Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Purification and properties of poly(adenosine diphosphate ribose) synthetase.

Poly(ADP-ribose) synthetase has been purified approximately 5000-fold from rat liver nuclei. The activity of the purified enzyme is absolutely dependent upon the presence of native or synthetic DNA, and the further addition of histone(s) stimulates the activity 3- to 5-fold. When the ADP-ribosylated material synthesized in the absence or presence of various histones is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the major product in all cases migrates between histones H1 and H3-H2B with the same RF value of 0.58 relative to the marker dye. No ADP-ribose was found to co-electrophorese with any of thehistones. The addition of histones does not affect the chain number of the poly(ADP-ribose) synthesized but does result in an increase in the average chain length of the polymer. In the presence of histones, the Km for NAD+ decreases from 80 micron to 25 micron and the Vmax doubles. These results indicate that, in the purified poly(ADP-ribose) synthetase system, histones are not ADP-robosylated but act as allosteric activators.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

Possible participation of glucocorticoid in elevation of 3',5'-cyclic AMP levels through inhibition of cyclic AMP phosphodiesterase.

Administration of prednisolone and cholate to rats elevated levels of cAMP (adenosine 3',5'-cyclic monophosphate) by 1.5- to 2.0-fold. Compounds such as prednisolone, hydrocortisone, cholate, and deoxycholate were found to be potent inhibitors of partially purified cAMP phosphodiesterase prepared from rat liver. Kinetic analysis showed that the prednisolone inhibition was noncompetitive with a Ki of 8.9 x 10(-4) M. These results suggest that in addition to increasing DNA-dependent RNA polymerase activity in vivo, a large application of glucocorticoid may incur elevation of intracellular cAMP levels.     

Enzyme systems involved in phosphorylation and dephosphorylation of two endogenous phosphoproteins in neuronal membranes.

Adenosine 3′:5′-monophosphate-dependent protein kinase and phosphoprotein phosphatases were solubilized by Triton X-100, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes, and partially purified. The properties of these partially purified enzymes were studied using two substrates, Protein I and Protein II, prepared from the synaptic membrane fraction, as well as the substrates protamine and histone. The results suggest that the phosphorylation of Protein I and Protein II, as well as protamine and histone, are catalyzed by a single species of cAMP-deperident protein kinase. Thus, a single peak of protein kinase activity was observed, upon DEAE-cellulose hromatography of the Triton X-100 extract of the synaptic membrane preparation, which catalyzed the phosphorylation of all four substrate proteins. Moreover, the activity of this partially purified protein kinase toward the various substrate proteins was altered in a parallel fashion, either when the protein kinase preparation was subjected to heat inactivation or pH inactivation, or when the enzyme was assayed in the presence of various concentrations of cyclic nucleotides or of a protein kinase modulator. The individual protein substrates acted as competitive inhibitors with respect to one another. Upon sucrose density gradient centrifugation, the protein kinase activity toward the various substrates sedimented as a single peak. Finally, the relative specific activities toward the various substrates did not change significantly during a 2000-fold purification of the enzyme. In contrast to these observations with protein kinase, two peaks of protein phosphatase activity, with markedly different specificities toward Protein I and Protein II, were found upon DEAE-cellulose and Bio-Gel P-200 column chromatography of the Triton X-100 extract of the synaptic membrane fractions. One peak catalyzed the dephosphorylation of Phosphoprotein I but not of Phosphoprotein II, whereas the other peak catalyzed the dephosphorylation of Phosphoprotein II but not of Phosphoprotein I. The dephosphorylation of Phosphoprotein I by Phosphoprotein I phosphatase was not affected by adenosine 3':5'-monophosphate, whereas the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase required the presence of this nucleotide. Moreover, the two phosphatases differed from one another with respect to Stokes' radius as well as sedimentation coefficient.     

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues

An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 bp was designed and constructed from 44 oligodeoxynucleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease alpha-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectroscopy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells.(ABSTRACT TRUNCATED AT 250 WORDS)