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PubMed2003

Electrophoretic dynamics of large DNA stars in polymer solutions and gels.

We have developed a procedure for synthesizing large stable branched DNA structures that enables visualization via fluorescence microscopy. Using this procedure we have synthesized large DNA stars and observed their electrophoretic behavior in polymer solutions and gels. In dilute polyacrylamide solutions, the DNA stars move as random coils and appear to experience only brief collisions with the polymer chains in solution. The effect of polymer solution concentration on the electrophoretic mobility of stars in the dilute regime is found to be in good accord with predictions of the transient entanglement coupling (TEC) model. In semidilute polymer solutions, the star arms extend in the field direction and drag the core through the matrix. The star arms form several U-shaped conformations as they collide and engage with polyacrylamide chains. The U-shaped conformations occasionally evolve into J-shaped conformations as the star arms slide off the matrix chains they engage during electrophoretic migration. In concentrated polymer solutions, the arms of the star extend and form V-shaped structures with the core as the apex. The arms then pull the core through the matrix. These V-shaped conformations are much longer-lived than U-shaped ones and, unlike the latter, do not transform to J-shaped conformations. In polyacrylamide and agarose gels, where matrix entanglements are fixed, DNA stars become trapped when entanglements with matrix molecules prevent the core from being pulled through the matrix by the extended arms. This trapping was observed at all gel concentrations and electric fields studied.

Base Sequence↗

PubMed1990

Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers.

After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase. We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine. These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture. The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized. Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated. Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE. Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum). It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.

Blotting, Western↗

PubMed1985

Synthesis of basement membrane components by differentiated thyroid cells.

Morphological studies indicate that basement membrane formation or maintenance can be achieved in cultures of thyroid cells. In the present investigation we have studied the biosynthesis of this extracellular matrix by differentiated porcine thyroid cells in culture. They were prepared by two procedures: (1) thyroid cells isolated by dispase digestion of the thyroid gland were maintained in serum-free medium on poly(L-lysine) coated dishes; (2) thyroid follicles released by collagenase treatment of the gland were isolated by differential filtration and cultured in suspension on agarose-coated dishes. In both cases, functional follicular-like structures were obtained as shown by their ability to organify Na125I and to respond to thyrotropin stimulation (250 microU/ml). After incubating the cells with radiolabeled proline or methionine, collagen synthesis was observed with the two types of culture, as shown by the formation of radioactive hydroxyproline and by the synthesis of peptides with electrophoretic properties identical to those of authentic collagen molecules and susceptible to collagenase. Besides variable amounts of type I and type III collagen-like peptides, significant proportions of labeled peptides migrated with type IV collagen chains and were precipitated by anti-type IV collagen antibody; thyrotropin had no significant effect either on the total collagen synthesis or on the relative amounts of the different collagen peptides. When thyroid cells were incubated with [35S]sulfate, a labeled glycosaminoglycan with chromatographic properties analogous to that of heparan sulfate could be obtained in both culture conditions; here again, no effect of thyrotropin was observed. The ability of differentiated porcine thyroid cells to synthesize basement membrane was suggested by their production of type IV collagen and heparan sulfate, two of its potential components. Thyrotropin, which drastically enhanced the functional property of the cells, did not seem to regulate this synthesis.

Animals↗

PubMed2000

Chemoenzymatical grafting of acrylamide onto lignin.

Laccase (E.C.1.10.3.2.) from white-rot basidiomycete Trametes versicolor and dioxane peroxides were essential in the copolymerization of acrylamide and a derivative with lignin in a dioxane-H(2)O (7:3) mixture. Both a solubility test and an elemental analysis of the eluted and separated fractions provided evidence of grafting. Gel permeation chromatography on Sephadex G-100 showed that the side chain and the lignin backbone migrate as one unit through the column. Copolymers obtained were characterized by UV-VIS-spectroscopy as well as FT-IR, and 13C-CPMAS-NMR spectroscopy. Freeze-dried copolymers of lignin and acrylamide appeared as homogeneous fibril-like particulate. The mechanism of the enzymatical grafting is discussed.

Acrylamide↗

PubMed2003

Membrane-type 1 matrix metalloproteinase: a key enzyme for tumor invasion.

