Marijke Fràter-Schröder

Transforming growth factor-beta inhibits endothelial cell proliferation

Transforming growth factor-beta (TGF-beta) is an inhibitor of the proliferation of bovine aortic endothelial cells in culture. Basal cell growth in serum-containing medium and cell proliferation stimulated by fibroblast growth factor (FGF) are inhibited by TGF-beta in a dose-dependent manner. Half-maximal inhibition occurs at an inhibitor concentration of 0.5-1.0 ng/ml. TGF-beta does not appear to be cytotoxic and cells treated with the inhibitor grow normally after removal of TGF-beta. High concentrations of FGF are ineffective in overcoming TGF-beta-induced inhibition of cell proliferation, suggesting that antagonism of growth factor-induced cell proliferation by TGF-beta is of a noncompetitive nature.

INCREASED UNSATURATED TRANSCOBALAMIN II IN ACTIVE AUTOIMMUNE DISEASE

Measurements of transcobalamin II (T.C. II) concentrations in 26 patients with lupus erythematosus, 4 with dermatomyositis, 2 with autoimmune haemolytic anaemia, and in 40 immunosuppressed renal-transplant patients showed that T.C. II levels were raised during active phases of autoimmune disease. Changes in T.C. II levels correlated better with the clinical course of autoimmune disease than did changes in C3, the titre of antinuclear antibody, or native D.N.A.-binding capacity.

Genetic patterns of transcobalamin II and the relationships with congenital defects

SummaryThe vitamin B 12-binding protein, transcobalamin 11, is a trace component of plasma with a rapid turnover. This protein is essential for absorption, transport, cellular uptake and for recycling of vitamin B12 (cobalamin). Congenital transcobalamin 11 deficiency, an inborn error of metabolism is inherited as a recessive trait. The homozygous form of the deficiency is accompanied by severe clinical, hematological and immunological disturbances in the first months of life. Analytical, genetic, biochemical and clinical aspects of transcobalamin II in man and in vertebrates have been reviewed here. A genetic polymorphism for the protein has been found in man, rabbits and mice. Family studies revealed that the genetic patterns in man are determined by four polymorphic and several rare alleles. This genetic variability has been applied in paternity testing and in population studies. Transcobalamin 11 typing in families of patients with the inherited functional deficiency has led to identification of various deficient alleles in heterozygous carriers of the defects. Applying transcobalamin 11 typing after bone marrow transplantation demonstrated that this protein originates partly in the bone marrow. Subsequent investigations in cell culture have shown that human skin fibroblasts and cultured bone marrow synthesize and secrete isotypes of a transport protein corresponding to the genetic isotypes observed in plasma. Comparison of transcobalamin 11 types in umbilical cord serum with the maternal types, has proven that the transcobalamin 11 activity in the cord serum is derived from the fetus. This finding will be of crucial importance in the early diagnosis of the deficiency syndrom.

Synthesis and secretion of the human vitamin B12-binding protein, transcobalamin II, by cultured skin fibroblasts and by bone marrow cells

Human skin fibroblasts and bone marrow cells were tested for their ability to synthesize the cobalamin-binding protein transcobalamin II. Cobalamin binders secreted in the media of cultured fibroblasts and of dextran-sedimented bone marrow cells in liquid culture could be identified as transcobalamin II on the basis of immunological, electrophoretical and chromatographical identity with serum transcobalamin II. The net secretion of transcobalamin II increased linearly with time of culture, up to 30 days after confluence. The reversible inhibition of transcobalamin II secretion by cycloheximide demonstrated that human fibroblasts are capable of de novo transcobalamin II synthesis. Addition of cyanocobalamin to the fibroblast culture medium induced a reduction of transcobalamin II net secretion, most likely due to preferred uptake of transcobalamin II saturated with cobalamin, as opposed to unsaturated protein. Addition of lysozymal enzyme inhibitors, ammonium chloride and chloroquine, resulted in a markedly increased secretion of transcobalamin II. In the culture medium of fibroblasts, obtained from two transcobalamin II-deficient patients, functionally deficient transcobalamin II was demonstrated on the basis of strongly reduced secretion of immunoreactive transcobalamin II, and the absence of apotranscobalamin II. Individual phenotypes in the culture media of the fibroblasts and bone marrow cells were identical to the corresponding serum transcobalamin II types.

