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E Boel

Publications and source records attributed to E Boel.

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

Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells.

The enzyme L-glutamic acid decarboxylase is a major autoantigen of the beta cell. Autoantibodies against this enzyme are observed before the onset of insulin-dependent diabetes mellitus (IDDM) in man and may be of predictive value. There is evidence that this enzyme is involved in the development of autoimmune diabetes in animals. In order to facilitate the investigation of the role of L-glutamine acid decarboxylase in IDDM, we expressed the 65 kDa isoform of human islet L-glutamic acid decarboxylase in insect cells using a baculovirus-based vector. The material was expressed at high levels (up to 50 mg/l of cells). Partially purified metabolically labelled L-glutamic acid decarboxylase bound to immunoglobulins in the sera from 20 of 49 subjects with newly-diagnosed IDDM. The enzyme was isolated in high yields (up to 26 mg/l cell culture) with fully maintained enzymatic activity by either ion-exchange chromatography or immunoaffinity chromatography. Purified L-glutamic acid decarboxylase inhibited the binding of radioactive L-glutamic acid decarboxylase, prepared by in vitro translation of mRNA, to immunoglobulins in the sera of subjects with IDDM. Recombinant human islet L-glutamic acid decarboxylase, isolated from Sf9 cells, is a suitable material for the large scale investigation of the utility of this enzyme in the prediction and prevention of autoimmune diabetes.

Adolescent↗
PubMed1994

Actions of phenylglycine analogs at subtypes of the metabotropic glutamate receptor family.

The functional effects of phenylglycine analogs on metabotropic glutamate receptor (mGluR) subtypes mGluR1 alpha, mGluR2 and mGluR4 were examined. (S)-4-Carboxyphenylglycine (IC50 = 65 +/- 5 microM), (R,S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 155 +/- 38 microM) and (S)-3-carboxy-4-hydroxyphenylglycine (IC50 = 290 +/- 47 microM) competitively antagonized glutamate-stimulated phosphoinositide hydrolysis in baby hamster kidney (BHK) cells stably expressing mGluR1 alpha. (S)-4-Carboxyphenylglycine and (R,S)-alpha-methyl-4-carboxyphenylglycine competitively antagonized glutamate-induced inhibition of forskolin-stimulated cAMP-formation in BHK cells stably expressing mGluR2 with IC50 values of 577 +/- 74 microM and 340 +/- 59 microM, respectively. (R,S)-4-carboxy-3-hydroxyphenylglycine, (R)-3-hydroxyphenylglycine and (S)-3-carboxy-4-hydroxyphenylglycine were agonists at mGluR2 with EC50 values of 48 +/- 5 microM, 451 +/- 93 and 97 +/- 12 microM, respectively. In parallel experiments, no activities of these phenylglycine analogs at mGluR4 were observed. The present report demonstrates that phenylglycine analogs possess differential functional activities at subtypes of the mGluR family.

Animals↗
PubMed1994

Thiol ester role in correct folding and conformation of human alpha 2-macroglobulin. Properties of recombinant C949S variant.

To determine the role of the thiol ester in the folding of human alpha 2-macroglobulin (alpha 2M) in the active conformation, we have characterized a recombinant variant of alpha 2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S alpha 2M behaves like methylamine-treated plasma alpha 2M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric alpha 2M does not require a pre-formed thiol ester. Active alpha 2M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S alpha 2M followed by thiol ester formation and a conformational change that gives the native active state.

Animals↗
PubMed1994

Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2.

Starting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3' and 5' cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences.

Amino Acid Sequence↗
PubMed1994

A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific glutamic acid decarboxylase antibodies in childhood IDDM.

Insulin-dependent diabetes mellitus (IDDM) is associated with autoreactivity against GAD but the diagnostic sensitivity (positivity in disease) and specificity (negativity in health) of isoform-specific GAD antibodies have yet to be defined in assay systems suitable for screening large number of samples. One set of IDDM patient (n = 10) and control (n = 50) standard sera were used to develop quantitative antibody assays with in vitro synthesized recombinant 35S-methionine-labelled GAD65 and GAD67, respectively, and protein A-Sepharose to separate free from antibody-bound ligand. Binding levels were not normally distributed (p < 0.0001) and therefore, the diagnostic accuracy of GAD antibodies was analysed by the ROC plots in population-based, consecutively-diagnosed, recent onset, 0-14 year-old patients (n = 105), and matched, healthy control subjects (n = 157). The ROC plots showed that the diagnostic sensitivity of GAD65 antibodies was 77% and the specificity 92% compared with 8% and 98%, respectively for GAD67 antibodies. In the IDDM sera, GAD65 and GAD67 antibodies were concordant in 7% (6 of 81) and GAD65 antibodies and ICA in 89% (72 of 81) without a correlation between the autoantibody levels. Autoantibodies to recombinant human islet GAD65 are specific and sensitive markers for childhood IDDM in this immunoassay with in vitro synthesized 35S-methionine-labelled recombinant GAD.

