A monoclonal antibody fluorescent polarization immunoassay for cyclosporine.
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Antibodies, Monoclonal↗
Biomedical subjects
E Simpson
Publications and source records attributed to E Simpson.
A glycophospholipid anchor is required for Qa-2-mediated T cell activation.
A number of lymphocyte surface proteins are anchored in the cell membrane by glycophosphatidyl inositol (known as GPI) linkages instead of hydrophobic protein domains. Treatment of mouse T lymphocytes with antibodies specific for two such proteins, Thy-1 and Ly-6, are known to induce proliferation. We have found that antibodies specific for Qa-2, a GPI-anchored class I histocompatibility antigen, can also activate mouse T cells. To determine whether the GPI-anchor is important for this pathway of cell activation, we produced transgenic mice expressing either normal GPI-anchored Qa-2, or Qa-2 molecules with a membrane-spanning protein domain derived from H-2. Our studies show that only lymphocytes from transgenic mice carrying GPI-anchored forms of Qa-2 can be activated in vitro by Qa-2-specific antibodies. We also show that transgenic mouse T cells expressing a GPI-anchored form of H-2Db can be activated by anti-H-2Db antibodies. These results strongly indicate that the GPI-anchor is critical for this pathway of T cell activation.
Animals↗
Antistasin, a leech-derived inhibitor of factor Xa. Kinetic analysis of enzyme inhibition and identification of the reactive site.
Antistasin is a 119-amino acid protein isolated from the salivary glands of the Mexican leech Haementeria officinalis. The determination of the primary structure of antistasin revealed that the protein is highly disulfide-bonded with a 2-fold internal homology. Antistasin exhibits a potent anticoagulant activity purportedly due to the selective inhibition of Factor Xa (Tuszinsky, G. P., Gasic, T. B., and Gasic, G. J. (1987) J. Biol. chem. 262, 9718-9723). In the present study a detailed kinetic analysis of the inhibitory interaction between antistasin and Factor Xa was performed. In addition, the specificity of antistasin was examined by testing its ability to inhibit a variety of serine proteinases. Utilizing purified antistasin and a tripetidyl p-nitroanilide substrate, antistasin was shown to act as a reversible inhibitor of Factor Xa which exhibits slow-tight binding kinetics. Antistasin reacts stoichiometrically with Factor Xa with inhibition displaying a mixed, primarily competitive type. The inhibition is partial in the presence of Ca2+ and becomes complete in the absence of Ca2+. The estimated dissociation constant for the enzyme-inhibitor complex is between 0.31 and 0.62 nM. After binding to Factor Xa, antistasin is cleaved at a single site to yield a modified inhibitor. Automated gas-phase sequence analysis of the modified inhibitor indicates the arginine residue at position 34 in antistasin occupies the P1 position of the reactive site. These data indicate that the leech has evolved a highly selective and potent inhibitor of coagulation Factor Xa that shares several mechanistic similarities with other serine proteinase inhibitors.
Amino Acid Sequence↗
Isolation and structural characterization of a potent inhibitor of coagulation factor Xa from the leech Haementeria ghilianii.
The present work reports the discovery and characterization of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalis, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host's blood.
Amino Acid Sequence↗
Florid radiological appearance of megakaryoblastic leukaemia--an aid to earlier diagnosis.
Accurate diagnosis of leukaemia is essential to enable appropriate treatment. The diagnosis is made by cytological and cytochemical techniques but radiology may, however, serve a very useful role in first suggesting a diagnosis. We present here four cases of megakaryoblastic leukaemia (AML FAB classification M7), three of which have strikingly similar unusual radiological changes. A review of the literature suggests this condition has previously been diagnosed as acute or malignant myelofibrosis, and the megakaryoblastic nature of the disease was not recognised. The presence of these radiological features may therefore prompt specific immunocytochemical testing, in a disease that is otherwise difficult to diagnose early.
Bone and Bones↗
Minor transplantation antigens: their role in shaping the T cell repertoire.
Minor transplantation, or histocompatibility (H), antigens are the targets of host-versus-graft (hvg) and graft-versus-host (gvh) reactions that occur when organs or tissues are exchanged between members of the same species who, although genetically not identical, are matched for their major histocompatibility complex (MHC) encoded transplantation antigens. Genes encoding minor H antigens map outside the MHC, on a number of different chromosomes. Whilst gvh and hvg reactions against individual minor H antigens are relatively weak, certainly in comparison with such reactions against MHC antigens, the presence of multiple minor H differences (the situation encountered in man) gives rise to very vigorous reactions that can endanger the survival of graft or host, or both. This is the pathological role of minor H antigens and, indeed, it was this role which was first designated to the MHC antigens, before their physiological role as guidance molecules for T lymphocytes was discovered. Recently, a potential physiological role for minor H antigens has been uncovered by the finding that the presence of certain minor H alleles in mice leads to removal in the thymus (negative selection) of all those T cells expressing a particular T cell receptor (TCR) gene. Such cells therefore never reach the periphery, where they might otherwise give rise to autoimmune reactions. The T cell repertoire is thus moulded by at least some minor H antigens, which may therefore be regarded as non-MHC immune response genes. Furthermore, T cell receptor usage by T cells specific for allogeneic minor H antigens appears not to be representative of T cell receptor usage in the peripheral pool.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Quantitative computed tomography reflects vertebral fracture morbidity in osteopenic patients.
