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Biomedical subjects

A L Brown

Publications and source records attributed to A L Brown.

At least 55 records · Page 3Linked to original sources
PubMed1993

Three clustered Sp1 sites are required for efficient transcription of the TATA-less promoter of the gene for insulin-like growth factor-binding protein-2 from the rat.

Insulin-like growth factor-binding protein-2 (IGF-BP-2) transcription in rat liver varies with developmental age and fasting. To define the DNA elements required for efficient expression of the TATA-less rat IGFBP-2 gene, the native or mutated promoter was fused to a promoterless luciferase reporter gene and transfected into BRL-3A rat liver and 293 human embryonic kidney cell lines. Luciferase activity decreased approximately 25-fold with progressive 5' promoter deletions from nucleotide (nt) -581 to nt -189 (relative to ATG, +1). The smallest construct, however, still had > 21-fold greater luciferase activity than the promoterless construct. In DNase I foot-printing assays using native nt -276 to -36 promoter fragments or fragments containing block substitution mutations, BRL-3A nuclear extract and purified human transcription factor Sp1 protected a region from nt -234 to -215 containing one GC box and a broad region from nt -189 to -125 that contained three clustered but independent GC boxes. In gel retardation assays using an Sp1 oligonucleotide probe, BRL-3A extract formed two closely migrating complexes that were immunologically related to Sp1; Sp1 gave a single complex that co-migrated with the more retarded BRL-3A complex. Binding was competitively inhibited by oligonucleotides corresponding to each of the four GC boxes. The proximal three GC boxes were sufficient to allow trans-activation of the IGFBP-2 promoter by Sp1 in Drosophila SL2 cells. Independent block mutations indicated that all three of the GC boxes are required for promoter activity and are equally important. Thus, binding of Sp1 or Sp1-related proteins to three clustered GC boxes in the proximal IGFBP-2 promoter is essential for promoter activity. Multiple upstream regions also contribute to the full expression of the IGFBP-2 gene.

Animals↗
PubMed1993

The effect of short-term low-dose cyclosporin on renal function and blood pressure in patients with psoriasis.

The effect of short-term (mean 2.4 months), low-dose (5 mg/kg) cyclosporin A (CyA) on renal function and blood pressure was studied in eight patients with psoriasis. Studies were undertaken before, during and after treatment. Glomerular filtration rate (GFR) post-treatment was significantly higher than pretreatment or during treatment (pre, 119 +/- 7; during, 113 +/- 9; post, 133 +/- 11 ml/min per 1.73 m2; pre vs. during, NS; during vs. post, P < 0.01; pre vs. post, P < 0.05); effective renal plasma flow (ERPF) was unchanged (pre, 515 +/- 38; during, 485 +/- 49; post, 560 +/- 45 ml/min per 1.73 m2). There was no change in the urinary excretion of either albumin or the enzymes N-acetyl-glucosaminidase, lactate dehydrogenase, alanine aminopeptidase and alkaline phosphatase. There was a decrease in exchangeable sodium which persisted post-treatment (pre, 56 +/- 3; during, 49 +/- 3; post, 49 +/- 3 mmol/kg LBM; pre vs. during, P = 0.07; during vs. post, NS; pre vs. post, P = 0.06). Blood pressure assessed as either a single reading, or the mean of a 24 h ambulatory recording, increased during treatment (single reading: pre, 113/73; during, 126/83; post, 114/70 mmHg; mean 24 h: pre, 114/71; during, 123/76; post, 120/72 mmHg). Thus, short-term use of CyA at a dose of 5 mg/kg for the treatment of psoriasis is associated with a significant increase in blood pressure, but only a transient mild reduction in GFR, which did not reach significance.

Adult↗
PubMed1993

Resistance of a human serum-selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120.

We have selected an HXB2 variant which can replicate in the presence of a neutralizing human serum. Sequencing of the gp120 region of the env gene from the variant and parental viruses identified a single amino acid substitution in the third conserved region of gp120 at residue 375 (AGT-->AAT, Ser-->Asn; designated 375 S/N). The escape mutant was found to be resistant to neutralization by soluble CD4 (sCD4) and four monoclonal antibodies (MAbs), 39.13g, 1.5e, G13, and 448, binding to epitopes overlapping that of the CD4 binding site (CD4 b.s.). Introduction of the 375 S/N mutation into HXB2 by site-directed mutagenesis confirmed that this mutation is responsible for the neutralization-resistant phenotype. Both sCD4 and three of the CD4 b.s. MAbs (39.13g, 1.5e, and G13) demonstrated reduced binding to the native 375 S/N mutant gp120. The ability to select for an escape variant resistant to multiple independent CD4 b.s. MAbs by a human serum confirms the reports that antibodies to the discontinuous CD4 b.s. are a major component of the group-specific neutralizing activity in human sera.

