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Biomedical subjects

R M Greene

Publications and source records attributed to R M Greene.

At least 19 recordsLinked to original sources
PubMed2002

Expression of the nuclear coactivators CBP and p300 in developing craniofacial tissue.

cAMP regulatory element-binding protein (CREB)-binding protein (CBP) and its functional homolog, the adenovirus E1A -associated 300-kDa protein (p300) are nuclear coactivators and histone acetyltransferases that integrate signals from disparate pathways by bridging specific transcription factors to the basal transcription apparatus. Their role in patterning and development was suggested by studies in mice in which CBP and p300 expression was disrupted and by the human Rubinstein-Taybi syndrome, which is associated with mutations of CBP. The cAMP signal transduction pathway plays a critical role during development of the palate. The linkage between cAMP and expression of specific genes is mediated via activation of trans-acting deoxyribonucleic acid-binding proteins such as the nuclear CREB. For genes regulated by CBP- or p300-containing transcriptional complexes, rates of transcription will depend in part on cellular levels and distribution of CBP/p300. We have thus determined the temporal and spatial expression of CBP and p300 in murine embryonic palatal tissue. Both CBP and p300 proteins and messenger ribonucleic acids are expressed in palatal tissue on each d of palate development (days 12-14 of gestation), as measured by Western blotting and reverse-transcription polymerase chain reaction. Expression of both CBP and p300 was greatest on day 12 of gestation, suggesting that these transcriptional coactivators are developmentally regulated. Immunohistochemical analysis of CBP and p300 expression in the murine embryonic craniofacial region revealed a ubiquitous distribution for both proteins. These studies lay the groundwork for further investigations into the role of CBP and p300 in cellular signaling during craniofacial development.

Animals↗
PubMed2001

Convergence of cAMP, TGF-beta and retinoic acid signaling pathways in cells of the embryonic palate.

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-beta) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-beta1, cyclic adenosine 3', 5'-monophosphate (cAMP) and RA signaling systems. TGF-beta1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-beta mRNA in MEPM cells, though only TGF-beta1 inhibited RA-induced RAR-beta protein expression. Forskolin, but not TGF-beta1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-beta gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-beta-responsive, element. Thus, a putative TGF-beta Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-beta promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.

Animals↗
PubMed2001

Expression of the E2F and retinoblastoma families of proteins during neural differentiation.

Development of the brain is determined by a strictly orchestrated program of proliferation, migration, apoptosis, differentiation, synaptogenesis, tract formation, and myelination. The E2F family of transcription factors, whose activity and functions are regulated in large part through interactions with the retinoblastoma (Rb) family of tumor suppressor proteins, has been implicated as a key regulator of proliferation, differentiation, and apoptosis in a variety of tissues. We have examined levels of the E2F and Rb families of proteins during both brain development and neural differentiation of P19 cells, and found the expression profiles during these two processes of neural development and maturation to be quite similar, i.e., strong up-regulation of p130, pronounced down-regulation of p107, moderate up-regulation of pRb, and significant down-regulation of most species of E2F and dimerization protein (DP). However, several specific isoforms, namely a 30 kDa form of DP-2, a 57 kDa species of E2F-3, a 59 kDa form of E2F-5 and the isoforms of E2F-1 recognized by the E2F-1 (KH-95) antibody were up-regulated suggesting that these particular isoforms of E2F and DP play a tissue-specific function in differentiation and maturation of nervous tissue. The potential role of the E2F/DP family of transcription factors in aspects of neural development and differentiation are considered.

Animals↗
PubMed2000

Taenia solium: molecular cloning and serologic evaluation of 14- and 18-kDa related, diagnostic antigens.

