Bidirectional Activity Research Articles

サリドマイドをリードとした新規生物応答調節剤の創製

There has been a resurgence of interest in thalidomide (N-α-phthalimidoglutarimide), a teratogenic hypnotic/sedative agent, in recent years due to its potential usefulness of the treatment of various diseases. The effectiveness of the drug has been attributed to its regulating activity on tumor necrosis factor alpha production. The regulating activity has been found to be bidirectional, depending on both cell-type and the cell-stimulator. The bidirectional activity was separated by structural development of the drug, resulting in novel, potent, and structurally new thalidomides. By further structural development, novel types of androgen antagonists and nonpeptide protease inhibitors have been derived. Some of the developed compounds have been found to elicit potent antiangiogenic activity and/or potent inhibitory activity on tumor cell invasion. The studies on these structural development and the expansion of biological activities is reviewed in relation to the structure-activity relationships.

Activity of spinal interneurons and their effects on forearm muscles during voluntary wrist movements in the monkey.

We studied the activity of 577 neurons in the C6-T1 spinal cord of three awake macaque monkeys while they generated visually guided, isometric flexion/extension torques about the wrist. Spike-triggered averaging of electromyographic activity (EMG) identified the units' correlational linkages with </=12 forearm muscles. One hundred interneurons produced changes in the level of average postspike EMG with onset latencies consistent with mono- or oligosynaptic connections to motoneurons; these were classified as premotor interneurons (PreM-INs). Most PreM-INs (82%) produced postspike facilitations in forearm muscles. Earlier spike-related features, often beginning before the trigger spike, were seen in spike-triggered averages from 72 neurons. Postspike effects were present in one muscle for 64% of the PreM-INs. Neurons with divergent linkages to larger "muscle fields" usually generated postspike effects in synergistic muscles. Fifty-eight percent of the PreM-INs had postspike effects in flexor muscles only and 29% in extensor muscles only. Postspike effects were distributed relatively evenly among the primary flexor and extensor muscles studied. The mean percent change in EMG level from baseline and the mean onset latencies for postspike facilitations and postspike suppressions were similar. PreM-INs exhibited a variety of response patterns during the generation of isometric wrist torque. The response patterns and output effects of 24% of the PreM-INs were consistent with a strict reciprocal organization of flexor and extensor muscle control. For another 60% of the PreM-INs, there was a congruent relation between activity and output effects for only one direction of torque production. These neurons were active for both flexion and extension torques, including 37 neurons that exhibited bidirectional increases in discharge rate. The relatively small number of postspike suppressions observed suggests that inhibitory interneurons were silent when their target muscles were recruited. Compared with premotor neurons in the motor cortex, the red nucleus and the C8-T1 dorsal root ganglia, spinal PreM-INs affected flexor muscles in greater proportions and had smaller muscle fields. The magnitudes of postspike facilitations were similar in all premotor populations. Bidirectional activity, common for PreM-INs, was rare for corticomotoneuronal and premotor dorsal root ganglion cells, which discharge only for torques in their preferred direction.

Response patterns and force relations of monkey spinal interneurons during active wrist movement.

