Sentence denotes To examine how BMP-6 exerts its functional effects in B cells, we analysed BMP receptor expression by western blot analysis.
T7822 13676-13885 Sentence denotes Human peripheral B cells were found to express the BMP type I receptors Act-RIA and BMP-RIB, and the type II-receptors BMP-RII and Act-RIIb, which signal after binding of several BMPs, including BMP-6 [16,13].
T111 13676-13885 Sentence denotes Human peripheral B cells were found to express the BMP type I receptors Act-RIA and BMP-RIB, and the type II-receptors BMP-RII and Act-RIIb, which signal after binding of several BMPs, including BMP-6 [16,13].
T7823 13886-13974 Sentence denotes To further explore BMP-6 induced signalling, activation of several pathways is possible.
T112 13886-13974 Sentence denotes To further explore BMP-6 induced signalling, activation of several pathways is possible.
T7824 13975-14052 Sentence denotes The major signalling pathway known to date, is activation of R-Smads [13,27].
T113 13975-14052 Sentence denotes The major signalling pathway known to date, is activation of R-Smads [13,27].
T7825 14053-14183 Sentence denotes In that respect, BMPs have been shown to exert antiproliferative effects in B lineage cells via phosphorylation of R-Smad [11,28].
T114 14053-14183 Sentence denotes In that respect, BMPs have been shown to exert antiproliferative effects in B lineage cells via phosphorylation of R-Smad [11,28].
T7826 14184-14269 Sentence denotes Furthermore, BMP-2 has been shown to induce activation of STAT3 in myeloma cells [9].
T115 14184-14269 Sentence denotes Furthermore, BMP-2 has been shown to induce activation of STAT3 in myeloma cells [9].
T7827 14270-14340 Sentence denotes However, phosphorylation of R-Smad was not investigated in that study.
T116 14270-14340 Sentence denotes However, phosphorylation of R-Smad was not investigated in that study.
T7828 14341-14405 Sentence denotes BMP-2 has also been shown to induce phosphorylation of p38 [29].
T117 14341-14405 Sentence denotes BMP-2 has also been shown to induce phosphorylation of p38 [29].
T7829 14406-14603 Sentence denotes Thus, phosphorylation of p38, STAT3 and Smad1/5/8 represent important BMP-signalling pathways that mediated the effects of BMPs and even cross-talk between these pathways has been reported [29,30].
T118 14406-14603 Sentence denotes Thus, phosphorylation of p38, STAT3 and Smad1/5/8 represent important BMP-signalling pathways that mediated the effects of BMPs and even cross-talk between these pathways has been reported [29,30].
T7830 14604-14749 Sentence denotes In the present study, we were not able to detect BMP-6-induced changes in the phosphorylation status of STAT3 or p38 in human peripheral B cells.
T119 14604-14749 Sentence denotes In the present study, we were not able to detect BMP-6-induced changes in the phosphorylation status of STAT3 or p38 in human peripheral B cells.
T7831 14750-14820 Sentence denotes Instead, a rapid and marked phosphorylation of Smad1/5/8 was revealed.
T120 14750-14820 Sentence denotes Instead, a rapid and marked phosphorylation of Smad1/5/8 was revealed.
T7832 14821-14953 Sentence denotes In a parallel study, we have found that other BMPs also induced phosphorylation of Smad1/5/8 in peripheral B cells (data not shown).
T121 14821-14953 Sentence denotes In a parallel study, we have found that other BMPs also induced phosphorylation of Smad1/5/8 in peripheral B cells (data not shown).
T7833 14954-15120 Sentence denotes We are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various BMPs in human B cells.
T122 14954-15120 Sentence denotes We are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various BMPs in human B cells.
T7834 15121-15338 Sentence denotes Upregulation of Id1 via Smad1/5/8 phosphorylation is a known mechanism for BMP-6 signalling in other cell systems [31,32] and regulation of Id-proteins is thought to be an important mechanism for Smad-signalling [17].
T123 15121-15338 Sentence denotes Upregulation of Id1 via Smad1/5/8 phosphorylation is a known mechanism for BMP-6 signalling in other cell systems [31,32] and regulation of Id-proteins is thought to be an important mechanism for Smad-signalling [17].
T7835 15339-15514 Sentence denotes In the present study, real-time RT-PCR experiments revealed a specific four-fold upregulation for Id1 in BMP-6-treated B cells, while the amount of Id2–Id4 remained unchanged.
T124 15339-15514 Sentence denotes In the present study, real-time RT-PCR experiments revealed a specific four-fold upregulation for Id1 in BMP-6-treated B cells, while the amount of Id2–Id4 remained unchanged.
T7836 15515-15672 Sentence denotes In agreement with this, western blot analysis demonstrated an upregulation of Id1 protein, while the amount of Id2 and Id3 protein levels remained unchanged.
