Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Binding of aluminium to DNA of DNP in pea root nuclei

The association of absorbed aluminium (Al) with nuclei in Alaskapea roots was shown by zonal centrifugation of isolated nuclei.In nuclei, 73% of the total incorporated Al was detected inthe DNP fraction. Furthermore, Al in DNP was found to be boundspecifically to DNA by gel filtration of dissociated DNP. (Received March 19, 1977; )     

Identification of true thymidine kinase in Tetrahymena pyriformis ST.

Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg²⁺, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Although the mitochondrial fraction also exhibited ATP-dependent phosphorylation, it is not stimulated by Mg²⁺ and not significantly inhibited by dTTP. Nucleoside phosphotransferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphotransferase activities were separated by polyacrylamide gel electrophoresis, establishing the presence of both enzymes in this organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.     

Regulation of lettuce hypocotyl elongation by gibberellic acid. Correlation between cell elongation, stress-relaxation properties of the cell wall and wall polysaccharide content

Lettuce hypocotyl elongation caused by gibberellic acid wasstrongly inhibited by coumarin and dichlobenil, known inhibitorsof cellulose biosyndiesis. Stress-relaxation analysis of thecell wall revealed that gibberellic acid induces a decreasein both minimum relaxation time (To) and relaxation rate (b)and an increase in maximum relaxation time (Tm), when gibberellicacid stimulates hypocotyl elongation. Both coumarin and dichlobenilnullified the effect of gibberellic acid on changes in To, Tmand b values. The content of pectic, hemicellulosic and cellulosic substancesin the cell wall increased per hypocotyl but decreased per unithypocotyl length, in response to gibberellic acid treatment.Particularly, gibberellic acid caused a substantial increasein cellulose content per hypocotyl but a decrease per unit length.A good correlation existed between the decrease in To and thedecrease in hemicellulose content per unit lengdi of the cellwall. The increase in Tm was correlated with the decrease incellulose content per unit length of the cell wall. The decreasein b was correlated with the decrease in the content of bothcellulose and hemicellulose per unit length. Based on these results, we discuss the role of polysaccharidemetabolism of the cell wall in gibberellic acid-induced lettucehypocotyl elongation and the nature of gibberellic acid-inducedbiochemical modifications of the cell wall, which are representedby changes in stress-relaxation properties of the cell wall. ¹Present address: Department of Anatomy, Aichi Medical University,Nagakutecho, Aichigun, Aichi 480-11, Japan. (Received September 22, 1975; )     

Stimulation of auxin-induced elongation of cucumber hypocotyl sections by dihydroconiferyl alcohol. Dihydroconiferyl alcohol inhibits indole-3-acetic acid degradation in vivo and in vitro

The mechanism by which dihydroconiferyl alcohol (DCA) stimulatesindole-3-acetic acid (IAA)-induced elongation of cucumber hypocotylsections was studied. Although DCA did not affect the uptakeof IAA-5-³H by hypocotyl sections, the endogenous level of IAA-5-³Hin DCA-treated sections was much higher than in DCA untreatedones. IAA-5-³H in the incubation medium was degraded in thepresence of hypocotyl sections, and this degradation of IAAwas inhibited by DCA. An in vitro experiment with horseradishperoxidase revealed that DCA inhibited the IAA degrading activityof the oxidase, as did caffeic acid and ferulic acid. Theseresults suggested that DCA enhances IAA-induced cucumber hypocotylelongation by acting as an antioxidant of IAA. (Received June 4, 1975; )     

Indole-3-acetaldehyde oxidase of pea seedlings

Indole-3-acetaldehyde oxidase was partially purified from the epicotyl of Pisum satiyum seedlings by column chromatography using CM-Sephadex and Sephadex G-150. The enzyme was only active in the presence of molecular oxygen. The activity was maximal at pH 8.0, and the Km value for indole-3-acetaldehyde was 1.4 × 10⁻³ M . The enzyme was inhibited strongly by p -hydroxymercuribenzoate, cyanide and hydroxylamine, suggesting that it contains sulfhydryl group(s) and a metal component such as iron.     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)