The activity of C6-T1 spinal cord neurons was recorded in three macaques while they generated isometric wrist flexion and extension torques in visually guided step-tracking tasks. Electromyographic activity (EMG) was recorded in </=12 independent forearm muscles. Spike-triggered averages (STAs) of rectified and unrectified EMG were used to classify neurons into four groups. Motoneurons (MNs) had a clear postspike motor unit signature in the unrectified STA of one muscle. Premotor interneurons (PreM-INs) had postspike effects in at least one muscle, with onset latencies of >/=3.5 ms from the trigger. Synchrony interneurons (Sy-INs) were non-PreM-Ins that had spike-related features with latencies <3.5 ms in at least one muscle. Unidentified interneurons (U-INs) showed no features in any of the STAs. A total of 572 task-related spinal neurons were studied; 29 cells were MNs, 97 PreM-INs, 32 Sy-INs, and 414 U-INs. MNs were activated predominantly in a tonic fashion during the ramp-and-hold torques and were active in one direction only. The most common response pattern for interneurons, irrespective of their class, was phasic-tonic activity, followed by purely tonic and purely phasic activity. Most interneurons (77%) were bidirectionally active in both flexion and extension. For all classes of interneurons, units with phasic response components tended to be activated first, before torque onset, followed by tonic units. The onset times of PreM-INs relative to onsets of their target muscles were distributed broadly, with a mean of -25 +/- 128 (SD) ms. For most neurons with tonic response components (all MNs, 71% of PreM-INs, 67% of Sy-INs, and 84% of U-INs), activity during the hold period was correlated significantly with the magnitude of static torque exerted by the monkey. The rate-torque regressions generally had positive slopes with higher mean slopes for extension than for flexion. The phasic response components were correlated significantly with rate of change of torque for a smaller percentage of tested PreM-Ins (50%), Sy-INs (83%), and U-INs (77%). In contrast to other premotor neurons [corticomotoneuronal (CM), rubromotoneuronal (RM), and dorsal root ganglion (DRG) afferents] previously characterized under similar conditions, a larger proportion of the spinal PreM-INs were activated after onset of their target muscles, probably reflecting a larger proportion of PreM-INs driven by peripheral input. The rate-torque slopes of PreM-INs tended to be less steep than those of CM and RM cells. Unlike the CM and DRG PreM afferents, which were activated unidirectionally, most spinal PreM-INs showed bidirectional activity, like RM cells.

A 69-Base Pair Fragment Derived from Human Transcobalamin II Promoter Is Sufficient for High Bidirectional Activity in the Absence of a TATA Box and an Initiator Element in Transfected Cells: ROLE OF AN E BOX IN TRANSCRIPTIONAL ACTIVITY

A 69-base pair (bp) (-581/-513) fragment derived from human transcobalamin II distal promoter constructed upstream of a chloramphenicol acetyltransferase reporter gene demonstrated high bidirectional promoter activity in transfected epithelial Caco-2 cells. DNase I footprinting, gel mobility shift, supershift, and mutagenesis studies with the 69-bp fragment demonstrated that a GC box (-568/-559) and an E box (-523/-528), which interacted with Sp1/Sp3 and USF1/USF2 (where USF is upstream stimulatory factor), respectively, were required for the full transcriptional activity of this fragment. Whereas mutations in the GC box reduced the promoter activity by 50%, mutations in the E box alone or in both the E box and GC box resulted in 90% loss of transcriptional activity. The essential role of the E box in the bidirectional promoter activity was further demonstrated by transient transfection in Caco-2, K-562, and HeLa cells using a 29-bp (-541/-513) fragment that contained only the E box. Based on these results we suggest that 1) the E box is essential for both the GC box-dependent and -independent promoter activity of the 69-bp fragment, 2) cooperative interactions between Sp1/Sp3 and USFs are required for the full activation of the 69-bp promoter activity, and 3) the single E box is able to mediate bidirectional transcription in transfected cells in the absence of an obvious TATA box or a known initiator element.

Novel Biological Response Modifiers Derived from Thalidomide

Thalidomide (N-alpha-phthalimidoglutarimide) was used widely as a hypnotic/sedative agent in the late 1950s and the early 1960s, but had to be withdrawn from the market because of its severe teratogenicity. In spite of this, there has been a resurgence of interest in the drug in recent years due to its potential usefulness for the treatment of various diseases, including acquired immunodeficiency syndrome (AIDS) and graft-versus-host disease (GVHD). The effectiveness of the drug in these diseases has been attributed to its specific inhibitory activity on tumor necrosis factor-alpha (TNF-alpha) production. Because TNF-alpha, a cytokine mediating host defence and immune regulation, with a wide range of activities, has deleterious pathophysiological effects in various diseases, including AIDS, tumors, rheumatoid arthritis and diabetes, its production-regulators are attractive lead compounds for novel biological response modifiers. The regulatory effect of thalidomide on TNF-alpha production has been found to be bidirectional, depending on both the cell-type and the TNF-alpha production-inducer; i.e., thalidomide possesses both enhancing and inhibiting activities on TNF-alpha production. Structural modification of thalidomide aiming at the creation of superior TNF-alpha production-regulators has afforded a number of phenyl- and benzylphthalimide analogs possessing more potent activity than thalidomide itself. The structure-activity relationships of these analogs has been investigated. The bidirectional TNF-alpha production-regulating activity is electronic state- and enantio-dependent, and both pure inhibitors and pure enhancers of TNF-alpha production has been obtained. Further structural development of the phthalimide analogs has yielded potent non-steroidal androgen antagonists.

