Search PubMed⌕ Search

Biomedical subjects

U Sohn

Publications and source records attributed to U Sohn.

17 recordsLinked to original sources
PubMed2000

Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I.

An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd). A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.

Animals↗
PubMed1999

DNA computing the Hamiltonian path problem.

The directed Hamiltonian path (DHP) problem is one of the hard computational problems for which there is no practical algorithm on a conventional computer available. Many problems, including the traveling sales person problem and the longest path problem, can be translated into the DHP problem, which implies that an algorithm for DHP can also solve all the translated problems. To study the robustness of the laboratory protocol of the pioneering DNA computing for the DHP problem performed by Leonard Adleman (1994), we investigated how the graph size, multiplicity of the Hamiltonian paths, and the size of oligonucleotides that encode the vertices would affect the laboratory procedures. We applied Adleman's protocol with 18-mer oligonucleotide per node to a graph with 8 vertices and 14 edges containing two Hamiltonian paths (Adleman used 20-mer oligonucleotides for a graph with 7 nodes, 14 edges and one Hamiltonian path). We found that depending on the graph characteristics such as the number of short cycles, the oligonucleotide size, and the hybridization conditions that used to encode the graph, the protocol should be executed with different parameters from Adleman's.

Algorithms↗
PubMed1999

Generation and chromosome mapping of expressed sequence tags (ESTs) from a human infant thymus.

In an effort to identify novel genes that are expressed differentially in an infant thymus, we constructed an oligo-d(T) primed cDNA library from a human infant thymus followed by single-run partial sequencing to generate expressed sequence tags (ESTs). Characterization of more than 1400 sequences enabled us to convert human thymus transcripts into 1223 useful ESTs. These ESTs consisted of 613 (50.1%) showing homology to known human genes, 51 (4.2%) matching to genes from other species, 289 (23.6%) matching ESTs of unknown functions, and 182 (14.9%) being novel transcripts. The expression profile of an infant thymus features a high number of genes related to cell division-DNA synthesis and gene-protein expression, indicating the active growth stage of an infant thymus. To identify the chromosomal localization of 43 thymus ESTs, PCR-based mapping was performed using a human-rodent somatic cell hybrid or radiation hybrid mapping panel. The results indicated that several novel genes were determined to be located in the vicinity of previously mapped disease loci; histidinemia loci, plasminogen Tochigi disease loci, Ehlers-Danlos syndrome, hypertriglyceridemia, thyroid resistance locus, ocular albinism, galactosemia, porphyria variegata, Charcot-Marie-tooth disease, FEOM (fibrosis of extraocular muscles), Prader-Willi syndrome.

Animals↗
PubMed1997

Cloning and expression of human mitotic centromere-associated kinesin gene.

The human homologue of the hamster mitotic centromere-associated kinesin (HsMCAK) gene containing a central type motor domain was isolated from a Jurkat T-cell derived cDNA library. The HsMCAK gene has a predicted 723 amino acid open reading frame, encoding a 81 kDa protein that shares 79.2% homology with hamster MCAK. Unstimulated T lymphocytes contained no detectable HsMCAK-specific mRNA. Activation of resting T-cells by immobilized anti-CD3 resulted in the expression of a 2.9-kb transcript during the S phase of the cell cycle. The TPA-induced monocytic differentiation of U937 which also results in growth-arrest abruptly downregulates the expression of HsMCAK. Removal of TPA restored the growth of the cell through the retrodifferentiation process and the subsequent expression of HsMCAK. HsMCAK is expressed in tissues containing dividing cells, such as thymus, testis, small intestine, colon (mucosal lining), and placenta. These results suggest that the expression of HsMCAK is first detected in early S phase to support the proliferative response and is strictly regulated at the transcriptional level.

Amino Acid Sequence↗
PubMed1996

Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter.

The intracellular pathways responsible for maintenance of tone in the lower esophageal sphincter (LES) are not well understood. We show that the protein kinase C (PKC) antagonists (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride) and calphostin C reduce spontaneous resting tone in LES muscle strips, whereas the calmodulin antagonist N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide hydrochloride) has no effect, which suggests that LES tone is maintained by a PKC-mediated mechanism. In addition, U73122, an inhibitor of phosphatidylinositol-4,5-bisphosphate (PIP2)-specific phospholipase C, and D609, an inhibitor of phosphatidylcholine-specific phospholipase C, reduced diacylglycerol formation and LES tone in a concentration-dependent manner. Finally diacylglycerol levels and PKC activity were reduced during relaxation of the LES induced by the inhibitory neurotransmitter vasoactive intestinal peptide. These data suggest that resting LES tone is associated with elevated diacylglycerol levels and PKC activity, which are reduced during relaxation. Diacylglycerol is derived from at least two different sources. Hydrolysis of PIP2 by PIP2-specific phospholipase C produces equimolar amounts of inositol 1,4,5-triphosphate and diacylglycerol, which may interact synergistically to activate PKC and develop tone. Furthermore, PKC-mediated contraction may be augmented by additional diacylglycerol production arising from the hydrolysis of phosphatidylcholine by phosphatidylcholine-specific phospholipase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1985

Genes encoding the T-cell antigen receptor.

