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Biomedical subjects

G Ehrlich

Publications and source records attributed to G Ehrlich.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.

Amino Acid Sequence↗
PubMed1992

Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center.

Amino Acid Sequence↗
PubMed1992

Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries.

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1990

Immunodetection of human T-cell lymphotropic virus type I core protein in biological samples by using a monoclonal antibody immunoassay.

A monoclonal antibody-based enzyme immunoassay (EIA) has been developed for detection of human T-cell lymphotropic virus type I (HTLV-I) core protein. The monoclonal antibody (clone 6.11) specifically recognizes the p19 gag gene-encoded protein of the virus. The EIA was over 100 times more sensitive than reverse transcriptase measurement and was capable of responding to less than 500 pg of whole-virus lysate. The assay exhibited type specificity in that HTLV-II antigens failed to produce a positive signal. In addition, a panel of other viruses demonstrated no antigenic cross-reactivity. These included herpesviruses, measles virus, human immunodeficiency viruses, and others. Viral p19 was followed during the course of density gradient ultracentrifugation in the presence of detergent, where it was noted to associate with viral membrane proteins. In comparison, reverse transcriptase activity localized in fractions of higher density containing envelope-free cores. Of clinical interest, the EIA was used to detect HTLV-I antigen in the viral cultures of patients with HTLV-I-associated myelopathies and from symptom-free individuals with proviral integration.

Antibodies, Monoclonal↗
PubMed1990

Acetylcholinesterase and butyrylcholinesterase genes coamplify in primary ovarian carcinomas.

The genes for acetylcholinesterase (ACHE) and butyrylcholinesterase (CHE) are expressed in multiple tumor tissues, including ovarian carcinomas. Both CHE and ACHE genes coamplify in leukemias. To examine the relationship of gene amplification to the expression of these genes in tumors, ACHE and CHE genes and their expression were studied in primary ovarian carcinomas. DNA blot hybridization demonstrated a significant amplification and mutagenesis of both genes in 6 of 11 malignant tumors studied. This was greater or of the same order of magnitude as the amplification of the oncogenes c-rafi, v-sis, and c-fes in these tumors. No amplification was found in normal ovarian tissues or benign ovarian cysts. Xenopus oocyte microinjections, blot and in situ hybridizations, and immuno- and cytochemical staining revealed translatable CHEmRNA and its active protein product in discrete tumor foci. The frequent coamplification in ovarian carcinomas of ACHE and CHE genes implicates cholinesterases in neoplastic growth and/or proliferation.

Acetylcholinesterase↗
PubMed1988

Detection of human T-cell lymphoma/leukemia virus type I DNA and antigen in spinal fluid and blood of patients with chronic progressive myelopathy.

The presence of antibodies to human T-cell lymphoma/leukemia virus Type I (HTLV-I) has been associated with chronic progressive myelopathy. We attempted to isolate the virus from the blood and spinal fluid of patients with chronic progressive myelopathy and to define the clinical, radiologic, and electrophysiologic features of this disease. Ten of 13 patients from tropical countries and 2 of 8 from the United States had serum antibodies to HTLV-I. The virus was detected in cultures of peripheral-blood lymphocytes from three of seven patients by means of Southern blot hybridization. Using a sensitive in vitro enzymatic gene-amplification technique, we detected HTLV-I sequences in fresh peripheral-blood mononuclear cells of all of 11 patients tested who were positive for the antibody, and in cell cultures of the spinal fluid from 3 of the 11 tested. Magnetic resonance imaging of the cranium revealed periventricular lesions in the white matter of 3 of the 12 antibody-positive patients. Five of these patients had mild axonal sensorimotor polyneuropathy, and one had bilateral lumbar radiculopathy. Visual evoked potentials were abnormal in three seropositive patients, and brain-stem evoked responses were abnormal in two. The detection of the DNA and proteins of HTLV-I strengthens the proposition that this virus is involved in the pathogenesis of a subset of cases of chronic progressive myelopathy.

Adult↗
PubMed1988

The short arm of chromosome 11 is a "hot spot" for hypermethylation in human neoplasia.