T125 15515-15672 Sentence denotes In agreement with this, western blot analysis demonstrated an upregulation of Id1 protein, while the amount of Id2 and Id3 protein levels remained unchanged.
T7837 15673-15875 Sentence denotes Previously, Id1 has been considered not to be expressed in later developmental stages than pro-B cells [33,34], and its constitutive expression has been reported to impair mouse B cell development [35].
T126 15673-15875 Sentence denotes Previously, Id1 has been considered not to be expressed in later developmental stages than pro-B cells [33,34], and its constitutive expression has been reported to impair mouse B cell development [35].
T7838 15876-16021 Sentence denotes Therefore, our demonstration of the time-dependent upregulation of Id1 mRNA and protein in mature normal human B cells is of particular interest.
T127 15876-16021 Sentence denotes Therefore, our demonstration of the time-dependent upregulation of Id1 mRNA and protein in mature normal human B cells is of particular interest.
T7839 16022-16180 Sentence denotes In that respect, it is noteworthy that TGF-β signalling in early and mature B cells induces both Id2 and Id3 expression [36,37], but not Id1 (data not shown).
T128 16022-16180 Sentence denotes In that respect, it is noteworthy that TGF-β signalling in early and mature B cells induces both Id2 and Id3 expression [36,37], but not Id1 (data not shown).
T7840 16181-16289 Sentence denotes Interestingly, these results show that various members of the TGF-β family regulate Id proteins differently.
T129 16181-16289 Sentence denotes Interestingly, these results show that various members of the TGF-β family regulate Id proteins differently.
T7841 16290-16379 Sentence denotes Id2 and Id3 are considered to be the Id proteins mainly expressed in mature B cells [38].
T130 16290-16379 Sentence denotes Id2 and Id3 are considered to be the Id proteins mainly expressed in mature B cells [38].
T7842 16380-16496 Sentence denotes The present study also found Id2 and Id3 protein in B cells to be more highly expressed than Id1 in resting B cells.
T131 16380-16496 Sentence denotes The present study also found Id2 and Id3 protein in B cells to be more highly expressed than Id1 in resting B cells.
T7843 16497-16588 Sentence denotes However, BMP-6 did not induce significant changes in the protein expression of Id2 and Id3.
T132 16497-16588 Sentence denotes However, BMP-6 did not induce significant changes in the protein expression of Id2 and Id3.
T7844 16589-16699 Sentence denotes It is believed that Id proteins block differentiation and promote proliferation in various cell types [39,33].
T133 16589-16699 Sentence denotes It is believed that Id proteins block differentiation and promote proliferation in various cell types [39,33].
T7845 16700-16850 Sentence denotes Id proteins act as dominant-negative inhibitors of E-proteins and Pax5 function by forming dimers with these proteins, making them unable to bind DNA.
T134 16700-16850 Sentence denotes Id proteins act as dominant-negative inhibitors of E-proteins and Pax5 function by forming dimers with these proteins, making them unable to bind DNA.
T7846 16851-16983 Sentence denotes It has been proposed that the balance among E-proteins, Pax5 and Id proteins might have an important role in activated B cells [38].
T135 16851-16983 Sentence denotes It has been proposed that the balance among E-proteins, Pax5 and Id proteins might have an important role in activated B cells [38].
T7847 16984-17149 Sentence denotes In that respect, E-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development [40].
T136 16984-17149 Sentence denotes In that respect, E-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development [40].
T7848 17150-17419 Sentence denotes The antiproliferative and death inducing effect of BMP-6 in B cells with concomitant upregulation of Id1 protein is therefore in line with the view that Id proteins are required for the induction of growth arrest and apoptosis in B-lymphocyte progenitors by TGF-β [40].
T137 17150-17419 Sentence denotes The antiproliferative and death inducing effect of BMP-6 in B cells with concomitant upregulation of Id1 protein is therefore in line with the view that Id proteins are required for the induction of growth arrest and apoptosis in B-lymphocyte progenitors by TGF-β [40].
T7849 17420-17556 Sentence denotes Furthermore, Id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis [32].
T138 17420-17556 Sentence denotes Furthermore, Id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis [32].
T7850 17557-17817 Sentence denotes It is well established that various members of the Id family are overexpressed in a range of human tumours and generally, Id1 appears to be the family member most widely overexpressed in a variety of human malignancies [41], including multiple myeloma [42,32].
T139 17557-17817 Sentence denotes It is well established that various members of the Id family are overexpressed in a range of human tumours and generally, Id1 appears to be the family member most widely overexpressed in a variety of human malignancies [41], including multiple myeloma [42,32].
