Differential Inhibitory Effects Research Articles

A Target-Site-Specific Screening System for Antifungal Compounds on Appressorium Formation inMagnaporthe grisea

ABSTRACT Chemical fungicides are a major method of control for plant diseases in spite of potential negative effects on the environment and the appearance of resistant strains. Development of new chemical fungicides has been largely dependent upon in vivo efficacy tests in the greenhouse or in fields, which is in contrast to target-oriented in vitro screening systems widely used in the pharmaceutical field. To establish a target-site-specific screening system for antifungal compounds, specific inhibition on appressorium formation of the rice blast fungus Magnaporthe grisea was employed. For many plant-pathogenic fungi, including M. grisea, appressorium formation is an essential step to infect host plants. Among 1,000 culture filtrates of members of the class Actinomycetes and fungi, five (A5005, A5008, A5314, A5387, and A5397) from the class Actinomycetes showed differential inhibitory effects on appressorium formation of M. grisea in a dosage-dependent manner. Three (A5005, A5314, and A5387) of these were further fractionated into ethyl acetate and water fractions. The ethyl acetate fraction of A5005 and both the ethyl acetate and water fractions from A5314 and A5387 inhibited appressorium formation, while conidial germination remained little affected. Inhibition of appressorium formation by the ethyl acetate or water fraction was reversed by the exogenous addition of cyclic AMP. Significantly reduced numbers of conidia with appressoria were observed on rice leaves in the presence of culture filtrates. Furthermore, these culture filtrates also exhibited significant disease control of rice blast in the greenhouse. This rapid and target-oriented screening system could be adopted to screen candidate compounds for rice blast control and could be applicable for other appressorium-forming, plant-pathogenic fungi.

The effect of fentanyl, DNQX and MK-801 on dorsal horn neurones responsive to colorectal distension in the anaesthetized rat.

Certain dorsal horn neurones respond in a graded manner to noxious colorectal distension (CRD). Morphine inhibits these responses in the spinalized rat, but the role of excitatory amino acids in baseline visceral nociceptive transmission is less clear. This study examines the effect of the mu-opiate receptor agonist fentanyl, and the non-NMDA and NMDA antagonists DNQX and MK-801, respectively, on such responses to CRD in the sodium pentobarbitone-anaesthetized rat. Male rats were prepared for extracellular recording from the lumbosacral spinal cord. 90 neurones responsive to CRD, located throughout the dorsal horn, were classified according to their response duration and latency to 60 mmHg distension, as SL-A (short latency-abrupt; 59%), SL-S (short latency-sustained; 23%), L-L (long-latency; 10%) and Inhib (inhibited; 8%). Convergent cutaneous receptive fields were mapped for 79/90 neurones and classified as LT (low threshold), WDR (wide dynamic range) or HT (high threshold). CRD (20-100 mm Hg) elicited graded responses in most neurones. In 6/6 SL-S neurones, fentanyl (1-8 microg kg-1) dose-dependently inhibited the response to 60 mm Hg CRD, in a naloxone-sensitive manner, with an ID50 value (+/-95% confidence limits) of 2.48 (1.7-3. 7) microg kg-1. In 6/6 SL-A neurones, fentanyl had no significant effect on the response to CRD. DNQX (0.03-3 mg kg-1) produced a dose-dependent inhibition of the response to CRD in 5/5 SL-A neurones, with an ID50 value of 0.32 (0.01-41.1) mg kg-1. MK-801 (0. 03-0.3 mg kg-1) had no significant effect on responses to CRD in 6/6 SL-A neurones. The differential inhibitory effects of fentanyl on two neuronal subtypes may indicate functional differences. In SL-A neurones AMPA/kainate, but not NMDA receptors are involved in mediating baseline nociceptive neurotransmission.

TGF-beta autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells.

