Transcriptional termination and coupled polyadenylation in vitro.

Using a coupled, in vitro transcription and polyadenylation system we have investigated the molecular mechanism of transcriptional termination by RNA polymerase II (PolII). We showed previously that specific G-rich sequences pause transcription and then activate polyadenylation. We show that physiological pause sites activate polyadenylation in our in vitro system. We also investigate the mechanism of PolII transcriptional termination, and show that these transcripts are either directly released from the transcription complex or are 3' end processed while still attached to the complex. We also show that 3' product (generated by cleavage/polyadenylation) remains associated with the transcription complex, but is rapidly degraded on it.

Paraquat-induced membrane dysfunction in pulmonary microvascular endothelial cells.

Membrane dysfunction monitored by lactate dehydrogenase release from cultured pulmonary microvascular endothelial cells of pigs, which were exposed to paraquat at different concentrations (0.1-2 mM), was examined. Paraquat caused a time-dependent increase in lactate dehydrogenase release. Lactate dehydrogenase releases after 72 hr, 32, 58, and 84% by 0.1, 0.5, and 2 mM paraquat, respectively, were well correlated with cell viability measured by cell adherence. In contrast, reductions of two tetrazolium compounds were depleted profoundly by 72 hr after exposure to 0.5 mM paraquat, suggesting depletion of intracellular reductive substances. Extracellular hydrogen peroxide began to significantly increase 56 hr or 32 hr after exposure to 0.5 mM or 1.5 mM paraquat, respectively, preceding the initial increase of lactate dehydrogenase release (64 hr by 0.5 mM or 48 hr by 1.5 mM). Lactate dehydrogenase release 72 hr after exposure to 0.5 mM paraquat was prevented strongly by catalase (1000 units/ml), but weakly by superoxide dismutase (1000 units/ml). These enzymes failed to restore the reduced acid phosphatase activity. Also, 0.1 mM desferal or alpha,alpha'-dipyridyl protected lactate dehydrogenase release. Similarly, 1 mM thiourea or dimethylthiourea, and 0.5 mM alpha-tocopherol or trolox, were effective, but diethylenetriaminepentaacetic acid (0.1 mM) and probucol (5 or 10 microM) were ineffective. Exposure of 0.5 or 1.5 mM paraquat suppressed levels of lipid peroxidation. These results indicate that membrane dysfunction by paraquat is ascribed to an iron-catalyzed reaction of extracellularly increased hydrogen peroxide. A deleterious species for the membrane dysfunction is discussed.

Superoxide production from paraquat evoked by exogenous NADPH in pulmonary endothelial cells.

Superoxide production from paraquat in a pulmonary microvascular endothelial cell (PMEC) suspension was demonstrated using 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a chemiluminescence probe, to detect superoxide anions. Increased rates of superoxide production from paraquat, which were sensitive to superoxide dismutase (SOD), required the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the reaction medium, and occurred instantaneously after the addition of NADPH, which is impermeable to cell membranes. NADH as an electron donor was not as effective, and xanthine or succinate had no influence. Paraquat was anaerobically reduced in the presence of NADPH and PMECs to yield a one-electron reduced radical, and the reduction was inhibited by NADP+. Diphenyleneiodonium, an inhibitor of flavoprotein reductases, also markedly inhibited both paraquat reduction and superoxide production. These results indicate that NADPH-dependent superoxide production from paraquat probably occurs by a flavoprotein with NADPH-dependent reductase activity in cell membranes. NADPH-dependent superoxide production from paraquat was also reproduced using adherent PMECs on wells. Under these conditions, superoxide production was enhanced with agonists, including interleukin-1beta, A23187, and phorbol 12-myristate 13-acetate. The effect of the former two was blocked with staurosporine, while the latter's effect was suppressed with calyculin A.

Specific transcriptional pausing activates polyadenylation in a coupled in vitro system.

We have developed a coupled in vitro transcription-polyadenylation system to investigate RNA polymerase II (Pol II) termination, which depends on active polyadenylation of the nascent RNA. Specific G-rich sequences originally identified as binding sites for the transcription factor MAZ both pause Pol II and activate polyadenylation of an upstream poly(A) signal. They do not affect polyadenylation efficiency in an uncoupled cleavage assay. In contrast, pausing of Pol II elongation induced by a high-affinity DNA-binding protein does not activate polyadenylation, indicating that G-rich MAZ sequences have a specific effect on polyadenylation. They also promote intrinsic pausing of purified Pol II, indicating a general role in the modulation of cotranscriptional RNA processing events.