We are attempting to design a simpler assay based on synthetic or recombinant antigens to replace the labor-intensive enzyme-linked immunoelectrotransfer blot (EITB-C), which is currently used to diagnose Taenia solium cysticercosis. From the lentil lectin-bound fraction of cyst glycoproteins (the LLGP fraction used in the EITB-C), we previously identified and purified 2 related polypeptides of 14- and 18-kDa that demonstrated diagnostic usefulness. Using degenerate oligonucleotide primers corresponding to amino acid sequences of these polypeptides and a cDNA library prepared from T. solium cysticerci, we amplified cDNA clones that represent the 14- and 18-kDa polypeptides. These clones share sequence homology at the nucleotide and amino acid levels. Synthetic polypeptides that represented the full-length, mature proteins (sTS14 and sTS18) were assessed for serologic potential using an ELISA. sTS14, but not sTS18, demonstrated utility as a diagnostic antigen. sTS14 was recognized by antibodies in a majority of the sera from patients with cysticercosis and none of the sera from persons with other helminth infections or uninfected human sera. Furthermore, polyclonal antibodies to sTS14 reacted with 6 discrete proteins present in the LLGP cyst fraction, suggesting that TS14 is a subunit of other previously described antigens used for diagnosing cysticercosis.

Amino Acid Sequence↗
PubMed2000

Expression of the E2F family of transcription factors during murine development.

The E2F family of transcription factors plays a crucial role in the control of cell cycle progression and regulation of cellular proliferation, both processes fundamental to mammalian development. In the present study, we have examined the levels of expression of the six currently identified E2F proteins in murine embryos/fetuses as a function of gestational age, compared the expression of these six proteins in selected developing and adult tissues, and examined E2F expression in the embryonic murine palate, a tissue in which perturbation of proliferation is associated with induction of cleft palate. Our results indicate that: 1) multiple forms of individual E2F family members are present in embryonic, fetal and adult cells/tissues; 2) each of the six E2Fs is expressed in a tissue specific manner in both adult and embryonic/fetal organs; 3) certain forms of individual E2F family members are preferentially detected in adult tissues, whereas others are preferentially expressed in embryonic/fetal tissues; 4) expression of the various E2Fs and their isoforms follows distinct temporal patterns during murine gestation; and 5) individual E2F family members also exhibit differential patterns of temporal expression during murine palatogenesis.

Animals↗
PubMed1999

Differential display identification of plunc, a novel gene expressed in embryonic palate, nasal epithelium, and adult lung.

We have identified a novel gene transcript of approximately 1.1 kilobases in length that is expressed in the presumptive nasal epithelium of the mouse embryo. In situ hybridization analysis shows discrete regions of expression associated with the palate, nasal septum, and nasal conchae. This transcript is also expressed strongly in the trachea and bronchi of the adult lung. Screening of a mouse heart cDNA library yielded several overlapping clones to give a continuous sequence of 1113 bases, containing an open reading frame of 278 codons comprising the complete mRNA. No significant homologies with known genes were observed at the nucleotide level; limited amino acid homology with two salivary gland-specific proteins was noted. A search for functionally significant protein motifs revealed consensus sequences for N-glycosylation, protein kinase C and casein kinase phosphorylation, and a leucine zipper. Additionally, we observed a unique amino acid sequence pattern, consisting of the residues Gly-(Leu/Pro/Gln)-(Pro/Leu)-Leu-Pro-Leu, repeated four times near the amino-terminal portion of the protein with two amino acid residues separating the repeats. Based on these observations, we propose that we have identified a new gene, which we call plunc (for palate, lung, and nasal epithelium clone; GenBankTM accession number U69172).

Amino Acid Sequence↗
PubMed1999

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse.

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.

Animals↗
PubMed1998

Selective modulation of MAP kinase in embryonic palate cells.