Transforming growth factor beta (TGF) is a well-known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF-beta autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF-beta, thus decreasing the concentration of active TGF-beta to which the cells are exposed. This event is mediated by the TPA-induced alteration of the uPA/PAI serine-protease system. Complete removal of TGF-beta, mediated by the ectopic expression of a soluble type II TGF-beta receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF-beta concentration, to a range of 0.14-0.20 x 10(-2) ng/ml (which is similar to that measured in TPA-treated cells), mimics TPA-induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF-beta; furthermore, they show that a 'critical' concentration of TGF-beta is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF-beta autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF-beta is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF-beta independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF-beta in the regulation of both the transformed and differentiated phenotype.

Differential growth inhibitory effect of melatonin on two endometrial cancer cell lines.

The effect of melatonin on endometrial cancer cell growth was investigated using two cell lines, SNG-II and Ishikawa, which are different in their estrogen receptor status. A physiological concentration of melatonin (10(-9) M) showed no growth inhibitory effect on SNG-II cells, which are estrogen receptor-negative at all cell densities and incubation times. In contrast, melatonin significantly inhibited Ishikawa cells, which are estrogen receptor-positive at all cell densities tested after 96 hr incubation. The greatest inhibition of Ishikawa cell growth was observed at 10(-9) M melatonin, compared with other supra (10(-6), 10(-8) M) or subphysiological concentrations (10(-10), 10(-12) M). This growth inhibitory effect of melatonin on Ishikawa cells was completely blocked by 10(-10) to 10(-8) M concentrations of 17-beta estradiol administration. Pretreatment with luzindole, which is a selective melatonin receptor antagonist, prior to the addition of melatonin also blocked the inhibitory effect of melatonin on Ishikawa cells. This is the first study to demonstrate an anti-proliferative effect of physiological melatonin on endometrial cancer cells in vitro. The present study revealed that melatonin also inhibits the growth of estrogen receptor positive endometrial cancer cells and that this effect of the pineal indole may be mediated by both steroid and melatonin receptors.

Inhibitory effect of luteinising hormone-releasing hormone analogues on human endometrial cancer in vitro

We studied the effects of luteinising hormone-releasing hormone (LHRH) agonist leuproreline (1 μM for 96 h) and LHRH antagonist cetrorelix on the cell growth of primary cultures from nine human endometrial cancers using the sulphorhodamine colorimetric test. Histological examinations and reverse transcription and polymerase chain reaction amplification (RT–PCR) for LHRH receptors were also performed. The endometrial cancers examined had a medium to high degree of proliferative activity and a low degree of apoptotic power; furthermore, they expressed the LHRH receptor RNA variably, detectable in 71% of cases. The addition of leuproreline or cetrorelix to cell cultures inhibited growth in a statistically significant way compared to untreated control cells; nevertheless, the percentage of cell growth inhibition obtained was very variable. These data suggest that LHRH analogues can exert differential inhibitory effects on the growth of endometrial cancer, which seems to be independent of the expression of specific LHRH receptors.

Influence of polyaspartic acid and phosphophoryn on octacalcium phosphate growth kinetics

Polyaspartic acid (PAA) and phosphophoryn (PPn) have been suggested to adsorb specifically on the (100) and (010) faces, respectively, of octacalcium phosphate (Ca 4H(PO 4) 3 · 2.5H 2O, OCP). In this study, the extent of adsorption and influence of these molecules on OCP crystal growth has been investigated. For kinetic studies, protein effects on crystal growth were examined in solutions sustained at a constant level of supersaturation at pH 6.00 and ionic strength of 0.08 mol l −1. The maximum adsorbed mol surface concentration for PPn was 100-fold less than that for PAA. Inhibitory effects interpreted in terms of mol surface coverage showed PPn to retard OCP growth more effectively than PAA. However, when considering percentage of crystal face covered by protein, PAA and PPn showed similar maximum adsorption concentrations onto the (100) and (010) crystal faces, respectively. PAA inhibited OCP growth by 20% when only 1% of the (100) face (1% total crystal area) was covered. PPn had to reach over 200% (010) face coverage (or 28% total crystal area) before a similar level of crystal growth inhibition was obtained. This difference in inhibitory effect may be the result of a more effective β-strand conformation of the shorter PAA molecule or may indicate that growth at the (100) face is rate controlling and, therefore, less than 1% coverage of this face is needed before a significant decrease in rate is observed.

Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex

We analysed the inhibitory effects in vitro and in vivo of several metal ions on aldosterone binding to the rat kidney mineralocorticoid receptor with the purpose of assessing possible toxic effects of those ions on sodium retention, as well as to obtain information on receptor structural requirements for ligand binding. For the assays in vitro, the inhibitory effects of 20 metal ions were analysed on steroid-binding capacity for renal receptor cross-linked to 90-kDa heat-shock protein (hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking prevented the artifactual dissociation of hsp90 (and, consequently, the loss of steroid binding) from the mineralocorticoid receptor due to the presence of high concentrations of salt in the incubation medium. Cross-linked heterocomplex showed no difference in ligand specificity and affinity with respect to native receptor, but increased stability upon thermal- or ionic-strength-induced destabilization was observed. Treatments in vitro with metal ions in the range 10(-8)-10(-1) M resulted in a differential inhibitory effect for each particular ion on aldosterone binding. Using the negative logarithm of metal concentration for 50% inhibition, the ions could be correlated with their Klopman hardness constants. The analysis of this relationship led us to postulate three types of reaction: with thiol, imidazole and carboxyl groups. The essential role played by these residues in steroid binding was confirmed by chemical modification of cysteines with dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and acidic amino acids with Woodward's reagent (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic effects of some metal ions were also observed by treatments in vivo of adrenalectomized rats on both steroid-binding capacity and aldosterone-dependent sodium-retaining properties. We suggest that those amino acid residues are involved in the activation process of the mineralocorticoid receptor upon steroid binding. Thus toxic effects observed with these metal ions may be a consequence of modifications of those essential groups. Our results support the notion that toxicity of metals on renal mineralocorticoid function may be predicted according to their chemical hardness.

Erythropoietin production: evidence for multiple oxygen sensing pathways.

The production of erythropoietin (Epo), the glycoprotein hormone which controls red blood cell formation, is regulated by feedback mechanisms sensing tissue oxygenation. The mechanism of the putative oxygen sensor has yet to be elucidated. There is evidence that at least two pathways participate in hypoxia signal transduction. One appears to involve a specific haem protein, and a second implicates reactive oxygen species (ROS). Iron catalyses the generation of intracellular ROS and therefore alters the cellular redox state. We have investigated the effect of modulating intracellular iron content on Epo production in Hep 3B cells. Iron chelation stimulates Epo production at 20% O2 and enhances Epo production at 1% O2, but it has no additive effect on cobalt-induced Epo production. Excess molar iron inhibited Epo production in response to hypoxia, desferrioxamine (DFO) and cobalt chloride and inhibited the DFO-enhancing effect of hypoxia-induced Epo production. We found that sulphydryl oxidising agents exert a differential inhibitory effect on hypoxia-induced versus DFO-induced Epo production, providing further evidence that multiple pathways of oxygen sensing exist.

Differential inhibitory effects of protoberberines on sterol and chitin biosyntheses in Candida albicans.

The anti-Candida potentials of 12 Korean medicinal plants were explored: methanol extracts from Coptis rhizoma and Phellodendron amurense caused significant inhibition of growth of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The predominant active components of the extracts were the protoberberines berberine and palmatine; the most potent inhibition of growth was exhibited by berberine on C. krusei (MIC <4 mg/L) and palmatine on C. parapsilosis (MIC 16 mg/L). Both berberine and palmatine inhibited the in-vivo rate of incorporation of L-[methyl-14C]methionine into C-24 of ergosterol in C. albicans (50% inhibition concentration (IC50 values), 25 microM and 300 microM, respectively); this result suggests that sterol 24-methyl transferase (24-SMT) is one of the cellular targets for the antifungal activity of the protoberberines. In-vitro 24-SMT activity in microsomes from the yeast growth form of C. albicans was inhibited by both berberine (inhibition constant (Ki) 232 microM) and palmatine (Ki 257 microM) in a non-competitive manner; inhibition of 24-SMT was more marked for the mycelial form than for the yeast growth form of this organism. Palmatine inhibited chitin synthase from both the yeast and mycelial growth phases of C. albicans in a non-competitive manner (Ki 780 microM). The effects of protoberberines, extracted from established medicinal plants, on both sterol and cell wall biosyntheses in pathogenic fungi indicate that the potential of these compounds, or their semi-synthetic derivatives, as a novel class of antifungal agents should be investigated more fully.