T7851 17818-18000 Sentence denotes Additionally, our findings that BMP-6 activates intracellular signalling pathways in human B cells might be of potential pathophysiological significance in lymphoma and inflammation.
T140 17818-18000 Sentence denotes Additionally, our findings that BMP-6 activates intracellular signalling pathways in human B cells might be of potential pathophysiological significance in lymphoma and inflammation.
T7852 18001-18094 Sentence denotes High BMP-6 mRNA expression in DLBCL has been shown to correlate to unfavourable outcome [12].
T141 18001-18094 Sentence denotes High BMP-6 mRNA expression in DLBCL has been shown to correlate to unfavourable outcome [12].
T7853 18095-18291 Sentence denotes In this respect, it is of interest that targeted expression of Id1 to B-lymphocytes resulted in aberrant B cell development, massive apoptosis, and subsequent development of B cell lymphomas [35].
T142 18095-18291 Sentence denotes In this respect, it is of interest that targeted expression of Id1 to B-lymphocytes resulted in aberrant B cell development, massive apoptosis, and subsequent development of B cell lymphomas [35].
T7854 18292-18465 Sentence denotes Moreover, BMP-6 has been suggested to play a role in rheumatoid arthritis (RA) [43,44] and elevated levels of Id1 and Id3 have been found in the synovia of RA-patients [45].
T143 18292-18465 Sentence denotes Moreover, BMP-6 has been suggested to play a role in rheumatoid arthritis (RA) [43,44] and elevated levels of Id1 and Id3 have been found in the synovia of RA-patients [45].
T7855 18466-18626 Sentence denotes Altogether, these results point to an important role for Id proteins in the regulation of normal B cell homeostasis and in diseases, where B cells are involved.
T144 18466-18626 Sentence denotes Altogether, these results point to an important role for Id proteins in the regulation of normal B cell homeostasis and in diseases, where B cells are involved.
T7856 18627-18780 Sentence denotes It will therefore be important to further elucidate the role of Id-1 in human B cells by selective over expression or inhibition of Id-1 gene expression.
T145 18627-18780 Sentence denotes It will therefore be important to further elucidate the role of Id-1 in human B cells by selective over expression or inhibition of Id-1 gene expression.
T7857 18781-19021 Sentence denotes Given the role of BMP-6 in mature human B cells demonstrated here, identification of BMP-6 producing cells in vivo with possibility of interaction with naïve and memory B cells might contribute to the understanding of mature B cell biology.
T146 18781-19021 Sentence denotes Given the role of BMP-6 in mature human B cells demonstrated here, identification of BMP-6 producing cells in vivo with possibility of interaction with naïve and memory B cells might contribute to the understanding of mature B cell biology.
T7858 19022-19110 Sentence denotes High BMP-6 mRNA expression in DLBCL has been detected by gene expression profiling [12].
T147 19022-19110 Sentence denotes High BMP-6 mRNA expression in DLBCL has been detected by gene expression profiling [12].
T7859 19111-19215 Sentence denotes Furthermore, production of BMP-6 transcripts in normal activated B cells was detected in the same study.
T148 19111-19215 Sentence denotes Furthermore, production of BMP-6 transcripts in normal activated B cells was detected in the same study.
T7860 19216-19323 Sentence denotes Of note, an autocrine BMP-6 loop has been reported by others in chondrocytes and in the ovarium [46,19,18].
T149 19216-19323 Sentence denotes Of note, an autocrine BMP-6 loop has been reported by others in chondrocytes and in the ovarium [46,19,18].
T7861 19324-19417 Sentence denotes Therefore, we wanted to explore the possibility for an autocrine BMP-6 loop in human B cells.
T150 19324-19417 Sentence denotes Therefore, we wanted to explore the possibility for an autocrine BMP-6 loop in human B cells.
T7862 19418-19642 Sentence denotes We analysed the expression BMP-6 mRNA in peripheral blood B cells by real-time PCR, and report here the upregulation of endogenous BMP-6 transcripts after stimulation with FCS, human AB-serum and, most importantly, anti-IgM.
T151 19418-19642 Sentence denotes We analysed the expression BMP-6 mRNA in peripheral blood B cells by real-time PCR, and report here the upregulation of endogenous BMP-6 transcripts after stimulation with FCS, human AB-serum and, most importantly, anti-IgM.
T7863 19643-19865 Sentence denotes However, our attempts to study BMP-6 protein levels were unsuccessful due to problems with unspecific binding of the anti-BMP-6 antibodies tested, and lack of specific staining in control cells known to express BMP-6 mRNA.
T152 19643-19865 Sentence denotes However, our attempts to study BMP-6 protein levels were unsuccessful due to problems with unspecific binding of the anti-BMP-6 antibodies tested, and lack of specific staining in control cells known to express BMP-6 mRNA.
