Biomedical subjects
R J White
Publications and source records attributed to R J White.
RNA polymerase III transcription factor IIIB is a target for repression by pocket proteins p107 and p130.
RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.
Activation of RNA polymerase III transcription in cells transformed by simian virus 40.
RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses.
Chest volume and shape and intrapleural pressure in microgravity--reply [letter ; comment].
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Emergency department telephone advice.
OBJECTIVE: To evaluate telephone advice given in an emergency department. DESIGN: Prospective, observational study. SETTING: A community-based emergency department in a semi-rural/outer metropolitan setting, between August and November 1995. PARTICIPANTS: All people telephoning the emergency department for medical advice. METHODS: Details of all calls, callers and patients were recorded. Within 72 hours, a follow-up call was initiated seeking replies to a series of standardised questions. MAIN OUTCOME MEASURES: Number, timing and duration of calls; appropriateness of the advice given; compliance with the advice; and callers' satisfaction with the service. RESULTS: Over the four-month period, 1682 calls were received, 58% between 4pm and midnight. There were 33 telephone calls per 100 emergency department attendances. The mean call duration was 3.9 minutes (range, 0.25-25 minutes); 49% of patients were less than 14 years old, and 72% of callers phoned because of spontaneous illness. The advice given was considered inappropriate in only 1.4% of calls. Follow-up calls were made to 1132 people (67%), revealing a non-compliance rate of only 6.9% and a high level of caller satisfaction, with 99% of callers affirming a need for such a service. CONCLUSIONS: The provision of telephone advice by emergency department staff is rated highly by the community and compliance with the advice is strong. Paediatric problems, arising as a result of spontaneous illness, predominate and there is a large bias towards after-hours use of the service. Experienced staff provide better advice.
Regulation of a TATA-binding protein-associated factor during cellular differentiation.
RNA polymerase III transcription is down-regulated when F9 embryonal carcinoma cells differentiate into parietal endoderm. This reflects a decrease in the activity of TFIIIB, a multisubunit complex that is required for all class III gene expression. Two essential components of TFIIIB are the TATA-binding protein (TBP) and an associated polypeptide called BRF that is specific to this complex. The abundance of both TBP and BRF decreases during F9 cell differentiation. Whereas the amount of TBP assembled into TFIIIB is down-regulated, this is not the case for all TBP-containing complexes. BRF levels show a more dramatic decline that appears sufficient to account for the overall change in transcriptional activity. Developmental regulation of a specific class of genes may therefore be achieved through changes in the availability of a TBP-associated factor.
Human cloning research.
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p53 is a general repressor of RNA polymerase III transcription.
p53 is a major tumour suppressor that is inactivated in a large proportion of human cancers. We show that p53 serves as a general repressor of transcription by RNA polymerase (pol) III. It can inhibit the synthesis of a range of essential small cellular RNAs including tRNA, 5S rRNA and U6 snRNA, as well as viral products such as the adenovirus VAI RNA. Fibroblasts derived from p53 knock-out mice display a substantial increase in pol III transcriptional activity. Endogenous cellular p53 is shown to interact with the TATA-binding protein (TBP)-containing general factor TFIIIB, thereby compromising its function severely. However, assembly of TFIIIB into a pre-initiation complex confers substantial protection against the inhibitory effects of p53. Since TFIIIB is an essential determinant of the biosynthetic capacity of cells, its release from repression by p53 may contribute to a loss of growth control during the development of many tumours.
Transcription by RNA polymerases I and III: a potential link between cell growth, protein synthesis and the retinoblastoma protein.
The rate of protein synthesis is a critical determinant of cellular growth. Abnormal activation of this process is a frequent feature of transformed and tumour cells. Several distinct components of the translation apparatus have been shown to be deregulated in response to malignant transformation. Indeed, overexpression of certain translation factors has been found to predispose cells to transformation or even initiate it. The latest twist to this story comes from the discovery that the retinoblastoma protein RB plays a major role in restricting the production of tRNA and rRNA. RB is an important tumour suppressor. Its ability to limit the synthesis of these principle determinants of biosynthetic capacity could provide a mechanism for restraining cell growth. The loss of this control may constitute a significant step towards tumour progression.
Neurosurgery and the Nobel Prize.
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Weightlessness and the human body.
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Effect of exercise induced hypoxaemia on myocardial repolarisation in severe chronic obstructive pulmonary disease.
BACKGROUND: Exercise training is being promoted increasingly for patients with chronic obstructive pulmonary disease (COPD). Many of these patients experience exercise related arterial desaturation but the clinical importance of these hypoxaemic episodes is not known. QTc dispersion is a marker of myocardial repolarisation abnormalities and there has been much interest in its role as a non-invasive predictor of cardiac arrhythmias and sudden death. However, little is known about the dynamic effects that exercise and hypoxaemia have on QTc dispersion in patients with COPD. METHODS: 20 patients with severe COPD (FEV1 < 40% predicted) undertook two 15 minute treadmill tests at a speed calculated to produce a constant workload of 50% maximum oxygen consumption (VO2max) during which they were blindly given either air or 35% oxygen in random order. Physiological measurements taken throughout exercise included 12 lead electrocardiograms from which QTc dispersion values were calculated according to standard criteria. Nine of the patients who desaturated with exercise were studied further. A similar degree of hypoxaemia was induced at rest by giving them a titrated mixture of air and oxygen and the changes in QTc dispersion were recorded. RESULTS: 11 of the 20 patients developed significant hypoxaemia (desaturation by > or = 5% to < 90%) with exercise breathing air. There were no significant changes in QTc dispersion with either exercise or hypoxaemia. There were no significant changes in QTc dispersion when comparing those who did and did not desaturate, and those with and without a high baseline QTc dispersion values (60 ms). Induced hypoxaemia without exercise also failed to worsen QTc dispersion. CONCLUSIONS: No evidence was found to suggest that exercise, even when associated with hypoxaemia, causes myocardial repolarisation abnormalities in patients with COPD.