The role of 11 beta-hydroxysteroid dehydrogenase in the pathogenesis of hypertension.

The two 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) isozymes catalyze the interconversion of cortisol and cortisone. Type 1 11 beta-HSD (11 beta-HSD1) has bidirectional activity, while type 2 11 beta-HSD (11 beta-HSD2) mainly converts cortisol into cortisone. Of these two hormones only cortisol has affinity to mineralocorticoid receptors (MRs) and thus induces mineralocorticoid effects. A normal activity of 11 beta-HSD2 is crucial for prevention of mineralocorticoid activity of cortisol. Absent or decreased 11 beta-HSD2 activity results in cortisol-mediated hypermineralocorticoid hypertension. In several hypertensive syndromes a decreased 11 beta-HSD2 activity has been described as the pathogenetic mechanism of the increased blood pressure. In the apparent mineral corticoid excess (AME) syndrome type 1, absence of 11 beta-HSD2 activity is caused by mutations in the gene coding for 11 beta-HSD2. In licorice-induced hypertension glycyrrhetinic acid, the active substituent of licorice, inhibits 11 beta-HSD2 resulting in an acquired hypermineralocorticoid state. 11 beta-HSD2 activity is not decreased in glucocorticoid hypertension (Cushing's syndrome). In essential hypertension some evidence for decreased systemic and skin activity of 11 beta-HSD1 and/or 11 beta-HSD2 has been found, while renal activity of both isozymes appears to be normal. 11 beta-HSD2 activity is also present in cardiovascular myocytes of humans and dogs, and inhibition of 11 beta-HSD potentiates the vascular response to catecholamines. Although MRs in the central nervous system have been incriminated in the pathogenesis of mineralocorticoid hypertension, a pathophysiological role for 11 beta-HSD2 has not yet been described. Finally, in the placenta 11 beta-HSD2 reduces fetal exposure to maternal glucocorticoids and a decreased activity of this isozyme may result in low birth weight and increased risk of high blood pressure at adult age.

Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes

We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and ∼290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of ∼190 bp of upstream and ∼170 bp of transcribed DNA (exon 1 and most of intron 1). This ∼360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5′-most CCAAT box decreased activity ∼5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Sp1 site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had ∼5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.

Truncation of the N- and C-terminal regions of the human 11 β-hydroxysteroid dehydrogenase type 2 enzyme and effects on solubility and bidirectional enzyme activity

The 11 β-hydroxysteroid dehydrogenase type II enzyme (11 βHSD2) endows specificity on the mineralocorticoid receptor by metabolising glucocorticoids. Sequence comparisons with other microsomal proteins showed the strongly preferred topology of a lumenal pentapeptide followed by three transmembrane helices with residues beyond Ala 73 on the cytoplasmic side of the membrane, suggesting that 11 βHSD2 is anchored to the endoplasmic reticulum by the N-terminal region. However, deletion of the N-terminus (11 βHSD2ΔN) and expression of the construct in mammalian cells showed that the enzyme remained bound to the microsomal fraction, indicating that other regions are also involved in membrane anchoring. Crosslinking studies and nonreducing SDS-PAGE demonstrated that 11 βHSD2 is a non-covalently linked dimer. Deletion of the non-conserved C-terminal region (11 βHSD2ΔC) resulted in an enzyme with a K m of 215 nM for cortisol in whole cell assays, while 11 βHSD2 and 11 βHSD2ΔN displayed a K m of 62 and 74 nM, respectively. In homogenates 11 βHSD2 and 11 βHSD2ΔC displayed maximal activity at 140 mM NaCl or KCl, but showed a marked decrease in enzyme activity with increasing salt. 11 βHSD2 was more stable than 11 βHSD2ΔC in the presence of NaSCN, suggesting that the C-terminal region plays a role in enzyme stability. There was no detectable activity in homogenates containing 11 βHSD2ΔN, while 11 βHSD2ΔC and 11 βHSD2 displayed a K m of 135 and 46 nM, respectively. Although 11 βHSD2 is conventionally considered a unidirectional dehydrogenase all constructs converted 11-dehydrodexamethasone to dexamethasone in whole cell assays, providing an explanation for the potency of the synthetic glucocorticoid in the face of a powerful inactivator of natural glucocorticoids.