Differential Inhibition of Smad6 and Smad7 on Bone Morphogenetic Protein- and Activin-mediated Growth Arrest and Apoptosis in B Cells

Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-beta family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells.

Differential inhibitory effect on human nociceptive skin senses induced by local stimulation of thin cutaneous fibers

It is known that stimulation of thin cutaneous nerve fibers can induce long lasting analgesia through both supraspinal and segmental mechanisms, the latter often exhibiting restricted receptive fields. On this basis, we recently developed a new method, termed cutaneous field stimulation (CFS), for localized stimulation of A δ and C fibers in the superficial part of the skin. In the present study, we have evaluated the effects of CFS on non-nociceptive and nociceptive skin senses. We compared the effects of CFS with those of conventional transcutaneous electrical nerve stimulation (TENS), known to preferentially activate coarse myelinated fibers. A battery of sensory tests were made on the right volar forearm of 20 healthy subjects. CFS (16 electrodes, 4 Hz per electrode, 1 ms, up to 0.8 mA) and TENS (100 Hz, 0.2 ms, up to 26 mA) applied either on the right volar forearm (homotopically), or on the lower right leg (heterotopically) were used as conditioning stimulation for 25 min. The tactile threshold was not affected by either homo- or heterotopical CFS or TENS. The mean thresholds for detecting warming or cooling of the skin were increased by 0.4–0.9°C after homo- but not heterotopical CFS and TENS. Regarding nociceptive skin senses, homo- but not heterotopical CFS, markedly reduced CO 2-laser evoked A δ- and C fiber mediated heat pain to 75 and 48% of control, respectively, and mechanically evoked pain to 73% of control. Fabric evoked prickle, was not affected by CFS. Neither homo- nor heterotopical TENS induced any marked analgesic effects. It is concluded that different qualities of nociception can be differentially controlled by CFS.

Differential non-redox inhibitory effects of glutathione against protein kinase C isozyme family members.

Glutathione (GSH) and the GSH metabolic precursor N-acetylcysteine (NAC) are potent antioxidants that have clear potential either as cancer chemopreventive agents or as lead compounds for new cancer chemopreventive agents. The potential efficacy of GSH and NAC in clinical cancer chemoprevention is suggested by their antagonism of tumor promotion in animal models. Protein kinase C (PKC) is an isozyme family that plays a critical role in phorbol ester-mediated tumor promotion. We recently found that GSH and NAC exert direct inhibitory effects against a purified PKC isozyme mixture by a mechanism that did not involve their antioxidant properties. In this report, we characterize non-redox inhibitory effects of glutathiones on PKC isozymes that have been shown to produce partially or fully transformed phenotypes in mammalian cells. We show that GSH, NAC, and oxidized GSH analogs exert potent inhibition of the isozyme cPKC-ç and are somewhat less effective against cPKC- 1. In contrast, the oncogenic isozyme nPKC-â was unaffected by NAC, and it was inhibited by GSH and oxidized GSH analogs very modestly. Our results suggest that the potential impact of non-redox GSH/NAC-mediated PKC inhibition on cellular responses to tumor promoters and indeed, on cell growth regulation in general, may depend upon the pattern of PKC isozyme expression in the cells.

