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Biomedical subjects

N A Kalsheker

Publications and source records attributed to N A Kalsheker.

At least 19 recordsLinked to original sources
PubMed1998

Enzymatically active papain preferentially induces an allergic response in mice.

Human exposure to papain, a cysteine proteinase, is associated with hypersensitivity reactions. We demonstrate in mice that enzymatically active papain preferentially induces an IgG1 response and results in mast cell degranulation, both features typical of an allergic reaction. Inactive papain, blocked with E-64, appears to desensitize mice to subsequent challenge by active enzyme. These results suggest that the enzymatic activity of papain is critical in inducing an allergic response and that the use of inactive allergens may be a possible strategy for desensitizing allergic individuals.

Animals↗
PubMed1997

Mutation in an alpha1-antitrypsin enhancer results in an interleukin-6 deficient acute-phase response due to loss of cooperativity between transcription factors.

We previously reported that a mutation in a 3' enhancer region of the alpha1-antitrypsin (AAT) gene is associated with chronic obstructive airways disease (COAD). During the acute-phase response the plasma concentration of AAT increases approximately 3-fold and this effect is mediated primarily by interleukin-6 (IL-6). We demonstrate, by transfection of Hep G2 cells, that the AAT gene contains at least two enhancers, one at the 5' end of the gene which is dominant under basal conditions, and another at the 3' end of the gene which exhibits weak basal activity. However, both enhancers must be present to modulate the IL-6-induced response which is diminished as a consequence of the 3' enhancer mutation. These results suggest that the 3' enhancer modulates the IL-6 response through an interaction with the 5' enhancer. The mutation occurs at a DNA binding site for the ubiquitous transcription factor octamer-1 (Oct-1) and results in a loss of cooperativity between Oct-1 and the tissue-specific transcription factor, NF-IL6 (C/EBPbeta), a member of the C/EBP family of transcription factors. NF-IL6 is a key transcription factor for a major pathway through which IL-6 mediates its effects. These observations provide a novel mechanism for a diminished IL-6-induced response.

Acute-Phase Reaction↗
PubMed1997

Microsatellite polymorphism of the alpha 1-antichymotrypsin gene locus associated with sporadic Alzheimer's disease.

A variant of the apolipoprotein E gene, APOE*4, is associated with both sporadic Alzheimer's disease (AD) and a subset of familial AD and this association is stronger with early as opposed to late onset AD. Both APOE*4 and alpha 1-antichymotrypsin (ACT) will accelerate the rate of amyloid filament formation and are major constituents of the plaques associated with AD. We now show that a dinucleotide microsatellite allele in the 5'-flanking sequence of the ACT gene, designated A10, in association with APOE*4 significantly increases the risk of developing sporadic AD, which accounts for the majority of AD cases.

Aged↗
PubMed1997

Regulation of the serine proteinase inhibitor (SERPIN) gene alpha 1-antitrypsin: a paradigm for other SERPINs.

alpha 1-antitrypsin (AAT) is the archetypal member of the serine proteinase inhibitor (SERPIN) gene family. AAT is an acute-phase reactant and the plasma concentration increases three- to four-fold during the inflammatory response. In hepatocytes this increase is mediated primarily by the cytokine interleukin-6 (IL-6) via the transcription factor NF-IL6. The AAT gene contains at least two enhancer elements, one at the 5' end of the gene and the other at the 3' end. Functional studies performed in mammalian hepatoma cells (Hep G2) using constructs containing these AAT enhancer regions linked to a reporter gene have demonstrated that the 5' enhancer is dominant under basal conditions and that, following stimulation with IL-6, both enhancers are essential and the 3' enhancer plays a major role. We have identified a mutation associated with lung disease which occurs in the 3' AAT enhancer; the mutation occurs at a binding site for the ubiquitous transcription factor Oct-1. The functional significance of this mutation is a deficient IL-6 response. Using the AAT gene as a model, we describe the interactions which occur between transcription factors within the 3' enhancer and also those which take place between the 5' and 3' enhancers. These studies shed light on the molecular mechanism of the acute-phase response which could possibly be extended to other members of the SERPIN gene family.

