Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Myosin has been isolated from cloned mouse fibroblasts, line L-929. Fibroblast myosin: (i) binds to rabbit muscle actin and is dissociated from it by ATP, (ii) has an ATPase activity that is suppressed by Mg(2+) in 0.6 M KCl and is activated by rabbit muscle actin in the presence of Mg(2+) in 14 mM KCl, (iii) forms thin bipolar aggregates in 0.1 M KCl when viewed in the electron microscope, (iv) possesses a heavy chain with the same mobility as muscle myosin (molecular weight 200,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these respects, fibroblast myosin appears to be similar to muscle myosin in structure and function.     

Human Pituitary Growth Hormone Isolation and Properties of Two Biologically Active Fragments from Plasmin Digests

Two biologically active fragments have been isolated from plasmic digests of human pituitary growth hormone. It was shown that these two fragments were derived from the cleavage of the Arg-Thr (positions 134-135) and the Lys-Gln (positions 140-141) bonds of the hormone: one has 134 amino acids and the other 51 amino acids, respectively. The two fragments were active in the rat tibia and pigeon crop-sac tests, as well as in complement fixation experiments.     

Isolation and Properties of a Low Molecular Weight Antiplasmin of Human Blood Platelets and Serum

A low molecular weight antiplasmin has been detected in human blood platelets. The antiplasmin is dialysable and a similar material is present in normal plasma. A simple method for rapid isolation of the antiplasmin based on ultrafiltration is described. The inhibition of plasmin by these materials under different conditions has been studied. In the ultracentrifuge, both antiplasmins showed a single broad peak with an approximate sedimentation coefficient of 0.5S. Paper chromatography and paper electrophoresis indicated the isolated material to be heterogeneous. The individual components were isolated by paper chromatography and the antiplasmin activity was measured by the fibrin plate method. Preliminary studies on the fraction with maximal antiplasmin activity suggest that the inhibitory effect might be due to enzyme-inhibitor complex formation. Based on the present data, it is concluded that the platelet antiplasmin and the serum antiplasmin are very similar. This antiplasmin material may be useful in fibrinolytic therapy.     

Calpains and Calpastatin in Human Blood Platelets

Calpain, a Ca²⁺ activated intracellular protease and its endogeneous protein inhibitor, calpastatin are abundant in platelets. The structure and enzymological properties of calpain, including its isozymes, and of calpastatin in platelets have been fully characterized. Also, platelet calpain has been shown to cleave various endogeneous polypeptides. However, the mode of activation and the physiological function of platelet calpains have not been clarified. Our recent investigations on platelet calpains with cell permeable calpain antagonists and specific antibodies reveal that calpains are not involved in the early phases of platelet activation such as shape change and aggregation, but in the later phases of platelet activation such as cytoskeletal reorganization and Ca²⁺ uptake.     

Glycogen in Human Blood Platelets. Isolation by Ultracentrifugation and Characteristics of the Isolated Particles

Platelet glycogen was studied under various conditions of ultracentrifugationand an isolation procedure devised.

Glycogen could be separated from the majority of soluble protein andcellular particulates by centrifugation into dense sucrose solutions. Minorcontaminants were removed by differential centrifugation and detergent treatment. Characteristics of the isolated particles were consistent with the presumed "native" particle (glycosome) as measured by spectrophotometry,analytical ultracentrifugation, and protein: glycogen ratios.

Submitted on August 8, 1969 Accepted on October 31, 1969     

Papain-Solubilized HL-A Antigens from Cultured Human Lymphocytes Contain Two Peptide Fragments

HL-A 2 and HL-A 7 antigens have been solubilized from cultured human lymphocytes by papain and purified on a small radioactive scale after formation of immune complexes, or on a large scale by column chromatographic methods. In both cases, it has been shown that the materials obtained consist of two noncovalently bound fragments. One fragment is a glycopeptide of molecular weight 30,000-31,000, the other is a peptide of molecular weight 11,000-12,000. In polyacrylamide gels containing 8 M urea, the large fragments of HL-A 2 and HL-A 7 exhibited different electrophoretic mobilities, while the small fragments were the same. The relationship of these materials to the native HL-A molecule is discussed.     

Biochemical Characterisation of A Blood Group Activity on Human Platelets

A butanol extraction procedure has been used to isolate the blood group A antigen determinants from platelet membranes and to determine their biochemical structure-glycolipid and/or glycoprotein. The blood group A serological activity was found to reside entirely in the butanol (organic) fraction. When this fraction was analyzed by thin-layer chromatography, the A-inhibitory activity was found to comigrate with extracts of erythrocyte type Aa, Ab and Ac variant structures. The results of this study indicate that the ABO blood group A determinants on human platelets are glycolipid, similar in structure to glycolipid A determinants on erythrocytes.     