Amino Acid Sequence↗
PubMed1993

Bacteraemia due to recurrent reinfection with Staphylococcus epidermidis associated with defective opsonisation and procidin function in serum.

AIMS: To differentiate between reinfection and relapsing infection with Staphylococcus epidermidis in a middle-aged woman with defective opsonisation and procidin function in serum. METHODS: Microbiological typing was done by biotyping, phage typing, and polyacrylamide gel electrophoresis of radiolabelled bacterial proteins (radioPAGE method). Polymorphonuclear cell function was assessed in vitro by phagocytosis and killing of Candida albicans; measurement of neutrophil random locomotion and chemotaxis; reduction of nitroblue tetrazolium after stimulation by opsonised Candida and a radiometric saccharomyces opsonisation assay. The effect of plasma infusions on opsonic activity was assessed by chemiluminescence using control polymorphonuclear leucocytes with a laboratory strain of S epidermidis opsonised with either patient or control serum. RESULTS: Recurrent reinfection with different strains of Staphylococcus epidermidis rather than relapsing infection was confirmed as having occurred by typing bacterial strains. The RadioPAGE method detected all the S epidermidis strains involved in this patient's illness. The patient's serum was shown to be defective in both opsonin and procidin function. The defects were correctable in vitro by the addition of normal serum. Clinical recovery occurred after repeated infusions of normal fresh frozen plasma and prolonged antibacterial treatment; antibacterial treatment alone was insufficient. CONCLUSIONS: The radioPAGE method is useful in distinguishing recurrent reinfection with S epidermidis from relapsing infection with this organism. Elucidation of the nature of, and underlying predisposition to, infection in the patient studied allowed a rational treatment plan of plasma infusion combined with antibacterial treatment to be devised which ultimately proved successful.

Autoradiography↗
PubMed1993

Studies on the acute lethality of diethylcarbamazine in the rat.

In spite of its effectiveness against microfilariae, very little is known about diethylcarbamazine's (DEC) therapeutic mechanism of action or the toxic sequelae which can result from overdose. In preliminary studies, a precipitous decrease in heart rate was noted in rats receiving 1000 mg DEC/kg ip. This effect was less pronounced at 750 mg/kg and was non-existent at 500 mg/kg. In the present study, attempts to attenuate DEC's cardiopulmonary insult by pretreating animals with cyproheptadine failed. Atropine pretreatment failed to block the negative chronotropic effects of DEC, but did restore respiratory function and reduce the lethality associated with the drug. Biochemical studies showed that ATP:ADP ratios in the hearts from rats given high dosages of DEC were elevated over those in controls (11:1 versus 5:1). Inosine levels decreased in cardiac tissues taken from DEC-treated rats. Subsequent enzyme studies revealed that DEC has a potent inhibitory effect on calcium-dependent ATPases from a variety of tissues. Taken together, our data indicate that the mode of acute DEC-lethality involves cardiopulmonary suppression. Furthermore, the cardiac depressant effect of DEC appears related to inhibition of calcium ATPases in cardiac myocytes. To our knowledge, this is the first report of ATPase sensitivity to DEC, a finding that has interesting toxicologic and pharmacologic ramifications.

Adenosine Diphosphate↗
PubMed1992

Transcription of the insulin-like growth factor-binding protein-2 gene is increased in neonatal and fasted adult rat liver.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of proteins that specifically bind IGF-I and IGF-II, determine their bioavailability to tissues, and modulate their actions in target tissues. Levels of IGFBPs in plasma and IGFBP mRNAs in liver are highly regulated with developmental age and metabolic status. We now demonstrate that the increase in IGFBP-2 mRNA in fasted adult rat liver and in the liver of normal neonatal rats reflects an increased rate of transcription. When adult rats were fasted for 2-3 days, IGFBP-2 mRNA was increased in liver, but not in brain or kidney. The increase in hepatic IGFBP-2 mRNA was observed after only 1 day of fasting. Levels decreased by half after 6 h of refeeding and returned to their low starting values after 2 days of refeeding. Transcription-elongation experiments indicated that transcription of the IGFBP-2 gene was increased in fasted liver. The rate of transcription increased 9.2- +/- 3.5-fold for transcripts labeled in exon 1 and 6.6- +/- 2.4-fold for transcripts labeled in exons 2, 3, and 4, suggesting that fasting causes a uniform increase in the number of RNA polymerase II molecules along the length of the IGFBP-2 gene. We infer from these results that the regulation of IGFBP-2 gene transcription in fasting occurs at the level of initiation rather than elongation. IGFBP-2 gene transcription also was increased 3.8- +/- 1.2-fold (exon 1) and 2.9- +/- 0.9-fold (exons 2, 3, and 4) in nuclei from 2-day postnatal rat liver compared with adult rat liver, consistent with the greater abundance of IGFBP-2 mRNA in neonatal rat liver.