T7864 19866-19924 Sentence denotes In contrast, the recombinant protein was readily detected.
T153 19866-19924 Sentence denotes In contrast, the recombinant protein was readily detected.
T7865 19925-20035 Sentence denotes In that respect, few investigators have detected BMP-6 protein in humans, especially in non-pathogenic tissue.
T154 19925-20035 Sentence denotes In that respect, few investigators have detected BMP-6 protein in humans, especially in non-pathogenic tissue.
T7866 20036-20245 Sentence denotes The possibility of BMP-6 production in human B-cells is in line with a recent work that reported the production of BMP-6 in mouse B cells, infiltrating the bone marrow of mice with inflammatory arthritis [43].
T155 20036-20245 Sentence denotes The possibility of BMP-6 production in human B-cells is in line with a recent work that reported the production of BMP-6 in mouse B cells, infiltrating the bone marrow of mice with inflammatory arthritis [43].
T7867 20246-20333 Sentence denotes In this study, a role for BMPs in the inflammatoric process of arthritis was suggested.
T156 20246-20333 Sentence denotes In this study, a role for BMPs in the inflammatoric process of arthritis was suggested.
T7868 20334-20436 Sentence denotes The upregulation of the BMP-6-transcripts after IgM-crosslinking is of pathophysiologic interest [12].
T157 20334-20436 Sentence denotes The upregulation of the BMP-6-transcripts after IgM-crosslinking is of pathophysiologic interest [12].
T7869 20437-20616 Sentence denotes A loss of TGF-β-responsiveness has been suggested to be a critical contribution to malignant transformation [47,48] and similar oncogenic mechanisms have been postulated for BMPs.
T158 20437-20616 Sentence denotes A loss of TGF-β-responsiveness has been suggested to be a critical contribution to malignant transformation [47,48] and similar oncogenic mechanisms have been postulated for BMPs.
T7870 20617-20773 Sentence denotes Lines of evidence suggest [49] that at early stages of carcinogenesis, BMP-6 is not a tumour promoter, but suppresses benign and malignant tumour outgrowth.
T159 20617-20773 Sentence denotes Lines of evidence suggest [49] that at early stages of carcinogenesis, BMP-6 is not a tumour promoter, but suppresses benign and malignant tumour outgrowth.
T7871 20774-20997 Sentence denotes These findings are in good agreement with previous findings for other TGF-β family members, including TGF-β1 and BMP-4 [50], indicating that cellular context of the BMP target cell might define the various observed effects.
T160 20774-20997 Sentence denotes These findings are in good agreement with previous findings for other TGF-β family members, including TGF-β1 and BMP-4 [50], indicating that cellular context of the BMP target cell might define the various observed effects.
T7872 20998-21298 Sentence denotes In contrast to the upregulation of BMP-6 transcript in B cells, we were not able to detect BMP-6 transcripts in human peripheral blood CD4+ or CD8+ T cells (resting or stimulated with anti-CD3 and anti-CD28; data not shown), consistent with the findings in T cell lines [20] and T cells in mice [43].
T161 20998-21298 Sentence denotes In contrast to the upregulation of BMP-6 transcript in B cells, we were not able to detect BMP-6 transcripts in human peripheral blood CD4+ or CD8+ T cells (resting or stimulated with anti-CD3 and anti-CD28; data not shown), consistent with the findings in T cell lines [20] and T cells in mice [43].
T7873 21299-21449 Sentence denotes Other potential BMP-6 sources for mature B cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system.
T162 21299-21449 Sentence denotes Other potential BMP-6 sources for mature B cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system.
T7874 21450-21546 Sentence denotes One well recognized source for BMP-6 production is the human bone and bone marrow stroma [51,8].
T163 21450-21546 Sentence denotes One well recognized source for BMP-6 production is the human bone and bone marrow stroma [51,8].
T7875 21547-21726 Sentence denotes Furthermore, it is noteworthy that human umbilical vein endothelial cells (HUVEC) highly express BMP-6 mRNA [52], and vascular endothelium has been reported to produce BMP-6 [53].
T164 21547-21726 Sentence denotes Furthermore, it is noteworthy that human umbilical vein endothelial cells (HUVEC) highly express BMP-6 mRNA [52], and vascular endothelium has been reported to produce BMP-6 [53].
T7876 21727-21811 Sentence denotes These studies might imply a role for BMP-6 in transendothelial migration of B cells.
T165 21727-21811 Sentence denotes These studies might imply a role for BMP-6 in transendothelial migration of B cells.
T7877 21812-21901 Sentence denotes BMP-6 mRNA has been demonstrated in murine macrophage cell lines, but not in humans [54].
T166 21812-21901 Sentence denotes BMP-6 mRNA has been demonstrated in murine macrophage cell lines, but not in humans [54].
