A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-chain gene, causing defective calcium binding and impaired fibrin polymerization

A congenitally abnormal fibrinogen (Vlissingen) was isolated from the blood of a young woman suffering from massive pulmonary embolism. Fibrinogen Vlissingen showed an abnormal clotting time with both thrombin and Reptilase. The release of the fibrino-peptides A and B by thrombin was normal, but fibrin polymerization was impaired both in the presence and absence of Ca2+ ions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed according to Laemmli the gamma-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1,500 daltons. The previously described protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain of normal fibrinogen was only partially detectable in fibrinogen Vlissingen. In addition the binding of Ca2+ ions was decreased. Fibrinogen Vlissingen bound 2.4 Ca2+ ions per fibrinogen molecule at pH 7.4, whereas normal fibrinogen bound 3.1 Ca2+ ions. At pH 5.8 fibrinogen Vlissingen bound 1.1 Ca2+ ions, whereas normal fibrinogen bound 2.0 Ca2+ ions per molecule fibrinogen in the D-domains, again indicating a structural change in the carboxyl terminus of fibrinogen. The structural defect was determined by sequence analysis of DNA amplified by use of the polymerase chain reaction. Exons VIII, IX, and X of the gamma-chain gene were amplified and the DNA sequence of the amplified fragments was determined. A 6-base deletion was found in 50% of the fragments corresponding to exon VIII, indicating that the patient was heterozygous for the mutation. This deletion codes for amino acids Asn-319 and Asp-320 in the normal fibrinogen gamma-chain. The data indicate that Asn-319 and Asp-320 are crucial for maintaining the integrity of the carboxyl-terminal polymerization sites, the protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen.     

Haemoglobin Titusville: alpha94 Asp replaced by Asn. A new haemoglobin with a lowered affinity for oxygen.

A new haemoglobin variant with a decreased oxygen affinity is described, in which the substitution, alpha 94 (G1) Asp replaced by Asn, affects the alpha1beta2 contact alpha1G1-beta2G4. The relevance of this variant to our understanding of the importance of the hydrogen bond between alpha1G1 and beta2G4 in Perutz's model of oxyhaemoglobin A is discussed.     

Antibody to fibrinogen/fibrin products (Anti-F) in malaria, babesiosis, and trypanosomiasis of rodents.

Antibody to fibrinogen/fibrin related products (Anti-F) was stimulated during the course of Plasmodium chabaudi, Babesia rodhaini, and Trypanosoma lewisi infections in rats. Titers of this autoantibody remained elevated in serum from rats that had recovered from each of the infections. Column chromatographic studies indicated that Anti-F was a 19S globulin, possibly IgM. During acute infections high titers of Anti-F were associated with elevated titers of cold-active hemagglutinin (CAH) and immunoconglutinin (IK) and all were associated with anemia and elevated parasitemia. Titers of Anti-F and IK, but not CAH, remained elevated in serum of recovered rats. The presence of Anti-F indicated that the coagulation system had been activated during each infection to release fibrinogen/fibrin-related products (FRP) to serve as antigen(s) for Anti-F. Since IK is antibody to the third component of fixed complement, it could be assumed that complement fixing antigen-antibody complexes were also present during the acute stage of each infection. The possibility that complexes of FRP and Anti-F could have contributed to anemia in each infection is discussed.     

Macrophage migration in fibrin gel matrices. II. Effects of clotting factor XIII, fibronectin, and glycosaminoglycan content on cell migration

We investigated the migration of oil-induced, guinea pig peritoneal macrophages in three-dimensional fibrin matrices, with particular attention to variables which modified fibrin gel structure and/or its adhesive properties for cells. The variables studied were fibrin concentration, gel cross-linking, and fibronectin and glycosaminoglycan content. Macrophage migration was an inverse linear function of fibrinogen concentration. Little or no fibrinolysis accompanied macrophage migration; rather, macrophages migrated through fibrin gels by an active process associated with marked distortions of cell shape and specialized plasma membrane contacts with fibrin strands. Fibrin matrices prepared from fibrinogen that had been depleted of clotting factor XIII and/or fibronectin provided a superior matrix for macrophage migration. Both the number of migrating cells and distance of migration were reduced when the gel matrix included fibronectin and was cross-linked by factor XIII. A hexapeptide containing the fibronectin cell-binding RGDS sequence reversed this migration inhibition, suggesting that fibronectin immobilized by cross-linking to fibrin may have bound macrophages and restricted cell migration. Hyaluronic acid, heparin, and heparan sulfate inhibited macrophage migration in cross-linked fibrin-fibronectin gels over a range of concentrations. These data are relevant to an understanding of macrophage migration in vivo where cross-linked fibrin-fibronectin gels containing variable amounts of glycosaminoglycans are deposited in tissues in immunologic reactions and in many other types of pathology.     

An amino acid substitution (Gly853-->Glu) in the collagen alpha 1(II) chain produces hypochondrogenesis.