We studied the relationship between spontaneous vertebral compression fractures and lumbar vertebral trabecular bone density in 69 consecutive patients with suspected osteopenia. Seven had biopsy-confirmed osteomalacia. The remaining 62 were divided into three groups: group 1--asymptomatic patients suspected of having osteopenia on plain films, but with no vertebral compression fractures (N = 24); group II--those with one to five vertebral compression fractures (N = 16); and group III--those with six or more vertebral compression fractures (N = 22). A quantitative computed tomographic (QCT) scan of the lumbar spine was performed on all patients. Patients in group I had QCT values of 94 +/- 23 mg/cm3 (mean +/- SE); those in group II had QCT values of 66 +/- 28 mg/cm3; and those in group III had values of 34 +/- 28 mg/cm3. There were significant differences among all groups (P less than .001), although there was considerable overlap of individuals among the groups. There was no significant difference between the mean QCT value of patients with one compression fracture and the value of those with between two and five compression fractures. Patients with biopsy-proven osteomalacia had higher vertebral trabecular bone density than patients with osteoporosis and compression fractures. Our study provides evidence suggesting a strong inverse relationship between QCT-measured vertebral bone density and the presence of vertebral compression fractures in a group of osteopenic patients.
Absorptiometry, Photon↗
Mapping minor H genes.
The manner in which minor histocompatibility (H) antigens have been defined in mouse and man, in vivo and in vitro, is considered. Chromosomal mapping of minor H genes using T-cell clones is illustrated, with particular reference to the H-Y antigen gene, using the sex-reversing translocation Sxr of mouse and the Sxr' mutation derived from it. A number of minor H antigen-specific T-cell clones restricted by class I or class II major histocompatibility complex (MHC) molecules are described, together with information about their phenotypes and T-cell receptor usage.
Animals↗
Suppression of the immune response by cytotoxic T cells.
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Animals↗
The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure.
Antistasin is a 15-kDa protein from the salivary glands of the Mexican leech, Haementeria officinalis, which manifests anticoagulant activity by inhibiting factor Xa. Previous work demonstrating the presence of this activity in salivary gland extracts and its partial purification has been reported (Tuszynski, G. P., Gasic, T. B, and Gasic, G.J. (1987) J. Biol. Chem. 262, 9718-9723). The present study includes further purification to homogeneity of antistasin and its subsequent fragmentation and complete amino acid sequence determination. The protein, which possesses 119 amino acid residues, is blocked at its amino terminus by the presence of a pyroglutamic acid residue and has an unusually high cysteine content, with 20 cysteine residues. The primary structure of antistasin shows no homology to hirudin, a 65-residue anticoagulant protein from the medicinal leech, Hirudo medicinalis. Of great interest is the finding of significant internal homology within antistasin where a 2-fold internal repeated structure is observed. At least four isoforms of antistasin have been identified in leech salivary gland extracts by high performance liquid chromatography analysis, and partial amino acid sequence analysis of these isoforms indicates they differ by 1 or 2 amino acid residues.
Amino Acid Sequence↗
Identification of novel proteins synthesized in bone cells by antibody screening of a cDNA expression library.
Novel proteins synthesize predominantly in bone have been identified by antibody screening of bone cell cDNA expression libraries. Two unique cDNAs were identified whose structures do not match any known nucleic acid or protein sequence in the NIH computer bank. The first cDNA clone, BP-I, encoded a mRNA of 2300 bases in size which was expressed at high levels in 17/2.8 rat osteosarcoma cells, rat calvarial bone cells and placenta. A second clone, BP-II, encoded a mRNA of 1500 bases which was expressed at high levels in 17/2.8 osteosarcoma cells and in salivary gland. Expression of both mRNAs in osteosarcoma cells was modulated by the calciotropic hormone, vitamin D. Southern blot analyses indicated that the two cDNAs represented distinct, single copy genes in the rat genome. These novel gene products may serve as potential new markers to study bone turnover in metabolic bone disease.
Amino Acid Sequence↗
Rearrangement of T-cell receptor and immunoglobulin heavy chain genes in childhood acute mixed lineage leukaemia.
