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D J Bergsma

Publications and source records attributed to D J Bergsma.

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

Comparison of the enzymatic activities of human galactokinase GALK1 and a related human galactokinase protein GK2.

The GALK1 cDNA encoding human galactokinase was recently cloned and its cognate GALK1 gene shown to be involved in galactokinase deficient galactosemia. Previously, a separate human galactokinase cDNA, GK2, was cloned by complementation of a galactokinase deficient yeast mutant; however, the galactokinase activity of GK2 was not demonstrated in mammalian cells. To compare the relative galactokinase activity of GALK1 and GK2, their corresponding cDNAs were expressed in COS cells. Northern blot analysis indicated that both cDNAs were transcribed into mRNA transcripts of the expected size; however, only the GALK1 cDNA produced high levels of galactokinase activity. This result would suggest that GALK1 is the major enzyme for galactose metabolism while the role of GK2 remains uncertain.

Base Sequence↗
PubMed1995

Cloning, expression and functional characterization of type 1 and type 2 steroid 5 alpha-reductases from Cynomolgus monkey: comparisons with human and rat isoenzymes.

The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two known human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the suitability of this species at the level of the molecular targets, a Cynomolgus monkey prostate cDNA library was prepared and screened using human SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA sequences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with their human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corresponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Subsequently, both monkey cDNAs were individually expressed in a mammalian cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and rat enzymes, the monkey type 1 and type 2 SRs were most active at neutral and low pH, respectively. The results of inhibition studies with over 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocity and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest that the monkey SR isoenzymes are structurally and functionally comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of SR inhibitors.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

Cloning of the galactokinase cDNA and identification of mutations in two families with cataracts.

Galactokinase is an essential enzyme for the metabolism of galactose and its deficiency causes congenital cataracts during infancy and presenile cataracts in the adult population. We have cloned the human galactokinase cDNA, which maps to chromosome 17q24, and show that the isolated cDNA expresses galactokinase activity in bacteria and mammalian cells. We also describe two different mutations in this gene in unrelated families with galactokinase deficiency and cataracts. The availability of the cloned galactokinase gene provides an important reference to identify mutations in patients with galactokinase deficiency and cataracts.

Adult↗
PubMed1995

Mouse galactokinase: isolation, characterization, and location on chromosome 11.

Elevated galactose levels can be caused by several enzyme defects, one of which is galactokinase. Galactokinase deficiency cause congenital cataracts during infancy and presenile cataracts in the adult population. We have isolated the mouse cDNA for galactokinase, which shares extensive amino acid sequence homology, 88% identity, with a recently cloned human galactokinase. It is expressed in all tissues examined. In an interspecific backcross analysis galactokinase maps to the distal region of mouse chromosome 11, a region that is homologous to human chromosome 17q22-25. The availability of the mouse gene provides an opportunity to make a knockout model for galactokinase deficiency.

Alleles↗
PubMed1994

Differentiation between binding sites for angiotensin II and nonpeptide antagonists on the angiotensin II type 1 receptors.

To characterize binding sites for nonpeptide angiotensin antagonists on the human angiotensin II receptor type 1 (AT1 receptor) we have systematically exchanged segments of the human receptor with corresponding segments from a homologous Xenopus laevis receptor, which does not bind the nonpeptide compounds. Substitution of transmembrane segment VII of the human AT1 receptor dramatically reduced the binding affinity of all of the 11 nonpeptide antagonists tested (55- to > 2000-fold) with no effect on the binding of angiotensin. The affinity for the nonpeptide compounds decreased additionally one order of magnitude when transmembrane segment VI and the connecting extracellular loop 3 from the Xenopus receptor were also introduced into the human AT1 receptor. Exchanges of smaller segments and single residues in transmembrane segments VI and VII and extracellular loop 3 revealed that the binding of nonpeptide antagonists was dependent on nonconserved residues located deep within the transmembrane segments VI and VII, in particular Asn295 in transmembrane segment VII. Surprisingly, all exchanges in transmembrane segment VII, including the Asn295 to Ser substitution, had a more pronounced effect on the binding of the competitive antagonists relative to the insurmountable antagonists. It is concluded that the binding mode for peptide and nonpeptide ligands on the AT1 receptor is rather different and that competitive and insurmountable antagonists presumably bind to overlapping but distinct sites located in transmembrane segments VI and VII.

Amino Acid Sequence↗
PubMed1994

Human AT1 receptor is a single copy gene: characterization in a stable cell line.

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430 +/- 15 fmol/mg] exhibiting high affinity [KD = 0.15 +/- 0.02 nM] for [125I][SAR1, Ile8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed receptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5'-(gamma-thio) triphosphate [GTP gamma S]. Angiotensin II evoked a rapid efflux of 45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.

Animals↗
PubMed1994

Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.

Base Sequence↗
PubMed1994

Epristeride is a selective and specific uncompetitive inhibitor of human steroid 5 alpha-reductase isoform 2.