Inactivation of normally expressed genes may play a role in the formation and/or progression of human cancers. Methylation of cytosine in DNA could potentially participate in such alterations of gene expression. Abnormalities in DNA methylation are a consistent feature of human neoplasms, and we now show that these include not only previously recognized widespread genomic hypomethylation, but also regional increases in gene methylation. A hot spot for abnormal methylation of C + G-rich areas has been detected on the short arm of chromosome 11 in an area known to harbor tumor suppressor genes. This change occurs consistently in common forms of human cancer and appears early during the transformation of cells with viruses including members of the human T-cell leukemia (HTLV) family. Furthermore, in one chromosome 11 gene examined, calcitonin, the increased methylation in somatic tumor cells coincides with the presence of an "inactive" chromatin pattern in the transcriptional regulatory area. The increased regional DNA methylation demonstrated may then participate in or mark chromosomal changes associated with gene inactivation events that are central to the genesis and/or progression of human cancers.

Calcitonin↗
PubMed1988

Characterization of a sequence of human T cell leukemia virus type I from a patient with chronic progressive myelopathy.

DNA from peripheral blood mononuclear cells of individuals with chronic progressive myelopathy (CPM) were extensively analyzed for the presence of human T cell leukemia virus (HTLV) type I-like sequences by using the polymerase chain reaction. The DNA samples were amplified with oligonucleotides from three separate regions of HTLV viral genomes. A portion of the amplified viral genome from a representative patient was sequenced after molecular cloning into bacteriophage M13. Sequence data indicate that HTLV type I and not a related virus is associated with CPM.

Aged↗
PubMed1988

Enzymatic amplification of HTLV-I viral sequences from peripheral blood mononuclear cells and infected tissues.

Human T-cell lymphotropic virus type I (HTLV-I) and human T-cell lymphotropic virus type II (HTLV-II) have been associated with adult T-cell leukemia/lymphoma (ATL) and a rare T-cell variant of hairy cell leukemia, respectively. Direct detection of viral nucleic acid in peripheral blood lymphocytes (PBLs) and infected tissues in carrier patients and those with chronic disease has proven refractory due to viral transcriptional dormancy and the small number of infected cells present. The investigators report here the successful application of the DNA amplification procedure, termed PCR, to the detection of these human oncoviruses. Judicious selection of specific oligonucleotides for primers and probes provides type-specific and simultaneous detection of these two retroviruses. The ability to amplify and detect highly conserved regions of these medically relevant viruses may facilitate the identification of, as yet, uncharacterized retroviruses.

Base Sequence↗
PubMed1987

Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection.

Human immunodeficiency virus (HIV) has been associated with acquired immunodeficiency syndrome and related disorders. Assays to detect antibodies to HIV proteins have been developed and used to screen sera for the identification of individuals who have been exposed to the virus. Although these serological tests have significant sensitivity and specificity for detecting exposure to the virus, they do not provide direct identification of HIV. We report here the application of recently developed nucleic acid amplification and oligonucleotide-based detection procedures for the identification of HIV sequences in established infected cell lines and in cells cultured from infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Specific adsorption of HTLV-I to various target human and animal cells.

In this report, we describe a flow cytometric analysis of HTLV-I specific binding to fresh and cultured cells on a single cell basis. This assay uses rhodamine hydrocarbon tagged, purified HTLV-I virions according to the procedure originally described for avian retroviruses. Successful HTLV-I transmission was detected by analysis of integrated HTLV-I DNA, virion-associated reverse transcriptase, and/or intracellular HTLV-I core antigen p19 expression. Only a specific virus-cell interaction was detected because nonrhodamine-tagged homologous virus or related HTLV-II interfered with tagged HTLV-I binding. In contrast, an unrelated, nonlabeled animal retrovirus was unable to block tagged HTLV binding. Of the cell lines tested, 2 nonlymphoid mammalian and 3 human lymphoid bound significantly high to moderate levels of HTLV-I-tagged virions. The other three human lymphocyte cell lines were insensitive to HTLV-I adsorption. A direct correlation was observed between HTLV-I binding sites and infectivity of human lymphoid cells alone and not other nonlymphoid animal cells. Fresh normal human mononuclear cells bound low levels of HTLV-I virions. As expected, T lymphocytes demonstrated more binding than did the non-T cell population. Enhancement of HTLV-I cell binding in a subpopulation of mononuclear target cells was achieved with phytohemagglutinin (PHA) activation and interleukin 2 (IL2) stimulation, which correlates well with previously published infectivity studies.