The significance of platelet-derived growth factors for proliferation of vascular smooth muscle cells

Platelets are important in acute thrombotic occlusion of injured vessels, e.g., subsequent to angioplasty. In contrast to these acute events of thrombus formation, much less is known about the significance of platelets for the control of smooth muscle cell (SMC) proliferation. A body of experimental and clinical evidence indicates an involvement of platelets in the pathology of atherosclerosis and restenosis. However, the precise role of platelet-derived growth factors for SMC proliferation in atherosclerotic and restenotic vessels is not clear and many questions remain unresolved. Platelet-dependent SMC mitogenesis is determined by a coordinate action of several classes of mitogenic factors which are either released from storage pools or generated upon platelet activation. Although platelet-derived growth factor (PDGF) is considered to be the most important platelet mitogen it is very likely that yet unknown factors and mechanisms are involved. Differential (stimulatory or inhibitory) effects on SMC growth and differentiation have been reported for different platelet-derived growth factors. Thus, for the overall response, complex interactions between multiple factors need to be considered. In addition, multicellular interactions, e.g., between platelets and endothelial cells may modulate the effects of platelet-derived factors on SMC mitogenesis. Taken together, the mechanisms of platelet-dependent SMC proliferation need to be reevaluated. The assessment of the precise role of platelet mitogens in the complex proliferative repair mechanisms of an injured vessel wall clearly requires further studies.

Differential Inhibitory Effects on Human Endometrial Carcinoma Cell Growth of Luteinizing Hormone-Releasing Hormone Analogues

In addition to its function as a key hormone in the regulation of the pituitary–gonadal axis, luteinizing hormone-releasing hormone (LHRH) probably also affects various extrapituitary tissues. LHRH binding sites andin vitroantiproliferative effects of LHRH analogues have been reported in human endometrial cancer. The effects of the LHRH agonist leuproreline and LHRH antagonist antide were studied on the cell growth, DNA synthesis, and cell cycle distribution of the human endometrial cancer cell lines HEC-1A and HEC-1B by the sulforhodamine B (SRB) method, [3H]thymidine assay incorporation, and propidium iodide DNA staining, respectively. In the presence of 1.0–100 μM leuproreline the proliferation of HEC-1A cells was significantly reduced as early as 3 days after drug exposure, with a minimum growth value of 69.9 ± 3.6% (mean ± SE) at the highest concentration tested (100 μM). Similar antiproliferative effects were obtained following a 6-day treatment with the LHRH antagonist antide. Also, inhibitory effects on [3H]thymidine incorporation into the DNA of the HEC-1A cell line were noted after a 6-day exposure to both LHRH analogues, in the above-mentioned concentration range. Cell cycle analysis of HEC-1A cells cultured in the presence of 10 μM leuproreline and antide showed a slight accumulation of cells in the G0/G1phase, while the proportions of cells in the S and G2/M phases concomitantly decreased. No significant effects on proliferation, DNA synthesis, and cell cycle distribution were observed in HEC-1B cells with either leuproreline or antide (up to 100 and 10 μM, respectively) after a 6-day exposure. Both Northern blot analysis and reverse transcription polymerase chain reaction failed to detect expression of mRNA for the LHRH receptor in both HEC-1A and HEC-1B cell lines. In addition, the LHRH analogues did not affect the intracellular free calcium concentration, indicating that the classic signal transduction for LHRH is absent or impaired in HEC-1A cells. The observed direct inhibitory actions on HEC-1A cells support the concept that the two LHRH analogues may exert biological effects via cellular effectors distinct from the “classic” LHRH receptor. Although the mechanism by which these direct actions are produced is still obscure, these results might help to establish the basis for new approaches to the therapy of endometrial cancer.

Differential inhibitory effects of thiopental, thiamylal and phenobarbital on both voltage-gated calcium channels and NMDA receptors in rat hippocampal slices

