[Treatment of hemophilia A and von Willebrand's disease with 1-deamino-8-D-arginine vasopressin (DDAVP). Hemophilia and DDAVP].
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Clinical Trials as Topic↗
Biomedical subjects
T Kristensen
Publications and source records attributed to T Kristensen.
Isolated HeLa cell nuclei synthesize meaningful DNA.
DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The results obtained indicate that the isolated nuclei make "meaningful" DNA, as judged by the melting characteristics of the hybrids between rRNA and the in vitro replicated DNA. Hybridization of the labelled DNA against rRNA fractionated by electrophoresis and blotted onto nitrocellulose verified the presence of sequences complementary to 18 S and 28 S rRNA.
Base Sequence↗
Isolation and sequence analysis of a cDNA clone encoding the fifth complement component.
We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.
Adult↗
Common evolutionary origin of alpha 2-macroglobulin and complement components C3 and C4.
A comparison of the sequence of the subunit of human alpha 2-macroglobulin (alpha 2M; 1451 amino acid residues) with that of murine complement component pro-C3 (1639 amino acid residues) reveals eight extended regions of sequence similarity. These regions contain between 19% and 31% identically placed residues and account for 75% and 67%, respectively, of the polypeptide chains of alpha 2M and pro-C3. Published sequence data for complement component C4 show that segments of this protein match well with corresponding stretches in alpha 2M and pro-C3. It is proposed that alpha 2M, C3 and C4, which all contain a unique activatable beta-cysteinyl-gamma-glutamyl thiol ester, have a common evolutionary origin and are homologous proteins. Several larger regions of low sequence similarity indicate the presence of structural domains in each of these proteins that specifically modify an underlying common gross structure. The quartets of basic residues in pro-C3 and pro-C4, at which cleavage takes place to produce the mature subunits of these proteins, and most of the residues forming the anaphylatoxin peptides of C3 and C4 (C3a and C4a) are absent in alpha 2M. In addition, C3 and C4 contain large portions, which extend beyond the COOH terminus of alpha 2M.
Amino Acid Sequence↗
Immunogenetic composition of 94 consecutive Danish families investigated with respect to bone marrow transplantation.
The immunogenetic composition of 94 patients needing bone marrow transplantation and their core families primarily investigated to select family bone marrow donors have been further analysed to test for association between disease and HLA-region markers. From this material it is shown, that in the primary immunogenetic analysis of the family, inclusion of mixed lymphocyte culture analysis increases donor possibilities by approximately 14% when a reciprocal negative MLC response and phenotypic HLA-DR compatibility are accepted as criteria for transplantation. Further, the results indicate, that no association between HLA and leukemia seems to exist.
ABO Blood-Group System↗
Micropore filters for sterile filtration may leach toxic compounds affecting cell cultures (HL 60).
Reduction in HL-60 cell growth could be traced unexpectedly to sterile filtration procedures. To circumvent this problem, 11 available brands of micropore filters (five prepacked and six to be packed and autoclaved) were investigated with the aim of finding the least toxic product. Samples of 10 ml of RPMI 1640 medium with 10% fetal calf serum added were filtered through four filters of each brand to detect even small amounts of leached toxic compounds. HL-60 cells were cultured in these filtered media for three days and the results compared with cultures using unfiltered medium. A large variation in growth inhibition was found between the filters investigated, ranging from 0% to about 90%. The growth inhibition was due to leaching of toxic compounds, as revealed by viability test, reseeding, and direct microscopy. Adsorption of essential proteins to the micropore filters was found for only one of the filters investigated in this study.
Cell Survival↗
Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments.
The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.
Amino Acid Sequence↗
Primary structure of human alpha 2-macroglobulin. IV. Primary structure of two large CNBr fragments, located in the COOH-terminal part and accounting for 337 residues.
The amino acid sequences have been determined for two CNBr fragments of human alpha 2-macroglobulin which, due to the presence of an uncleaved Hse-Thr bond, form an Mr = 40,000 fragment. These fragments are located in the COOH-terminal part of alpha 2-macroglobulin (CB21, residues 955-1185 and CB22, residues 1186-1291). CB21 contains one glucosamine-based carbohydrate group attached to Asn-14 and one internal disulfide bridge (Cys-102 bound to Cys-150). CB21 and CB22 account for 337 of the 1451 residues of the subunit of alpha 2-macroglobulin.
Amino Acid Sequence↗
Primary structure of human alpha 2-macroglobulin. V. The complete structure.