Cell Line↗
PubMed1986

Can a diagnosis be made in retrospect? The case of Desiderius Erasmus.

Review of data in very old charts sometimes brings new insights and perspectives, and suggests new hypothesis for illnesses of patients long deceased. Such applications can be made if enough information is available, e.g., autobiographical letters, skeletal remains, pictorial representations. Finally the process must be discussed in the context of the major disease processes occurring at that time. When such an approach was applied to Erasmus (Rotterdam c 1466--Basle 1536), the differential diagnosis included gout, syphilitic arthritis and enteric rheumatism.

Arthritis↗
PubMed1984

Investigations on the age-dependence of T-lymphocyte subpopulations and lymphocyte reactivity.

The percentage T-lymphocyte subpopulations were determined with the aid of radioactively-labelled monoclonal antibodies to the antigens of total T-lymphocytes and to suppressor T-lymphocyte surface antigens and the responsiveness of the lymphocytes in the presence of ConA and PHA was investigated in groups of 8 subjects aged either between 20 and 30 years or over 75 years. It was found that there was no difference with respect to the response of the lymphocytes to the plant mitogens ConA and PHA. The percentage of suppressor T-lymphocytes, on the other hand, was significantly reduced in the older subjects, whilst the percentage of total T-lymphocytes did not differ significantly from the percentage in younger subjects. These results are discussed in connection with published data.

Adult↗
PubMed1984

Efficient transformation of previously activated and dividing T lymphocytes by human T cell leukemia-lymphoma virus.

Modifying previously reported techniques, we attempted to increase the efficiency of human T cell leukemia-lymphoma virus (HTLV) transformation of human T lymphocytes. Lethally irradiated donor cells (DCs) were cultured with target mononuclear cells (TMCs). DCs included ten HTLV+ T cell lines with varying degrees of virus expression or seven cell lines that do not express HTLV. TMCs were prepared from 20 cord and 16 adult peripheral blood samples, including eight patients with acquired immunodeficiency syndrome (AIDS). TMCs were either added directly to the DCs or were first stimulated with phytohemagglutinin (PHA) (5 micrograms/mL) and grown in T cell growth factor (TCGF) prior to exposure to DCs. The presence of integrated HTLV proviral DNA in the transformed cells was determined by dot blot hybridization, utilizing a cloned probe to the HTLV-I genome. HTLV production by transformed TMCs was assessed for HTLV p19, reverse transcriptase, and virus particles. No transformation occurred with T cell donor lines that do not express HTLV. Low virus expressor DCs could only, with rare exception, transform preactivated TMCs. High-titer virus-producing DCs could transform activated and nonactivated cord blood cells and activated adult TMCs. Only MT-2 could routinely transform nonactivated normal adult and activated AIDS TMCs. HUT 102 B2 could transform only one activated AIDS sample, the cells of which initially expressed HTLV-like proteins and virions. Transformed cell lines contained subsets of mature T lymphocytes with variable HTLV expression. Prior activation and culture of the T lymphocytes increases the probability and rate of transformation by HTLV, allowing for biologic detection of low HTLV-producing cells and for in vitro expansion of T lymphocyte subsets from selected patients.

Acquired Immunodeficiency Syndrome↗
PubMed1984

[Intestinal invagination in adulthood].

Intussusception in adults is rare. Symptoms are often uncharacteristic. Therefore diagnosis is difficult. Diagnosis can be confirmed by radiologic and endoscopic measures. Surgical treatment consists in the resection of the struck part of the intestine according to the principles of tumor surgery. The danger of perforation rises if desinvagination is tried. It is reported about 11 own cases in which 7 neoplasms were the cause of intussusception. Two patients died of aspiration pneumonia and intracerebral hemorrhage caused by rupture of an aneurysm of the cerebri media artery.

Adolescent↗