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Biomedical subjects

A McLachlan

Publications and source records attributed to A McLachlan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Regional drug delivery II: relationship between drug targeting index and pharmacokinetic parameters for three non-steroidal anti-inflammatory drugs using the rat air pouch model of inflammation.

PURPOSE: To quantify the advantage gained by direct administration to a target site for two non-steroidal anti-inflammatory drugs (NSAIDs) piroxicam and diclofenac in the rat air pouch model of inflammation. To derive a model relating drug targeting index (DTI) to the pharmacokinetic parameters of the target and systemic sites, and to compare predictions with observations. METHODS: DTI was calculated based on area under the concentration time curve at target (pouch) and systemic site (venous blood) following administration into and sampling from both sites. A model was derived relating DTI to systemic clearance, target permeability, plasma protein binding and fraction of the targeted dose that is systemically available. RESULTS: Both NSAIDs exhibited linear pharmacokinetics over the dose ranges studies. They differed primarily in total body clearance which was approximately 16 fold greater for diclofenac (213 ml hr-1 per 250 g) than piroxicam (13 ml hr-1 per 250 g). Observed DTIs (11, 114 and 276 for piroxicam, S[+]ibuprofen [studied previously] and diclofenac) were ranked in order of total body clearance but were approximately 7.5 fold lower than predicted (101, 700 and 2214 respectively). CONCLUSIONS: The discrepancy was explained by the influx of the plasma binding protein, albumin, into the target site due to increased vascular permeability associated with the inflammatory response. The originally derived equation for DTI, which assumed only unbound drug diffuses across the target site, was modified to take into account the simultaneous flux of bound drug.

Animals↗
PubMed1995

Regulation of transcription from the hepatitis B virus large surface antigen promoter by hepatocyte nuclear factor 3.

The influence of hepatocyte nuclear factor 3 (HNF3) on the level of transcriptional activity from the four hepatitis B virus promoters was investigated by transient-transfection analysis in the dedifferentiated hepatoma cell line, HepG2.1. It was found that the large surface antigen promoter and, to a much lesser extent, the nucleocapsid promoter were transactivated in the presence of HNF3. DNase I footprinting analysis demonstrated that purified recombinant HNF3 alpha protects one region of the large surface antigen promoter. Gel retardation analysis showed that a double-stranded oligonucleotide containing this HNF3-binding site formed a specific complex with DNA-binding proteins in the differentiated hepatoma cell lines, Huh7 and HepG2. The complex formed with Huh7 cell extract comigrated with exogenously expressed HNF3 beta in HeLa S3 extracts and was specifically inhibited from forming by the addition of HNF3 beta antiserum. The promoter element which appears to mediate the HNF3 transactivation was functionally mapped by mutational analysis to a region between nucleotides -65 and -54 relative to the transcriptional start site. This regulatory sequence is within the region protected from DNase I digestion by HNF3 alpha and contains 10 of 12 nucleotides homologous to the HNF3-binding-site consensus sequence. A synthetic promoter construct containing this HNF3-binding site was able to mediate transactivation by HNF3 beta. These and previous results suggest that the hepatitis B virus large surface antigen promoter is regulated by at least two liver-enriched transcription factors, HNF1 and HNF3, which together may contribute to the differentiated liver cell type specificity of this promoter.

Base Sequence↗
PubMed1994

Novel clustering of Sp1 transcription factor binding sites at the transcription initiation site of the human muscle phosphofructokinase P1 promoter.

The regulatory sequence elements of the human muscle phosphofructokinase (HPFKM) p1 promoter from -655 to +78 were cloned and characterized. In the human cervical carcinoma cell line, HeLa S3, the HPFKM type C RNA initiated from a single predominant transcription initiation site and the HPFKM p1 promoter displayed transcriptional activity in transient transfection assays. The HPFKM p1 promoter region was shown to possess eight binding sites for the Sp1 transcription factor by DNase I footprinting and gel retardation analysis. The functional importance of these interactions was examined by transient transfection analysis in Drosophila SL2 and HeLa S3 cells. This analysis demonstrated that the HPFKM p1 promoter sequence between +12 and +78 retained Sp1-dependent transcriptional activity in Drosophila SL2 cells and retained promoter activity in HeLa S3 cells. These results suggest that the Sp1 binding site (site 8 between +12 and +21) immediately adjacent to the transcription initiation site represents an important regulatory element of this promoter at least in the context of the minimal HPFKM p1 promoter. However mutagenesis of the Sp1 site 8 demonstrated that, in the context of a larger HPFKM p1 promoter region containing Sp1 sites 1 to 7, it now contributed very little to the total promoter activity. Therefore it appears the Sp1 sites in the HPFKM p1 promoter display functional redundancy.

