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M Eberle

Publications and source records attributed to M Eberle.

At least 19 recordsLinked to original sources
PubMed1997

Identification of a new HLA-B*08 variant, HLA-B*0804.

The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA-B*08 variant, HLA-B*0804, found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B*51 in JH and HLA-B*4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B*08 variant, HLA-B*0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B*0804 differed from HLA-B*0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR-SSP. This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles.

Amino Acid Sequence↗
PubMed1997

HLA-B typing by allele separation followed by direct sequencing.

Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous molecular typing method for the alleles at this locus. Here we describe a technique for the direct sequencing of HLA-B alleles. Initially, HLA-B alleles were PCR-amplified after locus-specific reverse transcription of RNA. Alleles were then separated using denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplified and directly sequenced. The derived sequences were aligned to a database of published HLA-B sequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 known HLA-B alleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE-separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood samples from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities.

Alleles↗
PubMed1997

Further diversification of the HLA-B locus in Central American Amerindians: new B*39 and B*51 alleles in the Kuna of Panama.

Several new HLA-B locus alleles have been discovered in South American Amerindians. By contrast, analysis of the MHC class I alleles of North American native populations has revealed few new HLA-B alleles. This suggests that the HLA-B locus is evolving rapidly in South American populations. Here we describe the HLA-B locus alleles present in individuals from a Central American tribe, the Kuna of Panama. Using a sequence-based typing technique that separates alleles by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we determined the HLA-B alleles from eight Kunas. Two of the HLA-B alleles present in the Kuna have been previously described in other South American Amerindian populations; one allele has been characterized in a Mexican-American. We characterized two new HLA-B alleles in the Kuna, HLA-B*3911 and HLA-B*5110. HLA-B*3911 differed from HLA-B*3905 by only a single nucleotide substitution in exon 3. This substitution resulted in an amino acid replacement of leucine by arginine at residue 156 in the alpha 2 domain. Such a change may affect the repertoire of peptides that are bound by this molecule. HLA-B*5110 differed significantly from other HLA-B*51 alleles in that it is the result of an unusually large intra-locus recombination event of minimally 216 nucleotides. This recombination results in an allele that is part HLA-B*51 and part HLA-B*40. Thus, more dramatic recombination events may also play a role in the rapid evolution of the HLA-B locus in Amerindians.

Alleles↗
PubMed1994

Differential polymerase chain reaction assay of cyclin D1 gene amplification in esophageal carcinoma.

The cyclin D1 gene, located on chromosome 11q13, is frequently rearranged in parathyroid neoplasms and amplified in some carcinomas of other organs. Recent studies have detected amplification of cyclin D1 and other markers on chromosome 11q13 (evaluated by Southern or slot blot assays) in approximately 25-50% of squamous cell carcinomas of the esophagus and noted that amplification was associated with lessened survival time. We applied the technique of differential polymerase chain reaction to the evaluation of cyclin D1 gene amplification in squamous cell carcinomas of the esophagus. Cyclin D1 was found to be amplified in 10 of 45 (22%) primary tumors and three of 12 (25%) lymph node metastases. Lymph node metastases tended to be more common in patients with cyclin D1 amplification (70%) than in those without amplification (37%). In 36 patients with follow-up, cyclin D1 amplification was associated with decreased 1 year survival (28% vs. 59%). Cyclin D1 gene amplification in esophageal carcinomas can be evaluated by differential polymerase chain reaction and may provide useful prognostic information.

Base Sequence↗
PubMed1993

Prevalence of human herpesvirus 6 variant A and B infections in bone marrow transplant recipients as determined by polymerase chain reaction and sequence-specific oligonucleotide probe hybridization.

An oligotyping methodology was devised by using the polymerase chain reaction and sequence-specific oligonucleotide probe hybridization in order to discriminate the A and B variants of human herpesvirus 6 (HHV-6). Comparative DNA sequence analysis of portions of the U1102 (variant A) and Z29 (variant B) genomes revealed polymorphic regions which allowed for the synthesis of variant-specific and consensus oligonucleotide probes. These probes were found to hybridize exclusively to their respective HHV-6 variants. This strategy was then further tested by evaluating 16 clinical isolates derived from patients undergoing bone marrow transplantation to determine the subtype prevalence of HHV-6 infection in these patients. All clinical isolates were documented to be of variant B, indicating that the majority of bone marrow transplantation patients may be preferentially infected with this HHV-6 subtype. This oligotyping strategy may be useful in defining the relative prevalence of HHV-6A and HHV-6B infections in patient populations potentially at risk for HHV-6 disease.

Adult↗
PubMed1992

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.

Actins↗
PubMed1992

Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan.

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.

Acetylglucosaminidase↗
PubMed1992

Molecular analysis of HLA-DRB1*08/12 alleles: identification of two additional alleles.