Matrix metalloproteinases (MMPs) are believed to play a pivotal role in malignant behavior of cancer cells such as rapid tumor growth, invasion, and metastasis by degrading extracellular matrix (ECM). Different types of synthetic inhibitors against MMPs (MMPIs) were developed as candidates for anti-cancer therapeutics and so far clinical trials had led to no significant success. However, this does not diminish the importance of MMPs in the malignancy of cells. Details about MMPs, specifically when and how they take part in the development of cancer are necessary for more advanced application of MMPIs. In this paper, we summarize recent knowledge about membrane-type 1 matrix metalloproteinase (MT1-MMP) which is expressed on cancer cell surface as an invasion-promoting proteinase. By localizing at the leading edge of invasive cancer cells, MT1-MMP degrades components of the tissue barriers. One of the major targets is type I collagen, the most abundant ECM component. Although MT1-MMP itself cannot degrade type IV collagen in the basement membrane, it binds to and activates proMMP-2, one of the type IV collagenases. However, degradation of the ECM is not the sole function of MT1-MMP. MT1-MMP also regulates cell-ECM interaction by processing cell adhesion molecules such as CD44 and integrin alphav chain, and eventually promotes cell migration as well. In addition to the transcriptional regulation, invasion-promoting activity of the MT1-MMP is also strictly monitored at the post-translational level. Precise knowledge about the regulation will give us insight to develop new methods for treating invasive cancer patients.

Animals↗

PubMed2003

Molecular phylogenetics of the RrmJ/fibrillarin superfamily of ribose 2'-O-methyltransferases.

Recent analyses identified a putative catalytic tetrad K-D-K-E common to several families of site-specific methyltransferases (MTases) that modify 2'-hydroxyl groups of ribose in mRNA, rRNA and tRNA (designated the RrmJ class after one of the structurally characterized members; 1eiz in Protein Data Bank) [Genome Biol. 2(9) (2001) 38]. Subsequently, three residues of the tetrad (K-D-K) were shown to be essential for catalysis in RrmJ [J. Biol. Chem. 277 (2002) 41978]. Here, we report identification of a similar conserved tetrad (K-D-K-H) in the family of snoRNA-guided ribose 2'-O-MTases related to fibrillarin (represented by the Mj0697 protein structure; 1fbn in PDB). The corresponding functional groups of putative catalytic tetrads of RrmJ and Mj0697 may be superimposed in space. However, one of the invariant residues (K(164) in RrmJ and K(179) in Mj0697) is observed in two distinct locations in the primary sequence, suggesting an interesting case of 'migration' of the conserved side chain within the framework of the active site. RrmJ and Mj0697 sequences were used as starting points to carry out comprehensive sequence database searches, resulting in identification of a similar conserved tetrad (and hence, prediction of a ribose 2'-O-specificity) in several families of putative MTases, including TlyA hemolysins, novel proteins from Trypanosoma, and large multidomain proteins from Flaviviriruses, Nidoviruses, and Alphaviruses. The results of our analysis of phylogenetic relationships in the RrmJ/fibrillarin superfamily provide insight into the evolution of site-specific and snoRNA-guided ribose 2'-O-MTases from a common ancestor.

Amino Acid Sequence↗

PubMed1981

Purification and properties of poly(ADP-ribose) polymerase from lamb thymus.

Poly(ADP-ribose) polymerase was purified 2900-fold from lamb thymus with a recovery of 5%. Addition of exogenous DNA was essential for activity of the purified enzyme, and the reaction was stimulated by the addition of either a mixture of histones or purified histone H1. The enzyme is inhibited by sulfhydryl binding agents such as phenylmethanesulfonyl fluoride or N-ethylmaleimide. It does not require magnesium or other metal ion cofactors for activity. The enzyme migrated as a single polypeptide chain with an apparent molecular weight of 135 000 when gel electrophoresis was performed in the presence of sodium dodecyl sulfate. The apparent molecular weight was 175 000 when determined by gel filtration on Sepharose CL-6B-200. The isoelectric point was pH 9.6, and the pH optimum for activity was 8.6-8.8. The apparent Km for NAD+ was 160 microM at 37 degrees C. The activity of the purified polymerase was unaffected by the presence of ADP-ribose, 3',5'-cAMP, or NaF. Nicotinamide, 5-methyl-nicotinamide, theophylline, and thymidine markedly inhibited enzyme activity. Lamb thymus DNA, originally associated with the enzyme, was more effective than commercially obtained calf thymus DNA as an enzyme activator.

