Biomedical subjects

J A Lees

Publications and source records attributed to J A Lees.

13 recordsLinked to original sources

PubMed1992

A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F.

The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein.

Amino Acid Sequence↗

PubMed1992

Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors.

The oestrogen receptor stimulates transcription by means of at least two distinct transcriptional activation domains, TAF-1 in the N-terminal domain and TAF-2 in the hormone binding domain. Here we show that TAF-2 activity requires a region in the C-terminus of the hormone binding domain between residues 538 and 552 in the mouse oestrogen receptor which is conserved among many nuclear hormone receptors. Point mutagenesis of conserved hydrophobic and charged residues significantly reduced ligand dependent transcriptional activation but had no effect on steroid or DNA binding. Mutation of the corresponding residues in the glucocorticoid receptor also abolished transcriptional activation. We therefore propose that the conserved region may be essential for ligand dependent transcriptional activation by other members of the nuclear receptor family.

3T3 Cells↗

PubMed1992

The retinoblastoma protein physically associates with the human cdc2 kinase.

The protein product (pRB) of the retinoblastoma susceptibility gene functions as a negative regulator of cell proliferation, and its activity appears to be modulated by phosphorylation. Using a new panel of anti-human pRB monoclonal antibodies, we have investigated the biochemical properties of this protein. These antibodies have allowed us to detect a pRB-associated kinase that has been identified as the cell cycle-regulating kinase p34cdc2 or a closely related enzyme. Since this associated kinase phosphorylates pRB at most of the sites used in vivo, these results suggest that this kinase is one of the major regulators of pRB. The associated kinase activity follows the pattern of phosphorylation seen for pRB in vivo. The associated kinase activity is not seen in the G1 phase but appears in the S phase, and the levels continue to increase throughout the remainder of the cell cycle.

Antibodies, Monoclonal↗

PubMed1991

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.

Amino Acid Sequence↗

PubMed1990

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Amino Acid Sequence↗

PubMed1990

The effect of patterns of rumen fermentation on the response by dairy cows to dietary protein concentration.

Four groups of seven dairy cows were given hay plus high-fibre concentrates based on sugar-beet feed (hay-concentrate, 40:60 w/w) or high-starch concentrates based on flaked maize (hay-concentrate, 20:80 w/w), with a crude protein (nitrogen x 6.25) content of either 160 or 220 g/kg dry matter, over weeks 4-18 of lactation. Performance during week 3 of lactation, when all cows were fed on a standard ration, was used as a covariate. For diets with a high-fibre content, higher protein concentrations led to increases in yields of milk and milk fat, with no effect on live-weight loss. For diets with a high-starch content, higher protein concentrations did not affect milk yield or composition but resulted in an increase in live weight rather than a decrease. Diets with a high-starch content led to increased proportions of propionic acid in the rumen and increased concentrations of insulin in the blood. It is concluded that the source of carbohydrate needs to be taken into account when predicting the response to protein supply by dairy cows.

Animals↗

PubMed1990

A 22-amino-acid peptide restores DNA-binding activity to dimerization-defective mutants of the estrogen receptor.

We have identified residues within the estrogen receptor that are required for dimerization and high-affinity DNA binding. A 22-amino-acid peptide encompassing these residues was sufficient to restore DNA-binding activity to a mutant receptor lacking most of the hormone-binding domain. Point mutagenesis of the fusion protein confirmed that this sequence continued to mediate dimerization in a manner similar to that within the native receptor, although its position relative to the DNA-binding domain was appreciably altered.

Amino Acid Sequence↗

PubMed1989

Identification of two transactivation domains in the mouse oestrogen receptor.

We have identified two discrete transactivation domains within the mouse oestrogen receptor whose relative activities vary according to the target promoter. One domain lies within the N-terminal region and is active in the absence of oestradiol. The second domain is contained within the C-terminal portion of the protein and depends upon oestrogen binding for its activity. The location and oestrogen dependence of this domain has been confirmed using chimaeric receptors containing the Lex A DNA binding domain. Although transactivation by the C-terminal domain is dependent upon ligand binding the analysis of receptor deletion mutants has demonstrated that these two functions are not entirely coincident.

Animals↗

PubMed1989

Identification of constitutive and steroid-dependent transactivation domains in the mouse oestrogen receptor.