Identification of a kinetically distinct activity of 11beta-hydroxysteroid dehydrogenase in rat Leydig cells.

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11beta-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11beta-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11beta-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11beta-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11beta-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11beta-HSD and MR messenger RNAs (mRNAs), and for type I 11beta-HSD protein. Leydig cell 11beta-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11beta-HSD is present in rat Leydig cells. However, unlike the type I 11beta-HSD in liver parenchymal cells, the Leydig cell 11beta-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (Km) NADP-dependent oxidative activity with a Km of 41.5 +/- 9.3 nM and maximum velocity (Vmax) of 7.1 +/- 1.2 pmol x min x 10(6) cells. The second component consisted of high Km activities that were consistent with type I:NADP-dependent oxidative activity with Km of 5.87 +/- 0.46 microM and Vmax of 419 +/- 17 pmol x min x 10(6) cells, and NADPH-dependent reductive activity with Km of 0.892 +/- 0.051 microM and Vmax of 117 +/- 6 pmol x min x 10(6) cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11beta-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low Km NADP-dependent oxidative activity of 11beta-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low Km component could allow for specificity in MR-mediated responses.

The sheep kidney contains a novel unidirectional, high affinity NADP+-dependent 11β-hydroxysteroid dehydrogenase (11β-HSD-3)

The 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes convert corticosterone and cortisol to 11-dehydrocorticosterone and cortisone, and are thought to convey extrinsic specificity to the mineralocorticoid receptor by limiting access of the relatively more abundant glucocorticoids to it. Two different 11β-hydroxysteroid dehydrogenases (11β-HSD) have been described and cloned. The liver-type, NADP +-dependent 11β-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD +-dependent 11β-HSD-2 has a higher affinity, in the nanomolar range, and exhibits only oxidase activity. 11β-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor, is likely to serve as the “gatekeeper” for the mineralocorticoid receptor in the kidney. Although the rat kidney expresses both isoforms, only the high-affinity, NAD +-dependent 11β-HSD-2 has been reported in the sheep kidney. We found both 11β-HSD NAD +- and NADP +-dependent activities in sheep kidney to be present. The NAD +-dependent activity exhibited a Km similar to that reported in the literature, 3.85 ± 1.28 nM for corticosterone and 21.3 ± 5.8 for cortisol, was distributed in approximately equal amounts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited prominent substrate inhibition. The NADP +-dependent activity had a Km for corticosterone of 4 ± 1.3 nM and a Km for cortisol of 35.2 ± 2 nM, 100-fold lower than that described for the 11β-HSD-1 in the liver of sheep and other species, and was more prevalent in the microsomes than the nuclei. This enzyme was not inhibited by its substrate. The NAD +-dependent activity was approximately 3–10 times greater than the NADP +-dependent activity when incubated with 5nM corticosterone substrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster ovary cell line) transiently transfected with the sheep 11β-HSD-2 plasmid exhibited a marked preference for NAD + as co-factor. Oxidation of corticosterone by transfected cells in the presence of NADP + was present, but minimal; NADP + did not support the metabolism of cortisol, the primary glucocorticoid of sheep. These data suggest the existence of another NADP +-dependent enzyme, 11β-HSD-3, which, because of its high affinity and unidirectional oxidase activity, may play a physiological role in the modulation of glucocorticoid binding to both the mineralocorticoid and glucocorticoid receptors.

Differential expression of 11 beta-hydroxysteroid dehydrogenase types 1 and 2 in human placenta and fetal membranes.

Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) are present in mammals. 11 beta HSD1 interconverts biologically active cortisol and inactive cortisone, whereas 11 beta HSD2 only converts cortisol to cortisone. Placental 11 beta HSD has been proposed to protect the fetus from high level of maternal glucocorticoids. Although bidirectional activity of 11 beta HSD has been demonstrated in homogenized human placental tissues, the tissue and cellular distribution of 11 beta HSD1 has not been resolved. In this study, the cellular localization of 11 beta HSD1 protein and levels of its messenger ribonucleic acid (mRNA) in human placenta and fetal membranes were determined by immunohistochemistry and Northern blot analysis, respectively. We found that 11 beta HSD1 immunoreactivity was present in the placental extravillous intermediate trophoblasts, chorion trophoblasts, amnion epithelial cells, and stromal cells of the decidua vera. Positive staining was also observed in the endothelium of the blood vessels in both placental villous tissue and umbilical cord. However, in contrast to previous reports of immunoreactive 11 beta HSD2 localization, 11 beta HSD1 immunoreactivity was undetectable in placental syncytiotrophoblast. Using a human 11 beta HSD1 complementary DNA as probe, a 1.5-kilobase mRNA transcript was detected in the chorion, amnion, and placental tissue, with the greatest amount in the chorion. In contrast, the 1.9-kilobase mRNA of 11 beta HSD2 was observed only in the placenta, not in the chorion and amnion. The process of labor had no significant effect on levels of 11 beta HSD1 or 11 beta HSD2 mRNA in the chorion or placenta. We conclude that there is a striking difference in the tissue localization of 11 beta HSD1 and 11 beta HSD2 expression in the late gestation human placenta and fetal membranes, which may discretely determine the accessibility of bioactive glucocorticoid to specific cell types.

11β-Hydroxysteroid Dehydrogenase in the Brain: A Novel Regulator of Glucocorticoid Action?

Corticosteroids (glucocorticoids and mineralocorticoids) have multiple actions in the brain which are mediated via specific intracellular receptors. Recently, a novel and important control of glucocorticoid action has been identified in peripheral tissues; prereceptor metabolism by 11β-hydroxysteroid dehydrogenase (11β-HSD). This enzyme catalyses the conversion of the active glucocorticoids corticosterone and cortisol to inert 11 keto-products (11-dehydrocorticosterone, cortisone), thus regulating access of glucocorticoids to receptors. Two distinct isozymes occur. 11β-HSD-1 is a widespread, NADP(H)-dependent enzyme, which shows bidirectional activity in tissue homogenates and microsomal preparations, but may predominantly function as an 11β-reductase (regenerating active glucocorticoids) in intact cells. 11β-HSD-2 is a much higher affinity, NAD-dependent, exclusive 11β-dehydrogenase (glucocorticoid inactivating enzyme), which, when colocalized with otherwise nonselective mineralocorticoid receptors (MR), ensures selective access for aldosterone in vivo.Accumulating evidence indicates widespread expression of 11β-HSD-1 in the brain. The highest levels are found in cerebellum, hippocampus, cortex, and pituitary, but detectable activity is also present in the hypothalamus (including the paraventricular nucleus) and other regions of neuroendocrine interest. 11β-HSD-1 protein has been detected on Western blots of brain and immunostaining is widespread, localized predominantly in neurons and their processes. The mRNA encoding 11β-HSD-1 is also widely expressed in the brain, its distribution broadly paralleling enzyme bioactivity and immunostaining. 11β-HSD-1 expression is regulated during late prenatal and postnatal ontogeny and by glucocorticoids and stress, prompting suggestions that this isoform may play a role in protecting the brain from the deleterious consequences of glucocorticoid excess. However, in primary cultures of hippocampal neurons, 11β-HSD-1 functions as a predominant 11β-reductase, reactivating inert corticoids and thus potentiating neurotoxicity. The functions of 11β-HSD-1 in the CNS are not defined, but may relate to mood, neuronal survival, and glucocorticoid feedback. The identification of aldosterone-selective actions in the brain (upon blood pressure and salt appetite) predict the presence of 11β-HSD-2. This isozyme has very limited expression in the adult brain, probably confined to the subregions of the brain stem and the subcommissural organ, where some aldosterone-selective actions may be mediated. However, the midgestation fetal brain highly expresses 11β-HSD-2, which might modulate glucocorticoid effects on CNS development. Studies with licorice-derived enzyme inhibitors indicate functional effects for 11β-HSD in the adult brain, notably in the periventricular hypothalamus and limbic system. Thus, 11β-HSD represents a novel and potentially important level of control of glucocorticoid action in the CNS. Enzyme modulation by pharmacological or other agents may provide a useful means to target increased or attenuated glucocorticoid action to specific sites in the brain.

