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Biomedical subjects

A L Brown

Publications and source records attributed to A L Brown.

At least 19 recordsLinked to original sources
PubMed1995

Immunogenic HIV variant peptides that bind to HLA-B8 can fail to stimulate cytotoxic T lymphocyte responses.

Cytotoxic T lymphocyte responses in HIV infection can be impaired through variation in the epitope regions of viral proteins such as a gag. We report here an analysis of variant epitope peptides in three gag epitopes presented by HLA B8. Fifteen variant peptides were examined for their binding to HLA-B8; all but one bound at concentrations comparable to known epitopes. All except two of those that bound could be recognized by CTL from an HLA-B8 positive HIV-1-infected patient and were therefore immunogenic. However, in a hemophiliac patient studied in detail, there was a failure to respond to two immunogenic peptide epitopes representing virus present as provirus in the patient's peripheral blood. In one case, the patient's CTL had previously responded to the peptide; in the other case, there was a good response to a peptide of closely related sequence. Thus there was a selective failure of the CTL response to some proviral epitopes. This impaired reaction to new variants could contribute to the loss of immune control of the infection.

Animals↗
PubMed1995

Use of reverse ligation-PCR to identify transcriptional start sites in GC-rich TATA-less genes: application to the rat IGFBP-2 gene.

TATA-less genes are often GC-rich in the region of transcriptional initiation and the corresponding mRNAs are prone to the formation of secondary structure. These properties have made it difficult to determine unambigously the start sites of transcription for some of these genes by conventional assays such as primer extension and nuclease protection. Using the TATA-less rat IGFBP-2 gene, we demonstrate that tobacco acid pyrophosphatase-reverse ligation polymerase chain reaction (TAP-RLPCR), a novel and sensitive assay, can be used to map the start sites of these genes. First, the validity of TAP-RLPCR was demonstrated by mapping the transcription start site of the rat insulin-like growth factor 1 (IGFBP-1) gene to the correct position (nucleotides -173 relative to ATG, +1). Using total RNA obtained from the rat liver cell line BRL-3A, the transcription start sites of the rat IGFBP-2 gene were mapped to a narrow cluster extending from nucleotides -86 to -90 (ATG, +1), 39 bp downstream of three adjacent GC boxes that are essential to the transcriptional activity of the gene. The assay was also used to map the start sites of a luciferase reporter gene driven by the fragment -1,295 to -32 of the rat IGFBP-2 promoter after transfection in the human embryonic kidney cell line 293. The hybrid gene utilized the same transcription start sites as the rat IGFBP-2 gene, indicating that the elements required for positioning of the transcription initiation complex are contained within the 3' end terminating at nucleotide -32.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Widening of the tracheal bifurcation on chest radiographs: value as a sign of left atrial enlargement.

OBJECTIVE: This study evaluated widening of the tracheal bifurcation (subcarinal and interbronchial) angle on posteroanterior chest radiographs as a sign of left atrial enlargement. The purpose was to determine the diagnostic sensitivity and specificity of the sign. MATERIALS AND METHODS: The chest radiographs and echocardiograms of 122 clinically stable patients were retrospectively reviewed. The mean interval between examinations was 10 days (range, 0-30 days). The left atrial dimension was measured on the two-dimensional echocardiograms. Sixty-five patients had a normal left atrial dimension (mean, 34 mm), and 45 patients had an enlarged atrium (mean, 47 mm) on echocardiography. Subcarinal and interbronchial angles were jointly measured by two observers who were unaware of the echocardiographic findings. Correlation analysis was used to determine which angle measurement best predicted the left atrial size. Discriminant analysis was used to derive a threshold angle that predicted left atrial enlargement. RESULTS: The carina was inadequately seen on 14 radiographs. Left atrial size correlated poorly with both the interbronchial (r = .33) and the subcarinal (r = .25) angel values. An interbronchial angle of 76.4 degrees and a subcarinal angle of 65.4 degrees were the best discriminators between patients with normal and those with enlarged left atrial dimensions (sensitivities, 61% and 51%, respectively; specificities, 63% and 66%). CONCLUSION: Our findings show that widening of the tracheal bifurcation angle on chest radiographs is an insensitive and nonspecific sign of left atrial enlargement. This sign is of little value in diagnosing left atrial enlargement.

Adult↗
PubMed1994

The effect of inhibition of glutathione S-transferase P on the growth of the Jurkat human T cell line.