Specificity of an enzyme inhibitor can have profound implications upon the compound's therapeutic potential, utility and safety profile. As potent inhibitors of human steroid 5 alpha-reductase (SR) the 3-androstene-3-carboxylic acids, or steroidal acrylates, may be useful in treatment of diseases such as benign prostatic hyperplasia for which 5 alpha-dihydrotestosterone (DHT) appears to be a causative agent. To determine its specificity profile, the interactions of a representative compound from this class, N-(t-butyl)androst-3,5-diene-17 beta-carboxamide-3-carboxylic acid (epristeride, SK&F 105657), have been studied with 7 other steroid processing enzymes and 5 steroid hormone receptors. The affinity of epristeride for each of these 12 potential targets was found to be at least 1000-fold weaker than that for SR, the intended target. In addition, using samples of the individually expressed two known forms of human SRs, epristeride has been shown to be a selective inhibitor of the recombinant human SR type 2, the predominant activity found in the prostate of man. Nonetheless, the mechanisms of SR inhibition for both isoenzymes involve formation of a ternary complex with epristeride, NADP+, and enzyme. Epristeride, consequently, has been shown to be an uncompetitive inhibitor versus steroid substrate of both human SR isoenzymes. These results suggest that this 3-androstene-3-carboxylic acid is a specific and selective inhibitor of the human type 2 SR, and that epristeride is an attractive compound for further investigation as a safe and effective therapeutic agent in the potential treatment of disease states associated with DHT-induced tissue growth.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1994

Mutations in transmembrane segment VII of the AT1 receptor differentiate between closely related insurmountable and competitive angiotensin antagonists.

Chimeric constructs between the human and the Xenopus laevis AT1 receptor have demonstrated, that the binding of non-peptide angiotensin antagonists is dependent on non-conserved residues located deep in transmembrane segment VII of the AT1 receptor. Here we have studied four pairs of closely related antagonists each consisting of a competitive and an insurmountable compound differentiated by one out of three different types of minor chemical modifications. None of the antagonists bound to the Xenopus receptor and the binding of all of the compounds to the human receptor was severely impaired by the introduction of non-conserved residues from transmembrane segment VII of the Xenopus receptor. In all four pairs of antagonists the competitive compound was affected more by these substitutions than the corresponding insurmountable one (209 vs. 22, 281 vs. 29, 290 vs. 29 and 992 vs. 325-fold increase in Ki values). A similar pattern was observed in response to substitution of a single non-conserved residue in transmembrane segment VII, Asn295 to Ser. These results indicate that a common molecular mechanism distinguishes the interaction of insurmountable and competitive antagonists with the AT1 receptor.

Amino Acid Sequence↗
PubMed1994

Characterization of a functional angiotensin II receptor in Xenopus laevis heart.

High-affinity (104 +/- 18 pmol/l) and high-density (204 +/- 25 fmol/mg) angiotensin II (AII) binding sites have been identified in Xenopus laevis heart membranes. Competition binding of [125I]Sar1,Ile8 angiotensin (SIA) to these receptors by peptide analogs selective for the mammalian AII receptor subtypes AT1 and AT2 suggested that the amphibian AII binding sites were more closely related to the AT1 receptor subtype. Also in common with AT1 receptors, dithiothreitol and GTP gamma S inhibited [125I]SIA binding to Xenopus heart receptors, exhibiting IC50 values of 600 and 0.95 mumol/l, respectively. In addition, Xenopus oocytes injected with Xenopus heart mRNA were capable of mobilizing calcium when exposed to AII, demonstrating that Xenopus AII receptors are functionally linked to a second-messenger system similar to that coupled to mammalian AT1 receptors. However, in contrast to both AT1 and AT2 receptor subtypes, nonpeptide antagonists DUP 753 and SK&F 108566 (AT1 receptor selective) and PD123319 (AT2 selective) did not bind the Xenopus AII receptors, thus establishing that the amphibian receptors were pharmacologically unique. Together, these results demonstrate that Xenopus heart AII receptors are functionally similar to mammalian AT1 receptors but are pharmacologically distinct from both AT1 and AT2 receptors.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1993

The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding.

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent RmR rbp1 mutants and screened them for both RBP1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm.(ABSTRACT TRUNCATED AT 250 WORDS)

Antifungal Agents↗
PubMed1993

Molecular characterization and regulation of the human endothelin receptors.

Endothelin receptors (ETRs) are distributed throughout a variety of tissues. Two human cDNAs were identified which encode distinct ETR proteins. One cDNA encoded a 427-amino acid protein that shared 91% identity to rat ETAR. The second cDNA encoded a 442-amino acid protein that was 88% identical to rat ETBR. Ligand binding studies of the cloned receptors expressed in COS cells confirmed that they were pharmacologically ETAR and ETBR subtypes; although the selective antagonist BQ123 showed a potency similar to ET-3 in displacing 125I-ET-1 binding to ETAR. This observation contrasts with rat ETAR pharmacology where BQ123 has a 100-fold higher affinity than ET3. Chinese hamster ovary cells expressing the human ETAR displayed equal potencies in displacing 125I-ET-1 binding, which indicates that rat and human ETAR are pharmacologically distinct. Electrophysiological studies of both ETRs expressed in Xenopus oocytes revealed that they are functional. Northern analysis indicated that the two ETRs are differentially expressed in many tissues. Marmosets maintained on a high fat/high cholesterol diet exhibited 3-fold increase in ETBR mRNA levels with little change in ETAR mRNA levels. Availability of cDNA clones for ETR subtypes can open avenues for future analysis of their role in pathophysiology of various diseases.

