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R M Gill

Publications and source records attributed to R M Gill.

30 records · Page 2Linked to original sources
PubMed1993

Molecular cloning and characterization of the mouse RB1 promoter.

We report the isolation and characterization of the mouse RB1 promoter and surrounding DNA sequences, and the identification of elements required for basal transcriptional activity. The mouse RB1 promoter, like the human homologue, has a high G + C content, constitutes a CpG island and is devoid of typical TATA and CAAT boxes. The first 235 base-pairs upstream of the translation initiation codon in the mouse promoter exhibit 80% sequence homology with the human sequence. This homology includes a region which contains putative binding sites for the transcription factors Sp1, ATF and E2F/DRTF1. Four major transcription initiation sites were identified downstream of this conserved region. Mutational analysis revealed that the Sp1 and ATF binding sites, but not the putative E2F/DRTF1 binding site, are critical for promoter activity. Complete disruption of the putative Sp1 and ATF sites abrogated transcription, whereas the introduction of point mutations, previously identified in the Sp1 and ATF sites in two low penetrance retinoblastoma families, reduced promoter activity in a cell type specific manner. Less reduction in activity occurred in retinoic acid induced differentiated P19 cells and NIH3T3 mouse fibroblasts than in undifferentiated embryonal carcinoma P19 cells. Activity of the RB1 promoter was found to be stimulated in retinoic-acid induced differentiated P19 cells compared to undifferentiated P19; this stimulation required intact Sp1 and ATF sites but not the putative E2F/DRTF1 binding site. Our results indicate that basal level of RB1 expression is governed by Sp1 and ATF.

Animals↗
PubMed1993

Regulated expression of the retinoblastoma gene in differentiating embryonal carcinoma cells.

The expression of the retinoblastoma susceptibility (RB) gene was investigated in P19 embryonal carcinoma cells and in these cells induced to differentiate with retinoic acid (RA) or with dimethyl sulfoxide (DMSO). In undifferentiated cells very low levels of RB mRNA and protein were present. DMSO-treated P19 cell cultures develop into mesodermal and endodermal cells and RB expression increased only slightly in these differentiating cells. RA-treated P19 cells develop into neuroectoderm and this differentiation was accompanied by a marked increase in RB expression with mRNA levels increasing 15 fold by 4-6 days following initiation of RA treatment. No such increase occurred in mutant cells that fail to respond to RA. The RB promoter did not appear to be directly activated by RA. Nevertheless, the increase in RB expression in RA-treated cells appeared to be due to enhanced initiation of transcription because cells transfected with a reporter gene driven by the RB promoter expressed the reporter gene with kinetics similar to that of the RB gene. Thus the activation of the RB gene appears to be achieved indirectly by RA-induced factor(s) in differentiating neuroectodermal cells. The post-mitotic neurons that developed in RA-treated cultures contained only the hypophosphorylated form of the RB protein. Recent studies (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992) have shown that mice lacking the RB gene have abnormalities in early brain development suggesting that the rapid rise in RB expression and the hypophosphorylation of the protein are essential for neuronal cell differentiation.

Animals↗
PubMed1992

Transcriptional repression of the E2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1 gene.

