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PubMed1992

Transfer of plutonium to rat embryos in vivo and in vitro.

The 239Pu distribution in the 12.5-day-old rat conceptus was compared between in vivo and in vitro experimental systems to establish a possible mechanism of cross-placental transfer of this radionuclide. In the in vivo study, plutonium citrate solution was injected intravenously to pregnant Wistar rats. In the in vitro study, either plutonium citrate or plutonium hydroxide colloid was administered, as a solution of Eagle MEM and FCS containing 239Pu at the concentration used in the maternal serum in the in vivo experiments, to rat conceptuses maintained by the whole-embryo culture method. The concentration of 239Pu in the yolk sac (239Pu activity per gram wet weight) were much higher than in the embryo in both the in vivo and in vitro experiments, suggesting that the yolk sac may be an effective barrier against the transfer of plutonium to the embryos. The ratios of the 239Pu concentration in the yolk sac to that in the embryo were relatively constant with time after administration in the in vitro system; 18-27 for plutonium citrate and 67-84 for plutonium hydroxide. In the in vivo experiment, these ratios changed with time after injection; 15 at 5 min and 62 and 60 min after injection. This suggests that in the in vivo system, the chemical form of 239Pu changed with time after injection, probably to a macromolecular form such as the hydroxide colloid or plutonium-protein complex although 239Pu was injected to the maternal blood as citrate.

Animals↗
PubMed2005

Advances in prodrug design.

The background of prodrug design is presented herein as the basis for introducing new and advanced latent systems, taking into account mainly the versatility of polymers and other macromolecules as carriers. PDEPT (Polymer-Directed Enzyme Prodrug Therapy); PELT (Polymer-Enzyme Liposome Therapy); CDS (Chemical Delivery System); ADEPT(Antibody-Directed Enzyme Prodrug Therapy); GDEPT/VDEPT (Gene-Directed Enzyme Prodrug Therapy/Virus-Directed Enzyme Prodrug Therapy); ODDS (Osteotropic Drug Delivery System) and LEAPT (Lectin-directed enzyme-activated prodrug therapy) are briefly described and some examples are given.

Animals↗
PubMedDate not supplied

Protection of cells from complement-mediated attack: CD59 receptor clustering for the entry of macromolecules into hematopoietic cells.

CD59 is a glycosylphosphatidylinositol anchored protein (GPI-protein) that is expressed on surface membranes to protect host cells from complement-mediated attack. CD59 may also serve as a receptor for the endocytosis of macromolecules into nucleated cells. Here we investigate the effects of primary clustering of CD59 with anti-CD59 monoclonal antibody and the secondary clustering of biotinylated anti-CD59 with avidin on red blood cells and erythroleukemic K562 cells. On red blood cells, CD59-targeted antibodies remained evenly distributed on the external membranes. In contrast, clustering, capping and endocytosis of the CD59-targeted complexes was detected on K562 cells. Secondary clustering appeared more efficient and resulted in endosomal localization of the fluorescently labeled complexes within 2 hours. The endocytosis of CD59-bound complexes did not affect K562 cell viability or growth and the surface level of CD59 was constant during the process. These result suggest clustering and subsequent endocytosis of CD59 may enable the entry of macromolecules to the endosomal compartments of hematopoietic cells.

CD59 Antigens↗
PubMed1982

Creatine kinase.

Creatine kinase is present in significant concentrations in skeletal muscle and cardiac muscle and to a lesser extent in gastrointestinal tract and brain tissue. The enzyme has been purified from a variety of tissues and an examination of its kinetic and physical properties reveal that the enzyme consists of two subunits and can exist as three isoenzymes containing essential cysteine residues. These properties are important in understanding its stability, the assay conditions, and the techniques used to identify the different isoenzymes. The relationship between the properties and the determination of the enzyme in biological fluids will be a main thrust of the review. Creatine kinase activity in serum rises rapidly in conditions such as acute myocardial infarction and trauma to skeletal muscle. However, the interpretation of such increases is dependent upon a sound knowledge of the factors which influence both the total and isoenzyme activities. The nature of these factors will be discussed in detail.

Animals↗
PubMed2000

Efficient introduction of macromolecules and oligonucleotides into brain capillary endothelial cells using HVJ-liposomes.

In this study, we examined the feasibility of introducing macromolecules into cultured mouse brain capillary endothelial cells (MBEC4 cells) by utilizing the hemagglutating virus of Japan (HVJ)-liposomes with fusogenic activity. We used fluorescein isothiocyanate dextran (FITC-Dextran) and FITC-labeled oligodeoxynucleotide (FITC-ODN) as models of a macromolecule and an ODN, respectively. Intracellular fluorescence appeared rapidly after the exposure of MBEC4 cells to FITC-Dextran-containing HVJ-liposomes, and remained detectable for at least 3 days. Only a control level of intracellular fluorescence was seen after treatment with FITC-Dextran alone, FITC-Dextran with empty HVJ-liposomes or FITC-Dextran-containing liposomes without fusogenic activity. In the early phase after administration (0-30 min), the introduction of FITC-Dextran into MBEC4 cells by the HVJ-liposome method resulted in a rapid and time-dependent increase of intracellular fluorescence intensity. Moreover, FITC-ODN was also introduced into MBEC4 cells by the HVJ-liposome method, although FITC-ODN alone was not introduced. These results indicate that the HVJ-liposome method is useful for the efficient introduction of macromolecules, including ODN, into brain capillary endothelial cells.