Acute-Phase Reaction↗
PubMed1996

The house dust mite allergen Der p1 catalytically inactivates alpha 1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma.

Der p1, a cysteine proteinase derived from the house dust mite (HDM) Dermatophagoides pteronyssinus, is a major component of the allergic immune response in HDM atopic individuals. Recent evidence suggests that cysteine proteinase activity is important in the disease process as it increases the permeability of the allergen in the respiratory tract and disrupts the regulation of IgE synthesis. Der p1 is found in high concentrations in the faecal pellets of mites which are aerosolised and inhaled via the respiratory tract. The serine proteinase inhibitor, alpha 1-antitrypsin, protects the lower respiratory tract against damage by proteinases released in the lung during inflammation. Der p1 catalytically inactivates alpha 1-antitrypsin by a thiol-dependent mechanism involving specific cleavage of the reactive centre loop and we propose that this mechanism may be important in the pathogenesis of asthma.

Allergens↗
PubMed1996

Alpha 1-antichymotrypsin.

alpha 1-Antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family, inhibits neutrophil proteinase cathepsin G and mast cell chymases, and protects the lower respiratory tract from damage by proteolytic enzymes. It contains a reactive centre loop, which interacts with cognate proteinases, resulting in loop cleavage and a major conformational change. Recently, alpha 1-antichymotrypsin has been identified as a major constituent of the neurofibrillary plaques associated with Alzheimer's disease, and in vitro studies have shown that it enhances the rate of amyloid-fibril formation. These observations and recent genetic evidence suggest that alpha 1-antichymotrypsin is important in the pathogenesis of Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Development of a rapid DNA screening procedure for the Factor V Leiden mutation.

Aim-To develop a rapid, simple and highly specific DNA screening procedure based on the amplification refractory mutation system (ARMS) to detect the Leiden mutation in whole blood.Methods-ARMS PCR amplification primers with additional mismatches at either -2 or -3, which greatly improves specificity, were constructed to detect the normal Factor V gene and the Leiden mutation in whole blood samples from patients with abnormal clotting results.Results-Construction of ARMS primers with either an additional mismatch at -2 or -3 at the 3' end of the primer could be used to detect the Leiden mutation in 0.5 mu1 whole blood in under three hours. Primers destabilised at position -3 could be used at a lower annealing temperature, which gave greater sensitivity and are now routinely used. A control set of primers was included in the same reaction to act as a positive control.Conclusions-This rapid and specific assay for the factor V Leiden mutation is a useful addition to the investigation of patients with or at risk from thrombovascular disease.

Journal Article↗
PubMed1996

Greater frequency of K-ras Val-12 mutation in colorectal cancer as detected with sensitive methods.

Assays for tumor mutations need to be sufficiently sensitive that a mutation can be readily detected in a high background of wild-type sequence. We have evaluated competitive allele-specific oligonucleotide (cASO) hybridization, the amplification refractory mutation system (ARMS), and a combination of cASO with mutant-enriched polymerase chain reaction (ME-PCR) for their respective sensitivities in detecting the K-ras Val-12 mutation. cASO hybridization detected 1 mutant allele in 100 wild-type alleles and identified 6 of 74 (8%) colorectal tumors as having the Val-12 mutation. ARMS detected 1 mutant in 1000 and 12 of 74 (16%) tumor samples, and the ME-PCR with cASO hybridization detected 1 in 5000 and 20 of 74 (27%) tumor samples. The mutation was not detected in normal bowel mucosa from selected Val-12 tumor-positive patients. ME-PCR combined with cASO resulted in a threefold higher detection rate than has previously been reported and suggests that other studies may have underestimated the frequency of K-ras mutations.

Base Sequence↗
PubMed1994

Comparative modelling of major house dust mite allergen Der p I: structure validation using an extended environmental amino acid propensity table.