The protein product of the retinoblastoma susceptibility gene, p110RB1, is a nuclear phosphoprotein [W.H. Lee, J.Y. Shew, F.D. Hong, T.W. Sery, L.A. Donoso, L.J. Young, R. Bookstein, and E.Y. Lee, Nature (London) 329:642-645, 1987] with properties of a cell cycle regulator (K. Buchkovich, L.A. Duffy, and E. Harlow, Cell 58:1097-1105, 1989; P.L. Chen, P. Scully, J.Y. Shew, J.Y. Wang, and W.H. Lee, Cell 58:1193-1198, 1989; J.A. DeCaprio, J.W. Ludlow, D. Lynch, Y. Furukawa, J. Griffin, H. Piwnica-Worms, C.M. Huang, and D.M. Livingston, Cell 58:1085-1095, 1989; and K. Mihara, X.R. Cao, A. Yen, S. Chandler, B. Driscoll, A.L. Murphree, A. TAng, and Y.K. Fung, Science 246:1300-1303, 1989). Although the mechanism of action of p110RB1 remains unknown, several lines of evidence suggest that it plays a role in the regulation of transcription. We now show that overexpression of p110RB1 causes repression of the adenovirus early promoter EIIaE and the promoters of two cellular genes, c-myc and RB1, both of which contain E2F-binding motifs. Mutation of the E2 element in the c-myc promoter abolishes p110RB1 repression. We also demonstrate that a p110RB1 mutant, which is refractory to cell cycle phosphorylation but intact in E1a/large T antigen-binding properties, represses EIIaE with 50- to 80-fold greater efficiency than wild-type p110RB1. These data provide evidence that hypophosphorylated p110RB1 actively represses expression of genes with promoters containing the E2F-binding motif (E2 element).

Adenoviridae↗
PubMed1992

The retinoblastoma protein and the cell cycle.

Although mutations of the retinoblastoma gene (RB1) contribute to malignant progression in many types of tumor, the role of RB1 mutation in cancer initiation is highly restricted to the rare embryonic tumor, retinoblastoma. However, RB1 is expressed and its product, p110RB1, is regulated through the cell cycle in many tissues. This apparent paradox may be clarified by the emerging evidence that p110RB1 functions as a regulator of transcription of cell cycle genes. Tissue specific effects may be determined by the cellular proteins that interact with p110RB1, or by cell type-specific expression of target genes regulated by p110RB1.

Cell Cycle↗
PubMed1992

Regions controlling hyperphosphorylation and conformation of the retinoblastoma gene product are independent of domains required for transcriptional repression.

Cell cycle-dependent phosphorylation appears to control the function of the retinoblastoma gene product, p110RB1. We have previously shown that both the N-terminal portion and the portion corresponding to exon 23 are important in the hyperphosphorylation of p110RB1. To gain further insight into the domains involved in these phosphorylations, we have created a series of mutations in the murine cDNA and investigated their effects on phosphorylation, protein conformation and transcription of genes controlled by an E2 element. Three series of p110RB1 constructs were made: (1) mutations and/or deletions in the cluster of potential p34cdc2 consensus sequences of exon 23, (2) deletions for the entire region C-terminal to the large T antigen (TAg)-binding domains or (3) mutations in eight potential p34cdc2 sites, including four in exon 23. Following transfections into COS cells, phosphorylation, TAg binding and protein conformation were evaluated. Constructs were also tested for their ability to repress transcription from the adenovirus early promoter. All mutant proteins bound to SV40 TAg. Mutation of Ser-781, Ser-787 and Ser-788 had no detectable effect on the phosphorylation pattern of p110RB1, producing both the fast-migrating, hypophosphorylated and slower migrating, hyperphosphorylated bands on SDS-PAGE gels. However, mutation or deletion of Ser-800 and Ser-804 resulted in proteins which failed to show the characteristic shift in molecular weight associated with the hyperphosphorylated form of p110RB1. A protein mutated only at Pro-805, the residue adjacent to Ser-804, behaved similarly to mutants with both Ser-800 and Ser-804 altered; mutation of Pro-801 had no effect on the mobility of the hyperphosphorylated species. While differences were seen in the ability of these proteins to be hyperphosphorylated, the wild-type function of repression of adenovirus early promoter activity was retained. These results are consistent with the large shift in apparent molecular weight of p110RB1 upon hyperphosphorylation being due to phosphorylation of Ser-804. Phosphorylation of this p34cdc2 consensus sequence and the subsequent conformational change in p110RB1 detected in denaturing gels is not necessary, however, for the phosphorylation-dependent modulation of p110RB1-TAg interaction.

Amino Acid Sequence↗
PubMed1991

Characterization of refractory period extension by transcardiac shock.