The search for the elusive and controversial T-cell antigen receptor is over. It is now clear that gene complexes for both alpha and beta chains are distinct from those for immunoglobulin genes. They are, however, related to Ig genes as well as to other class I and class II major histocompatibility complex (MHC) gene products. Therefore, they belong to the immunoglobulin super gene family.

DNA, Circular↗
PubMed1983

7S-K nuclear RNA from simian virus 40-transformed cells has sequence homology to the viral early promoter.

Previous findings in our laboratory have identified a specific small nuclear RNA (7S-K) that promotes transcription initiation by RNA polymerase II in isolated mammalian nuclei. The present study was designed to investigate the homology between 7S-K RNAs and host and viral sequences in simian virus 40 (SV40)-transformed and in untransformed mouse 3T3 cells, with the object of testing the hypothesis that these RNAs take part in the transcription initiation complex by base pairing to promoter/enhancer regions of active genes. DNA . RNA hybridization experiments, using either Southern or RNA blotting techniques, indicated that both 7S-K and 7S-L RNAs hybridize to midrepetitive fractions of the mouse genome. However, only 7S-K RNA from transformed cells hybridized to SV40 DNA. Restriction mapping and nuclease S1 treatment of the hybridized region of SV40 yielded a 45-nucleotide-long hybrid duplex. Partial sequence analysis of the 5' end of the DNA in this duplex revealed sequence homology with the 21-base-pair repeat sequence, identified as the SV40 early promoter. Because the viral early gene is expressed in transformed cells, we conclude that 7S-K RNAs in these cells contain a species that has sequence homology to a promoter of an active gene. Taking these results together with previous ones, we postulate that the observed stimulatory activity of 7S-K RNA on transcription initiation is due to its recognition of promoter sequences, either to facilitate the formation or as part of the transcription initiation complex.

Animals↗
PubMed1979

The role of H-2 and Ia antigens in graft-versus-host reactions (GVHR). Presence of host alloantigens on donor cells after GVHR and suppression of GVHR with an anti-Ia antiserum against hose Ia antigens.

By using an indirect immunofluorescence technique, the presence of host cell derived H-2K, H-2D, and Ia alloantigens on donor cells recovered from recipient spleens after a graft-versus-host response (GVHR) was demonstrated. Mapping studies indicated that only host K, D, and I-A region gene products could be identified on the donor cells. Host I-E/C- and I-J-subregion products were not absorbed by donor cells. Treatment of activated donor cells with anti-Ly sera plus C' revealed that donor cells carrying host Ia antigens have a Ly-1+,2-,3- phenotype, whereas donor cells carrying H-2K and H-2D host antigens have a Ly-1-,2+,3+ phenotype. A GVHR that resulted from only an I-region incompatibility was suppressed by the injection of recipient mice with an anti-Ia antiserum directed against self Ia antigens. The degree of suppression was proportional to the amount of anti-Ia antiserum administered.

Animals↗
PubMed1979

In vitro analysis of allogeneic lymphocyte interaction. III. Generation of a helper allogeneic effect factor (AEF) across an I-J subregion disparity.

Allogeneic effect factors (AEF) were produced across an I-J subregion incompatibility. The helper activity of these AEFs is H-2 restricted since they help B cells only of the stimulator haplotype and of other haplotypes that carry the same I-J subregion gene(s) as the stimulator haplotype. Immunoadsorption studies demonstrate that they consist of I-J determinants derived initially from the GVHR host and MLR stimulator cells and not the GVHR donor and MLR responder cells used to generate AEF. It is postulated that the genetic restriction of AEF helper activity is mediated in part by the ability of the GVHR activated donor T cells to acquire, in vivo, recipient T cell and/or macrophage derived I-J determinants. Cellular adsorption studies indicate that AEF helper activity may be adsorbed by B cells, but neither T cells nor macrophages, of the stimulator haplotype. The results suggest that an I-J-positive AEF interacts with an I-J subregion controlled complementary recognition structure on a target B cell and, after antigenic stimulation, activates that B cell to IgG antibody synthesis.

