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Biomedical subjects

D Meek

Publications and source records attributed to D Meek.

17 recordsLinked to original sources
PubMed2001

Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteasome. Stress signals which impinge on p53, leading to its activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.

Amino Acid Sequence↗
PubMed1999

Protein kinase CK2-dependent regulation of p53 function: evidence that the phosphorylation status of the serine 386 (CK2) site of p53 is constitutive and stable.

The p53 tumour suppressor protein is regulated by several mechanisms including multisite phosphorylation. One of the protein kinases which has an established role in regulating p53 function is the protein kinase CK2. The regulation by CK2 occurs both through interaction of p53 with CK2 itself (the regulatory beta subunit) and phosphorylation at the penultimate residue of p53, serine 386 (murine p53). Strikingly, this phosphorylation event controls several independent functions of p53 including site-specific DNA binding, strand renaturation, transcriptional repression and the anti-proliferative function of p53. However, CK2 is a constitutively-active enzyme and therefore the mechanism by which the phosphorylation of p53 at serine 386 is itself regulated, or indeed the question as to whether phosphorylation of this site is regulated at all, remains unresolved. In this paper we provide evidence that serine 386 is highly resistant to dephosphorylation in cultured cells, even though this site can be dephosphorylated in vitro by recombinant protein phosphatase 1. These data suggest that, once phosphorylated at the CK2 site, a p53 molecule remains in this modified form throughout its lifespan. To address the issue of whether the level of serine 386 phosphorylation may be regulated through controlling the subcellular compartmentalisation of p53 and CK2, we examined the subcellular localisation of p53 and CK2alpha in C57MG cells and Rat-1 fibroblasts by immunofluorescence staining. Both proteins were present in the cytoplasm and enriched in the nucleus, with minor variations in the intensity of subcellular location over the course of the cell cycle. Similarly, activation of p53 by UV irradiation or DNA damage-inducing drugs had no effect on either the localisation or levels of CK2alpha, even although significant nuclear p53 accumulation was observed. A striking observation arising from these studies was the intense staining of CK2alpha with the centrosomes, suggesting a potentially important role for this kinase in microtubule formation and/or chromosomal segregation.

Amino Acid Sequence↗
PubMed1998

Construction and characterisation of Salmonella typhimurium aroA simultaneously expressing the five pertussis toxin subunits.

Pertussis toxin (PT) is a major protective antigen of Bordetella pertussis, the causative agent of whooping cough. Current vaccines against whooping cough are administered parenterally, and generate a systemic immune response. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunisation with live vaccine strains of Salmonella spp. We have individually expressed the five PT subunits in Salmonella typhimurium aroA and subsequently expressed a synthetic pertussis toxin operon (pDP16), via the use of a temperature inducible expression system. In S. typhimurium aroA containing individual subunit expression plasmids the S5 subunit was found to be completely processed, while all other subunits were partially processed. In S. typhimurium aroA (pDP16) S5 was completely processed, S1 was partially processed, whilst S2, S3 and S4 remained unprocessed. Induction of the synthetic PT operon decreased the in vitro invasiveness of S. typhimurium aroA, compared to vector-only and plasmid-less strains. Following oral immunisation of Balb/c mice with S. typhimurium aroA (pDP16), statistically significant IgG (p < 0.05) specific to PT was detected in the serum of mice. Nonetheless, despite a statistically significant anti-PT serum antibody response in immunised mice, vaccination with S. typhimurium aroA (pDP16) failed to protect mice from virulent challenge.

Administration, Oral↗
PubMed1996

p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage.

The p53 tumour suppressor protein plays a central role in the cellular defence against agents which cause genetic damage. The activity of p53 is regulated at different levels and is subject to multi-site phosphorylation by a variety of different protein kinases. In this paper we have characterised p53 N-terminus-targeted protein kinase (p53NK) activities, present in a range of cell lines, following fractionation of cellular lysates by ion exchange chromatography on HiTrap Q and Mono Q resins. Three peaks of p53NK activity were observed following fractionation of HeLa cell lysates; these activities were each able to catalyse phosphorylation of up to three residues (serines 4, 6 and 9 in murine p53) within the N-terminus of the p53 protein. Similarly, multiple p53NK activities were detected in the MethAp53(ts) cell line (which expresses the valine 135 temperature-sensitive p53 protein). Strikingly, when these cells were shifted from 38 degrees C (the non-permissive temperature) to 28 degrees C, at which the p53 adopts a wild type conformation, a fivefold stimulation of kinase activity was detected. Moreover, when the DNA damage-inducing drugs etoposide or camptothecin were added to the cells, a further stimulation of kinase activity was observed following growth at 28 degrees C, but not 38 degrees C. These data are consistent with a regulatory model in which p53 is sensitive to stress or DNA damage through phosphorylation at its N-terminus.

Animals↗
PubMed1992

Mitral valve replacement in patients with severe mitral regurgitation and impaired left ventricular function.

We have studied short- and mid-term effects of preservation and excision of the mitral subvalvar support during mitral valve replacement in 40 patients, who had developed moderate to severe degree of left ventricular function impairment, secondary to pure severe mitral regurgitation. These patients had valve replacement, because valve anatomy was unsuitable for reconstructive procedures. Mitral subvalvar support was excised and valve replaced with a Björk-Shiley prosthesis, in 10 patients with moderately impaired left ventricular ejection fraction (mean 32% +/- 1.2%) and in 18 patients with severely impaired left ventricular function (left ventricular ejection fraction: 22% +/- 0.8%). In 12 patients with severely impaired left ventricular function (left ventricular ejection fraction: 20% +/- 1%) posterior subvalvar apparatus was preserved and valve replaced with a bioprosthesis. Prognosis of patients with moderately impaired left ventricular function was not influenced by the loss of chordopapillary support. Actuarial survival at 8 and 10 years was 46% +/- 7.8% for patients with severely impaired left ventricular function with chordopapillary support excised and 70% +/- 10% for patients with severely impaired left ventricular function with chordopapillary support preserved (p less than 0.01). Preservation of mitral subvalvar support is important in patients with severely impaired left ventricular function (left ventricular ejection fraction less than 25%).