Characterization and Significance of Collagen Induced Biphasic Aggregation of Human Platelets

Biphasic collagen-induced platelet aggregation, resembling that induced by epinephrine, was noted in platelet-rich plasma (PRP) of 11 stroke and 1 coronary disease patients. Similar pattern of aggregation was not observed in normal PRP. The occurrence of the biphasic collagen aggregation does not appear to relate to platelet count, smoking habit, medication, or other abnormalities such as hypertension, diabetes, and elevated serum lipid levels. However, platelets of these patients were very sensitive to aggregating agents including epinephrine and adenosine diphosphate. The concentration of collagen that elicited biphasic aggregation in these platelets was too weak to aggregate platelets of normal subjects. We believe that the release threshold of these platelets is reduced to such an extent that minute amounts of collagen, which would be insufficient to induce release from normal platelets, are capable of inducing release from these platelets. Both phases of collagen induced aggregation are probably resulted from the activation of the release mechanism.     

Isolation and Characterization of Two Glycoproteins from Patients with Alveolar Proteinosis

The saline-insoluble particulate material obtained by pulmonary lavage from four patients with alveolar proteinosis was analyzed for total lipid (52%), protein (44%), and carbohydrate (4%). Sodium dodecyl sulfate-gel electrophoresis revealed only three major peptides in these patients. Molecular weights (from the gels) were 69,000, 62,000, and 36,000, and the latter two peptides were periodic acid-Schiff stain-positive. Amino-acid and sugar analyses were performed on the peptides cut from the gels and on peptides purified by Sephadex G-200 chromatography. The 69,000-molecular-weight peptide contained no carbohydrate. Antibody studies and aminoacid analysis indicated that it was albumin. The 62,000-molecular-weight peptide contained 1% hydroxyproline, 1% hydroxylysine, 10% glycine, and 9% carbohydrate. The 36,000-molecular-weight peptide contained 1.2% hydroxyproline, 1% hydroxylysine, 13% glycine, 1.4% sialic acid, 2.6% glucose, 2.4% galactose, 2% mannose, 0.8% fucose, 0.4% glycosamine, and 0.6% galactosamine. Proteins extracted from kidney glomeruli with similar amino-acid and carbohydrate composition have been observed previously.     

Lipids of Human Platelets and Their Action on the Blood Coagulation Process

(1) Purification of lipid extracts from human platelets yielded non-lipidmaterial which exhibited anti-thromboplastic and/or anti-thrombic properties.In one instance, both uronic acids and hexosamines were absent; consequently, the anticoagulant activity could not be attributed to the presence ofacid mucopolysaccharides. The purified lipid fraction contained most of thethromboplastic activity of human platelets.

(2) The neutral lipid fraction of human platelets contained neither vitamins A, D, E, and K nor steroid hormones. The neutral lipids of humanplatelets exhibited no thromboplastic activity when tested individually orin combination with one another or with lecithin.

(3) Human platelet phospholipids were resolved into five major fractionsby means of silicic acid column chromatography and countercurrent distribution technics. The purified lecithin, sphingomyelin, and inositol phosphatides were void of any thromboplastic activity when tested individuallyor in combination with one another. It was confirmed that both phosphatidylserine and phosphatidylethanolamine exhibited thromboplastic activity andthat phosphatidylserine at high concentrations had a pronounced anticoagulanteffect.

(4) When phosphatidylethanolamine and phosphatidylserine were combined, synergistic thromboplastic activity was observed. When phosphatidylserine was combined with lecithin, the thromboplastic activity of phosphatidylserine was greatly enhanced.

Submitted on September 4, 1963 Accepted on December 23, 1963     

Isolation and Characterization of Gastric Carcinomaassociated DNA Fragments by U~ng Optimized AP-PCR

The molecular mechanism of Gastric cancer (GC) is gaining ground rapidly. Cytological study has identified the multiple changes of gene, to isolate GC associated genes, optimized arbitrarily primed polymerase chain reaction (AP-PCR) is used in present study matching GC tissues and non-cancerous gastric tissues. The present study includes three parts: the first step is acquirement of the differential bands. The DNA is isolated from matched GC tissues and non-cancerous gastric tissues. DNA fingerprinting generated by using optimized APPCR. The second step is identification of the DNA. The DNA extracted from the differential bands are amplified and identified by dot blot hybridization,     

Quantitative Isolation and Purification of Blood Group-Active Glycosphingolipids from Human B Erythrocytes

11.4 mg of a ceramide hexahexoside (B-I) and 16.4 mg of a ceramide octahexoside (B-II) as blood group B-active glycosphingolipids composed of glucose, galactose, N-acetylglucosamine and fucose (molar ratios 1:3:1:1 and 1:4:2:1 respectively) have been isolated from 6,400 ml of packed human B erythrocytes. This yield is greater by more than the 13-fold amount of B-I glycosphingolipid and the 20-fold amount of B-II glycosphingolipid which has hitherto been isolated from human erythrocytes. The B-active glycosphingolipids isolated represent about 0.04% of the erythrocyte membrane and in consequence must be regarded as the main representants of B properties of the erythrocytes as far as they have been investigated up to this time. This high yield was achieved by a simple and conservative erythrocyte membrane preparation without loss of serological activity and by the improvement of some chromatographical methods which permitted a high purification without the acetylation-deacetylation procedure. Purity was checked by gas-liquid chromatographical analysis of the sugars as their alditol acetates and by the hemagglutination inhibition technique. 1.7 x 10-8 g of each of these glycosphingolipids completely inhibit the agglutination of human B erythrocytes by 4 hemagglutination units of normal human anti-B sera. A ceramide tetrahexoside and a glycolipid fraction with a high H activity could also be isolated which possibly are blood group-intermediate substances. Lewis blood group-active glycosphingolipids characterized by the hemagglutination inhibition test and by passive hemagglutination are trace constituents of other glycosphingolipid fractions.     

