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Biomedical subjects

G G Brownlee

Publications and source records attributed to G G Brownlee.

At least 19 recordsLinked to original sources
PubMed1992

Recovery from hemophilia B Leyden: an androgen-responsive element in the factor IX promoter.

One form of the inherited, X-linked, bleeding disorder, hemophilia B, resolves after puberty. Mutations at -20 and -26 in the clotting factor IX promoter impair transcription by disrupting the binding site for the liver-enriched transcription factor LF-A1/HNF4. The -26 but not the -20 mutation also disrupts an androgen-responsive element, which overlaps the LF-A1/HNF4 site. This explains the improvement seen in patients with the -20 mutation and the failure of the -26 patient to recover.

Base Sequence↗
PubMed1992

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.

Amino Acid Sequence↗
PubMed1992

A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo.

The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus.

Base Sequence↗
PubMed1992

Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus.

The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates. Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side. Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers. Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA. The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase. Both enzymes failed to show primer-independent synthesis of vRNA. The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed.

Base Sequence↗
PubMed1992

Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.

Amino Acid Sequence↗
PubMed1992

Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro.

The 12 nucleotide conserved sequence at the 3' end of influenza A virion RNA is sufficient to function as a promoter in vitro. By introducing point mutations in all 12 positions of this promoter in model RNA templates and studying the efficiency of RNA synthesis in vitro, we show that only three nucleotides, residues 9, 10 and 11, are crucial for activity, although other nucleotides play a significant but less important role. Additions or deletions within the promoter are tolerated, resulting in either an increase or a decrease in promoter activity, depending on the mutation introduced; in some cases premature termination is caused. Taking these observations into account, a model for RNA polymerase binding and copying of the promoter is discussed.

Base Composition↗
PubMed1992

Subcutaneous injection of factor IX for the treatment of haemophilia B.

Haemophilia B patients are normally treated, either prophylactically or in response to bleeding episodes, by frequent intravenous injections of factor IX purified from blood donors. Here we show in model animal experiments that purified human factor IX, when injected subcutaneously, is rapidly (in 3-11 h) and reasonably efficiently (30-40% of an equivalent intravenous dose) transported at least partly by the lymphatic drainage of the skin into the bloodstream, mostly in a biologically active form. This suggests that patients could be treated prophylactically by subcutaneous rather than intravenous injection, where the short delay in raising plasma factor IX to haemostatic levels would be clinically acceptable. More generally, our studies emphasize that the subcutaneous route of injection should be useful for other therapeutic proteins, including other clotting factors, which have to be delivered to the bloodstream, as long as their half-life is at least a few hours allowing time for transport into the general circulation.

Animals↗
PubMed1991

Key residues involved in calcium-binding motifs in EGF-like domains.

Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a beta-hydroxylated residue). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gln 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations in this domain, Asp 47----Glu, and Asp 64----Asn, present in patients with haemophilia B, reduce calcium binding to the domain greater than 4-fold and greater than 1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gln 50----Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs.

Amino Acid Sequence↗
PubMed1991

Protein engineering of the propeptide of human factor IX.

Vitamin-K-dependent plasma proteins contain a highly conserved propeptide sequence located between the classical hydrophobic leader sequence and the N-terminus of the mature protein. This acts as a recognition sequence for the vitamin-K-dependent carboxylase which catalyses the conversion of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in the adjacent Gla domain. Protein engineering of the 18 residue propeptide from human factor IX has highlighted the importance of residues -16Phe and -10Ala with respect to carboxylase recognition. In addition, studies of haemophilia B patients have shown that C-terminal propeptide residues -4Arg and -1Arg are required for proteolysis of the propeptide from the mature protein. To extend these previous studies we have introduced two novel mutations into the propeptide of human factor IX at positions -17(Val----Asp) and -6(Leu----AsP), and studied the effect of these changes on gamma-carboxylation and proteolytic processing. Both mutations reduce the expression of a calcium-dependent epitope in the Gla domain; however, only -6Leu----Asp shows reduced binding to barium sulphate. In addition, this latter mutation prevents proteolytic processing of the propeptide. These data support the current hypothesis that the propeptide contains two recognition elements: one for carboxylase recognition located towards the N-terminus, and one for propeptidase recognition located near the C-terminus.

Amino Acid Sequence↗
PubMed1991

Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin.

Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy-terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes.

Amino Acid Sequence↗
PubMed1991

Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes.

Retroviral vectors were used to transduce recombinant DNA encoding firefly luciferase, Escherichia coli beta-galactosidase or human factor IX into fetal rat hepatocytes in primary culture. Hepatocytes were transduced optimally during a restricted time interval, 2-4 days post-plating. Although efficient and stable expression of reporter gene products was observed in vitro, it was affected differentially by culture conditions (plating density, media constituents) and chemical modulators of hepatocyte growth and differentiation (gelatin, hydrocortisone, isobutylmethylxanthine). Cultured cells, mock-infected or infected with a luciferase-expressing vector, were harvested non-enzymatically and injected subcutaneously into the dorsal neck fascia of neonatal syngeneic rats. Tissue isolated from injection sites one week later contained hepatocyte foci. In animals transplanted with infected cells, the preliminary results suggest that luciferase activity was present at these sites in proportion to the numbers of injected cells. These findings and previous observations made with hepatocytes from neonatal and adult primary cultures, indicate that from day 19 in utero through maturity the transient temporal 'period of susceptibility' to infection in vitro is independent of the developmental state of starting tissue. Transplantability of cultured fetal hepatocytes infected with retroviral vectors and stably expressing reporter gene products suggests that such cells might provide promising models for liver gene therapy.

Animals↗
PubMed1990

Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B.

Haemophilia B (or Christmas disease) is an inherited, X-linked bleeding disorder caused by mutations in the gene for clotting factor IX. There is a rare class of patients, exemplified by haemophilia B Leyden, who suffer from haemophilia B as children but improve after puberty. In these patients, plasma factor IX concentrations are less than 10% of normal during childhood, but after puberty they gradually rise to between 40 and 80% of normal. Mutations clustered around the main transcription start point (defined as +1 (ref.2)) have been reported in seven of these patients (at -20 (refs 1, 3, 4); -6 (refs 5, 6) and +13 (refs 7, 8)). To determine how these mutations interfere with factor IX expression, we have assayed for transcription factors binding to this area and have identified a nuclear factor-1 liver (NF1-L) binding site (-99 to -76) and a binding site for the CCAAT/enhancer binding protein (C/EBP) (+1 to +18). We show that the A----G mutation at +13 prevents the binding of C/EBP to this site. Furthermore, we show that C/EBP is capable of transactivating a cotransfected normal factor IX promoter but not the mutant promoter. This is the first natural mutation to be reported which disrupts a C/EBP binding site and is an illustration of the importance of this transcription factor in humans.

Age Factors↗
PubMed1990

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

It has been suggested that epidermal growth factor-like (EGF-like) domains, containing conserved carboxylate residues, are responsible for the high-affinity calcium binding exhibited by a number of vitamin K-dependent plasma proteins involved in the control of the blood coagulation cascade. These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S. To test this hypothesis we have expressed the first EGF-like domain from human factor IX (residues 46-84) using a yeast secretion system, and examined calcium binding to the domain. Using 1H-NMR to measure a calcium-dependent shift assigned to Tyr69 we have detected a high-affinity calcium binding site (Kd = 200-300 microM). We suggest that other EGF-like domains of this type may have similar calcium binding properties. In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.

Amino Acid Sequence↗
PubMed1989

RNA-binding properties of influenza A virus matrix protein M1.

The matrix protein (M1) of influenza A virus, which has a critical role in viral assembly and can inhibit the viral transcriptase complex, has the ability to bind RNA. The RNA-binding property of M1 is specific for single-stranded RNA, like that of influenza nucleoprotein (NP) and shows similar sensitivity to pH and to salt concentration. M1:RNA complexes are stable, once formed, to competition from excess single-stranded RNA. The possible location of the RNA-binding regions in the M1 protein is discussed.

Animals↗
PubMed1989

A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes.

A recombinant vaccinia has been designed to express amino acids 366-379 of influenza nucleoprotein, previously shown to be the minimal epitope recognized by a class I-restricted cytotoxic T cell clone. Target cells infected with the recombinant vaccinia virus expressing this peptide are recognized by CTL as efficiently as target cells expressing the complete nucleoprotein. The results imply the existence of a peptide transport system that constitutively passes the products of degraded proteins from the cytoplasm into a membrane-bound compartment of the cell.

Animals↗