We have identified two transactivation domains in the mouse oestrogen receptor whose activities depend on the target promoter. The major domain is contained within the C-terminal portion of the protein and depends upon oestrogen binding for its activity. The location and oestrogen dependence of this domain has been confirmed using chimaeric receptors containing the Lex A DNA binding domain. Although transactivation by the C-terminal domain is dependent upon ligand binding the analysis of receptor deletion mutants has demonstrated that these two functions are not entirely coincident. The second transactivation domain lies within the N-terminal region and is active in the absence of oestradiol. The differences in oestrogen requirement for the activity of the two transactivation domains may account for the partial agonist activity of certain antihormones.

Animals↗

PubMed1989

A proposed consensus steroid-binding sequence--a reply.

A consensus sequence has been proposed as necessary for steroid binding on the basis of sequence homology in steroidogenic enzymes, steroid binding proteins and steroid receptors. We have mapped the limit of sequences important for steroid binding in the mouse estrogen receptor using Scatchard analysis of deletion mutants. Since a mutant lacking the entire proposed consensus is still able to bind estradiol with essentially wild type affinity, the functional significance of this motif is unclear.

Amino Acid Sequence↗

PubMed1987

Structural organization and expression of the mouse estrogen receptor.

Complementary DNA clones corresponding to the mouse uterus estrogen receptor mRNA have been isolated and characterized. Nucleotide sequence analysis predicts that full-length cDNA has the potential to code for a polypeptide of 599 amino acids, and comparison with the protein sequences of the rat, human, and chicken estrogen receptors reveals overall homologies of 97%, 88% and 77%, respectively. Genomic clones for the mouse estrogen receptor have been isolated from a cosmid library and used in conjunction with the cDNA clones to study the expression of the receptor in vivo by RNase mapping, primer extension, and Northern blotting. These analyses demonstrate that transcription initiates at multiple sites which span a region of at least 62 base pairs and that the estrogen receptor is encoded by mRNA of approximately 6.5 kilobases in size. There are 10 major starts in total, one of which is situated 31 nucleotides downstream from a TATA box-like motif and coincides with the start of the cDNA clone pMOR8. The ability of the cDNA clone to produce a functional protein was verified by transfection into COS-1 cells which lack endogenous estrogen receptor. The mouse estrogen receptor, in a SV40-based expression vector, was cotransfected with a chimeric marker plasmid consisting of an estrogen response element from the vitellogenin A2 gene linked to the thymidine kinase promoter and the chloramphenicol acetyl transferase gene. In the presence of estradiol chloramphenicol acetyl transferase activity is stimulated by up to 80-fold, while tamoxifen and 4-hydroxytamoxifen act primarily as antiestrogens in this in vitro assay.

Amino Acid Sequence↗

PubMed1986

Hypoglycemia in hospitalized patients. Causes and outcomes.

We analyzed 137 episodes of hypoglycemia (serum glucose less than or equal to 49 mg per deciliter) occurring in 94 adult patients hospitalized during a six-month period at a tertiary care hospital. Forty-five percent of the patients had diabetes mellitus, and administered insulin was implicated in 90 percent of episodes in diabetics. Hypoglycemia in diabetic patients occurred under a variety of circumstances, frequently because of decreased caloric intake related to illness or hospital routine. Insulin-induced hypoglycemia also occurred during treatment of hyperkalemia (eight patients) or during hyperglycemia related to total parenteral nutrition (six patients). Forty-six of the 94 patients had chronic renal insufficiency, and 20 of these 46 had underlying diabetes mellitus. Thus, renal insufficiency unrelated to diabetes mellitus was the second most frequent diagnosis associated with hypoglycemia. The majority of other cases of hypoglycemia were related to liver disease, infections, shock, pregnancy, neoplasia, or burns. Hypoglycemia was not the apparent cause of death in any patient, but the overall hospital mortality was 27 percent and was related to the degree of hypoglycemia and the number of risk factors for hypoglycemia. We conclude that hypoglycemia is a common problem in hospitalized patients, is common in renal insufficiency, is usually iatrogenic, and correlates with high mortality in severely ill patients.

Adult↗

PubMed1985

Pyocystis, pyonephrosis and perinephric abscess in end stage renal disease.

We report 8 patients with end stage renal disease on maintenance hemodialysis who had pyocystis, pyonephrosis or a perinephric abscess. Delay in diagnosis was frequent because the urine-deprived bladder and kidneys were dismissed as potential sites of infection. Treatment of pyocystis with bladder irrigations often was successful and obviated cystectomy. Pyonephrosis or perinephric abscess was a more serious problem and required unilateral nephrectomy.

Abscess↗