The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven intrachain disulfide bridges have been placed (Cys25-Cys63, Cys228-Cys276, Cys246-Cys264, Cys255-Cys408, Cys572-Cys748, Cys619-Cys666, Cys798-Cys826, Cys824-Cys860, Cys898-Cys1298, Cys1056-Cys1104, and Cys1329-Cys1444). Cys-447 probably forms an interchain bridge with Cys-447 from another subunit. The beta-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically regulated systems with particular reference to the complement components C3 and C4.
Amino Acid Sequence↗
Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobulin.
Human pregnancy zone protein (PZP) is a major pregnancy-associated protein. Its quaternary structure (two covalently bound 180-kDa subunits, which are further non-covalently assembled into a tetramer of 720 kDa) is similar to that of human alpha 2-macroglobulin (alpha 2M). Here we show, from the results of complete or partial sequence determination of a random selection of 38 tryptic peptides covering 685 residues of the subunit of PZP, that PZP and alpha 2M indeed are extensively homologous. In the stretches of PZP sequenced so far, the degree of identically placed residues in the two proteins is 68%, indicating a close evolutionary relationship between PZP and alpha 2M. Although the function of PZP in pregnancy is largely unknown, its close structural relationship to alpha 2M suggests analogous proteinase binding properties and a potential for being taken up in cells by receptor-mediated endocytosis. In this regard our studies indicate a bait region in PZP significantly different from that present in alpha 2M. PZP could be the human equivalent of the acute-phase alpha-macroglobulins (e.g., rat alpha 2M and rabbit alpha 1M) described earlier.
Amino Acid Sequence↗
Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase.
Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.
Adenosine Diphosphate Ribose↗
ADP-ribosylation in permeable HeLa S3 cells.
ADP-ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4-5-times greater in metaphase cells. 17-22% was in the soluble fraction which contained material released from the cells, 16-22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP-ribosylated proteins corresponding to 78 000, 54 000 and 36 000 Da. In addition metaphase cells contained several other ADP-ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32 000-39 000-Da region suggesting ADP-ribosylation of histone H1 with up to 10 residues of ADP-ribose and in the 17 000-20 000-Da region indicating ADP-ribosylation of core histones. The pattern of ADP-ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP-ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP-ribose) and oligo(ADP-ribose). The results do not lend support to a special function of ADP-ribosylated histones in the mitotic event while certain ADP-ribosylated non-histone proteins may be specific for metaphase cells.
Adenosine Diphosphate Ribose↗
Murine H-2Dd-reactive monoclonal antibodies recognize shared antigenic determinant(s) on human HLA-B7 or HLA-B27 molecules or both.
We have evaluated the serological relationships between the murine H-2Dd and human HLA molecules using four H-2Dd-reactive monoclonal antibodies (mAbs) produced in the A.BY (KbIbDb) anti-A.TL (KsIkDd) combination. In the mouse, these reagents exhibited three distinct reactivity patterns: Dd, Ks, and H-2u (mAb 81.L); Dd, H-2p, and H-2u (mAb 81.R); and Dd, Kd, H-2p, H-2u, and H-2v (mAbs 97.G and 97.H). Sequential immunoprecipitation and cross-competitive mAb binding experiments revealed that these mAbs recognized determinants in two spatially distinct polymorphic domains on the H-2Dd molecule of B10.A(5R) cells (defined by mAbs 81.L and 81.R, 97.H, and 97.G, respectively). MAbs 81.R, 97.G, and 97.H, but not 81.L, also defined an HLA-linked polymorphism in the human, the main characteristics of which can be summarized as follows: (i) on B lymphoblastoid cell lines, mAbs 81.R and 97.H bound to cells expressing the HLA-B7, HL-B27 or Bw40 cross-reacting specificities, (ii) on peripheral blood lymphocyte (PBL) panel mAb 81.R exerted C dependent cytotoxicity to 118 of 400 cells tested, including almost all HLA-B7 or HLA-B27 cells or both (r: 0.952), (iii) the expression of the 81.R cross-reacting determinant segregated in an informative family with the parental haplotype carrying the HLA-B7 allele, and (iv) mAbs 81.R, 97.G, and 97.H recognized topologically related determinants on the same class I molecule(s) of the human B lymphoblastoid cells JY (HLA-A2,2, -B7,7). These data support the view that some, but not all H-2Dd allotopes have been conserved throughout evolution and are associated in the human with the HLA-B7, -B27 cross-reacting specificities.