The Bacillus subtilisDNA Replication Terminator

The recent discovery of the Bacillus subtilisplasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B. subtilisreplication terminators in general. The minimal TerIand TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators. It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region. A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerIwith an opposed segment identical to that in the B site. The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator. TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator. It was found that the deviations introduced converted the terminator significantly towards polarity of action. The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence. The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB. A model to account for the generation of RTP – terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI- VIis presented.

Inducer-Specific Bidirectional Regulation by Thalidomide and Phenylphthalimides of Tumor Necrosis Factor-α Production

Regulation by thalidomide [N(α)-phthalimidoglutarimide] of tumor necrosis factor (TNF)-α production was found to be inducer-specific. Thalidomide enhances TNF-α production by human leukemia HL-60 cells induced with 12-O-tetradecanoylphorbol 13-acetate (TPA), while it inhibits TNF-α production induced with okadaic acid (OA) in the same cell line. Some phthalimide analogs, including PP-33 [2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione] and its 4,5,6,7-tetrafluoro derivative (FPP-33), also showed such an inducer-specific bidirectional TNF-α production-regulating activity. The structure–activity relationships of the compounds tested are similar, but not identical, in the TPA-stimulated HL-60 and OA-stimulated HL-60 assay systems.

Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20.

We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium. TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.

11β-Hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11β-hydroxysteroid dehydrogenases (11β-HSD) have been described. 11β-HSD-1 is NADP +-dependent and has a K m in the micromolar range and bidirectional activity. 11β-HSD-2 is NAD +-dependent, has a K m in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11β-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD +-dependent 11β-HSD-2. We found that the K m for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD +-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, K m = 40 nM, NADP +-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11β-HSD-1 nor the 11β-HSD-2 and have named it 11β-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5β-dihydroprogesterone, and 5α-dihydroprogesterone, but not bile acids.

Bidirectional Activity of the Endoplasmic Reticulum Ca2+-ATPase of Bovine Adrenal Cortex

It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor play major roles in the complex mechanisms by which agonists increase intracellular Ca2+ concentration. In these mechanisms, the endoplasmic reticulum Ca(2+)-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca(2+)-ATPase of bovine adrenal cortex was investigated. We show that the Ca(2+)-pumping activity of the Ca(2+)-ATPase is related to the ADP/ATP ratio. Our results also show that a brisk increase of the ADP/ATP ratio upon addition of exogenous ADP triggered a rapid release of Ca2+ from preloaded microsomes. ADP released Ca2+ in a dose-dependent manner with an EC50 of 2.98 +/- 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADP-induced Ca2+ release could not be attributed to the mere inhibition of the Ca(2+)-ATPase, since the rate of ADP-induced Ca2+ release was 20 times faster than the rate of Ca2+ release induced by a maximal concentration of thapsigargin (2 microM). ADP-induced Ca2+ release experiments performed in the presence of [32P]PO4 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 +/- 0.70 mM. ADP-induced [32P]ATP production was prevented by ionomycin (10 microM) and by high concentrations of extramicrosomal Ca2+. These results demonstrate that the microsomal Ca(2+)-ATPase of adrenal cortex possesses a bidirectional activity that depends on ADP concentrations, the Ca2+ gradient across the microsomal membrane, and probably also ATP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription termination of the streptokinase gene of Streptococcus equisimilis H46A: bidirectionality and efficiency in homologous and heterologous hosts.