Animals↗

PubMed1982

Purification and properties of a presynaptically acting neurotoxin, mandaratoxin, from hornet (Vespa mandarinia).

A hornet (Vespa mandarinia) neurotoxin, mandaratoxin (MDTX), was purified by simple procedures with column chromatography made on Sephadex G-50 and CM-Sephadex by using an acetate buffer. The molecular weight of homogeneous MDTX was calculated to be approximately 20000 by gel filtration, NaDodSO4 disc gel electrophoresis, and amino acid analysis. MDTX is a single-chain polypeptide. MDTX did not migrate electrophoretically in a basic buffer at pH 8.3 but did so when the buffer was acidic, at pH 4.3. The isoelectric point of the toxin was determined at 9.1 by isoelectric focusing. A relatively high amount of lysine was found in the amino acid analysis. A280nm1% was 15.1. Glucosamine and galactosamine were not detectable by amino acid analysis. MDTX had neither hemolytic nor enzymatic activity. The toxin was heat labile. By use of neuromuscular junctions of a lobster walking leg, it was found that the nanomole range of MDTX irreversibly blocked the excitatory postsynaptic potential without appreciable change in the resting conductance of the postsynaptic membrane. Intracellular recording from the presynaptic nerve fiber showed that MDTX blocked the action potential mainly by reducing the sodium current.

Action Potentials↗

PubMedDate not supplied

Characterization of lipophilic gemcitabine prodrug-liposomal membrane interaction by differential scanning calorimetry.

Gemcitabine is an anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine. Its stability as well as bioavailability can be increased by making prodrugs. A series of lipophilic prodrugs of gemcitabine were synthesized by linking the 4-amino group with valeroyl, lauroyl, and stearoyl linear acyl derivatives. We studied, by the differential scanning calorimetry technique, and compared the interaction of pure gemcitabine and its prodrugs with dimyristoylphosphatidylcholine and distearoylphosphatidylcholine vesicles with the aim of demonstrating if the gemcitabine prodrug is more able than the pure gemcitabine to interact with lipid vesicles employed both as model biomembranes and as carriers in the transport of antitumor drugs. These studies, carried out by static and kinetic calorimetric measurements, give evidence that the increase of the prodrug's lipophilic character improves the interaction with lipid bilayers, favoring the absorption through the lipid barriers and allowing the liposomes to work (when the prodrug is inserted inside the vesicles) as a lipophilic carrier which is able to deliver the drug near the cell surface. The use of different prodrugs modified in their lipophilic character, of different kinds of vesicles (multilamellar and unilamellar), and of different kinds of vesicles forming phospholipids permitted us to determine the better equilibrium between in-vesicle solubility and through-vesicle diffusion of the drug, important in the preformulative studies of antitumor carriers based on phospholipid formulations. Such studies suggest that the prodrug lipophilic tail should modulate the transport and the release of gemcitabine inside the cellular compartments, and the efficiency of the liposomal system is related to the length of the prodrug's acyl chain which has to match the phospholipid acyl chain allowing or retarding the migration through the lipid release device.

Calorimetry, Differential Scanning↗

PubMed1983

Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin.

Vitamin K-dependent protein S exists in two forms in human plasma, namely as the free protein and in complex with C4b-binding protein [Dahlbäck & Stenflo (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2512-2516]. Now reported is a simple purification procedure for human protein S that includes barium citrate adsorption, DEAE-Sephacel chromatography and chromatography on Blue Sepharose. The yield was approx. 30% relative to the concentration of free protein S in plasma, which was found to be approx. 10 mg/l. Purified protein S migrated as a single-chain band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions and as a doublet of Mr approx. 85 000 and 75 000 on reduction. A third band of Mr 16 000 was observed after electrophoresis of 125I-labelled protein S and radioautography of reduced samples. This band appears to be disulphide-linked to the 75 000-Mr chain before reduction. Thrombin converted the 85 000-Mr chain of protein S into a 75 000-Mr chain and an 8000-Mr fragment, the latter again being detectable only by radioautography of reduced samples. The 16 000-Mr fragment was not observed, suggesting its degradation by thrombin. Under non-reducing conditions, no change in apparent molecular weight of thrombin-treated protein S was observed, indicating disulphide linkage of the fragments. Thrombin also affected the mobility of protein S on agarose-gel electrophoresis in the presence of Ca2+, suggesting a decreased affinity to Ca2+ of the cleaved form of protein S as compared with the undegraded molecule. After activation of the complement system in human serum, protein S was found to be a constituent part of the complex formed by C4b-binding protein and component C4b.