To investigate the possible role of glutathione S-transferase P (GSTP) in carcinogenesis and cell proliferation, ethacrynic acid (EA) was used to inhibit GSTP in the human Jurkat T cell line. At lower doses (0-30 microM), EA led to a decreased rate of proliferation as assessed by the MTT assay. This was associated with a decreased DNA S+G2/M phase population and also a dose-dependent increase in apoptosis. At concentrations of EA > 30 microM, cells suffered non-specific cytotoxic injury and underwent necrosis. The total cell number fell over the time course of the experiment. A resistant subculture of cells which proliferated in the presence of EA at 30 microM was selected by continuous growth in the presence of EA. Although this had a higher basal rate of apoptosis than control cells, it also showed a significantly larger growth fraction as assessed by flow cytometry. GSTP is frequently overexpressed in human tumours and animal models of carcinogenesis, and is regarded as a marker of the 'drug-resistant phenotype' of initiated cells. Our findings suggest that the role of GSTP in models of chemical carcinogenesis and in tumours may be its permissive effect on cell cycle activity and downregulation of apoptosis, thus allowing expansion of a population of initiated cells.

Apoptosis↗
PubMed1994

Percutaneous aspiration thromboembolectomy: a preliminary experience.

Percutaneous aspiration thromboembolectomy (PAT) is a radiological alternative to surgical embolectomy or thrombolysis in the treatment of acute arterial thromboembolic disease. We report our experience in eight patients aged 63-83 years (mean 71 years). Indications were graft thrombosis (1) or emboli from atrial fibrillation (3), abdominal aneurysm (2) or proximal angioplasty (2). PAT was performed at 10 arterial sites; common iliac (1), profunda femoris (1), superficial femoral (2), femoro-popliteal graft (1), popliteal (2) and arteries of the trifurcation (3). PAT was used as an adjunct to thrombolysis or angioplasty in five patients and as the sole procedure in three patients. It was successful in six patients (seven sites) with mean ABI rising from 0.4 pre- to 0.8 post-procedure. Two of the failures required amputations. One of these was a completely thrombosed dacron femoro-popliteal graft with poor run-off, and the second case had a failed surgical embolectomy prior to amputation. There were no major complications, and no mortality on follow-up at 1 month. PAT is a useful adjunct to thrombolysis and balloon angioplasty in the radiological treatment of acute thromboembolic disease. In patients in whom thrombolysis is contraindicated, it offers an alternative to surgical embolectomy.

Aged↗
PubMed1994

Recombinant human insulin-like growth factor (IGF)-binding protein-6 inhibits IGF-II-induced differentiation of L6A1 myoblasts.

Insulin-like growth factor-binding protein-6 (IGFBP-6) is an O-linked glycoprotein that binds insulin-like growth factor-II (IGF-II) with marked preferential affinity over IGF-I. Recombinant human IGFBP-6 (rhIGFBP-6) was synthesized by COS-7 monkey kidney cells that were transiently transfected with a eukaryotic expression vector into which a complementary DNA for IGFBP-6 modified for optimal translation had been inserted. rhIGFBP-6 was similar to IGFBP-6 purified from human cerebrospinal fluid with respect to IGF binding and O-glycosylation. The effect of rhIGFBP-6 on IGF-induced L6A1 myoblast differentiation was studied using creatine kinase activity as an index of differentiation. rhIGFBP-6 inhibited differentiation initiated by IGF-II in a dose-dependent manner, inhibition was complete when rhIGFBP-6 was present in a slight molar excess. In contrast, rhIGFBP-6 had no effect on IGF-I-induced differentiation, even when coincubated in a 5-fold molar excess. These results are consistent with the preferential affinity of IGFBP-6 for IGF-II. As cell association and proteolysis have been associated with the potentiation, rather than the inhibition, of IGF action by IGFBPs, we investigated whether they occurred in the L6A1 myoblast system. After incubation of L6A1 myoblasts with rhIGFBP-6, IGFBP-6 was recovered from the medium, but not from cell lysates or extracellular matrix. In addition, [125I]IGFBP-6 did not bind to myoblast monolayers, and there was no evidence that proteolysis had occurred. Together, these results indicate that rhIGFBP-6 remains intact and soluble and, hence, inhibits IGF-II-induced differentiation. The fidelity of the IGFBP-6 expression system used for these studies will enable us to use this system to determine how structural modifications of the protein affect the modulation of IGF action by IGFBP-6.

Animals↗
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

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↗
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↗