A nonradioactive oligotyping method that takes advantage of selective amplification using the polymerase chain reaction (PCR) and oligonucleotide probe hybridization was developed to distinguish all reported HLA-DRB1*08/12 alleles. Selective amplification was achieved using a primer complementary to the sequence encoding YSTGECY at positions 10-16 in the first hyperpolymorphic region (HPMR). This selective amplification of the HLA-DRB1*08/12 subset of alleles provides a refinement in HLA oligotyping that permits unambiguous oligotyping of many heterozygotes that cannot be resolved using less selective amplification alternatives. The amplified DNA was hybridized with a panel of then digoxigenin-labeled probes to resolve oligotypes that correspond to all reported HLA-DRB1*08/12 alleles. Oligotyping of HLA-DRB1*08/12 samples revealed two previously unknown HLA-DRB alleles. One allele, DRB1*0805, differs from DRB1*0801 by a leucine to alanine substitution at position 74. This allele is of particular interest because it is very similar to HLA-DRB1*08 alleles (YSTGECY and lack of an associated HLA-DRB3 gene), but it lacks leucine at position 74, which is characteristic of all previously reported DRB1*08 alleles. The second HLA-DRB1*08 allele, DRB1*0804, differs from DRB1*0802 by a glycine to valine substitution at position 86.

Alleles↗
PubMed1991

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils.

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.

Calcium↗
PubMed1991

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts.

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3' deleted by cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5'----3' as well as 3'----5' RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

Bacillus thuringiensis↗
PubMed1990

Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils?

Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated [32P]PIP3 levels decayed toward initial values. However, recovery of [32P]PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of [32P] PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.

Actins↗
PubMed1989

Codon usage in plant genes.

We have examined codon bias in 207 plant gene sequences collected from Genbank and the literature. When this sample was further divided into 53 monocot and 154 dicot genes, the pattern of relative use of synonymous codons was shown to differ between these taxonomic groups, primarily in the use of G + C in the degenerate third base. Maize and soybean codon bias were examined separately and followed the monocot and dicot codon usage patterns respectively. Codon preference in ribulose 1,5 bisphosphate and chlorophyll a/b binding protein, two of the most abundant proteins in leaves was investigated. These highly expressed are more restricted in their codon usage than plant genes in general.

Amino Acid Sequence↗
PubMed1980

Partial ciliary process laser photocoagulation in the management of glaucoma.

partial ciliary processes argon laser photocoagulation (PCLP) in the management of glaucoma has been the subject of a carefully conducted, prospective 5+-year clinical trial in 22 eyes of 20 patients. The mean intraocular pressure (IOP) +/- SD was significantly lower (15.5 +/- 3.6 mm Hg) than before the procedure (29.9 +/- 10.1 mm Hg; 0.001 less than P less than 0.005, paired t-test). The mechanism of intraocular pressure reduction is due mainly to the reduction in the rate of aqueous humor formation with a mean outflow facility (C-value) +/- SD of 0.12 +/- 0.11 before and 0.12 +/- 0.06 microliter/min/mm Hg after the procedure. This new procedure, done on an out-patient basis, shows promise as a convenient, low-risk, and useful alternative procedure in selected glaucoma cases that are poorly controlled by medical or surgical measures. This new approach may also be used as an adjunct in the medical and/or surgical management of aphakic and ciliary block glaucoma.

Adult↗
PubMed1975

The long-acting Ocusert-pilocarpine system in the management of glaucoma.

The Ocusert-pilocarpine devides with seven days' therapeutic duration were compared with pilocarpine eye drops for efficacy and ocular tolerance in the management of glaucoma in 16 patients. Magnitude of pressure reduction with release rates of 20 mcg. and 40 mcg. of pilocarpine per hour were as effective in the treatment of glaucoma as 2 per cent and 4 per cent pilocarpine eye drops, respectively. Ocular tolerance and retention in the human eyes were good. There were no appreciable side effects other than the occasional sudden leakage phenomenon encountered. Further refinement of the Ocusert-pilocarpine device to reduce the incidence of leakage would not only ensure precision in the rate of drug release, but would also expand its clinical application.

Acetazolamide↗
PubMedDate not supplied

28-day inhalation study of a cross-linked polyacrylate superabsorbent in the hamster.

Male and female Syrian Golden hamsters (20 of each sex) breathed respirable cross-linked polyacrylate dust (0.3, 1.0 or 10 mg m-3) for 6 h a day, 5 days a week for 28 exposures. More than 99% of the particles were 3-4 microns or smaller. No differences in overall health or body weight were observed among the groups, nor were there deaths during exposure or during the post-exposure period. In animals killed within 24 h after the final exposure, foreign material was detected in the lungs in a dose-related manner. In animals examined 4 weeks post-exposure, much of this material had been cleared from the lungs. There was a significant alveolar macrophage and a non-significant inflammatory cell response following exposure; both responses appeared to decrease during the post-exposure period. There was no evidence of fibrosis in the low- or medium-exposure groups after the exposure or post-exposure periods. In the high-exposure group there was equivocal evidence of fibrosis in one set of lungs at the end of the exposure and in another set after the post-exposure period. These results indicate that subacute exposure of Syrian Golden hamsters to low concentrations of cross-linked polyacrylate dust in particle sizes capable of reaching deep lung structures does not cause pulmonary fibrosis in the hamsters and that resolution of the cellular response induced by these particles occurs. These results also indicate that the test material was cleared from the lung following termination of exposure.

Absorption↗