Animals↗
PubMed1998

Direct convective delivery of macromolecules to the spinal cord.

OBJECT: Because of the limited penetration of macromolecules across the blood-spinal cord barrier, numerous therapeutic compounds with potential for treating spinal cord disorders cannot be used effectively. The authors have developed a technique to deliver and distribute macromolecules regionally in the spinal cord by using convection in the interstitial space. METHODS: The authors designed a delivery system connected to a "floating" silica cannula (inner diameter 100 microm, outer diameter 170 microm) that provides for constant volumetric inflow to the spinal cord. A solution containing albumin that was either unlabeled or labeled with carbon-14 or gadolinium was infused at various volumes (3, 6, 10, 20, 40, or 50 microl) at a rate of 0.1 microl/minute into the spinal cord dorsal columns of nine swine and into the lateral columns of three primates (Macaca mulatta). Volume of distribution (Vd), concentration homogeneity, and percentage of recovery were determined using scintillation analysis, kurtosis calculation (K), and quantitative autoradiography (six swine), magnetic resonance imaging (one swine and three primates), and histological analysis (all animals). Neurological function was observed for up to 3 days in four of the swine and up to 16 weeks in the three primates. The Vd of 14C-albumin was linearly proportional (R2=0.97) to the volume of infusion (Vi) (Vd/Vi=4.4+/-0.5; [mean+/-standard deviation). The increases in Vd resulting from increases in Vi were primarily in the longitudinal dimension (R2=0.83 in swine; R2=0.98 in primates), allowing large segments of spinal cord (up to 4.3 cm; Vi 50 microl) to be perfused with the macromolecule. The concentration across the area of distribution was homogeneous (K=-1.1). The mean recovery of infused albumin from the spinal cord was 85.5+/-5.6%. Magnetic resonance imaging and histological analysis combined with quantitative autoradiography revealed the albumin infusate to be preferentially distributed along the white matter tracts. No animal exhibited a neurological deficit as a result of the infusion. CONCLUSIONS: Regional convective delivery provides reproducible, safe, region-specific, and homogeneous distribution of macromolecules over large longitudinal segments of the spinal cord. This delivery method overcomes many of the obstacles associated with current delivery techniques and provides for research into new treatments of various conditions of the spinal cord.

Animals↗
PubMed2001

[The software design for the molecular biological image processing system].

With the use of Visual Basic 6.0 language and molecular biological technology, we have developed a new molecular biological information processing system to detect the characteristic parameter of molecular biological electrophoresis image. The software can determine the molecular weight of the restriction endonuclease hydrolytic DNA fragment and protein or other macromolecules. The application indicates that the system with its accuracy reliability high efficiency and a large capacity of data to be stored, It is very useful to gene diagnosis and molecular biological researches.

Computer Systems↗
PubMed1992

Partial characterization of an autoantibody recognizing the secondary binding site(s) of thrombin in a patient with recurrent spontaneous arterial thrombosis.

A patient with an 18 year history of recurrent arterial thrombosis and no evidence of atherosclerosis or embolism of cardiac origin presented with a prolonged thrombin clotting time when performed with human thrombin. The bovine thrombin clotting time was only slightly prolonged. During 30 months of follow-up, the thrombin time fluctuated, but remained prolonged. The patient has been treated with an oral anticoagulant for the past 8 years, with no thrombotic recurrence. The inhibitor activity was due to the presence of polyclonal IgGs which bound to thrombin-Sepharose. The influence of IgGs purified from the patient's serum was compared to the influence of normal IgGs in several systems exploring the catalytic activity of thrombin and the binding of the enzyme to macromolecular substrates through secondary binding site(s). We found that the IgGs did not impair the catalytic activity toward small synthetic substrates, but inhibited the binding of thrombin to fibrinogen, thrombomodulin and heparin cofactor II. Such proteins are known to require a secondary binding site of thrombin to interact with the enzyme. The anti-thrombin antibody might have resulted from an abnormal generation of thrombin. This would be the consequence of the process favouring thrombosis. Alternatively, the autoantibody might have favoured thrombosis primarily, by impairing natural antithrombotic mechanisms triggered by thrombin.

Adult↗
PubMed1991

Assembly of adenovirus type 2 fiber synthesized in cell-free translation system.

Physicochemical and functional analyses of the translation products of fiber mRNA in rabbit reticulocyte lysate suggested that fiber polypeptide chains (monomers) were capable of self-assembling in vitro, forming trimeric fibers (trimers) without direct intervention of any other adenovirus-coded protein or cell nuclear matrix component. Kinetic studies showed that trimer formation occurred at a rate six times lower than that of fiber polypeptide synthesis. Fiber assembly was found to be relatively inefficient in vitro, with only 25-30% fiber polypeptides trimerized after 4-h translation reaction. The rate constant for fiber subunit assembly, extrapolated from the kinetic curves of trimer formation, was found to be in the order of magnitude of 10(5) M-1 s-1, with a t 1/2 of 1.3 h at 30 degrees C. A latence phase of approximately 40 min in the appearance of the first detectable trimers indicated that fiber assembly did not occur co-translationally, suggesting the existence of rate-limiting intermediate step(s) during assembly.