T7878 21902-22060 Sentence denotes In accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the BMP-6 transcript [20].
T167 21902-22060 Sentence denotes In accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the BMP-6 transcript [20].
T7879 22061-22156 Sentence denotes To our knowledge, there is currently no report about BMP-6 production of human dendritic cells.
T168 22061-22156 Sentence denotes To our knowledge, there is currently no report about BMP-6 production of human dendritic cells.
T169 22158-22168 Sentence denotes Conclusion
T13400 22169-22331 Sentence denotes In conclusion, our results show that BMP-6 induces activation of intracellular Smad signalling in mature human B-cells with consecutive production of Id1 protein.
T170 22169-22331 Sentence denotes In conclusion, our results show that BMP-6 induces activation of intracellular Smad signalling in mature human B-cells with consecutive production of Id1 protein.
T13401 22332-22485 Sentence denotes Furthermore, we report that BMP-6 has an antiproliferative effect in B cells stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L.
T171 22332-22485 Sentence denotes Furthermore, we report that BMP-6 has an antiproliferative effect in B cells stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L.
T13402 22486-22569 Sentence denotes Additionally, BMP-6 induces cell death in activated memory B cells and Ramos cells.
T172 22486-22569 Sentence denotes Additionally, BMP-6 induces cell death in activated memory B cells and Ramos cells.
T13403 22570-22779 Sentence denotes Taken together, these results provide a rationale to further examine the role of BMP-6 signalling in normal B cell biology as well as in pathologic conditions like B cell malignancies and autoimmune disorders.
T173 22570-22779 Sentence denotes Taken together, these results provide a rationale to further examine the role of BMP-6 signalling in normal B cell biology as well as in pathologic conditions like B cell malignancies and autoimmune disorders.
T174 22781-22788 Sentence denotes Methods
T13700 22790-22802 Sentence denotes Cell culture
T175 22790-22802 Sentence denotes Cell culture
T13701 22803-22937 Sentence denotes If not specified, all cells were cultured in X-VIVO 15™ (BioWhittaker, Verviers, Belgium) serum-free medium at 37°C and 5% CO2 in air.
T176 22803-22937 Sentence denotes If not specified, all cells were cultured in X-VIVO 15™ (BioWhittaker, Verviers, Belgium) serum-free medium at 37°C and 5% CO2 in air.
T13702 22938-23101 Sentence denotes Peripheral blood was provided by the Blood Bank at Buskerud Regional Hospital with formal agreement by the patients, and approval by the regional ethics committee.
T177 22938-23101 Sentence denotes Peripheral blood was provided by the Blood Bank at Buskerud Regional Hospital with formal agreement by the patients, and approval by the regional ethics committee.
T13703 23102-23295 Sentence denotes Highly purified resting human B-lymphocytes (CD19+ cells) were isolated from the peripheral blood by rosetting with immunomagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) as described [55].
T178 23102-23295 Sentence denotes Highly purified resting human B-lymphocytes (CD19+ cells) were isolated from the peripheral blood by rosetting with immunomagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) as described [55].
T13704 23296-23426 Sentence denotes This procedure yields less than 0.5% T cells, 0.1% NK cells, and 0.5% monocytes as judged by indirect immunofluorescence staining.
T179 23296-23426 Sentence denotes This procedure yields less than 0.5% T cells, 0.1% NK cells, and 0.5% monocytes as judged by indirect immunofluorescence staining.
T13705 23427-23842 Sentence denotes The following cell lines from human lymphoid malignancies were maintained in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% foetal bovine serum (FCS), 100 units/ml penicillin G, and 100 units/ml of streptomycin sulphate, but serum-starved for at least four hours and cultured in X-VIVO 15™ when included in experiments: EBV-negative BL cell lines Ramos (ECACC 85030802), HL60 (JCRB0085).
T180 23427-23842 Sentence denotes The following cell lines from human lymphoid malignancies were maintained in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% foetal bovine serum (FCS), 100 units/ml penicillin G, and 100 units/ml of streptomycin sulphate, but serum-starved for at least four hours and cultured in X-VIVO 15™ when included in experiments: EBV-negative BL cell lines Ramos (ECACC 85030802), HL60 (JCRB0085).
T14146 23844-23870 Sentence denotes Growth factors/supplements
T181 23844-23870 Sentence denotes Growth factors/supplements
T14147 23871-24385 Sentence denotes The following reagents were used at indicated concentrations: recombinant human (rhu) BMP-6 (1 μg/ml, if not specified otherwise), rhu BMP-RIB/ALK-6/Fc Chimera (5 μg/ml), rhu BMPR-II/Fc Chimera (5 μg/ml), and recombinant mouse Noggin (5 μg/ml) were purchased from R&D Systems (Abingdon, UK); Anti-IgM F(ab)2 fragments of rabbit polyclonal antibodies to human IgM heavy chain (37.5 μg/ml) was obtained from Dako, Copenhagen, Denmark and rhu CD40 ligand (CD40L, 10 ng/ml) was a gift from Immunex Corp. (Seattle, WA).