In Streptococcus equisimilis H46A, a hypersymmetrical transcription terminator with bidirectional activity was localized between the translational termination codons of the streptokinase gene, skc, and the rel-orf1 genes. These two transcription units are oriented towards each other, and under normal conditions the skc mRNA level exceeds that of the rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusion of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additionally, skc and cat were fused directly, with deletion of the terminator. The reporter vectors were designed to be capable of being studied either as multicopy plasmids in Escherichia coli or in single copy following integration, via skc, into the S. equisimilis chromosome. Chloramphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybridization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, with high terminator strength being observed in the homologous streptococcal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator orientation, with termination being more efficient in rel-orf1 polarity. The results suggest that, in S. equisimilis, transcription of both skc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, which otherwise would seem to be particularly detrimental to the weakly expressed rel-orf1 genes.

Bidirectional activity of 11β-hydroxysteroid dehydrogenase in vascular smooth muscle cells

Endogenous glucocorticoids (GC) can be metabolized through the enzyme 11β-hydroxysteroid dehydrogenase (11β-OHSD); in the rat, corticosterone (B) is converted to its inactive metabolite 11-dehydrocorticosterone (A). Since increased tissue concentrations of GCs may affect blood pressure by potentiating the vasoactive effects of α-adrenergic agonists and possibly other pressors, we studied the metabolism of corticosterone in freshly dissected aortae and cultured vascular smooth muscle cells (VSMC). Incubations were generally conducted for 60 min with 10 −8 M steroid; steroids were isolated and identified by HPLC. In aortic minces stripped of endothelium, the oxo-reductase reaction of A back to B was nearly 4 times greater than the dehydrogenase reaction of B to A (2.8 ± 0.5 × 10 −11 versus 7.3 ± 1.0 × 10 −12 mol/mg protein). This pattern was also seen in cultured VSMC during growth and quiescent states (growth A to B 3.2 ± 0.4 × 10 −12 versus B to A 9.7 ± 0.9 × 10 −13 mol/mg protein; quiescent A to B 8.8 ± 0.1 × 10 −12 versus B to A 1.2 ± 0.2 × 10 −12 mol/mg protein). Enzyme activity in either direction was less during growth, correlating with a decrease in mRNA for 11β-OHSD. In cell homogenates containing 200 μM NADP(H), the enzyme functioned equally in either direction at pH 7.4 with an apparent K m for corticosterone of approximately 2 × 10 −7 M. Carbenoxolone, an inhibitor of 11β-OHSD, suppressed the dehydrogenase reaction to a greater degree than the reverse oxo-reductase reaction. Thus, in VSMC 11β-OHSD is bidirectional; under physiologic conditions intact VSMC favor the oxo-reductase reaction. Quiescent cells show greater enzyme activity than actively growing cells.

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V alpha 1 gene.

Previous studies of the TCR alpha chain gene have located promoter elements 5' to the start of the various V alpha genes. The only fully characterized enhancer for the entire alpha chain gene (V, J and C genes) has been located approximately 3 kb from the 3' end of C alpha. We now report the existence of additional regulatory elements located in the introns of several murine V alpha genes (V alpha 1, V alpha 3 and V alpha B6.2.16). In the case of V alpha 1, this element appears to be a promoter with bidirectional activity that is not T cell specific. Interestingly, upstream of the promoter in the antisense strand, an open reading frame has been found that codes for a small molecular weight protein (approximately 60 amino acids) that contains a proline-rich region and a tyrosine-isoleucine motif that has homology to Ig beta (the B29 gene product). A rabbit antiserum made against this sequence has confirmed its existence by Western blot and immunoprecipitation. Thus this V alpha 1 intronic promoter has the potential not only to induce the formation of a truncated V alpha 1 gene product, but also regulates the expression of a small molecular weight protein that may be involved in lymphocyte antigen receptor signaling. The activity of this promoter is regulated by changes in intracellular calcium. In the presence of ionomycin the promoter is down-regulated in the sense direction and its activity is enhanced in the antisense direction. This result suggests that this promoter can act differentially to produce two very different gene products. The bidirectional V alpha 1 promoter appears to be the first in the Ig superfamily to induce potentially functional proteins in both directions.