Expression of three forms of thyroid hormone receptor in human tissues

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.     

Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.     

Electron-microscopic immunocytochemistry of neuropeptide Y immunoreactive innervation of vasopressin neurons in the paraventricular nucleus of the rat hypothalamus

The neuropeptide Y (NPY) immunoreactive synaptic input to neurons containing neurophysin II (NP II), the carrier protein of vasopressin (VP), was observed in the paraventricular nucleus (PVN) of the rat hypothalamus by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase (PAP) method with the postembedding immunogold staining method at the electron-microscopic level. NPY-like immunoreactivities were detected by the PAP method in the dense granular vesicles (70-100 nm in diameter) in the immunoreactive presynaptic axon terminals. NP II-like immunoreactive large neurosecretory granules labeled with gold particles were found in the neurons receiving synaptic input of the NPY-like immunoreactive terminals. This suggests that NPY may be a neurotransmitter or neuromodulator and that NPY neurons may, through synaptic contacts, regulate the secretion of VP neurons.     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.     

ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis.

During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes.     

The Role in Ozone Phytotoxicity of the Evolution of Ethylene upon Induction of 1-Aminocydopropane-1-carboxylic Acid Synthase by Ozone Fumigation in Tomato Plants

The rate of evolution of ethylene by tomato plants was rapidlyincreased by O₃ fumigation. The time course of the increasein 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activitywas the same as that in the rate of evolution of ethylene, suggestingthat ACC synthase activity might be a rate-limiting step inthe evolution of ethylene that is caused by O₃ fumigation. Therate of the O₃-induced evolution of ethylene was increased bythe application of ACC to tomato plants, suggesting the involvementof ACC oxidase in the O₃-induced evolution of ethylene. Treatmentof plants with tiron inhibited the evolution of ethane, butnot of ethylene. These results indicated that evolution of ethylenein O₃-treated tomato plants might result from enzymatic reactionscatalyzed by both ACC synthase and ACC oxidase, but not fromstimulation by O₃ of the peroxidation of lipids mediated byfree radicals. Pretreatment of leaves with aminoethoxyvinylglycine (AVG), aninhibitor of ACC synthase, significantly inhibited the evolutionof ethylene that was induced by O₃ and concomitantly reducedthe extent of O₃-induced visible damage to leaves. Treatmentwith 2,5-norbonadiene, an inhibitor of the action of ethylene,strongly reduced the extent of visible damage caused by O₃,even though it did not suppress the evloution of ethylene. Theseresults indicate that ethylene acts on certain metabolic processesto cause visible damage. (Received September 7, 1995; Accepted December 18, 1995)     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.