The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyra z in-3-one (MCLA), a chemiluminescence probe to detect superoxide anions.

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O2.-, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2'-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe2+-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.

Lucigenin reduction by NADPH-cytochrome P450 reductase and the effect of phospholipids and albumin on chemiluminescence.

To assess lucigenin, a chemilumigenic probe, as a detector of superoxide anion in microsomes, NADPH oxidation, lucigenin disappearance, and chemiluminescence in a system including purified NADPH-cytochrome P450 reductase were examined. NADPH oxidation was increased by adding lucigenin, and concurrently, its disappearance and oxygen consumption were also stimulated. Chemiluminescence, which is negligibly emitted in the presence of the reductase alone, was remarkably amplified with phospholipids and albumin. Menadione inhibited lucigenin disappearance resulting in suppression of chemiluminescence. Lucigenin chemiluminescence measured in microsomes appears not to reflect direct superoxide anion production from microsomal components and from quinones, such as menadione.

Cell-cycle-dependent phosphorylation of the basal transcription factor RAP74.

In this report, cell-cycle-dependent effects of TFIID on other basal transcription factors were investigated. We purified TFIID fractions from HeLa cells synchronized in the S/G2 phases and in early G1 phase, and show that RAP74 is phosphorylated more highly by the S/G2 phase TFIID fraction than by the early G1 phase TFIID fraction. Further analyses using deletion mutants of RAP74 revealed that amino acid residues 206-256 are phosphorylated by the TFIID fraction. Reconstitution of in vitro transcription activity indicates that the cell-cycle-dependent phosphorylation of RAP74 increases TFIIF transcription activity.

Hepatitis C transmission between spouses.

To examine inter-spouse transmission as one of the potential routes of infection for hepatitis C virus (HCV), 121 patients with HCV-related chronic liver disease who tested positive for antibodies to HCV (anti-HCV) and their spouses were studied. Of these, 21 (17.4%) patients had spouses with anti-HCV. In 12 couples, the HCV genotype matched (type II: 10 couples, type III: two couples). The genotype differed in six couples. One patient was positive for anti-HCV, but negative for HCV-RNA, while the spouse was positive for both. The remaining two couples were only positive for anti-HCV. Genetic heterogeneity in the hypervariable region 1 of HCV was analysed in 11 couples with matched genotypes. In two couples, no mutation was recognized in the putative E2/NS1 genes using the heteroduplex method. The present study provides evidence to verify household transmission of HCV between patients and their spouses. These results suggest that inter-spouse transmission may be a potential route of transmission of HCV infection. However, other environmental factors (e.g. the duration of the marriage) cannot be ignored.

Enzymatic and molecular aspects of the antioxidant effect of menadione in hepatic microsomes.

The enzymatic features and molecular species of the inhibitory action of menadione on lipid peroxidation in rat liver microsomes were examined. In an ascorbate-supported system or a NADH-supported reconstituted system containing NADH-cytochrome b5 reductase and cytochrome b5, menadione was not an inhibitor of lipid peroxidation at pH 7.5, while some antioxidant ability was observed at lower pH ranges. Lipid peroxidation in the presence of menadione in the NADH-supported reconstituted system at pH 7.5 was markedly inhibited by adding lipoamide dehydrogenase. NAD(P)H-supported lipid peroxidation in microsomes with increased DT-diaphorase activity from 3-methylcholanthrene-treated rats was highly susceptible to menadione. These inhibitions were abolished by dicoumarol, an inhibitor of DT-diaphorase. Cumene hydroperoxide-dependent lipid peroxidation in microsomes, with desferal and NADP+ to prevent nonheme iron-dependent reactions and oxygen radical generation, was inhibited by menadione in the presence of NADPH, and the inhibition was also more effective in the microsomes with increased DT-diaphorase activity. Menadiol reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol at a molar ratio of DPPH/menadiol at 1.9. In an iron-supported reconstituted enzymatic or a nonenzymatic system at pH 7.5, menadiol showed an antioxidant effect at an early stage, followed by a prooxidant effect, which was prevented by SOD, probably by protecting menadiol autooxidation. These results show that menadione exerts an antioxidant effect through participation of microsomal DT-diaphorase by generating menadiol with a radical scavenging ability, while menadiol also has a prooxidant property.