Amino Acid Sequence↗
PubMed1993

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Alleles↗
PubMed1993

Characterization of [3H]SK&F 108566 as a radioligand for angiotensin type-1 receptor.

Rat aortic smooth muscle cells were used as a model system to characterize the binding properties of [3H]SK&F 108566, an angiotensin type-1 (AT1) receptor antagonist. The binding was specific, saturable and reversible. The association and dissociation rates of [3H]SK&F 108566 binding to smooth muscle cells were monophasic and Scatchard analysis of equilibrium binding data yielded a linear plot indicating a homogenous population of binding sites. The maximum binding (Bmax) and apparent dissociation constant (Kd) were 22,000 +/- 6000 sites/cell and 0.83 +/- 0.08nM respectively. The pharmacological specificity of [3H]SK&F 108566 binding to smooth muscle cells is consistent with that observed for AT1 and confirms AT1 receptor specificity of this radioligand. High affinity binding was observed in membranes prepared from bovine adrenal cortex, rat liver and rat kidney glomeruli. COS cells transfected with cDNA encoding human AT1 angiotensin II receptors also displayed high affinity binding site for [3H]SK&F 108566. No specific binding could be detected on membranes prepared from bovine cerebellum, a tissue rich in the angiotensin type-2 (AT2) receptor. These observations indicate that [3H]SK&F 108566 binds to sites which have pharmacological characteristics of angiotensin II AT1 subtype receptors and can be used as a subtype-selective radioligand to characterize AII receptors in various systems.

Acrylates↗
PubMed1993

Isolation and expression of a novel angiotensin II receptor from Xenopus laevis heart.

A Xenopus laevis heart cDNA library was screened using the human angiotensin type 1 (AT1) receptor cDNA coding sequence as a hybridization probe. A cDNA was isolated that encodes a protein of 363 amino acids that shares 63% sequence identity with the human AT1 receptor. Radioligand binding studies with the cloned receptor expressed in COS cells indicated that it is an angiotensin II receptor that possesses pharmacological properties distinct from those of the two known mammalian receptor subtypes, AT1 and AT2. Electrophysiological studies with the recombinant receptor expressed in X. laevis oocytes revealed that the amphibian receptor, like the mammalian AT1 receptor, can functionally couple to a second messenger system, leading to the mobilization of intracellular stores of calcium. However, nonpeptide antagonists selective for the mammalian AT1 and AT2 receptors do not block angiotensin II-stimulated functional responses in injected oocytes, which confirms that the amphibian receptor is a pharmacologically unique angiotensin II receptor. Nevertheless, based on conservation of structural features and motifs and similarity in coupling mechanisms, we speculate that the cloned Xenopus receptor is the amphibian counterpart of the mammalian AT1 receptor, having acquired its unique pharmacology as a consequence of evolutionary divergence.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of a human angiotensin II type 1 receptor.

A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.

Amino Acid Sequence↗
PubMed1992

The yeast cyclophilin multigene family: purification, cloning and characterization of a new isoform.

Cyclophilins (Cyps) constitute a highly conserved family of proteins present in a wide variety of organisms. Historically, Cyps were first identified by their ability to bind the immunosuppressive agent cyclosporin A (CsA) with high affinity; they later were found to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the folding of oligopeptides at proline-peptide bonds in vitro and may be important for protein folding in vivo. Cells of Saccharomyces cerevisiae contain at least two distinct Cyp-related PPIases encoded by the genes CYP1 and CYP2. A yeast strain (GL81) containing genomic disruptions of three known yeast PPIase-encoding genes [CYP1, CYP2 and RBP1 (for rapamycin-binding protein); Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] was previously constructed and found to be viable. Soluble fractions of these cells possess residual CsA-sensitive PPIase activity (2-5% of that present in wild-type cells as assayed in vitro). We have purified an approx. 18-kDa protein exhibiting PPIase activity from a soluble fraction of GL81 cells and determined that its N-terminal amino acid (aa) sequence exhibits significant homology (but nonidentity) to the Cyp1 and Cyp2 proteins. We designate the gene for this new protein, CYP3. Using a degenerate oligodeoxyribonucleotide (oligo) based on the N-terminal aa sequence, plus an internal oligo homologous to a conserved region within the portion of CYP1 and CYP2 that had been deleted in the genome, a CYP3-specific DNA fragment was generated by the polymerase chain reaction (PCR) using GL81 genomic DNA as a substrate. This PCR fragment was used as a probe to isolate CYP3 genomic and cDNA clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