Adolescent↗
PubMed1994

Structure-function relationships in naturally occurring mutants of pancreatic lipase.

From primary structure comparison, the pancreatic lipase family is now divided into three subgroups: classical pancreatic lipases, pancreatic lipase-related proteins 1 (RPI) and pancreatic lipase-related proteins 2 (RP2). Among the RP2 subfamily, the guinea-pig and coypu enzymes share kinetic properties which differ from those of classical pancreatic lipases. Both enzymes display a high phospholipase activity and are not interfacially activated using a short chain triglyceride as substrate. Their activity towards insoluble triglycerides is inhibited by micellar concentrations of bile salts and is not restored by addition of colipase. These atypical kinetic properties are discussed in the light of amino acid sequence comparison between RP2 and classical pancreatic lipases, based on the closed and open conformations of the 3-D structure of human pancreatic lipase.

Amino Acid Sequence↗
PubMed1994

Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay.

Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P < 0.01). The prevalence of islet cell antibodies showed a similar pattern relative to age at onset. Because simultaneous occurrences of multiple autoimmune phenomena are common, we analyzed sera from patients with other autoimmune diseases. The frequency of GAD antibodies in sera positive for DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P < 0.025]). In conclusion, even large numbers of serum samples can now be tested for GAD65 antibodies in a relatively short time, allowing screening of individuals without a family history of IDDM for the presence of this marker.

Adolescent↗
PubMed1994

Identification of glutamic acid decarboxylase autoantibody heterogeneity and epitope regions in type I diabetes.

Glutamic acid decarboxylase (GAD) is an autoantigen of the islet cell antibodies (ICAs) present in type I diabetes. GAD autoantibodies are also found in patients with stiffman syndrome and in certain ICA-positive individuals who rarely develop diabetes on long-term follow-up. This latter subset of ICA has been termed restricted or beta-cell-specific ICA because the antibodies react with only the beta-cells of the islet. By immunoprecipitation of recombinant GAD65 and GAD67 protein and protein fragments, 83% of sera from individuals with new-onset diabetes or prediabetes (n = 30) had GAD65 autoantibodies, but only 26% had GAD67 autoantibodies. In contrast, all restricted ICA sera (n = 6) had both GAD65 and GAD67 autoantibodies. In both types of sera, the binding of GAD67 autoantibodies could be blocked by preincubation of the serum with GAD65 and GAD67, but the binding of GAD65 autoantibodies could not be blocked by preincubation with GAD67. The titer of GAD65 autoantibodies was much higher in the restricted ICA sera (titer > 1:1,000) than in the sera from individuals with new-onset diabetes or prediabetes (titer < 1:100) and was reflected by the greater amount of GAD65 protein immunoprecipitated by restricted ICA sera (2.61 +/- 1.39 U) compared with sera from individuals with new-onset diabetes (0.51 +/- 0.34 U). The restricted ICA sera immunoprecipitated equimolar amounts of GAD65 protein fragments, suggesting a non-conformational or linear epitope; epitope mapping localized the major epitope region to amino acids 361-442 and a second minor epitope region to amino acids 1-195.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase.

Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids. A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed. The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities. The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases. We propose that this deletion is directly responsible for the anomalous behavior of this enzyme. Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases.

Amino Acid Sequence↗
PubMed1993

A zinc-finger gene ZNF141 mapping at 4p16.3/D4S90 is a candidate gene for the Wolf-Hirschhorn (4p-) syndrome.

Chromosomal aneusomy is a major cause of reproductive wastage and congenital malformations in man. Zinc finger encoding genes would be good candidates for being involved in the multiple developmental defects associated with chromosomal aneusomy--by virtue of their role as transcriptional regulators, their abundance in the genome and their known association with specific developmental disorders. We have isolated and mapped a zinc finger encoding cDNA (ZNF141) of the C2-H2/KRAB subfamily to the 4p- (Wolf-Hirschhorn) syndrome (WHS) chromosome region. ZNF141 mapped to the distal end of the 2.2 Mb smallest region of deletion overlap of WHS, 300 kb from the 4p telomere on cosmid CD1 defining the anonymous locus D4S90. ZNF141 was expressed ubiquitously at low levels in the analysed tissue. The identification of a candidate gene for a chromosomal aneusomy syndrome belonging to a class of evolutionary conserved genes will provide options for studying its normal and abnormal expression during mammalian embryogenesis.

Abnormalities, Multiple↗
PubMed1993

Quantitative assay using recombinant human islet glutamic acid decarboxylase (GAD65) shows that 64K autoantibody positivity at onset predicts diabetes type.