BACKGROUND: To better understand the refractory period extension (RPE) produced by transcardiac shocks and its possible role in defibrillation, we measured RPE under various experimental conditions. METHODS AND RESULTS: Using ventricular pacing in pentobarbital-anesthetized dogs, we characterized RPE in relation to the anatomic site of pacing, the local voltage gradient (LVG) produced by the shocks at the pacing site, and the pacing rate and pacing current used to make the measurements. We also determined if RPE persisted into the next refractory period after the shock and measured RPE at the end of 30-second episodes of acute ischemia to the pacing site, which were caused by occluding the left anterior descending artery. Each anatomic site tested showed RPE, which increased sharply with increasing LVG at lower levels but less sharply at higher LVG. The RPE versus LVG was approximated with an exponential curve that had an exponential constant of about 5-6 V/cm. At faster pacing rates, RPE occurred earlier in the refractory period but was unchanged when expressed as a percent increase of refractory period. RPE did not vary with the pacing current and was present only in the refractory period during which the shock was delivered. The RPE was not significantly altered by acute ischemia. These results show that transcardiac shocks selectively extend the refractory period of tissue proportional to the LVG and the timing of the shock in the refractory period. They are consistent with the concept that RPE prevents depolarization from tissue directly excited by a shock from propagating to tissue that was refractory to that same shock. CONCLUSIONS: The insensitivity of RPE to short ischemic episodes and the presence of RPE at increased activation rates suggest that RPE might exist under conditions of fibrillation and be a major determinant of the success or failure of defibrillation.

Acute Disease↗
PubMed1990

Ventricular refractory period extension caused by defibrillation shocks.

In pentobarbital-anesthetized dogs, transcardiac shocks of up to 30 J or pacing stimuli were delivered to myocardial tissue at different times in the electrical cycle. When delivered midway or later into electrical systole, shocks, but not pacing stimuli, greatly extended the refractory period as determined by left ventricular pacing. There was a positive correlation between both the shock energy and timing and the amount of delay. A 30-J shock given 10 msec before the end of the refractory period extended the refractory period by 63 +/- 15 msec (p less than 0.001), whereas the same shock given 40 msec earlier produced only 25 +/- 10 msec (p less than 0.001) of extension. By comparison, a 5-J shock given at those times produced 36 +/- 18 (p less than 0.005) and 10 +/- 8 msec (p less than 0.001) of extension, respectively. When delivered early into electrical systole, both a pacing stimulus and a shock had no substantial effect on the tissue refractory period. Because the tissue that is late in electrical systole would otherwise be the first to repolarize if no shock were given, the selective refractory period extension may create a period after the shock during which no tissue is repolarized to a level sufficient for wavefront propagation. Thus, the energy- and time-dependent refractory period extension may help explain the mechanism by which ventricular defibrillation occurs during transcardiac shocks.

Animals↗
PubMed1988

Stress fracture in parachute regiment recruits.

A survey of stress fractures occurring in recruits at the Depot of The Parachute Regiment was carried out over a one year period from July 1985 to August 1986. A total of 44 fractures in 36 soldiers were diagnosed. The incidence was thus 4.84%, much lower than that found in a recent prospective Israeli study. Only one case of progression to complete fracture was observed during the study. In contrast to the previous experience with British recruits, the commonest site of stress fracture was the tibial midshaft.

Aviation↗
PubMed1986

Effects of sympathetic tone on vagally induced phasic changes in heart rate and atrioventricular node conduction in the anesthetized dog.