Localization of the gene for the vitamin B12 binding protein, transcobalamin II, near the centromere on mouse chromosome 11, linked with the hemoglobin alpha-chain locus

Somatic cell hybrids, recombinant inbred (RI) mouse strains, and backcross breeding experiments were used to locate the gene of transcobalamin II (Tcn-2), the vitamin B12 binding protein in mouse serum. TCN-2 was found to be a useful genetic marker in the somatic cell hybrids. Selected hybrid clones were derived from fusions between GR mouse cells and the Chinese hamster cell line E36. Analysis of mouse specific chromosomal enzyme markers in relationship to TCN-2 secretion, in the hybrid clones, provided provisional evidence for assignment of the Tcn-2 locus to chromosome 11. The strain distribution pattern of the TCN-2 variants S and F in the RI series CXS, constructed from the cross of BALB/cHeA (TCN-2S) with STS/A (TCN-2F), implied a close linkage with the hemoglobin alpha-chain locus (Hba) on chromosome 11. Backcross breeding using inbred strains confirmed these findings and located the Tcn-2 gene closest to the centromere, linked with waved 2 (wa-2) and Hba with recombination frequencies of 6.9 and 19.2% each. The linkage group Tcn-2/wa-2/Hba was established.

Granulocyte dysfunction in transcobalamin II deficiency responding to leucovorin or hydroxocobalamin-plasma transfusion

Granulocytes from a 6-year-old boy with congenital transcobalamin II (TCII) deficiency were found to have abnormally low antibacterial activity againstStaphylococcus aureus and very low intracellular levels of the cobalamin coenzymes. Transfusion of hydroxocobalamin (OH-Cbl) bound to normal plasma temporarily restored granulocyte bactericidal activity and increased cellular levels of the cobalamin coenzymes. Granulocyte function was also temporarily restored by oral Leucovorin. The defect appeared to be causally related to the patients TC II deficiency and indirectly to a deficiency of cobalamin and folate coenzymes.

Assignment of human transcobalamin II (TC2) to chromosome 22 using somatic cell hybrids and monosomic meningioma cells.

SummaryHuman transcobalamin II (TC2), a vitamin B12 binding serum protein, is synthesized and secreted into the medium by cells growing in vitro. Mouse-man somatic cell hybrids were analyzed in order to map the locus of TC2. The presence of human TC2 in the culture media was correlated with the results of genetic marker and chromosome analysis of the hybrid cells. Chromosome 22 showed 100% concordancy. However, chromosome 6 (90% concordancy) and chromosome 7 (96% concordancy) were not completely excluded. Meningioma cells obtained from patients heterozygous for TC2 showed a concomitant loss of one chromosome 22 and one of the TC2 alleles, strongly supporting the assignment to chromosome 22.

Standardization of nomenclature for transcobalamin II variants

Department of Pediatrics, University of Zt~rich, CH-8032 Zt~rich, Switzerland 2 Institute of Human Genetics, Free University, Faculty of Medicine, P.O. Box 7161, 1007 MC Amsterdam, The Netherlands 3 Center for Demographic and Population Genetics, The University of Texas Health Science Center, P.O. Box 20334, Houston, Texas 77025, USA 4 Department of Genetics, Stanford University Medical Center, Stanford, California 94305, USA

Solid-phase immunoassay for the vitamin B12-binding protein transcobalamin II in human serum

Abstract A solid-phase radio immunoassay was developed for total immunoreactive transcobalamin II (TC II). Rabbit antihuman TC II antiserum (which recognizes both apo- and holo-TC II), was immobilized by covalent binding to acrylamide-acrylic acid copolymer beads. A normal mean and SD for immunoreactive TC II in serum was determined in 130 healthy adult individuals and found to be 1150 ± 250 ng/liter cobalamin equivalent. Mean holo-TC II (N = 30), estimated by substraction of apo-TC II from total TC II, was 137 ng/liter bound cobalamin (or 12% of total TC II). Three patients with lack of functional TC II had immunoreactive TC II levels between 22 and 39% of normal mean, which demonstrated that the solid-phase bound antiserum recognized deficient TC II molecules, whereas the same antiserum in its soluble form did not. Eight out of nine individuals, recognized as heterozygous for TC II deficiency, had TC II levels below the normal range, on the order of 50% of the normal mean. The stability of immunoreactive TC II was strongly enhanced by the presence of an unknown serum factor not corresponding to serum albumin.

Inheritance of transcobalamin II (TC II) in two families with TC II deficiency and related immunodeficiency

Transcobalamin II (TC II) is an essential transport protein for vitamin B12 in blood. TC II can be separated into isoproteins by polyacrylamide gel electrophoresis. This method was used in combination with a specific radioimmunosorbent technique to evaluate genetic variants and inheritance of TC II-deficient genes in relatives of two children with congenital TC II deficiency. Both patients presented with impairment of haematopoietic and immunological functions. Seven heterozygous individuals for TC II deficiency, who are clinically normal, were detected in the two families. Two out of seven could be identified unambiguously by TC II isoprotein analysis, as carriers of a deficient gene, which does not express functional TC II. Application of this new method to detect heterozygous carriers of the deficient gene provides a valuable addition to genetic counselling.