Animals↗
PubMed1994

Differentiation-specific transcriptional regulation of the hepatitis B virus nucleocapsid gene in human hepatoma cell lines.

Transcription from the hepatitis B virus (HBV) nucleocapsid promoter is regulated in a cell-type-specific manner and can be modulated by the HBV enhancer I element. Mutagenesis analysis of the nucleocapsid promoter demonstrated that the two Sp1-binding sites (CpB and CpC) in the minimal promoter were the major determinants of transcriptional activity in the dedifferentiated hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3. In contrast, binding sites for transcription factors located immediately upstream (CpE) and downstream (CpF) of the two Sp1-binding sites were shown to be important determinants of nucleocapsid promoter activity in the differentiated hepatoma cell lines, Huh7 and HepG2. The role of these elements in the regulation of the nucleocapsid promoter activity correlated with the formation of specific DNA-protein complexes between Huh7 and HepG2 nuclear extracts and the CpE and CpF region sequences. Characterization of the influence of the nucleocapsid promoter mutations in the presence or absence of the enhancer I demonstrated that modification of individual transcription factor binding sites does not prevent enhancer-mediated activation of transcription from the nucleocapsid promoter. These results indicate that differentiated-hepatoma-specific transcription factors plus the Sp1 transcription factor interacting with the nucleocapsid promoter and the enhancer I regulatory region contribute to the level of transcription from this HBV promoter.

Base Sequence↗
PubMed1994

Stereochemical aspects of warfarin drug interactions: use of a combined pharmacokinetic-pharmacodynamic model.

OBJECTIVE: To apply a combined pharmacokinetic-pharmacodynamic model to data from warfarin drug interaction studies. METHODS: The pharmacokinetic model for warfarin enantiomers combined a common first-order absorption process with individual clearance and volume of distribution values and is based on unbound drug. The complete pharmacodynamic model comprised two components: that involving inhibition of prothrombin complex activity (PCA) synthesis described by a sigmoid maximum effect (Emax) model and that relating temporal changes in PCA to synthesis and degradation. The combined model was applied to prothrombin time and plasma concentration-time data obtained after oral administration of single doses of racemic warfarin to healthy subjects either alone or during multiple dosing with the metabolic enzyme inhibitor phenylbutazone or the inducer secobarbital. RESULTS: The five parameters associated with the complete pharmacodynamic model were kd (0.054 +/- 0.014 hr-1), the degradation rate constant of PCA; Cu50,S (0.0026 +/- 0.0015 mg.L-1) and Cu50,R (3.45 +/- 4.20 mg.L-1), the unbound concentrations of (S)- and (R)-warfarin required to produce a 50% reduction in PCA synthesis if administered individually; gamma (0.90 +/- 0.23), the slope parameter in the sigmoid Emax model; and td (8.2 +/- 0.3 hours), the observed delay in the onset of warfarin anticoagulant response. CONCLUSIONS: These findings qualitatively confirm the known potency difference between warfarin enantiomers. Furthermore, although phenylbutazone and secobarbital altered the pharmacokinetics of warfarin, these compounds do not appear to influence its pharmacodynamics. Simulation studies indicate that, after racemate administration, the continual presence of the more potent (S)-enantiomer precludes accurate assessment of Cu50,R. Analysis indicates that use of racemic (rather than enantiomer) warfarin concentration data in drug interaction studies may lead to misinterpretation of pharmacodynamic data.

Blood Coagulation↗
PubMed1994

Regulation of transcription from the human muscle phosphofructokinase P2 promoter by the Sp1 transcription factor.