A model of the 3-D structure of a major house dust mite allergen Der p I associated with hypersensitivity reactions in humans was built from its amino acid sequence and its homology to three known structures, papain, actinidin and papaya proteinase omega of the cysteine proteinase family. Comparative modelling using COMPOSER was used to arrive at an initial model. This was refined using interactive graphics and energy minimization with the AMBER force field incorporated in SYBYL (Tripos Associates). Compatibility of the Der p I amino acid sequence with the cysteine proteinase fold was checked using an environment-dependent amino acid propensity table incorporated into a new program HARMONY with a variable length windowing facility. A five-residue window was used to probe local conformational integrity. Propensities were derived from a structural alignment database of homologous proteins using a robust entropy-driven smoothing procedure. Der p I shares essential structural and mechanistic features with other papain-like cysteine proteinases, including cathepsin B. The active-site thiolate-imidazolium ion pair comprises the side chains of Cys34 and His170. A cystine disulfide not present in other known structures bridges residue 4 of an N-terminal extension and the core residue 117. Two conserved disulfide bridges are formed by residues 31 and 71 and residues 65 and 103. Model building of peptide substrate analogue complexes suggests a preference for phenylalanyl or basic residues at the P2 position, whilst selectivity may be of minor importance at the S1 subsite. The electrostatic influences on the Der p I active-site ion pair and extended peptide binding region are markedly different from those in known structures. A highly immunogenic surface exposed region (residues 107-131), comprising several overlapping T cell epitope sites, has no shared sequence identity with human liver cathepsin B and contains three insertion-deletion sites. The structure provides a basis for testing the substrate specificity of Der p I and the potential role of proteinase activity in hypersensitivity reactions. These studies may offer a new treatment strategy by hyposensitization with inactive mutants or mutants with significantly altered proteinase activity, either alone or complexed with antibody.

Algorithms↗
PubMed1994

Regulation of the alpha 1-antitrypsin gene and a disease-associated mutation in a related enhancer sequence.

Ten years ago a search was initiated for DNA variation in the alpha-1-antitrypsin gene (alpha 1-AT) to determine whether there were mutations more commonly associated with patients who had chronic obstructive airways disease (COAD) than with healthy individuals. Using the conventional approach of Southern blotting and searching for restriction fragment length polymorphisms, we identified a potentially useful polymorphism that resulted in the loss of a recognition site for the restriction enzyme, Taq I. The polymorphism occurred in about 17% of patients with COAD and about 5% of the general population (p = 0.0016). The normal sequence in the 3' flanking region of the alpha 1-antitrypsin gene had to be characterized, as it was not known. On the basis of homology, a number of closely clustered sequence motifs demonstrating the characteristics of an enhancer were identified that would potentially increase the transcription and expression of alpha 1-antitrypsin. The normal Taq I sequence occurred in a motif that demonstrated homology to a DNA sequence for octamer transcription factors. The mutation was characterized by in vitro amplification of the region and direct sequencing as a G to A transition (Taq I site TCGA < TCAA). Specific binding of nuclear proteins by gel-shift analysis and DNase I footprinting and increased in vivo transcriptional activity were demonstrated by transfection of mammalian cells containing DNA fragments corresponding to the region of interest. In contrast, the mutant sequence demonstrated loss of binding to nuclear proteins and reduced transcriptional activity. The latter finding was not confined to tissues known to express alpha 1-antitrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Molecular pathology of alpha 1-antitrypsin deficiency and its significance to clinical medicine.

alpha 1-Antitrypsin (AAT) deficiency is associated with predisposition to developing liver cirrhosis in early childhood, and chronic degenerative lung disease in early adult life. The probable molecular basis for the disease associations is known. One of the common variants, Z, has the propensity to form polymers, a phenomenon which is concentration- and temperature-dependent. This results in accumulation of the protein in hepatocytes, the predominant tissue source of AAT, and leads to cell damage. AAT deficiency results in loss of protection in the lung against neutrophil elastase (NE) the major target for AAT. NE is capable of destroying the architecture of the lung, leading to pulmonary emphysema. The disease process is exacerbated by cigarette smoke, which is capable of oxidizing a critical methionine residue at the active site, rendering AAT an inefficient inhibitor of NE. The combination of deficiency and cigarette smoking are critical to the development of pulmonary emphysema. We have identified a mutation in an enhancer sequence which, in all probability, predisposes to disease by a novel mechanism related to diminished expression of AAT during inflammation. Our understanding of the mechanisms of disease should lead to improved therapeutic interventions.