The spondyloepiphyseal dysplasia subclassification of bone dysplasias includes achondrogenesis, hypochondrogenesis, and spondyloepiphyseal dysplasia congenita. The phenotypic expression of these disorders ranges from mild to perinatal lethal forms. We report the detection and partial characterization of a defect in type II collagen in a perinatal lethal form of hypochondrogenesis. Electrophoresis in sodium dodecyl sulfate-polyacrylamide of CB peptides (where CB represents cyanogen bromide) from type II collagen of the diseased cartilage showed a doublet band for peptide alpha 1(II)CB10 and evidence for post-translational overmodification of the major peptides (CB8, CB10, and CB11) seen as a retarded electrophoretic mobility. Peptide CB10 was digested by endoproteinase Asp-N; and on reverse-phase high pressure liquid chromatography, fragments of abnormal mobility were noted. Sequence analysis of a unique peptide D12 revealed a single amino acid substitution (Gly-->Glu) at position 853 of the triple helical domain. This was confirmed by sequence analysis of amplified COL2A1 cDNA, which revealed a single nucleotide substitution (GGA-->GAA) in 5 of 10 clones. Electron micrographs of the diseased cartilage showed a sparse extracellular matrix and chondrocytes containing dilated rough endoplasmic reticulum, which suggested impaired assembly and secretion of the mutant protein. This case further documents the molecular basis of the spondyloepiphyseal dysplasia spectrum of chondrodysplasias as mutations in COL2A1.     

Glucose starvation leads in rat hepatoma cells to partially N-glycosylated glycoproteins including alpha 1-acid glycoproteins. Identification by endoglycolytic digestions in polyacrylamide gels

Within minutes of glucose starvation confluent monolayers of rat hepatoma cells synthesize glycoproteins, including alpha 1-acid glycoprotein, which appear on two-dimensional gels as size heterogeneous spot series. The longer the period of glucose starvation the more the production of the glycoproteins is shifted toward smaller molecular weight forms. To compare these forms with the corresponding glycoproteins synthesized either in a cell-free system or by nonstarved cells, a mapping of the N-glycan was done by endo-beta-N-acetylglucosaminidase digestion within a polyacrylamide gel. Glycoproteins from glucose-starved cells contain a reduced number of N-glycans which belong to both the endo H-sensitive and resistant type. The decrease of N-glycosylation may be correlated with the accumulation of truncated lipid-bound oligosaccharides, for the gel chromatography of the oligosaccharides released from the lipid and protein fractions of glucose-starved cells revealed a drastic reduction in their size. Most of the lipid-linked oligosaccharides synthesized during glucose starvation are resistant to endo H digestion. Under conditions of limiting glycosylation we were able to show by glycopeptide analysis, that in the case of alpha 1-acid glycoprotein, N-glycans are added randomly to the 6 possible N-glycosylation sites. Furthermore, non- or partially N-glycosylated proteins do not acquire additional oligosaccharide units after restoration of glucose although the proteins can undergo secondary modification and, in the case of the secretory proteins, can be exported.     

A naturally occurring nonpolymerogenic mutant of alpha 1-antitrypsin characterized by prolonged retention in the endoplasmic reticulum.

The classical form of alpha 1-antitrypsin (alpha 1-AT) deficiency is associated with a mutant alpha 1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) Z allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of alpha 1-AT in a family in which three affected members had severe liver disease associated with alpha 1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI Z allele and a second allele, the PI Z + saar allele, bearing the mutation that characterizes alpha 1-ATZ as well as the mutation that characterizes alpha 1-AT Saarbrucken (alpha 1-AT saar). The mutation in PI saar introduces a premature termination codon resulting in an alpha 1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the alpha 1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of alpha 1-AT saar and alpha 1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant alpha 1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in alpha 1-AT deficiency.     

Chromosomal localization of human and rat A alpha, B beta, and gamma fibrinogen genes by in situ hybridization

In situ hybridization of radiolabeled fibrinogen cDNAs to human and rat metaphase chromosomes has shown that the genes encoding the A alpha, B beta, and gamma fibrinogen subunits are syntenic in both species. Our data localize the human fibrinogen gene cluster to band q31 on chromosome 4, thereby confirming and extending previous map assignments of these genes in man. We have also assigned these genes to the q31----q34 region of rat chromosome 2. This is the first map assignment of these genes in the rat and also the first report to clearly establish linkage of the B beta subunit gene to the A alpha and gamma genes in this species.     

An arthritogenic monoclonal antibody to type II collagen, CII-C1, impairs cartilage formation by cultured chondrocytes

Antibodies to type II collagen (CII) cause articular damage in collagen-induced arthritis (CIA) in mice as judged by passive transfer to naive animals of mAb to CII. We tested the hypothesis that mAb degrade cartilage structure by reacting with functionally important regions of the collagen molecule by examining the effects of an arthritogenic mAb to CII, CII-C1, on cultured bovine chondrocytes at high density, at days 7 and 14. The effects were compared of CII-C1, an isotype-matched control mAb, or medium alone, on chondrocyte proliferation and viability, cell morphology, matrix structure by light and electron microscopy, and matrix synthesis by metabolic labelling with 3H-proline for collagen or 35SO4 for proteoglycans. Chondrocytes in culture remained viable, proliferated, and produced an extracellular matrix in which CII was the major collagen. The addition of CII-C1, but not a control mAb, increased the synthesis of CII and proteoglycan, and caused disorganization of the extracellular matrix and thin collagen fibrils ultrastructurally. Moreover, using a cell-free assay, CII-C1 inhibited the normal self-assembly of collagen fibrils from CII in solution. The finding that the mAb to CII, CII-C1 has striking degradative effects in vitro on cartilage synthesis suggests that antibodies to collagen perpetuate the chronic phase of CIA and that, in mice at least, such antibodies are an important component of pathogenesis.     