The human muscle phosphofructokinase (HPFKM) p2 promoter contains sequence elements that are similar to the Sp1 transcription factor binding site consensus sequence. DNase I footprinting identified four regions of the HPFKM p2 promoter that bound purified Sp1. Gel retardation analysis using HeLa S3 nuclear extracts and purified Sp1 protein demonstrated that each of the four recognition elements bound the Sp1 transcription factor. The function of the HPFKM p2 promoter elements was examined in transient transfection assays using these binding sites cloned into a minimal promoter element. In Drosophila Schneider line-2 cells, each of these regulatory regions trans-activated transcription from a minimal promoter element in response to exogenously expressed Sp1. In addition, transcription from the HPFKM p2 promoter was shown to be trans-activated by exogenously expressed Sp1 in Drosophila Schneider line-2 cells. Deletion analysis of the HPFKM p2 promoter demonstrated that the promoter region between -66 and +16 was sufficient to confer sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor and mediates the majority of the transcriptional activity from the HPFKM p2 promoter in the human cervical carcinoma cell line HeLa S3. This demonstrates that the HPFKM p2 promoter contains four functional Sp1 binding sites that may contribute to the level of transcription from this promoter in a variety of cell types.

Base Sequence↗
PubMed1994

Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter.

It has been demonstrated that the hepatocyte nuclear factor 1 (HNF1) binding site is critical for the majority of the hepatitis B virus (HBV) large surface antigen promoter activity in differentiated hepatoma cell lines. Examination of a series of clustered point mutations in the minimal large surface antigen promoter demonstrated that the HNF1 and TATA box binding sites are the major regulatory elements required for transcription from this promoter. Synthetic promoter constructs containing the large surface antigen promoter HNF1 binding site and TATA box element upstream of the luciferase open reading frame were tested for their transcriptional activities in HepG2. 1 cells in the absence or presence of an HNF1 expression vector. These synthetic promoter constructs displayed a similar level of transcriptional activity and induction by HNF1 in comparison with the full-length large surface antigen promoter, suggesting that additional HBV sequences are dispensable for full transcriptional activity. The distance between the HNF1 binding site and TATA box element in the synthetic promoter constructs appeared to influence the transcriptional activity modestly and in a periodic manner.

Base Sequence↗
PubMed1993

Characterization of functional Sp1 transcription factor binding sites in the hepatitis B virus nucleocapsid promoter.

The hepatitis B virus nucleocapsid minimal promoter contains sequence elements which are similar to the Sp1 transcription factor binding site consensus sequence. The interaction of these regulatory elements with Sp1 was examined by DNase I footprinting with purified Sp1 protein and DNase I footprinting and gel retardation analysis with nuclear extracts from human cell lines and was examined functionally with transient transfection assays in human hepatoma and Drosophila melanogaster Schneider line-2 cells. DNase I footprinting identified two regions of the nucleocapsid promoter, representing three recognition elements, that bound purified Sp1. Gel retardation analysis with Huh7 nuclear extracts demonstrated that each of the three recognition elements bound the same or similar transcription factor(s) as that recognized by the Sp1 consensus sequence recognition element. The function of the nucleocapsid promoter elements was examined by transient transfection assays in D. melanogaster Schneider line-2 cells by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. In addition, the second Sp1 site was shown to be an essential element of the nucleocapsid promoter in human hepatoma cells. This demonstrates that the hepatitis B virus nucleocapsid promoter contains three functional Sp1 binding sites which may contribute to the level of transcription from this promoter during viral infection.

Animals↗
PubMed1993

The serology of chronic hepatitis B infection revisited.

The serology of chronic hepatitis B infection has been established through the use of commercial immunoassays to measure the structural antigens of the hepatitis B virus and their respective antibodies in serum. However, the commercial assays have not been designed to detect serum antibodies in the presence of an excess of circulating antigens. A series of serum samples from 200 HBeAg-positive, chronically infected hepatitis B patients with varying degrees of liver disease were analyzed using novel immunoassays designed to detect antibodies in the presence of circulating viral antigens. All patients, regardless of their liver disease, were seronegative for antibodies specific for the envelope antigens or the secreted nucleoprotein antigen (HBeAg) when the commercial assays were used. In contrast, virtually all chronically infected patients with liver disease and approximately 50% of patients without liver disease demonstrated anti-HBe and anti-envelope antibodies when sera were tested in the more sensitive immunoassays. Furthermore, asymptomatic patients could be serologically distinguished from symptomatic patients based on antibody fine specificity, titer, and IgG subclass. This study revealed that the majority of chronically infected hepatitis B patients produce a variety of antibodies for many years, and are not immunologically unresponsive, as suggested by the current assays.