Although it is known that there are some pharmacological differences between the structurally similar barbiturates, the underlying mechanism of action remains unclear. We have compared the effects of thiopental, thiamylal and phenobarbital on both voltage-gated calcium channels (VGCC) and N-methyl-D-aspartate (NMDA) receptors in rat hippocampal slices by determining changes in intracellular calcium ([Ca2+]i). Experiments were performed in adult rat hippocampal slices perfused with Krebs solution (37 degrees C). Concentrations of [Ca2+]i in the pyramidal cell layer of the CA1 region were measured using a calcium indicator dye, fura-2. To activate VGCC and NMDA receptors, slices were exposed to K+ 60 mmol litre-1 (< or = 60 s) and NMDA 100 mumol litre-1 (30 s), respectively. Thiopental, thiamylal and phenobarbital were present 5 min before, during and 1 min after high K+ or NMDA application. Both thiamylal and thiopental (50-600 mumol litre-1) attenuated the increases in [Ca2+]i produced by high K+ or NMDA in a concentration-dependent manner, while phenobarbital 50-1000 mumol litre-1 only slightly attenuated the [Ca2+]i increase produced by high K+ at concentrations of more than 200 mumol litre-1 and was ineffective on the [Ca2+]i response produced by NMDA. Although the increases in [Ca2+]i caused by membrane depolarization with high K+ were reduced equally with thiamylal and thiopental, thiamylal was more effective in attenuating the increase in [Ca2+]i produced by NMDA receptor activation than thiopental. We conclude that the depressant effects of barbiturates on both VGCC and NMDA receptors varied between agents. Differential inhibition of both VGCC and NMDA receptors may determine the pharmacological properties of barbiturates and their ability to protect neurones against ischaemia.

Differential inhibitory effect of OK-1035 on DNA repair in L5178Y murine lymphoma sublines with functional or defective repair of double strand breaks

Radiosensitive L5178Y-S (LY-S) subline and its parental, more radioresistant L5178Y-R (LY-R) subline differ in DNA double strand break (DSB) rejoining. In this work we examined by comet assay the repair of X-ray-induced DNA damage in LY cells treated with OK-1035, a potent DNA-PK inhibitor. The unirradiated cells differ: the respective tail moment values for LY-R and LY-S cells were 9.62±2.84 and 3.52±0.1, reflecting the susceptibility to lysis conditions as well as the possible endogenous (oxidative) damage level. The level of initial DNA damage measured after irradiation (8 Gy) at DNA-denaturing pH was the same in both LY sublines: the mean tail moment values±SD were 92.93±10.39 for LY-R cells and 94.93±12.94 for LY-S cells. In LY-S cells the repair of 8 Gy X-ray-induced damage proceeded identically in the presence or absence of 2 mM OK-1035 to the same level of residual damage. In contrast, the level of residual damage in inhibitor treated LY-R cells was considerably higher than that in the untreated cells. Moreover, the inhibitor affected LY-R cells in G1 and S phases and not those in G2, in agreement with cell-cycle specificity of DNA-PK. These results may indicate that the DSB repair defect previously identified in LY-S cells is due to a lack of function of DNA-PK or its impaired activation in the irradiated cells.

Comparative induction of CYP3A and CYP2B in rat liver by 3-benzoylpyridine and metyrapone

3-Benzoylpyridine (3BP) is a major metabolite of HGG-12, an oxime that has been synthesized as a potential antidote to the toxic effects of soman and other anticholinesterases. Structural similarities exist between 3BP, the cytochrome P450 (CYP)-inducer metyrapone (MET) and other 3-substituted pyridines that interact with CYPs. The present study evaluated the regulatory effects of 3BP on CYP expression in rat liver. Both 3BP and MET (100 mg/kg) increased total hepatic microsomal holo-CYP content significantly 24 h after administration to male rats. Pronounced increases in activities mediated by CYP2B (androstenedione 16 β-hydroxylation and 7-pentylresorufin O-depentylation) were produced by 3BP and MET, which correlated with respective 9- and 14-fold increases in CYP2B immunoreactive protein. In addition, both agents slightly increased rates of microsomal CYP3A-dependent steroid 6 β-hydroxylation, troleandomycin metabolite complex formation and total CYP3A immunoreactive protein. Induction of the dexamethasone-inducible CYP3A23 mRNA to 4.5- and 2.5-fold of control was detected in liver of MET- and 3BP-induced rats; CYP3A2 mRNA levels were unchanged. Analogous in vitro studies revealed that MET was a preferential inhibitor of CYP3A-mediated steroid 6 β-hydroxylation activity, but 3BP was inactive against constitutive steroid hydroxylase CYPs. These findings indicate that the structurally related 3BP and MET elicit similar induction effects on CYPs 2B and 3A23 in rat liver after in vivo administration, but differential inhibitory effects of the chemicals on CYP activity in vitro. Recent reports have implicated a microsomal binding site in the induction of CYP3A1/3A23 in rat liver. In light of the present findings, substituted pyridines like 3BP may be useful tools in structure–activity studies to evaluate the physicochemical requirements for binding to this protein.

Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes.

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913-924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37 degreesC of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37 degreesC or 16 degreesC. We show that incubation at 16 degreesC blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16 degreesC. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16 degreesC is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.

Differential Inhibitory Effects of Three Nitric Oxide Donors on Ornithine Decarboxylase Activity in Human Colon Carcinoma Cells

Ornithine decarboxylase (ODC, EC 4.1.1.17) is the enzyme responsible for the synthesis of polyamines, which are absolutely necessary for cell proliferation. In the present work, we tested the effects of 3 nitric oxide (NO) donors, namely, sodium nitroprusside (SNP), (Z)-1-( N-methyl- N-[6-( N-methylammoniohexyl)amino] diazen-1-ium-1,2-diolate (MAHMA/NO) and 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA/NO), on ODC activity in human colon carcinoma cells (HT-29). SNP was the most effective inhibitor of ODC activity with a concentration of 8 μmol/L inducing 50% inhibition of basal activity. The effect of SNP was reversed by haemoglobin (Hb), but not by GSH or l-cysteine (CYS). Very little of the SNP in solution was degraded into nitrite, but the presence of cellular homogenate increased the production of nitrite. MAHMA/NO and DEA/NO were much less effective than SNP as ODC inhibitors, since the concentrations of these agents which induce 50% inhibition of basal activity were 20- to 60-fold higher than that of SNP. The effects of MAHMA/NO and DEA/NO were not reversed by haemoglobin. In solution, these latter 2 agents were totally degraded into nitrites. In conclusion, SNP on the one hand and MAHMA/NO and DEA/NO on the other appeared to release different NOx species with different efficiency on ODC activity.

Simultaneous cross-linking of CD6 and CD28 induces cell proliferation in resting T cells.

In the present study, we showed that simultaneous ligation of the monoclonal antibodies (mAb) against CD6 and CD28 induces T-cell proliferation in purified resting T lymphocytes in the absence of T-cell receptor (TCR) occupancy. No cell proliferation was observed when the mAb were cross-linked alone or used simultaneously in the soluble form. T-cell proliferation mediated through CD6/CD28 is accompanied by the up-regulation of interleukin-2 (IL-2) mRNA and expression of IL-2 receptors on the cell surface. In the presence of IL-2-neutralizing mAb the proliferative response of the T cell induced through CD6/CD28 was inhibited dose dependently. Cross-linking mAb to CD6 and CD28 alone or together did not down-regulate the CD3/TCR complex. T-cell proliferation mediated through CD6/CD28 was only partially blocked by the immunosuppressive drug, cyclosporin A (CsA), whereas anti-CD28-induced T-cell proliferation in the presence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was unaffected. In sharp contrast T-cell proliferation mediated by anti-CD6 in the presence of TPA was efficiently blocked by CsA. In addition, two protein kinase C (PKC) inhibitors, GF 109203X and H-7 dose-dependently inhibited T-cell proliferation mediated through CD6/CD28, suggesting that PKC activation may be involved. Furthermore, there was a marked differential dose-dependent inhibitory effect of the PKC inhibitors on T-cell proliferation mediated by the co-ligation of anti-CD6 or anti-CD28 in the presence of anti-CD3, with the former being more sensitive to PKC inhibition. Taken collectively, our results suggest that T-cell activation can occur through an antigen-independent pathway by cross-linking the accessory molecules, CD6 and CD28, and that these two cell surface antigens may have distinct signalling pathways.