Animals↗
PubMed1991

Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins.

The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.

Amino Acid Sequence↗
PubMed1991

Recurrent CAPD peritonitis caused by coagulase-negative staphylococci: re-infection or relapse determined by clinical criteria and typing methods.

Four hundred consecutive episodes of continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis in 105 patients were analysed. Of these episodes 161 (40.25%) were caused by coagulase-negative staphylococci (CNS). Thirty-seven patients developed recurrent attacks (3-10) of peritonitis and CNS accounted for 72 (60%) of these episodes. Classification of reinfection or relapse in 67 of these recurrent episodes of peritonitis was based on clinical criteria alone. This was compared with the results of three typing methods of CNS strains: biotyping plus antibiograms, immunoblotting and 35S-methionine-labelled protein patterns (radio-PAGE). Radio-PAGE was the most discriminatory method followed by biotyping with antibiograms and then immunoblotting. There was total agreement between clinical diagnosis and the three typing methods in 67.2% of episodes but there was total disagreement between the clinical diagnosis and the three typing methods in 11.9%, suggesting inaccurate clinical diagnosis, and in 20.8% typing by at least one method differed from the clinical criteria. Thus, clinical criteria alone are inadequate for the accurate distinction of reinfection from relapse in recurrent CNS peritonitis. This distinction is desirable for optimal management and accurate assessment of different therapies. We suggest that CNS strains from peritoneal dialysate are stored for future typing should the patient develop repeated episodes of peritonitis, to aid in the diagnosis and management of such patients.

Autoradiography↗
PubMed1991

Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted.

The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV). Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals. Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle. Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg. Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure. In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle. Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes.

Amino Acid Sequence↗
PubMed1991

Epidemiology of CAPD-associated peritonitis caused by coagulase-negative staphylococci: comparison of strains isolated from hands, abdominal Tenckhoff catheter exit site and peritoneal fluid.

We identified twenty patients maintained on continuous ambulatory peritoneal dialysis who suffered repeated episodes of peritonitis caused by coagulase-negative staphylococci. We documented hand and exist-site coagulase-negative staphylococcus-associated peritonitis over a total period of 32 months, and compared hand and exit-site strains with strains isolated from dialysate fluid using three typing methods: biotyping using the API Staph kit plus antibiograms, immunoblotting using sera raised in rabbits to three standard strains of coagulase-negative staphylococci, and 35S-methionine-labelled coagulase-negative staphylococcal profiles separated on sodium dodecylsulphate polyacrylamide gel electrophoresis and visualised by autoradiography (radioPAGE). In 5 of 84 episodes, strains isolated from skin were indistinguishable by all three typing methods from the dialysate strain. In a further two episodes, hand or exit-site isolates were indistinguishable by all three typing methods from the dialysate strain isolated in the subsequent, but not the same, episode. Thus in the majority of episodes, no inference of hand or exit-site origin of dialysate infection could be drawn.

Adult↗
PubMed1991

Pitfalls after total parathyroidectomy and parathyroid autotransplantation in chronic renal failure.

We have described 4 patients with chronic renal failure receiving regular haemodialysis treatment who underwent total parathyroidectomy with autotransplantation of parathyroid fragments into the forearm musculature for hypercalcaemic hyperparathyroidism. In all, there was an immediate and profound fall in plasma calcium levels. Hypercalcaemia recurred 1-5 years post-operatively and was resistant to resection of the autograft. In 3 cases, thallium-technetium subtraction scanning and multiple venous sampling for estimation of parathyroid hormone levels suggested multiple sites of hypersecretion of parathyroid hormone in the neck. In 1 case, these investigations revealed a mediastinal adenoma which was successfully removed. These cases reinforce previous suggestions that total parathyroidectomy is frequently incomplete and undermine the procedure of total parathyroidectomy with autotransplantation in patients with persisting uraemia.

Adult↗
PubMed1991

Thyrotropin inhibits while insulin, epidermal growth factor and tetradecanoyl phorbol acetate stimulate insulin-like growth factor binding protein secretion from sheep thyroid cells.

Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP-2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl-lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function.

Animals↗
PubMed1990

Melting chocolate and melting snowmen: analogical reasoning and causal relations.

Children's performance in the classical a:b::c:d analogy task is traditionally very poor prior to the Piagetian stage of formal operations. The interpretation has been that the ability to reason about higher-order relations (the relations between the a:b and c:d parts of the analogy) is late-developing. However, an alternative possibility is that the relations used to date in the analogies are too difficult for younger children. Two experiments presented children aged 3, 4 and 6 years with a:b::c:d analogies which were based on relations of physical causality such as melting and cutting, for example chocolate bar:melted chocolate::snowman:melted snowman. Understanding of these particular causal relations is known to develop between the ages of 3 and 4 years. It was found that even 3-year-olds could solve the classical analogies if they understood the causal relations on which they were based.

Cacao↗