Antibody Formation↗
PubMed1993

Insights from pharmacokinetic and pharmacodynamic studies of hydroxychloroquine.

There is wide variability between subjects in the pharmacokinetic parameters of hydroxychloroquine. A range of concentrations is achieved by individuals receiving the same dosage. In a cross-sectional study, mean hydroxychloroquine concentrations in patients with shorter duration and lower intensity of morning stiffness and no rheumatoid factor were significantly higher than in patients with worse disease activity. Variable kinetic parameters, causing variable concentrations, are likely to be contributing to the variability in response to hydroxychloroquine in rheumatic diseases.

Adult↗
PubMed1992

Characterization of the hepatitis B virus X- and nucleocapsid gene transcriptional regulatory elements.

The regulatory DNA sequence elements that control the expression of the hepatitis B virus X- and nucleocapsid genes in the differentiated human hepatoma cell lines, Huh7, Hep3B, PLC/PRF/5, and HepG2, the dedifferentiated human hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3, were analyzed using transient transfection assays. In this system, the hepatitis B virus enhancer I located between coordinates 1071 (-239) and 1238 (-72) increases transcription from the X-gene promoter located between coordinates 1239 (-71) and 1376 (+67) more than 30-fold in the differentiated hepatoma and the HeLa S3 cell lines. In the dedifferentiated hepatoma cell line, HepG2.1, the enhancer I sequence increases the level of transcription from the X-gene promoter approximately 10-fold. The enhancer I subregion between coordinates 1117 (-193) and 1204 (-106) appears to be important for enhancer function only in the differentiated hepatoma cell lines, whereas the enhancer I subregion between coordinates 1222 (-88) and 1238 (-72) is required for enhancer activity in each of the cell lines examined. In all of the cell lines, the X-gene minimal promoter element was within a 138-nucleotide sequence located between coordinates 1239 (-71) and 1376 (+67). The enhancer I sequence increases transcription from the nucleocapsid promoter approximately 3- to 10-fold in the Huh7, Hep3B, PLC/PRF/5, and HeLa S3 cell lines, whereas it had little influence on the level of transcription from this promoter in HepG2 and HepG2.1 cells. The minimal nucleocapsid promoter element was within a 105 nucleotide sequence located between coordinates 1700 (-85) and 1804 (+20). This indicates that the levels of transcription from the X- and nucleocapsid gene promoters are determined in a cell-type-specific manner, in part, by the hepatitis B virus enhancer I and the corresponding minimal promoter sequence.

Base Sequence↗
PubMed1992

Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor.

The DNA-binding proteins which recognize the regulatory sequence elements of the hepatitis B virus (HBV) major surface antigen promoter were examined by gel retardation analysis, using nuclear extracts from the human hepatoma cell line Huh7. Using this assay, we identified four regions (B, D, E, and F) of the promoter that interact with the same or similar transcription factor(s). In addition, the recognition sequence for the Sp1 transcription factor bound the same or similar transcription factor(s) present in Huh7 cell nuclear extracts, and this binding was inhibited by the four major surface antigen promoter elements, B, D, E, and F. Purified Sp1 transcription factor was shown to bind to three (B, D, and F) of the major surface antigen promoter regulatory sequence elements by DNase I footprinting. Using transient transfection assays with Drosophila Schneider line 2 cells, we found that transcription from the major surface antigen promoter was transactivated by exogenously expressed Sp1, whereas transcription from the other three HBV promoters was not. Deletion analysis of the major surface antigen promoter demonstrated that the promoter region between -35 and +157 was sufficient to confer Sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor. The function of the B, D, E, and F promoter elements was further examined by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. This finding demonstrates that the HBV major surface antigen promoter contains four functional Sp1 binding sites which probably contribute to the level of expression from this promoter during viral infection.

Antigens, Viral↗
PubMed1991

Autoantibody production in hepatitis B e antigen transgenic mice elicited with a self T-cell peptide and inhibited with nonself peptides.