T182 23871-24385 Sentence denotes The following reagents were used at indicated concentrations: recombinant human (rhu) BMP-6 (1 μg/ml, if not specified otherwise), rhu BMP-RIB/ALK-6/Fc Chimera (5 μg/ml), rhu BMPR-II/Fc Chimera (5 μg/ml), and recombinant mouse Noggin (5 μg/ml) were purchased from R&D Systems (Abingdon, UK); Anti-IgM F(ab)2 fragments of rabbit polyclonal antibodies to human IgM heavy chain (37.5 μg/ml) was obtained from Dako, Copenhagen, Denmark and rhu CD40 ligand (CD40L, 10 ng/ml) was a gift from Immunex Corp. (Seattle, WA).
T14407 24387-24455 Sentence denotes Antibodies used for flow cytometric analysis and immunoblot analysis
T183 24387-24455 Sentence denotes Antibodies used for flow cytometric analysis and immunoblot analysis
T14408 24456-24597 Sentence denotes Antibodies against the human BMP-receptors Act-RIA, BMP-RIB, BMPR-II, Act-RIIA and Act-RIIb were purchased from R & D Systems (Abingdon, UK).
T184 24456-24597 Sentence denotes Antibodies against the human BMP-receptors Act-RIA, BMP-RIB, BMPR-II, Act-RIIA and Act-RIIb were purchased from R & D Systems (Abingdon, UK).
T14409 24598-24910 Sentence denotes Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).
T185 24598-24910 Sentence denotes Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R & D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).
T14410 24911-25055 Sentence denotes Characterisation of BMP-signalling pathways was done by use of anti-phospho-Smad1, -5, -and 8 polyclonal antibody (Chemicon, Temecula, CA, USA).
T186 24911-25055 Sentence denotes Characterisation of BMP-signalling pathways was done by use of anti-phospho-Smad1, -5, -and 8 polyclonal antibody (Chemicon, Temecula, CA, USA).
T14411 25056-25249 Sentence denotes Expression levels of Id1–3 proteins were detected with polyclonal rabbit antibody and detection was blocked with blocking peptide from Santa Cruz Biotechnology (Santa Cruz, San Diego, CA, USA).
T187 25056-25249 Sentence denotes Expression levels of Id1–3 proteins were detected with polyclonal rabbit antibody and detection was blocked with blocking peptide from Santa Cruz Biotechnology (Santa Cruz, San Diego, CA, USA).
T14412 25250-25421 Sentence denotes As secondary antibodies served anti-mouse, anti-goat or anti-rabbit IgG- horseradish peroxidase (HRP) from Dakocytomation AS (Copenhagen, Denmark) for immunoblot analysis.
T188 25250-25421 Sentence denotes As secondary antibodies served anti-mouse, anti-goat or anti-rabbit IgG- horseradish peroxidase (HRP) from Dakocytomation AS (Copenhagen, Denmark) for immunoblot analysis.
T189 25422-25455 Sentence denotes Anti-β-actin was from Santa-Cruz.
T14413 25422-25536 Sentence denotes Anti-β-actin was from Santa-Cruz. From Becton Dickinson (San Jose, CA), we purchased anti-CD19-PE, anti-CD19-FITC.
T190 25456-25536 Sentence denotes From Becton Dickinson (San Jose, CA), we purchased anti-CD19-PE, anti-CD19-FITC.
T14414 25537-25716 Sentence denotes The antibodies used for cell sorting were anti-CD19 PC5 from Immunotech SA (Marseille, France) and anti-CD27 PE from Becton Dickinson, Biosciences Pharmingen (San Diego, CA, USA).
T191 25537-25716 Sentence denotes The antibodies used for cell sorting were anti-CD19 PC5 from Immunotech SA (Marseille, France) and anti-CD27 PE from Becton Dickinson, Biosciences Pharmingen (San Diego, CA, USA).
T15100 25718-25730 Sentence denotes Cell sorting
T192 25718-25730 Sentence denotes Cell sorting
T15101 25731-25951 Sentence denotes Highly purified CD19+CD27- or CD19+CD27+ cells were obtained by staining CD19+ cells with anti-CD27 PE and CD19 PC5 mAbs for 30 minutes at 4°C, followed by washing with PBS and sorting on FACS DiVa from Becton Dickinson.
T193 25731-25951 Sentence denotes Highly purified CD19+CD27- or CD19+CD27+ cells were obtained by staining CD19+ cells with anti-CD27 PE and CD19 PC5 mAbs for 30 minutes at 4°C, followed by washing with PBS and sorting on FACS DiVa from Becton Dickinson.