Effects of membrane charges and hydroperoxides on Fe(II)-supported lipid peroxidation in liposomes.

The processes in producing a lag phase in Fe2+-supported lipid peroxidation in liposomes were investigated. Incorporation of phosphatidylserine (PS) or dicetyl phosphate (DCP) into phosphatidylcholine [PC(A)] liposomes, which have arachidonic acid, produced a marked lag phase in Fe(2+)-supported peroxidation, where PS was more effective than DCP. Phosphatidylcholine dipalmitoyl [PC(DP)] with a net-neutral charge was still effective in producing a lag phase, though weak. Increasing concentrations of PS, DCP, and PC(DP) prolonged the lag period. Initially after adding Fe2+, slight oxygen consumption occurred in PC(A)/PS liposomes including hydroperoxides, followed by a lag phase. An increase in the hydroperoxide resulted in a shortening of the lag period. The initial events of Fe2+ oxidation accompanied by oxygen consumption were dependent on the hydroperoxide content, but significant changes in diene conjugation and hydroperoxide levels at this stage were not found. The molar ratios of both disappeared Fe2+ and consumed O2 to preformed hydroperoxide in liposomes with or without tert-butylhydroxytoluene were constant, regardless of the different amounts of lipid hydroperoxides. The antioxidant completely inhibited the propagation of lipid peroxidation in the lipid phase, following a lag phase. In a model system containing 2,2'-azobis (2-amidinopropane) dihydrochloride, Fe2+ were consumed. We suggest that Fe2+ retained at a high level on membrane surfaces play a role in producing a lag phase following the terminating behavior of a sequence of free radical reactions initiated by hydroperoxide decomposition, probably by intercepting peroxyl radicals.

Cell cycle-dependent regulation of RNA polymerase II basal transcription activity.

Regulation of transcription by RNA polymerase II (pol II) in eukaryotic cells requires both basal and regulatory transcription factors. In this report we have investigated in vitro pol II basal transcription activity during the cell cycle by using nuclear extracts from synchronized HeLa cells. It is shown that pol II basal transcription activity is low in the S and G2 phases and high in early G1 phase and TFIID is the rate limiting component of pol II basal transcription activity during the cell cycle. Further analyses reveal that TFIID exists as a less active form in the S and G2 phases and nuclear extracts from S and G2 phase cells contain a heat-sensitive repressor(s) of TATA box binding protein (TBP). These results suggest that pol II basal transcription activity is regulated by a qualitative change in the TFIID complex, which could involve repression of TBP, during the cell cycle.

The reducing ability of iron chelates by NADH-cytochrome B5 reductase or cytochrome B5 responsible for NADH-supported lipid peroxidation.

We studied iron reduction, NADH oxidation, and lipid peroxidation in the presence of iron chelates with a chelator, such as nitrilotriacetate, ADP, citrate, and pyrophosphate, in NADH-supported reconstituted system. The results showed the selectivity of NADH-cytochrome b5 reductase or cytochrome b5 towards iron chelates and the subsequent ability of this system to promote peroxidation. The lipid peroxidation was partially inhibited by superoxide dismutase. In the presence of any iron chelate, hydrogen peroxide was produced in the systems including the reductase, and the production was accompanied with an increase in NADH oxidation.

A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation.

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5'-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPH via NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.

Regulation of the human general transcription initiation factor TFIIF by phosphorylation.