Studies in hepatitis B e antigen (HBeAg)-expressing transgenic mice indicate that self tolerance to two T-cell determinants on the same transgenic self molecule can differ markedly. The dominant T-cell site on HBeAg is tolerogenic, whereas a proportion of T cells recognizing a second T-cell site evade tolerance induction, persist in the periphery, and can be activated in vivo by a single injection of a 12-residue T-cell self peptide. The self-reactive T cells mediate in vivo autoantibody production sufficient to neutralize detection of the autoantigen in serum. Furthermore, autoantibody production can be inhibited by nonself peptides that compete with the self peptide for binding to major histocompatibility complex molecules. This model illustrates that T cells specific for an immunogenic T-cell site on a nonsequestered autoantigen can escape tolerance induction and, more importantly, can mediate autoreactivity in vivo. Furthermore, these results suggest that synthetic T-cell sites may be useful as immunotherapeutic agents for the purpose of circumventing nonresponse to HBeAg during persistent hepatitis B virus infection.

Amino Acid Sequence↗
PubMed1991

Promoter-specific transactivation of hepatitis B virus transcription by a glutamine- and proline-rich domain of hepatocyte nuclear factor 1.

The cloned transcription factor hepatocyte nuclear factor 1 (HNF1) transactivates transcription from the hepatitis B virus (HBV) large surface antigen promoter but does not influence the transcriptional activities of the other three HBV promoters. This indicates that this transcription factor can differentially influence the activities of the HBV promoter. By using a transient-transfection system, the major domain of the HNF1 polypeptide involved in transcriptional activation of the large surface antigen promoter in the human hepatoma cell line HepG2.1 has been mapped to a region that is rich in glutamine and proline residues (9 of 18) and is different from the previously identified regions of this factor responsible for in vitro transcriptional activation of a promoter containing human albumin promoter HNF1 binding sites. The human albumin promoter HNF1 binding site mediates transcriptional activation through the same HNF1 polypeptide domain as the HBV large surface antigen promoter HNF1 binding site in transient-transfection assays with HepG2.1 cells, suggesting that HNF1 may possess multiple transcriptional activation domains.

Animals↗
PubMed1991

Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus.

Expression of the hepatitis B virus core antigen (HBcAg) in mouse NIH 3T3 fibroblasts has been shown previously (A. McLachlan et al., J. Virol. 61:683-692, 1987) to result in the nuclear localization of this polypeptide. Since the carboxyl terminus of HBcAg contains four clusters of arginine residues which resemble nuclear localization sequences identified in other nuclear proteins, a series of carboxyl-terminus-truncated HBcAg polypeptides were expressed in mouse fibroblasts to examine the role of these sequences in the cellular localization of HBcAg. By immunofluorescence and cell fractionation analysis, it was demonstrated that regions of the HBcAg polypeptide including the most carboxyl-terminal (cluster 1) and amino-terminal (cluster 4) clusters of arginine residues represent distinct and independent nuclear localization sequences for this polypeptide. Substitution of a threonine residue for the second arginine residue in cluster 4 inactivates the nuclear localization signal in this region of the HBcAg polypeptide, demonstrating the importance of this residue to this signal sequence. However, HBcAg fails to accumulate in the nucleus only when both nuclear localization signal sequences are simultaneously deleted or disrupted by mutation. The possible significance of the nuclear localization sequences identified in the HBcAg polypeptide is discussed in the context of the role of the nucleocapsid in the hepatitis B virus life cycle.

Amino Acid Sequence↗
PubMed1991

Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines.

A detailed mutational analysis of the regulatory DNA sequence elements that control expression of the hepatitis B virus major surface antigen gene was performed in the human hepatoma cell lines HepG2.1 and Huh7, using transient transfection assays. Seven regions (A to G) of the major surface antigen promoter located within 200 nucleotides of the RNA initiation site have been identified which influence the level of transcription from this promoter. The three distal regions (A to C), located between -188 and -68, appear to possess a level of redundancy in their ability to influence the transcriptional activity from the major surface antigen promoter. The simultaneous deletion of regions A, B, and C resulted in an approximately fourfold reduction in transcription from the major surface antigen promoter. Region D, located between -67 and -49, is an essential element of the major surface antigen promoter. The three proximal regions (E to G) are located within 45 nucleotides of the major transcription initiation site. Region E prevents the negative influence of region F and can compensate for the effect of mutation of region G on transcription from the major surface antigen promoter. Region G can compensate for the effect of the loss of a functional region E sequence on the transcriptional activity of the major surface antigen promoter only in the absence of a functional region F sequence. These results imply that the level of expression of the major surface antigen gene is controlled by the complex interplay between a minimum of six transcription factors which activate and one transcription factor which represses transcription from this gene.

Base Sequence↗