Murine embryonic palate mesenchyme (MEPM) cells are responsive to a number of endogenous factors found in the local embryonic tissue environment. Recently, it was shown that activation of the cyclic AMP (cAMP) or the transforming growth factor beta (TGFbeta) signal transduction pathways modulates the proliferative response of MEPM cells to epidermal growth factor (EGF). Since the mitogen-activated protein kinase (MAPK) cascade is a signal transduction pathway that mediates cellular responsiveness to EGF, we examined the possibility that several signaling pathways which abrogate EGF-stimulated proliferation do so via the p42/p44 MAPK signaling pathway. We demonstrate that EGF stimulates MAPK phosphorylation and activity in MEPM cells maximally at 5 minutes. Tyrosine phosphorylation and activation of MAPK was unaffected by treatment of MEPM cells with TGFbeta or cholera toxin. Similarly, TGFbeta altered neither EGF-induced MAPK tyrosine phosphorylation nor activity. However, the calcium ionophore, A23187, significantly increased MAPK phosphorylation which was further increased in the presence of EGF, although calcium mobilization reduced EGF-induced proliferation. Despite the increase in phosphorylation, we could not demonstrate induction of MAPK activity by A23187. Like EGF, phorbol ester, under conditions which activate PKC isozymes in MEPM cells, increased MAPK phosphorylation and activity but was also growth inhibitory to MEPM cells. The MEK inhibitor, PD098059, only partially abrogated EGF-induced phosphorylation. Likewise, depletion of PKC isozymes partially abrogated EGF-induced MAPK phosphorylation. Inhibition of both MEK and PKC isozymes resulted in a marked decrease in MAPK activity, confirming that EGF uses multiple pathways to stimulate MAPK activity. These data indicate that the MAPK cascade does not mediate signal transduction of several agents that inhibit growth in MEPM cells, and that there is a dissociation of the proliferative response and MAP kinase activation. Furthermore, other signaling pathways known to play significant roles in differentiation of palatal tissue converge with the MAPK cascade and may use this pathway in the regulation of alternative cellular processes.

Animals↗
PubMed1998

Differential expression and biological activity of retinoic acid-induced TGFbeta isoforms in embryonic palate mesenchymal cells.

The effect of retinoic acid (RA) on TGF-beta mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-beta bioassay in association with TGF-beta isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-beta protein released into the culture medium. RA had little or no effect on TGF-beta1 mRNA expression and protein production. In contrast, RA increased TGF-beta2 and beta3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-beta released was increased relative to the total increase in TGF-beta released, suggesting that RA treatment stimulated activation of latent TGF-beta. RA also increased TGF-beta2 mRNA expression; we have previously shown that RA upregulates TGF-beta3 mRNA in these cells. RA and TGF-beta individually inhibited 3H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit 3H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-beta2 or beta3-specific neutralizing antibodies partially relieved the inhibitory effect on 3H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-beta. Simultaneous treatment with RA and TGF-beta also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-beta treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-beta in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-beta in retinoid-induced cleft palate.

Animals↗
PubMed1998

Non-invasive assessment of bleeding pulmonary artery aneurysms due to Behçet disease.

Because of its ability to depict intravascular, intramural, and extramural pathology, non-invasive imaging is well suited to assessing life-threatening hemoptysis that may complicate Behçet disease. We made exclusive use of CT angiography supplemented by MR to identify pulmonary thromboembolism, mediastinal lymphadenopathy, and bilateral pulmonary artery aneurysms with signs of previous unilateral rupture. Two-dimensional reformatted CT images provided surgeons with a road map of upstream and downstream vascular relationships prior to aneurysm resection. Imaging findings were confirmed by surgery and pathology. Non-invasive imaging proved to be a useful alternative to standard catheter arteriography in the preoperative assessment of hemoptysis in this patient with Behçet disease.

Adult↗
PubMed1998

Cross-talk between signaling pathways in murine embryonic palate cells: effect of TGF beta and cAMP on EGF-induced DNA synthesis.

Signaling pathways utilized by EGF, cAMP, and TGF beta have been demonstrated to play critical roles in normal palate development. Stimulation of these pathways has been shown in palate cells and numerous other systems to affect cell growth. Because proper regulation of cell growth is critical to palate development, we speculate that fine regulation of palatal cell growth may be accomplished through crosstalk between these signaling pathways. We therefore set out to determine the effects of cAMP and TGF beta on EGF-induced cell proliferation in murine embryonic palate cells. We found that both TGF beta and cAMP inhibited the proliferative response of cells to treatment with EGF, whereas H89, a serine/ threonine protein kinase inhibitor with selectivity towards cAMP-dependent protein kinase, increased the cells' proliferative response to EGF. Genestein, a selective inhibitor of tyrosine kinases, at high doses abrogated the cells' proliferative response to EGF, confirming that EGF's ability to induce cell proliferation is critically dependent upon tyrosine kinase activity. Lower doses of genestein, however, actually enhanced cellular response to EGF. The data suggest that both the TGF beta- and cAMP-mediated signaling pathways may be involved in modulation of the effects of EGF on palate cell growth in vivo.