Central venous pressure and cardiac function during spaceflight.
Early in spaceflight, an apparently paradoxical condition occurs in which, despite an externally visible headward fluid shift, measured central venous pressure is lower but stroke volume and cardiac output are higher, and heart rate is unchanged from reference measurements made before flight. This paper presents a set of studies in which a simple three-compartment, steady-state model of cardiovascular function is used, providing insight into the contributions made by the major mechanisms that could be responsible for these events. On the basis of these studies, we conclude that, during weightless spaceflight, the chest relaxes with a concomitant shape change that increases the volume of the closed chest cavity. This leads to a decrease in intrapleural pressure, ultimately causing a shift of blood into the vessels of the chest, increasing the transmural filling pressure of the heart, and decreasing the central venous pressure. The increase in the transmural filling pressure of the heart is responsible, through a Starling-type mechanism, for the observed increases in heart size, left ventricular end-diastolic volume, stroke volume, and cardiac output.
Identification of a putative BRF homologue in the genome of Caenorhabditis elegans.
We have identified a putative gene within the Caenorhabditis elegans genome which has the potential to encode a protein homologous to BRF, an RNA polymerase III general transcription factor. The predicted protein shares very similar overall structure with human and yeast BRF. In particular, its N-terminal half comprises a zinc-ribbon motif and a TR domain which is also present in the cyclin box. The C. elegans protein is more similar to human BRF than to the yeast BRF proteins, as would be expected from an evolutionary standpoint. Alignment of the C. elegans protein with the four known BRF proteins reveals two blocks conserved between all five sequences within the diverged C-terminal region. Profile searches using these regions suggest that they may contain evolutionarily conserved motifs. These comparisons provide insight into the structure and function of an important transcription factor.
Transcription factor IIIB: An important determinant of biosynthetic capacity that is targeted by tumour suppressors and transforming proteins.
Transcription factor IIIB (TFIIIB) is an important determinant of the biosynthetic capacity of cells, controlling the production of essential products such as tRNA and 5S rRNA. It is therefore not surprising that this factor is subject to tight regulation, in order to tailor its activity to metabolic demands. For example, TFIIIB can be strongly regulated during differentiation and is also subject to cell cycle control. The unrelated tumour suppressors RB and p53 both exert inhibitory influences upon TFIIIB. In contrast, several viruses have been shown to activate TFIIIB, including HBV and HTLV-1. The fact that cells possess multiple mechanisms for restraining the activity of TFIIIB, whereas oncogenic viruses act to subvert these controls, provides a clear indication of the significance of this factor. Deregulation of TFIIIB may be a significant step towards tumour development.
Data on health economics of pulmonary rehabilitation programmes are needed.
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Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein.
The tumour suppressor protein RB restricts cellular growth. This may involve inhibiting the synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III. We have shown previously that RB can repress pol III transcription when overexpressed either in vitro or in vivo. We also demonstrated that pol III activity is elevated substantially in primary fibroblasts from RB-deficient mice. Here we address the molecular mechanism of this regulation. RB is shown to repress all types of pol III promoter. It can do this even if added after transcription complex assembly. Functional assays demonstrate that RB targets specifically the general pol III factor TFIIIB. A physical interaction between TFIIIB and RB is indicated by fractionation, pull-down and immunoprecipitation data. We show that TFIIIB activity is elevated in primary fibroblasts from RB-deficient mice. TFIIIB is a multisubunit complex that includes the TATA-binding protein (TBP) and a TFIIB-related factor called BRF. We show that RB itself contains regions of homology to both TBP and BRF and propose a model in which RB disrupts TFIIIB by mimicking these two components.
Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones.
1. In cultures of rat forebrain neurones, mitochondria buffer glutamate-induced, NMDA receptor-mediated Ca2+ influx. Here, we have used the fluorescent calcium indicator, indo-1 AM to record [Ca2+]i from single cells. We varied either the glutamate concentration or the duration of exposure to investigate the cellular mechanisms recruited to buffer [Ca2+]i within different stimulation protocols. 2. For a 15 s stimulus, the recovery time doubled as the glutamate concentration was raised from 3 to 300 microM. Changing the duration of exposure from 15 s to 5 min increased the recovery time tenfold even when the glutamate concentration was held at 3 microM. 3. We used a selective inhibitor of the mitochondrial Na(+)-Ca2+ exchange, CGP-37157. When applied immediately after a 15 s, 100 microM glutamate challenge, CGP-37157 consistently caused a rapid fall in [Ca2+]i followed by a slow rise after the drug was washed out. A similar pattern was seen with the 5 min, 3 microM glutamate stimulus. The effects of CGP-37157 are consistent with the release of substantial mitochondrial Ca2+ stores during recovery from an intense glutamate stimulus. 4. These studies suggest that mitochondria become progressively more important for buffering glutamate-induced Ca2+ loads as the stimulus intensity increases. The recovery of [Ca2+]i to baseline following glutamate removal is critically regulated by the release of Ca2+ from mitochondrial stores via mitochondrial Na(+)-Ca2+ exchange. The data highlight a previously under-appreciated role for [Na+]i in the regulation of [Ca2+]i in central neurones.