Animals↗
Human cytotoxic, specific anti HLA-A, -B, -C T-lymphocyte clones are OKT3 positive, but may change their OKT4 and 8 phenotypes during culture.
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Antibodies, Monoclonal↗
Intra HLA-D/DR region recombinant detected by primed lymphocyte typing (PLT).
The chromosome 6 markers, HLA-ABC, D, DR, MT, properdin factor Bf, and complement factors 2 (C2) and 5 (C4), were studied in three families, each of which included two HLA identical siblings, one or both of whom were known to be HLA-B: GLO recombinants. The families were also typed with primed lymphocyte typing (PLT) for HLA-D/DR region associated DP antigens. None of these studies gave evidence that the recombinations had occurred within the HLA region. Mixed leucocyte culture (MLC) tests within the families showed no detectable stimulation between the HLA identical siblings in two of the families, but a very weak stimulation between the HLA identical siblings (H and G) in the third family (GG). No reactive PLT reagents were generated when cells from the HLA identical siblings of the first two families were primed against each other. In contrast, priming between cells of H and G gave rise to reactive reagents. One of these (GHx), reacted with a determinant which segregated within the GG family as if child G was a paternal recombinant between the HLA-D, DR, DP, and C4 loci, on the one hand, and on the other hand one or more loci governing other HLA-D/DR region controlled lymphocyte activating determinants. This reagent was only restimulated by cells from two of 47 unrelated individuals. The other PLT reagent (HGx) did not give a clearcut pattern within the family because it was weakly positive with all family members (most of whom were D/DR2-positive) except the specific responder; in the panel it reacted with a determinant significantly associated with D/DR/DP2. Other PLT reagents could be generated within the family against lymphocyte activating determinants controlled by genes in the two paternal haplotypes telomeric to the assumed recombinational site. These reagents gave stronger reactions than the HGx and GHx reagents and reacted with two determinants in the unrelated panel strongly associated with D/DR/DP2 and D/DR/DP6, respectively. It seems likely that the GG family represents a third example of a recombination between the HLA-DR and SB loci. Our findings further support the assumption that the DR determinants may be immunodominant in lymphocyte activation.
Chromosomes, Human, 6-12 and X↗
Influence of HLA-D/DR antigen disparity in CTL generation in vitro.
This report describes the influence of HLA-D/DR antigen disparity upon the level of cytotoxicity in allogeneic in vitro cultures. Allogeneic cultures, between unrelated HLA-D/-DR full house donors, tested in CML gave three different levels of cytotoxicity, termed weak, intermediate and strong cytotoxicity. HLA-D/-DR compatibility predicts weak cytotoxicity and two HLA-B antigen incompatibility predicts strong cytotoxicity. On the contrary, HLA-A antigens have no major influence upon the strength of cytotoxicity. Accepting that the MLC/CML reaction is an in vitro parallel to the in vivo transplantation of allogeneic tissue, the observations are in accordance with the results of HLA-D/-DR matching for graft survival in human renal transplantation.
Cytotoxicity, Immunologic↗
One-way positive cellular reactions between two HLA-A, B, C, D/DR genotypically identical brothers following active allogeneic immunization.
The HLA-D/Dr region in man encodes major determinants which stimulate T lymphocytes to proliferation. The genetic organization of this region is apparently complex and is at present largely unknown. One obstacle is the scarcity and quality of available typing reagents. In an attempt to obtain high quality anti-DR sera, a series of active immunizations was performed between highly selected, healthy unrelated donors and recipients. One recipient (AR8) was immunized using cells incompatible for HLA-A2, B40 (w60), Cw3 and D/DRw6 and readily developed anti-A2 and B40 antibodies but no anti-C, CR, or other antibodies. When tested against his HLA genotypically fully identical brother using te cellular MLC, PLT, or CML techniques before immunization, results were mutually negative as expected. Following immunization, however, AR8 was able to mount MLC, PLT, and possibly CML responses against lymphocytes from the brother while the reverse combinations remained negative. When tested in the family the trait(s) thus identified seems to be maternally inherited. These results suggest the existence of minor histocompatibility determinants encoded from regions not closely linked to HLA. The brother of AR8 and the immunizing donor thus seem to share one or more determinants not possessed by AR8.
Antibodies, Anti-Idiotypic↗
The primary structure of alpha 2-macroglobulin and localization of a Factor XIIIa cross-linking site.
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Amino Acid Sequence↗