T15255 25953-25974 Sentence denotes Western-blot analysis
T194 25953-25974 Sentence denotes Western-blot analysis
T15256 25975-26161 Sentence denotes B cells from peripheral blood or cultured cell-lines were lysed in lysis buffer (glycerol 10%, β-mercaptoethanol 5%, 0.0625 M Tris-HCL [pH 6.8], sodium dodecyl sulphate [SDS] 2.5%w/vol).
T195 25975-26161 Sentence denotes B cells from peripheral blood or cultured cell-lines were lysed in lysis buffer (glycerol 10%, β-mercaptoethanol 5%, 0.0625 M Tris-HCL [pH 6.8], sodium dodecyl sulphate [SDS] 2.5%w/vol).
T15257 26162-26355 Sentence denotes Total protein (30–100 μg) from each sample was run on 10% or 12% SDS/polyacrylamide (SDS/PAGE) gels and blotted onto nitrocellulose filters (Protran; Schleicher &Schuell GmbH, Dassel, Germany).
T196 26162-26355 Sentence denotes Total protein (30–100 μg) from each sample was run on 10% or 12% SDS/polyacrylamide (SDS/PAGE) gels and blotted onto nitrocellulose filters (Protran; Schleicher &Schuell GmbH, Dassel, Germany).
T15258 26356-26505 Sentence denotes Blocking, washing and incubation of the filters with primary antibodies were done according to the manufacturer's protocols at room temperature (RT).
T197 26356-26505 Sentence denotes Blocking, washing and incubation of the filters with primary antibodies were done according to the manufacturer's protocols at room temperature (RT).
T15259 26506-26687 Sentence denotes After washing with TBS/0.1% Tween-20 (TBS-T), the filters were incubated with horseradish peroxidase (HRP) coupled to relevant secondary antibodies (see above) for 60 minutes at RT.
T198 26506-26687 Sentence denotes After washing with TBS/0.1% Tween-20 (TBS-T), the filters were incubated with horseradish peroxidase (HRP) coupled to relevant secondary antibodies (see above) for 60 minutes at RT.
T15260 26688-26802 Sentence denotes Enzyme activity was visualised by the enhanced chemiluminescence system, ECL+PLUS (Amersham, Buckinghamshire, UK).
T199 26688-26802 Sentence denotes Enzyme activity was visualised by the enhanced chemiluminescence system, ECL+PLUS (Amersham, Buckinghamshire, UK).
T15261 26803-26929 Sentence denotes Densitometric analysis was performed by scanning hyperfilms on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA).
T200 26803-26929 Sentence denotes Densitometric analysis was performed by scanning hyperfilms on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA).
T15262 26930-27093 Sentence denotes Quantification of Id1, Id2 and Id3 protein was calculated by normalizing the specific protein bands to β-actin using Image Quant 5.5 software (Molecular Dynamics).
T201 26930-27093 Sentence denotes Quantification of Id1, Id2 and Id3 protein was calculated by normalizing the specific protein bands to β-actin using Image Quant 5.5 software (Molecular Dynamics).
T15588 27095-27144 Sentence denotes Analysis of BMP-6 messenger RNA (mRNA) expression
T202 27095-27144 Sentence denotes Analysis of BMP-6 messenger RNA (mRNA) expression
T15589 27145-27249 Sentence denotes Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction.
T203 27145-27249 Sentence denotes Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction.
T15590 27250-27402 Sentence denotes Total RNA was isolated using Absolutely RNA™ RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions.
T204 27250-27402 Sentence denotes Total RNA was isolated using Absolutely RNA™ RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions.
T15591 27403-27504 Sentence denotes Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements.
T205 27403-27504 Sentence denotes Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements.
T15592 27505-27653 Sentence denotes Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan® Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A).
T206 27505-27653 Sentence denotes Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan® Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A).
T15593 27654-27762 Sentence denotes To measure mRNA expression of BMP6, Id1–Id4 and PGK1 PCR were carried out with TaqMan® universal master mix.
T207 27654-27762 Sentence denotes To measure mRNA expression of BMP6, Id1–Id4 and PGK1 PCR were carried out with TaqMan® universal master mix.
T15594 27763-27836 Sentence denotes Primers and probes were provided by Assay-on-Demand (Applied Biosystems).
T208 27763-27836 Sentence denotes Primers and probes were provided by Assay-on-Demand (Applied Biosystems).
T15595 27837-27918 Sentence denotes PCR reactions were carried out in a final volume of 25 μl (BMP-6) or 20 μl (ID1).
T209 27837-27918 Sentence denotes PCR reactions were carried out in a final volume of 25 μl (BMP-6) or 20 μl (ID1).