A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mutation hisJ5625 has altered the histidine-binding protein J of Salmonella typhimurium such that histidine transport is impaired, even though binding of histidine by the J protein is unimpaired [Kustu, S.G., & Ames, G.F. (1974) J. Biol. Chem. 249, 6976--6983]. We have determined by protein analytical methods that the only effect of this mutation has been the substitution of a cysteine residue for an arginine at a site in the interior of the polypeptide chain. This arginine residue is therefore potentially essential for the transport function of the protein. The mutant protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis more slowly than the wild type protein, as if its molecular weight were greater by as much as 2000. Since this behavior is apparently due to a single amino acid replacement, a molecular weight difference even between two closely related proteins should not be inferred solely on the basis of sodium dodecyl sulfate gel electrophoresis.     

An amino acid substitution in the pyruvate dehydrogenase E1 alpha gene, affecting mitochondrial import of the precursor protein.

A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.     

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.     

Plankton ecology in Long Pond,St. John's,Newfoundland: A polluted pond characterized by a high flushing rate

Plankton was studied seasonally and quantitatively in Long Pond, St. John's, Newfoundland. This pond receives both rural and urban runoff and is characterized by a high flushing rate. A comparison is made with Clarkes Pond, Hogans Pond and Bauline Long Pond. Long Pond phytoplankton was characterized by nannoplankton (particularly flagellated forms). The most important zooplankters were Bosmina coregoni and Daphnia catawba. Aspects of plankton ecology are discussed in relation to eutrophication and water renewal.     

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.     

Bactericidal substances in cytoplasm of leucocytes. An attempt to separate lysozyme by gel filtration

A substance with a bactericidal effect onEscherichia coli was isolated from the cytoplasm of polymorphonuclear leucocytes. It consists of a mixture of different active substances and therefore a gel filtration using s Sephadex G-100 column was used to separate lysozyme from other active substances. The extract was separated by this procedure into five peaks, out of which peak I. possesed a significant bactericidal activity, peak II. and III. had a weak one, whereas the lysozyme activity was present in peak III., IV. and V.     

An X-ray determination of the molecular interactions in hemoglobin C: a disease characterized by intraerythrocytic crystals.

The X-ray structure of cyanomet human hemoglobin C has been solved and refined, R ～27%. The molecular packing can be represented in two dimensions by two sets of parallel strands, one set in the $\text{b}$ direction and the other in the $\text{c}$ direction. Taken together the two sets of strands interconnect the molecules into square nets or layers where each molecule contacts its four nearest neighbors. Molecules in one layer are displaced in $\text{a}$ and $\text{b}$ so that they fit into the “holes” of the square arrays of the adjacent layers (normal to $\text{a}$) resulting in a pseudo body-centered cubic packing. This packing can account for the hemoglobin crystallization in and fragility of the erythrocytes. The aberrant β6A3 Lys residue is in a position to influence the crystal formation.     

Hemoglobin Tarrant: alpha126(H9) Asp leads to Asn. A new hemoglobin variant in the alpha1beta1 contact region showing high oxygen affinity and reduced cooperativity.

Hemoglobin (Hb) Tarrant was detected by its electrophoretic mobility on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). On cellulose acetate it moved as a band between hemoglobins F and S, and on citrate agar as a band at hemoglobin S. The test for solubility in 2 M phosphate buffer with Na2S2O4 was negative. The new variant has a substitution of asparagine for aspartic acid in position 126 of the alpha-chain, one of the sites involved in the alpha1beta1 contact. Furthermore, in deoxyhemoglobin aspartic acid 126 of each alpha chain also forms a non-covalent electrostatic salt bridge with arginine 141 of the corresponding alpha chain (Perutz, M. F. and Ten Eyck, L. F. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 295-310 and Perutz, M. F. (1970) Nature 228, 726-739). As a consequence of this substitution in hemoglobin Tarrant, the deoxy conformation or T state is destabilized because these two bridges cannot be formed. This condition is reflected in high oxygen affinity and low cooperativity.     

Hb J-Singa (alpha-78 Asn leads to Asp), a newly discovered hemoglobin variant with the same amino acid substitution as one of the two present in Hb J-Singapore (alpha-78 Asn leads to, alpha-79 Ala leads to Gly)

A new fast-moving alpha-chain Hb variant with an Asn leads to Asp substitution at position alpha-78 was found in a French-Acadian family living in Eastern Canada. The identical substitution was reported in Hb J-Singapore, which also had an additional Ala leads to Gly substitution at position alpha-79. The new variant, which did not result in any clinical symptoms, was named accordingly, Hb J-Singa.