Blood Coagulation↗

PubMed1985

Physicochemical characterization of human S-protein and its function in the blood coagulation system.

S-protein, the main inhibitor of the assembly of the membrane attack complex of complement, was isolated from human plasma by a simple purification procedure, which includes barium citrate adsorption, ammonium sulphate precipitation, chromatography on DEAE-Sephacel and Blue Sepharose and gel filtration on Sephacryl S-200. The homogeneous protein (sedimentation coefficient 4.6 S) was obtained in approx. 5% yield relative to its concentration in plasma, which was found to be 0.3-0.5 mg/ml. The final product did not cross-react with antisera against complement proteins or other proteinase inhibitors of human plasma. On polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, S-protein migrated as a single-chain band with an apparent Mr of 74000 under non-reducing conditions and as a doublet of Mr 78000 and 65000 upon reduction. In plasma or serum S-protein also existed in two forms of corresponding Mr values, as was evidenced by an immunoblot enzyme-linked immunosorbent assay technique. S-protein was found to be an acidic glycoprotein with 10% (W/W) carbohydrate content and several isoelectric points in the range pH 4.75-5.25, and it contained one free thiol group per molecule of protein. The functional properties of S-protein in the complement system were demonstrated by its ability to inhibit complement-dependent cell lysis in a concentration-dependent manner (Ki 0.6 microM) and by its incorporation into the nascent SC5b-7 complex. A new function for S-protein could be revealed in the blood coagulation system. The slow progressive inhibition of thrombin by antithrombin III was not affected by S-protein, whereas the purified protein interfered with the fast inactivation of thrombin clotting as well as amidolytic activity by antithrombin III-heparin complex. The acceleration of this inhibition reaction by heparin was counteracted by S-protein, indicating the ability of S-protein to neutralize heparin activity.

Antithrombin III↗

PubMed2002

Transplantation of an indigenous neural stem cell population leading to hyperplasia and atypical integration.

Astrocytes exhibit neural stem cell characteristics in vitro by generating multipotent clones of cells. In order to see if normal cues are present in vivo that can direct these astrocytes to generate cells of neuronal lineage, the astrocytes were transplanted into the persistently neurogenic mouse subependymal zone/rostral migratory stream. Grafted astrocytes assumed migratory profiles, joined chains of indigenous neuroblasts, and migrated into the olfactory bulb. Additionally, however, some grafted astrocytes "homed" to the lateral ventricle where they became hyperplastic, forming spherical structures composed of cells of mixed phenotype that attached to the ventricular wall, and eventually penetrated and dispersed within surrounding brain parenchyma. It is proposed that, with an interest in the use of stem cell transplants for neurological disease, findings of hyperplasia and apparent atypical integration of a native population of multipotent astrocytic stem cells suggest the need for caution before beginning even autologous neural stem cell transplants.

Animals↗

PubMed2006

Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development.

We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.

Alternative Splicing↗

PubMed2005

Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties.

Mucosal antibody defense depends on a complex cooperation between local B cells and secretory epithelia. Mucosa-associated lymphoid tissue gives rise to B cells with striking J-chain expression that are seeded to secretory effector sites. Such preferential homing constitutes the biological basis for local production of polymeric immunoglobulin A (pIgA) and pentameric IgM with high affinity to the epithelial pIg receptor that readily can export these antibodies to the mucosal surface. This ultimate functional goal of mucosal B-cell differentiation appears to explain why the J chain is also expressed by IgG- and IgD-producing plasma cells (PCs) occurring at secretory tissue sites; these immunocytes may be considered as 'spin-offs' from early effector clones that through class switch are on their way to pIgA production. Abundant evidence supports the notion that intestinal PCs are largely derived from B cells initially activated in gut-associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists concerning the relative importance of M cells, major histocompatibility complex class II-expressing epithelial cells, and professional antigen-presenting cells for the uptake, processing, and presentation of luminal antigens in GALT to accomplish the extensive and sustained priming and expansion of mucosal B cells. Likewise, it is unclear how the germinal center reaction in GALT so strikingly can promote class switch to IgA and expression of J chain. Although B-cell migration from GALT to the intestinal lamina propria is guided by rather well-defined adhesion molecules and chemokines/chemokine receptors, the cues directing preferential homing to different segments of the gut require better definition. This is even more so for the molecules involved in homing of mucosal B cells to secretory effector sites beyond the gut, and in this respect, the role of Waldever's ring (including the palatine tonsils and adenoids) as a regional inductive tissue needs further characterization. Data suggest a remarkable compartmentalization of the mucosal immune system that must be taken into account in the development of effective local vaccines to protect specifically the airways, eyes, oral cavity, small and large intestines, and urogenital tract.