Animals↗
PubMed1997

Prospective validation of a composite end point in thrombolytic trials of acute myocardial infarction (TIMI 4 and 5). Thrombosis In Myocardial Infarction.

Although the use of composite end points in clinical trials has increased in recent years, few data are available on the validity of such an approach. In the Thrombolysis In Myocardial Infarction (TIMI) 4 and 5 trials, we set out to validate prospectively the nonfatal components of the "unsatisfactory outcome" end point. This end point consisted of the in-hospital occurrence or observation of new-onset severe congestive heart failure/shock, left ventricular ejection fraction <40% (or <30% for patients with prior myocardial infarction), reinfarction, reocclusion by sestamibi perfusion imaging, TIMI flow grade <2 at 90 minutes or 18 to 36 hours, intracranial hemorrhage, major spontaneous hemorrhage, or anaphylaxis. Among 576 patients in TIMI 4 and 5 with 1-year follow-up, a nonfatal unsatisfactory outcome end point was reached in hospital in 45% of patients. Compared with patients without such an end point, patients with an end point had a relative risk of 1-year mortality of 2.5 (95% confidence interval 1.4 to 5.6, p = 0.001). For individual components, new-onset severe congestive heart failure/shock had a relative risk of 4.6 (p = 0.001), left ventricular ejection fraction <40% had a relative risk of 3.5 (p = 0.006), recurrent myocardial infarction had a relative risk of 2.2 (p = 0.047), and TIMI flow grade <2 at 90 minutes had a relative risk of 2.2 (p = 0.005). Our findings show that these nonfatal in-hospital end points and the composite end point are associated with an increased risk of 1-year mortality and as such are valid predictive survival markers for use in clinical trials.

Aged↗
PubMed1997

Rescue angioplasty in the thrombolysis in myocardial infarction (TIMI) 4 trial.

Rescue percutaneous transluminal coronary angioplasty (PTCA) has been used to establish reperfusion after failed thrombolysis, and the goal of this study was to examine the angiographic and clinical outcomes after rescue PTCA performed for an occluded artery 90 minutes after thrombolysis. Four hundred two patients with acute myocardial infarction were randomized to receive either anistreplase (APSAC), recombinant tissue plasminogen activator, or their combination in the Thrombolysis in Myocardial Infarction (TIMI) 4 trial. The angiographic and clinical outcomes of patients with a patent artery 90 minutes after thrombolysis were compared with those of patients with an occluded artery treated in a nonrandomized fashion with either rescue or no rescue PTCA. At 90 minutes, the number of frames required to opacify standard landmarks (corrected TIMI frame count) was significantly lower (i.e., flow was faster) after successful rescue PTCA (27 +/- 11) than that in patent arteries after successful thrombolysis (39 +/- 20, p < 0.001), and the incidence of TIMI grade 3 flow was correspondingly higher after successful rescue PTCA (87% vs 65%, p = 0.002). In-hospital adverse outcomes (death, recurrent acute myocardial infarction, severe congestive heart failure, cardiogenic shock or an ejection fraction <40%) occurred in 29% of successful rescue PTCAs and in 83% of failed rescue PTCAs (p = 0.01). Among all patients in whom rescue PTCA was performed (successes and failures combined), 35% of patients experienced an adverse outcome, which was the same as the 35% incidence observed in patients not undergoing rescue PTCA (p = NS) and tended to be higher than the 23% incidence observed in patients with patent arteries (p = 0.07). Although successful rescue PTCA for an occluded artery at 90 minutes results in restoration of flow that is superior to that of successful thrombolysis, the incidence of adverse events for the strategy of rescue PTCA as a whole was the same as that of undertaking no PTCA.

Age Factors↗