Aged↗
PubMed1990

The tymobox, a sequence shared by most tymoviruses: its use in molecular studies of tymoviruses.

The 5'-terminal sequences of the virion protein mRNAs of ononis yellow mosaic and kennedya yellow mosaic tymoviruses were determined, and also the positions in the genomes of the transcription initiation sites of those mRNAs. Comparisons of the available genomic sequences of tymoviruses revealed two conserved regions, one at the initiation site and another longer sequence of sixteen nucleotides to the 5' side of it. The longer sequence, which we call the tymobox, was tested as a target for a designed ribozyme, which cleaved appropriate genomic fragments of three tymoviruses. A synthetic oligonucleotide with sequence complementary to the tymobox was shown to be a tymovirus-specific probe for diagnosing and identifying tymoviruses, except for wild cucumber mosaic tymovirus. The tymobox sequence was also used as a primer for the second strand DNA synthesis of dsDNA representing the virion protein gene of cacao yellow mosaic tymovirus, a tymovirus with unknown sequence. Thus, the tymobox is a useful tool in molecular studies of tymoviruses.

Base Sequence↗
PubMed1988

Growth rate of hepatic metastases in colorectal carcinoma.

The rates of growth of 29 hepatic metastases from 15 patients with primary colorectal carcinoma were studied using serial computed tomography (CT). Eleven metastases were found by the surgeon at laparotomy (overt metastases); the remaining eighteen were not evident to the surgeon at laparotomy, but were detected by CT scan during the immediate postoperative period (occult metastases). An estimate of tumour volume doubling time was obtained from a semi-logarithmic plot of tumour cell number against time. The mean doubling time for the overt metastases was 155 +/- 34 days (+/- s.e.m.) compared with 86 +/- 12 days (P less than 0.05) for the occult metastases. The age of the metastases at the time of surgery was estimated by extrapolation of the observed growth curve assuming Gompertzian kinetics. The mean age of the overt metastases was 3.7 +/- 0.9 years (+/- s.e.m.) The corresponding age of the occult metastases was 2.3 +/- 0.4 years.

Cell Division↗
PubMed1985

The use of endoscopic retrograde choledocho pancreatography (ERCP) in the diagnosis of internal fistulae complicating severe acute pancreatitis.

Three patients are presented in whom internal pancreatic fistulae were accurately demonstrated by ERCP. It is recommended that this procedure be considered in the investigation of patients with continuing acute pancreatitis who are not settling on standard conservative treatment and in whom internal fistula formation is suspected. The procedure should also be considered in patients who present with unusual respiratory symptoms or with amylase-rich effusions.

Acute Disease↗
PubMed1984

Juvenile progressive dystonia: a new phenotype of GM2 gangliosidosis.

A 10-year-old boy developed progressive dystonia and dementia. His symptoms had begun at age 2 1/2 years, and he had been unable to walk by 8 years. At age 10 he was severely dystonic, unable to use his hands to feed himself, and almost anarthric . He had dysphagia and urinary incontinence, and functioned at a 4-year-old level of mental development. The mean percentages of beta-hexosaminidase A measured in serum, leukocytes, and fibroblasts by the heat denaturation method, each on three separate assays, were 5.9, 9.8, and 13.0%, respectively. These values are higher than in Tay-Sachs disease but are similar to levels seen in late-onset or adult cases of GM2 gangliosidosis. This patient appears to represent a new phenotype of juvenile GM2 gangliosidosis having dystonia as the dominant symptom.

Child↗
PubMed1981

Pre-operative localization of aldosterone-secreting adrenal adenomas.

Techniques for pro-operative localization of aldosterone-secreting adrenal adenomas were studied in thirty-seven patients, each with hypertension and biochemical evidence of primary hyperaldosteronism and each later having adrenal surgery (thirty-two adenomas, five bilateral hyperplasia). Bilateral adrenal vein catheterization was attempted in all cases; it was successful on the left side in all patients and in 92% of cases on the right. Adrenal vein plasma samples were obtained from the left side in 92% and from the right in 73% of cases. Adrenal vein plasma aldosterone measurements correctly indicated the presence of tumour in twenty-eight cases but falsely predicted unilateral adenoma in two cases of bilateral adrenal hyperplasia. Adrenal venography also correctly predicted unilateral adrenal adenomas in twenty-six cases but falsely suggested the presence of tumour in three cases of bilateral adrenal hyperplasia. Computed tomography (CT) was used in the last eight cases. In seven instances the predictions (six adenomas, one bilateral adrenal hyperplasia) were confirmed at surgery. However, the remaining patient harboured an adenoma 20 mm in diameter which was not detected by CT although diagnosed both by adrenal venography and adrenal vein aldosterone measurements. Ultrasound detected adenoma in only three of twenty-two cases examined. Although further comparative studies of the type described here are required, the results of computed tomography are promising and suggest that this non-invasive technique might well become the first choice procedure in localizing aldosterone-secreting adenomas.

Adenoma↗
PubMedDate not supplied

BCG in bladder cancer.

BCG is usually thought of as a vaccine for tuberculosis. This paper describes how it can be used as an alternative to cytotoxic agents in the treatment of bladder cancer.

Administration, Intravesical↗