The transcription initiation factor, TFIIF, is essential not only for the initiation of transcription but also for efficient elongation of mRNA synthesis by mammalian RNA polymerase II and is extensively phosphorylated in vivo. The possible regulation of TFIIF activity by protein phosphorylation was investigated by comparing the biochemical properties of alkaline phosphatase-treated HeLa TFIIF with those of native or bacterially expressed factor. Alkaline phosphatase treatment decreased the size of the large subunit (RAP74) of TFIIF to that of the recombinant protein but did not change the size of the small subunit (RAP30). Both the transcription initiation and elongation stimulating activities of the alkaline phosphatase-treated TFIIF decreased to 15-20% of the native form under conditions in which the amount of TFIIF was rate-limiting for transcription. Furthermore, phosphatase-treated TFIIF assembled the DBPolF complex and bound to RNA polymerase II less efficiently than the native protein. When hybrid TFIIFs were reconstituted using native or recombinant subunits, a native form of RAP74 stimulated both transcription and DBPolF complex formation activity regardless of whether native or recombinant RAP30 was used. We propose that TFIIF activity is regulated by protein phosphorylation, particularly of the RAP74 subunit. The functional role of RAP74 in assembling the preinitiation complex and modulating TFIIF activity is discussed.

Enhancement of bacterial transcription initiation in vitro by the 74 kDa subunit of human general transcription factor IIF (RAP74).

The human general transcription factor IIF (TFIIF) is required for an accurate transcription initiation by RNA polymerase II and shares some analogous features with the sigma subunit of bacterial RNA polymerase. As an attempt to analyze the function of TFIIF, we examined its effect on bacterial transcription in vitro. TFIIF significantly enhanced the initiation of transcription by the bacterial RNA polymerase while other general transcription factors, TATA-binding protein, TFIIB, and TFIIE, did not. The enhancement of the bacterial transcription was ascribed to the 74 kDa subunit of TFIIF (RAP74). RAP74 had an activity of enhancing the binding of the bacterial RNA polymerase to the promoter. The enhancing activity of RAP74 depended on a low molar ratio of the RNA polymerase to the template DNA. The action of RAP74 in the bacterial transcription may be related to a possible regulatory role of RAP74 in the eukaryotic transcription initiation.

Mechanism of the biphasic effect of ethylenediaminetetraacetate on lipid peroxidation in iron-supported and reconstituted enzymatic system.

The biphasic action of ethylenediaminetetraacetate (EDTA), depending on its concentration, on lipid peroxidation was examined in an iron-supported and reconstituted enzymatic system. In the presence of NADPH-cytochrome P450 reductase and NADPH, Fe(3+)-PPi or Fe(3+)-ADP, though not reducible in the absence of EDTA, was markedly reduced with increasing concentration of EDTA. Lipid peroxidation, in the reconstituted system containing negatively charged liposomes, showed the maximal rate at 0.5 molar ratio of EDTA/iron, but no peroxidation occurred in positively charged liposomes, suggesting production of a positively charged iron complex as the prooxidant. Isotachophoresis indicated production of net-negative charge, EDTA-Fe(3+)-PPi complex, from Fe(3+)-PPi and EDTA at 1.1 ratio of EDTA/iron. The complex quenched Fe(2+)-PPi-supported lipid peroxidation. We suggest that EDTA-iron complexes of different charges are generated, depending on the amount of EDTA in the enzymatic system and, consequently, there is a switch between prooxidant and inhibitory effect at some critical ratio of EDTA/iron.

Domain structure of a human general transcription initiation factor, TFIIF.

The structural and functional domains of a general transcription initiation factor, TFIIF (RAP30/74, FC), have been investigated using various deletion mutants of each subunit, both in vivo and in vitro. An in vivo assay showed that the N-terminal sequence containing residues of 1-110 of RAP30 that is located close to a sigma homology region interacts with a minimum sequence of residues 62-171 of RAP74 to form a heteromeric interaction. Reconstitution of in vitro transcription activity by deletion mutants of RAP74 clearly indicated that both N-terminal residues 73-205 and C-terminal residues 356-517 are essential for full activity, the former interacting with RAP30, thus complexing with RNA polymerase II. From these data, the functional significance of domain structure of TFIIF is discussed in terms of its sigma homology sequences and complex formation with RNA polymerase II in the initiation and elongation of transcription.

Antioxidant mechanism of Mn(II) in phospholipid peroxidation.

Antioxidant action of Mn2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomes or in microsomes was investigated. High concentrations of Mn2+ above 50 microM inhibited 2,2'-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn2+. However, in a lipid-soluble radical initiator, 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN)-supported lipid peroxidation of methyl linoleate in methanol Mn2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 microM, Mn2+ competitively inhibited Fe(2+)-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the beta-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn2+.

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.