Animals↗
PubMed1979

In vitro analysis of allogeneic lymphocyte interaction. IV. Dual recognition of B cell-associated Mls locus and I-region determinants by a helper allogeneic effect factor (AEF) generated across a minor H locus disparity.

A helper allogeneic effect factor (AEF) was produced across an incompatibility at the minor histocompatibility loci. This AEF is genetically restricted in its activity since it helps B cells only of the stimulator haplotype and of haplotypes that share both an Mls and I-region identity with the stimulator haplotype. The I-region genes involved here map to the I-A and/or I-C subregions. An anti-LyM immunoadsorbent column but neither an anti-H-2 nor an anti-Ia column absorbed AEF helper activity. It is suggested that the activation of T helper cells by a positive allogeneic effect across a minor H locus difference and their genetically restricted interaction with allogeneic B cells may in part be due to the acquisition by alloactivated T cells of stimulator cell-derived LyM and/or Mls determinants. The data presented indicate that helper T cells recognize Mls locus alloantigens in the milieu of self Ia antigens.

Animals↗
PubMed1977

Development of methods for the quantitative in vitro analysis of androgen-dependent and autonomous Shionogi carcinoma 115 cells.

Androgen-dependent and androgen-independent (autonomous), cloned, cultured cell lines of the androgen-dependent mouse mammary adenocarcinoma, Shionogi carcinoma 115, have been established. Growth of the dependent cells requires the presence of androgen, provided they are growth in suspension culture in medium containing dextran-charcoal-treated fetal calf serum. The growth rate of autonomous cells in the presence or absence of DHT is similar to that of dependent cells grown in its presence. An agar culture method has been developed that enables the proportion of dependent and autonomous cells in mixed populations to be determined. Autonomous cells appear in dependent clones, and their frequency increases with increasing time of subculture. Dependent cells form tumors preferentially in male animals and dependent cell cytosols contain significant amounts (approximately 300 femtomoles per mg protein) of a specific androgen-binding macromolecule. Autonomous cells formed tumors equally well in both male and female mice, and autonomous cell cytols contain very low levels (less than or equal to 7 femtomoles per mg protein) of the specific androgen-binding macromolecule(s). These studies delineate a system which can be used to investigate the mechanism of steroid hormone-dependent and autonomous tumor growth, and the transitions between the hormone-dependent and autonomous states.

Animals↗
PubMed1975

[Problems of hepatitis in dialysis patients, technical personnel and their relatives].

In a centre for dialytic treatment of the district Karl-Marx-Stadt during 16 months patients and staff underwent a continuous clinico-epidemiological observation. By means of determinations of serum enzymes and Au-antigen-investigation regularly carried out in these cases the special risk of hepatitis could be confirmed. Hygienic-antiepidemic and organization measures are discussed and recommendations are given concerning the prophylactically more aimed course. Further investigations are necessary in as far as certain basic diseases (i.g., renal insufficiency) have an influence on the Au-antigen-persistence by impairment of the immune system and which epidemiological effects may result from this.

Female↗
PubMed1975

DNA: DNA hybridization studies in black flies.

The phylogenetic relationships of six species of black flies were investigated using the hybridization of iodinated unique DNA sequences. The thermal stability of these labelled hybrids allowed the construction of a phylogenetic tree based on base mismatch between chains of the heterologous duplex.

DNA↗
PubMedDate not supplied

Organization and sequences of the variable, joining and constant region genes of the human T-cell receptor alpha-chain.

An essential property of the immune system is its ability to generate great diversity in antibody and T-cell immune responses. The genetic and molecular mechanisms responsible for the generation of antibody diversity have been investigated during the past several years. The gene for the variable (V) region, which determines antigen specificity, is assembled when one member of each of the dispersed clusters of V gene segments, diversity (D) elements (for heavy chains only) and joining (J) segments are fused by DNA rearrangement. The cloning of the beta-chain of the T-cell antigen receptor revealed that the organization of the beta-chain locus, which is similar to that of immunoglobulin genes, is also composed of noncontiguous segments of V, D, J and constant (C) region genes. The structure of the alpha-chain seems to consist of a V and a C domain connected by a J segment. We report here that the human T-cell receptor alpha-chain gene consists of a number of noncontiguous V and J gene segments and a C region gene. The V region gene segment is interrupted by a single intron, whereas the C region contains four exons. The J segments, situated 5' of the C region gene, are dispersed over a distance of at least 35 kilobases (kb). Signal sequences, which are presumably involved in DNA recombination, are found next to the V and J gene segments.

Amino Acid Sequence↗