Adenoviruses, Human↗
PubMed1988

Cloning of cDNA encoding a 32-kDa protein. An accessory polypeptide of the H+-ATPase from chromaffin granules.

The purified H+-ATPase from chromaffin granules is composed of several polypeptides, one of which has an apparent molecular weight of 39,000. Immunoblots with the antibody against this protein and various membrane preparations showed that similar or even identical polypeptides may be associated with the H+-ATPases from synaptic vesicle, kidney microsomes, and lysosomes. A cDNA library was constructed from bovine adrenal medulla, and the cDNA encoding the polypeptide was isolated and sequenced. Search in DNA and protein data banks revealed no significant homology to known genes. Hydrophobicity plot revealed no obvious transmembrane segments with the exception of one stretch of hydrophobic and neutral amino acid starting at leucine 16. The cDNA was shown to encode the entire polypeptide by the virtue of an amino acid sequence corresponding to the N terminus of the open reading frame and by subunit and site-specific antibodies. The cDNA was cloned into an expression vector, transcribed by T7 polymerase, and translated by reticulocyte lysate. Even though the cDNA encodes a protein with a molecular weight of 31,495, the translation product comigrated on sodium dodecyl sulfate gels with the subunit of the purified H+-ATPase. In line with several other subunits of vacuolar H+-ATPases, no signal sequence was detected in the translated gene. Northern blots revealed the presence of a single mRNA of about 1.6 kb in bovine adrenal medulla. However, liver, lung, and kidney may contain additional mRNA of about 1.7 kb.

Adrenal Medulla↗
PubMed1987

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement.

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.

Binding Sites↗
PubMed1985

The molecular shape of dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla.

The structural features of the soluble dopamine beta-hydroxylase from chromaffin granules of bovine adrenal medulla were studied using negative staining and platinum shadowing electron microscopic methods. The enzyme was shown to be highly asymmetric as suggested in earlier hydrodynamic studies. The tetramer of the enzyme appeared as four subunits arranged in the shape of a planar rose with an estimated width of 15 nm. A minimum thickness of 3.0 nm for the enzyme monomer was calculated from the shadow length of unidirectionally shadowed molecules. A model composed of four oblate ellipsoid monomers in a tetrameric rose arrangement is proposed for the shape of the dopamine beta-hydroxylase molecule. Two monomers associate edge to edge to form an in-plane dimer and two dimers associate side-by-side with their respective long axes at a slight angle to form a tetramer. Theoretical calculations based on the model are consistent with previous hydrodynamic studies.

Adrenal Medulla↗
PubMed1983

[Effect of cytochrome b5 on the functional activity and conformational state of cytochrome P-450].

Microsomal cytochromes P-450 and b5 were shown to form mixed complexes with the association constant of 0.24 microM in water solution. Such complex formation stabilizes cytochrome P-450 in the catalytically active conformational state characterized by increased conformational rigidity and temperature stability. This stabilization results in acceleration of the cumene hydroperoxide-dependent oxidation of p-nitroanisol catalyzed by cytochrome P-450. The thermodynamic parameters of O-demethylation of p-nitroanisol catalyzed by cytochrome P-450 and mixed haemoprotein complexes measured in water solution and in a membrane-bound state were found to be different.

Cytochrome P-450 Enzyme System↗
PubMed1980

Luteinizing and follicle-stimulating hormones. Cell-free translations of messenger RNAs coding for subunit precursors.

Polyadenylated RNA prepared from pituitary glands of normal and castrate rats was translated in membrane-free heterologous cell-free systems derived from wheat germ and rabbit reticulocytes. Sodium dodecyl sufate (SDS)-polyacrylamide gel electrophoresis and autoradiography demonstrated a major [35S]methionine-labeled translation product of castrate pituitary mRNA with apparent Mr = 14,000. This Mr = 14,000 product was specifically immunoprecipitated by anti-sera to the alpha subunit of rat and bovine luteinizing hormone (LH alpha) and was much reduced in the translation products of normal pituitary RNA. Additional minor translation products of normal pituitary RNA. Additional minor translation products with apparent Mr = 15,000 and 16,000 were identified by immunoprecipitation with antisera to rat LH beta and to the beta subunit of rat follicle-stimulating hormone (FSH beta), respectively. The immunological identity of the alpha, LH beta, and FSH beta precursors was confirmed by successful competition of these immunoprecipitation reactions by unlabeled homologous LH alpha, LH beta, and FSH, respectively. These results indicate that the gonadotropin subunits are synthesized as precursors from separate mRNAs. This system is responsive to endocrine manipulations and is appropriate for studies of the regulation of gonadotropin biosynthesis.

Animals↗