At and before onset, most insulin-dependent diabetics (IDDM) have islet GAD65 autoantibodies (GAD65Ab). Since IDDM also occurs in older patients where non-insulin-dependent diabetes is common, we studied GAD65Ab at onset to classify diabetes type. Our quantitative immunoprecipitation assay uses recombinant human islet GAD65 stably expressed in hamster fibroblasts. Electrophoretic mobility was identical to native islet GAD65. Like native antigen, recombinant GAD65 migrated as two bands during electrophoresis, but converted to one under stronger reduction. Immunoprecipitation was linear with respect to antibody or antigen concentration. In 120 population-based diabetic patients of all ages grouped by treatment at onset and after 18 mo, GAD65Ab were present in 70% on insulin (n = 37), 10% on oral agent (n = 62, P < 0.0001), 69% changing from oral agent to insulin (n = 16, P < 0.001), and 1 of 33 controls. 65% with GAD65Ab, versus 8% without, changed from oral agent to insulin (P < 0.01). The GAD65Ab quantitative index was remarkably stable, and only 2 of 32 patients changed antibody status during follow-up. Concordance between GAD65Ab and islet cell antibodies was 93%. Quantitative correlation was approximate but significant. This highly sensitive, quantitative, high capacity assay for GAD65Ab reveals treatment requirements better than clinical criteria, perhaps guiding immunomodulatory therapy.

Adult↗
PubMed1993

Human G(olf) alpha: complementary deoxyribonucleic acid structure and expression in pancreatic islets and other tissues outside the olfactory neuroepithelium and central nervous system.

G(olf) alpha is a G-protein originally believed to mediate signal transduction exclusively within the olfactory neuroepithelium and subsequently found to be a major stimulatory G-protein in the basal ganglia. Here we present evidence that G(olf) alpha is expressed in several other tissues. The human isoform of G(olf) alpha was isolated from two human insulinoma cDNA libraries. Comparison of the human sequence with rat G(olf) alpha shows 91% nucleotide identity (within the coding region) and 99% identity at the amino acid level. Northern and reverse transcriptase-polymerase chain reaction analyses indicated that G(olf) alpha is expressed in all human insulinomas examined thus far as well as in normal pancreatic islets. G(olf) alpha mRNA was also detected in testis, retina, brain, and liver. Western blot analysis of various mouse tissues demonstrated that the level of G(olf) alpha protein in islets is lower than that in the olfactory neuroepithelium and other parts of the brain; its expression in retina, lung, and spleen was moderately higher than that in islets, and its expression in testis approached that in olfactory neuroepithelium. G(olf) alpha was also detected by immunohistochemistry in mouse islets, human insulinomas, the epithelial lining of mouse epididymis, photoreceptor cells of mouse retina, and mouse lung alveoli. These findings suggest a role for G(olf) alpha in a diverse population of cells located outside the olfactory neuroepithelium and central nervous system.

Amino Acid Sequence↗
PubMed1993

Differential expression of glutamic acid decarboxylase in rat and human islets.

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were beta-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to beta-cells, also surprisingly localized to some alpha-cells, delta-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained beta-cell specific as observed in vivo, whereas GAD67 was localized not only to the beta-cells but also in the alpha-cells and delta-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.

Animals↗
PubMed1992

Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.

From a plaque biopsy of a patient with mycosis fungoides, two different continuous cell lines were established by including both IL-2 and IL-4 in the culture medium. Both continuous cell lines appeared with characteristic chromosome markers after approximately 40 cell population doublings. The initial karyotype recognized in T cells from the skin biopsy was 46,XY and the karyotypes of the continuous cell strains were 46,XY, -18, + i(18q) and another with multiple chromosome aberrations as described in Sezary T-cell leukemia. Phenotyping with monoclonal antibodies and T-cell receptor analysis indicates that the latter cell strain represents a minority of T-cells in the plaque. Due to its many chromosomal aberrations it probably represents the malignant cell, which may be a central cell in the immune stimulation taking place in the skin.

Aged↗
PubMed1992

The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA.

The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol. Biochem. Parasitol. 27, 71-82). Analysis of the entire 8-kb variable region sequence revealed the presence of a 5.2-kb cluster of imperfect, tandemly repeated sequences, flanked by DNA of unique sequence. Both repetitive and unique DNA evolve rapidly, but comparison to the closely related strain EATRO 164 indicated that the repetitive cluster is more prone to sequence and size divergence. The variable region is transcribed into RNAs of varying lengths but appears to be devoid of genes encoding mitochondrial proteins or tRNAs, as judged from computer analysis. Moreover, genes that could encode guide RNAs involved in producing the known edited mitochondrial mRNA sequences are also absent. The repetitive DNA cluster within this region consists of 14 blocks each containing one 130 bp repeat and a variable number of 19 bp repeats. A duplicated sequence was identified (5'-GGGGTTGGTGT) which proved to be identical to the eleven 5'-terminal residues of the universal minicircle dodecamer involved in initiation of leading strand synthesis. This suggests a role for these sequences in the initiation of maxicircle DNA replication. With the data presented in this report, the nucleotide sequence analysis of the 23016 bp maxicircle of T. brucei brucei EATRO strain 427 has been completed.

Animals↗