Isolation and Characterization of Normal Human Megakaryocytes

S ummary Human megakaryocytes have been isolated from marrow obtained from ribs removed at thoracotomy. All but one of the patients had normal pre-operative platelet and leucocyte counts. Megakaryocytes averaged 0·37% of all cells in marrow cell suspensions from nine consecutive subjects. A 283-fold purification (to 10·3%) was achieved by a density gradient centrifugation followed by two successive velocity sedimentations at unit gravity. The net yield, 12 800 megakaryocytes per specimen, was sufficient for many kinds of morphological study. Bright-field, phase contrast, and electron microscopy were used to characterize the younger and smaller megakaryocytes. Ploidy analyses were carried out on 100–235 megakaryocytes per specimen; 8N was the predominant ploidy class in isolated megakaryocyte populations from three individuals. The mean megakaryocyte diameter was 24 μm in three other specimens and the range was 10–48 μm. This data had a normal distribution and overlapped minimally with the size range of all other marrow cells. The presence of a distinct size threshold (at 11·5 μm) implied that size alone may be a sufficient objective criterion for identification of human megakaryocytes.     

Isolation of Mesenchymal Stem Cells from Cryopreserved Human Umbilical Cord Blood

Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages. Because mesenchymal stem cells (MSCs) from bone marrow have been regarded as good materials for cell/gene therapy as well as for tissue engineering because of their multidifferentiation potential, a number of trials have been undertaken to isolate MSCs from UCB. However, the results have been controversial, and little has become known about the effect of cryopreservation on the isolation of these stem cells. In this study, we examined the ability of cryopreserved UCB-derived cells to produce MSCs. Various culture conditions, including the seeding concentrations of cells and the media used, were investigated. We were able to obtain adherent cell populations after 3 to 5 weeks in our culture conditions from UCB-derived mononuclear cell fractions that had undergone cryopreservation for 0.1 to 5 years. These cells exhibited a fibroblast-like morphology and typical mesenchymal-like immunophenotypes. The results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and clinical applications as well as for tissue engineering.     

Fine Structural Localization of Adenosine Triphosphatase in Human Platelets and Other Blood Cells

Adenosine triphosphatase activity has been localized in human blood cellsby combined histochemical and electron microscopic technics. Deposits oflead phosphate, indicative of enzymic activity, were observed on the surfacemembranes of erythrocytes, lymphocytes and platelets. Intracellular ATPaseactivity was found in lymphocyte mitochondria and nucleoli. Blood plateletscontained organelles in their hyaloplasm with dense accumulations of theenzyme reaction product. The distribution of lead phosphate in platelet organelles suggested that they were mitochondria, but some platelet granules mayalso contain ATPase activity.

Submitted on October 26, 1964 Accepted on March 2, 1965     

Isolation and Analysis of Somatic Hybrids Derived from Two Human Diploid Cells

Lesch-Nyhan fibroblasts and normal human leukocytes with different glucose-6-phosphate dehydrogenase genotypes were fused by Sendai virus. Clones were isolated on the basis of their resistance to a medium containing hypoxanthine, amethopterin, and thymidine and ability to proliferate in monolayer culture. These mononuclear cells (1) incorporated [³H]hypoxanthine, (2) expressed the glucose-6-phosphate dehydrogenase heteropolymer, and (3) were polyploid. Therefore, hybrids can originate from the fusion of two diploid human cells. X chromosome inactivation did not occur in these hybrid cells of male origin. The hybrids expressed both parental genomes and exhibited the senescence and contact feeding characteristic of the human skin fibroblast.     

Studies on the Ultrastructure of Pseudopod Formation in Human Blood Platelets.

A method for quantitative evaluation of blood platelet pseudopod formation is presented. Pseudopod formation was studied in whole blood incubated for 0, 5, 10, 20 and 40 minutes at + 4°, + 15° or + 37° C. Samples were anticoagulated with EDTA or sodium citrate, or incubated without anticoagulant. In instantly fixed blood, platelets showed a pseudopod index of about 0.5. In samples incubated at + 4° C., the pseudopod index rose to about 3 after 20 minutes. After 40 minutes there was no further increase. At + 37° C. the pseudopod index did not increase during the first 20 minutes of incubation, but after 40 minutes it had risen to levels almost similar to those of the + 4° samples. EDTA and sodium citrate exerted no influence upon pseudopod formation at any temperature level tested. Mechanical forces operating during the preparation of platelet rich plasma caused pseudopod formation and enhanced temperature induced pseudopod formation. These results support the theory of Bull & Zucker (1965) that blood platelets maintain their lenticular shape by energy requiring processes.