Rearrangement of the beta and gamma chain genes of the TCR gene complex and of the Ig heavy chain genes were examined in three cases of childhood acute mixed lineage leukaemia. Blast cells, classified morphologically as acute lymphoblastic leukaemia (ALL) in one child and acute non-lymphocytic leukaemia (ANLL) in the other two, all co-expressed markers associated with both T (CD7, TdT) and myeloid (CD33) cells. Cytogenetic analysis detected abnormalities associated with myeloid leukaemia. Immunoglobulin heavy chain genes were not rearranged in two patients but a novel rearrangement was seen in the third. No rearrangement of the beta or gamma chains of the T-cell receptor complex were seen. Acute mixed lineage leukaemia may thus arise from a pluripotent precursor cell capable of both lymphoid and myeloid differentiation.
Antigens, Differentiation↗
Sir Peter Medawar 1915-1987.
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Allergy and Immunology↗
Delay in onset of insulitis in NOD mice following a single injection of CD 4 and CD 8 antibodies.
Non-obese diabetic (NOD) mice injected with 500 micrograms of both CD 4 and CD 8 antibodies at 5 weeks of age did not develop insulitis until 18 weeks of age, 12 weeks later than the onset of insulitis in parallel uninjected controls. Injection with both antibodies at 2 weeks or 4 weeks of age protected from insulitis for 10 and 14 weeks respectively. Insulitis was not delayed in onset in animals injected at any age with one antibody only, or who were injected at birth. Injection after the onset of insulitis achieved partial but incomplete clearing of islet infiltrates. Salivary gland infiltrates (sialitis) were also delayed in animals injected with both CD 4 and CD 8 antibodies though the degree of protection was less pronounced than that seen for insulitis.
Age Factors↗
Location of the genes controlling H-Y antigen expression and testis determination on the mouse Y chromosome.
Sex-reversed XX male mice that carry the variant form of the testis-determining Sxr region, Sxr', do not express male-specific H-Y antigen. In a stock of mice segregating for Sxr', we detected an exceptional XX male that proved positive for H-Y antigen. DNA fingerprinting revealed that the banding pattern characteristic of Sxr' had been replaced by the pattern associated with the native testis-determining region of the normal Y chromosome of that stock, presumably by pairing and crossing-over between the two testis-determining regions of the father's Y Sxr' chromosome. Pairing between the two ends of such a chromosome in a loop-like configuration has been observed by electron microscopy. However, an anomalous crossing-over event of this kind would only give rise to the observed result if the native homologue of the Sxr region were situated on the very minute short arm of the Y chromosome. We therefore conclude that the two linked genes Tdy and Hya, controlling testis determination and H-Y antigen expression, respectively, are located on the short arm of the mouse Y chromosome.
Animals↗
Function of the MHC.
The major histocompatibility complex (MHC) was discovered originally as a genetic locus controlling rapid rejection of tissue grafts. Subsequently, study of antibody responses in vivo and T-cell responses in vitro to MHC antigens identified the presence of a number of closely linked loci within the MHC. Immune response (Ir) genes also mapped to the MHC. The discovery of MHC restriction and the molecular identification of MHC genes and their products has led to a unified theory of the principal function of MHC molecules to act as guidance molecules for T-cell responses. Additional functions are suggested by their association with cell surface receptors.
Animals↗
MHC-unrestricted T-cell cytotoxicity against tumour cells.
F9 embryonal carcinoma cells (EC) grow as tumours in their strain of origin, 129/Sv, but can be rejected by mouse strains differing at the H-2 and/or non-H-2 loci. The presence of H-2 class I and/or minor H antigens on F9 and other EC cells is implied by (i) the rejection of EC cells by mice immunized with appropriate H-2 class I transfectants, and (ii) the ability of appropriate EC cells to prime mice for second-set in vivo skin-graft rejection responses to H-Y, and secondary MLC responses to multiple minor H antigens. However, EC cells express no H-2 class I antigens in vitro, and for in vivo rejection by T-cell responses directed either at allogeneic class I molecules or at minor H antigens restricted by self class I molecules, one would need to postulate that EC cells growing in vivo could express sufficient class I antigens for recognition by T cells. In the course of investigating this question, we found evidence for class I expression but also evidence for an additional antigen(s), shared by EC and tumour cells and recognized in a non-MHC-restricted manner.
Animals↗
Absence of any male-specific antigen recognized by T lymphocytes in X/XSxr' male mice.
Previous work has established that whereas X/X mice carrying the sex-reversing chromosomal fragment Sxr are positive for the male-specific transplantation antigen, H-Y, X/X mice carrying the variant Sxr', although they too develop as phenotypic males, are H-Y negative. In this paper we show that X/XSxr' male mice do not express any male-specific antigen that can induce skin-graft rejection.
Animals↗