Animals↗

PubMed2004

Odorant receptor gene expression changes during the parr-smolt transformation in Atlantic salmon.

The ability of salmon to home accurately to their natal stream to spawn has long intrigued biologists and has important consequences for the maintenance of population structure in these species. It is known that olfaction is crucial to homing, and that the transition from the freshwater to the marine environment (the parr-smolt transformation; PST) is a period of increased olfactory sensitivity and learning, resulting in a permanent memory of natal site odours that is retained, at least in part, in peripheral sensory neurones. These odours are then used as cues by sexually maturing fish on their homeward migration. We used quantitative polymerase chain reaction techniques to demonstrate transient increases in expression of odorant receptor transcripts (of up to fifty-fold over pre-PST levels) coincident with PST. Both olfactory (SORB) and vomeronasal receptors (SVRA and SVRC) are involved, which suggests that the fish learn both environmental odours and semiochemicals (pheromones). Receptor expression varies between families and changes over time indicating both genetic differences in odour stimuli and multiple periods of olfactory sensitivity. We suggest that changes in OR gene expression may have a role in homing behaviour and thus the maintenance of population structure in Atlantic salmon.

Amino Acid Sequence↗

PubMed1983

Purification and characterization of myosin from calf brain.

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.

Actins↗

PubMed1994

Identification of the ron gene product as the receptor for the human macrophage stimulating protein.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

Animals↗

PubMed1977

Factor IX antigen by radioimmunoassay. Abnormal factor IX protein in patients on warfarin therapy and with hemophilia B.

Factor IX, isolated from normal human plasma, was homogenous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. On the latter, it migrated as a single polypeptide chain with or without reducing agents and had an apparent mol wt of 62,000. After iodination by chloramine-T, a single peak of 125I was found on gels. Immunoelectrophoresis in agarose with rabbit antifactor IX sera gave a single arc against both isolated and partially purified factor IX preparations. The rabbit antibody was specific as it failed to inhibit the activities of prothrombin or factors VII or X in normal plasma. At an additional 20-fold dilution, factor IX activity was inhibited 50%. In a double-antibody radioimmunoassay, excess rabbit anti-human factor IX precipitated 90-95% of the 125I-human factor IX. Control without specific antibody gave 6-8%. Dilutions of a pool of normal human plasma paralleled dilutions of the isolated preparation and were used for the standard curve. Of 39 plasma samples from normal donors, the mean factor IX antigen level was 93% of that of a separate normal pool. The radioimmunoassay detected the abnormal factor IX produced in patients on warfarin therapy. After Al(OH)3 adsorption of warfarin treated patient's plasma, factor IX antigen, but not activity, was present in the supernate. Samples from 28 patients on warfarin gave a mean factor IX clotting activity of 27% with a mean antigen of 69%. The antigen level from the warfarin group was significantly lower than the antigen level of the normal group (P less than 0.001). The factor IX antigen level was then assessed in 36 patients from 29 pedigrees with hemophilia B. The median antigen level was 17% of normal. The distribution of the antigen level was wide with two patients around 100% of normal; only two had levels below the limits of resolution of the radioimmunoassay as currently performed (less than 2%). Within each of the five pedigrees in which more than one affected member was tested, activity and antigen levels were the same. The degree of neutralization of the antibody's inhibition of normal plasma by patient's plasma was highly correlated. Additional evidence for the detection of abnormal protein was provided by immunodiffusion of plasmas concentrated by lyophilization. Reactions of complete identity occurred between normal, a warfarin treated and a hemophilia B subject's plasmas.

Adolescent↗