We examined the effects of stellate ganglia stimulation on the phase-dependent chronotropic and dromotropic responses to brief vagal bursts in open-chest anesthetized dogs. Stellate stimulation affected the phasic vagal effects on heart rate by shortening the latent period, shifting the phase at which maximum decrease in heart rate occurred to earlier phases, and reducing the maximum decrease in heart rate. These effects were due primarily to an increase in the basic heart rate. No significant sympathetic-parasympathetic interaction occurred for heart rate, indicating that accentuated antagonism did not occur with brief vagal bursts. Stellate stimulation primarily decreased the amplitude of the phasic vagal effects on atrioventricular nodal conduction, regardless of the underlying heart rate, and a significant sympathetic-parasympathetic interaction was associated with this effect. The peak of the phase-dependent vagal effects on heart rate and atrioventricular nodal conduction were phase-shifted with one another. From these findings, we postulate the small changes in sympathetic tone might shift the predominant phase-dependent vagal effect from one on heart rate to one on atrioventricular nodal conduction. Furthermore, our results suggest that dynamic vagal control of heart rate and atrioventricular node conduction involves both phase-dependent and phase-independent factors. Sympathetic activity appears to affect only the phase-independent factor(s) in the control of heart rate, whereas it affects both phase-dependent and phase-independent factors in the control of atrioventricular node conduction.

Animals↗
PubMed1985

Inhibition of premature ventricular extrastimuli by subthreshold conditioning stimuli.

The purpose of this study was to determine whether trains of subthreshold high frequency conditioning stimuli (333 Hz, 1 ms duration, 2 ms interval) delivered to the canine ventricle inhibited the response to a premature stimulus (S2) more effectively than did a single subthreshold conditioning stimulus. It was found that trains of conditioning stimuli (mean 1.21 mA) inhibited the response to S2 152 ms beyond expiration of the ventricular effective refractory period, whereas a single conditioning stimulus inhibited S2 only 20 ms or less beyond the ventricular effective refractory period. In late diastole, trains of conditioning stimuli failed to inhibit S2 when the train of stimuli caused ventricular depolarization or the latter occurred in response to the next sinus impulse. Trains of conditioning stimuli did not induce ventricular arrhythmias. Lidocaine or autonomic blockade did not alter the response to trains of conditioning stimuli. Trains of conditioning stimuli or a single conditioning stimulus inhibited the response to S2 only when they were delivered at the same electrode site. By lengthening the ventricular effective refractory period, trains of conditioning stimuli could prevent or terminate tachycardias, but this possibility is constrained, at present, by the spatial limitations of the technique.

Animals↗
PubMed1985

Interruption of sympathetic and vagal-mediated afferent responses by transmural myocardial infarction.

We have demonstrated previously that sympathetic and vagal afferents travel in an apical-to-basal course in the heart, and can be stimulated selectively with epicardial applications of bradykinin and nicotine, respectively. In this study we tested the hypothesis that transmural myocardial infarction interrupts sympathetic and vagal afferent fibers traveling through the infarction and produces regions of afferent denervation in areas apical to the infarction. In open-chest, chloralose-anesthetized dogs, transmural myocardial infarction was created by embolizing a diagonal branch of the left anterior descending coronary artery with a vinyl latex solution that was injected directly into the artery and hardened rapidly. The transmural nature of the infarction was verified by the nitro blue tetrazolium staining technique for dehydrogenase enzymes. Epicardial applications of bradykinin (5 micrograms) and nicotine (50 micrograms) were used to stimulate chemically sensitive sympathetic and vagal afferent nerve endings, respectively. Twenty-nine dogs were studied before and 90 min after creation of transmural myocardial infarction. In 20 dogs, epicardial bradykinin applied before production of transmural myocardial infarction produced a maximal pressor response of 13 +/- 3 mm Hg 40 sec after application (p less than .01 vs preapplication values), while topical nicotine produced a maximal depressor response of 14 +/- 2 mm Hg (p less than .01 vs preapplication values) 20 sec after application at all sites tested. Ninety minutes after production of transmural myocardial infarction, epicardial sites basal to the infarction continued to respond normally to both drugs, while sites within the area of infarction and apical to the area (noninfarcted myocardium) no longer showed a pressor response to topical bradykinin or a depressor response to topical nicotine.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