Gene Expression↗
PubMed1993

Point mutation in a 3' flanking sequence of the alpha-1-antitrypsin gene associated with chronic respiratory disease occurs in a regulatory sequence.

A point mutation in the 3' flanking sequence of the alpha-1-antitrypsin gene is associated with chronic respiratory disease. This study demonstrates that the mutation occurs in a motif that binds a nuclear factor. A direct consequence of the mutation is the loss of specific binding. Functional studies with constructs containing this region downstream of a reporter gene in the sense orientation demonstrated that the wild type sequence increased expression compared with control promoter plasmid and there was a significant reduction in expression by the mutant sequence. These effects were demonstrated in three distinct cell lines suggesting an ubiquitous rather than a tissue-specific effect. However, transacting factors may influence the response in different tissues. The mutation does not appear to affect basal expression of the protein as the plasma concentration of alpha-1-antitrypsin is normal in individuals who carry the mutation. However, the binding and functional studies suggest that it may reduce the three- to four-fold rise in plasma alpha-1-antitrypsin concentration that occurs during inflammation.

Base Sequence↗
PubMed1990

Linkage disequilibrium between DNA polymorphisms within the porphobilinogen deaminase gene.

Three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, ScrFI/BstNI) within the human porphobilinogen deaminase (PBG-D) gene have been studied in 47 unrelated patients with the autosomal dominant disorder, acute intermittent porphyria (AIP), and in 92 control subjects. Each enzyme identified a two-allele polymorphism with allele frequencies close to 0.50: however, marked linkage disequilibrium limited the number of observed haplotypes to four, of which one is uncommon. No association was detected between any haplotype and AIP.

Acute Disease↗
PubMed1990

Acute intermittent porphyria caused by a C----T mutation that produces a stop codon in the porphobilinogen deaminase gene.

A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a C----T transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.

Codon↗
PubMed1990

Molecular characterisation of two alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) Null(Newport) (Gly115----Ser) and (Pi) Z Wrexham (Ser-19----Leu).

Two single point mutations in the alpha-1-antitrypsin gene, resulting in AAT deficiency, have been characterised in heterozygotes by DNA amplification and direct sequencing. The mutations result in amino acid substitutions, Gly115----Ser and Ser-19----Leu, in the leader sequence, respectively, and have been designated Pi Null(Newport) and Pi Z Wrexham. In the two families studied the mutations occur on chromosomes which also carry the common mutation causing Z deficiency. Individuals with such a deficiency are, therefore, compound heterozygotes. It is not known if these particular mutations would only cause a mild form of AAT deficiency in the absence of the Z mutation as they do not appear to cause predictable folding abnormalities. They do, however, result in severe deficiency when the Z mutation occurs in the same gene.

Amino Acid Sequence↗
PubMed1990

Characterisation of the alpha-1-antitrypsin M3 gene, a normal variant.

By sequence analysis of the complete protein-coding region of the human alpha-1-antitrypsin gene using polymerase chain reaction techniques, we have characterised one of the normal variants, M3. We have identified a single point mutation between M1 Val213 and M3 at codon position 376 which is a GAA(Glu) to GAC(Asp) transversion.

Amino Acid Sequence↗
PubMed1989

Molecular characterisation of three alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) nullcardiff (Asp256----Val); PiMmalton (Phe51----deletion) and PiI (Arg39----Cys).

Three mutations causing alpha-1-antitrypsin deficiency have been identified by gene amplification and direct DNA sequencing. In the Pi (proteinase-inhibitor) nullcardiff gene, the codon for aspartate at position 256 has mutated to encode valine. In PiMmalton and Pi I, the respective mutations are the deletion of the codon for a phenylalanine residue at position 51 or 52, and a single base substitution resulting in arginine being replaced by cysteine at position 39. Examination of the protein tertiary structure suggests that all of these mutations are likely to result in folding abnormalities that may explain the deficiency states.

Amino Acid Sequence↗