T15596 27919-28001 Sentence denotes The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA.
T210 27919-28001 Sentence denotes The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA.
T15597 28002-28173 Sentence denotes The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems).
T211 28002-28173 Sentence denotes The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems).
T15598 28174-28241 Sentence denotes The expression was then normalized to the expression level of PGK1.
T212 28174-28241 Sentence denotes The expression was then normalized to the expression level of PGK1.
T15599 28242-28352 Sentence denotes PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56].
T213 28242-28352 Sentence denotes PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56].
T15600 28353-28440 Sentence denotes Expression levels in B cells were then related to the expression levels in Ramos cells.
T214 28353-28440 Sentence denotes Expression levels in B cells were then related to the expression levels in Ramos cells.
T16230 28442-28460 Sentence denotes Cell proliferation
T215 28442-28460 Sentence denotes Cell proliferation
T16231 28461-28617 Sentence denotes For estimation of DNA synthesis, CD19+ cells (7.5 × 104 cells/0.2 ml) or Ramos cells (1 × 104 cells/0.2 ml) were cultured in triplicate in microtiter wells.
T216 28461-28617 Sentence denotes For estimation of DNA synthesis, CD19+ cells (7.5 × 104 cells/0.2 ml) or Ramos cells (1 × 104 cells/0.2 ml) were cultured in triplicate in microtiter wells.
T16232 28618-28742 Sentence denotes The cells were pulsed with 3.7 × 104Bq [3H]thymidine (Amersham, Buckinghamshire, UK) for the last 16 h of a 72-h incubation.
T217 28618-28742 Sentence denotes The cells were pulsed with 3.7 × 104Bq [3H]thymidine (Amersham, Buckinghamshire, UK) for the last 16 h of a 72-h incubation.
T16233 28743-28980 Sentence denotes The cells were harvested using an automated cell harvester (Packard Instrument Company, Meriden, CT, USA) and [3H]thymidine incorporation was determined in a scintillation counter (TopCount, Packard Instrument Company Inc., Meriden, CT).
T218 28743-28980 Sentence denotes The cells were harvested using an automated cell harvester (Packard Instrument Company, Meriden, CT, USA) and [3H]thymidine incorporation was determined in a scintillation counter (TopCount, Packard Instrument Company Inc., Meriden, CT).
T16473 28982-29009 Sentence denotes Determination of cell death
T219 28982-29009 Sentence denotes Determination of cell death
T16474 29010-29180 Sentence denotes Cell death was measured by vital dye exclusion test by staining cells with 5 μg/ml propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml) for one minute on ice.
T220 29010-29180 Sentence denotes Cell death was measured by vital dye exclusion test by staining cells with 5 μg/ml propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml) for one minute on ice.
T16475 29181-29257 Sentence denotes At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer.
T221 29181-29257 Sentence denotes At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer.
T16606 29259-29279 Sentence denotes Statistical analysis
T222 29259-29279 Sentence denotes Statistical analysis
T16607 29280-29471 Sentence denotes The statistical significance of differences between groups was determined using the paired two-tailed Wilcoxon nonparametric test, by applying SPSS10.1 software (SPSS Inc., Chicago, IL, USA).
T223 29280-29471 Sentence denotes The statistical significance of differences between groups was determined using the paired two-tailed Wilcoxon nonparametric test, by applying SPSS10.1 software (SPSS Inc., Chicago, IL, USA).
T16608 29472-29524 Sentence denotes P values less than 0.05 were considered significant.
T224 29472-29524 Sentence denotes P values less than 0.05 were considered significant.
T225 29526-29547 Sentence denotes List of abbreviations
T226 29548-29579 Sentence denotes BMP, Bone morphogenetic protein
T227 29580-29616 Sentence denotes DLBCL, diffuse large B cell lymphoma
T228 29617-29645 Sentence denotes Id, inhibitor of dna binding
T229 29646-29765 Sentence denotes Smad, the name Smad originates from the Drosophila protein, MAD and the Caenorhabditis elegans proteins, sma-2, 3 and 4
T230 29766-29820 Sentence denotes STAT, signal transducer and activator of transcription
T231 29822-29844 Sentence denotes Authors' contributions
T232 29845-29918 Sentence denotes CK designed and conducted experiments, oversaw research, and wrote paper.
T233 29919-29976 Sentence denotes EAS designed and conducted experiments, oversaw research.
T234 29977-30034 Sentence denotes MEH designed and conducted experiments, oversaw research.
T235 30035-30073 Sentence denotes LF designed and conducted experiments.
T236 30074-30134 Sentence denotes EBS designed experiments, oversaw research, and wrote paper.
T237 30135-30209 Sentence denotes JHM designed and conducted experiments, oversaw research, and wrote paper.