Abstract  PURPOSE: This study was designed to clarify the involvement of the prostanoid FP receptor in the intraocular pressure (IOP)-lowering effects of latanoprost, travoprost, bimatoprost, and unoprostone with the use of FP-receptor-deficient (FPKO) mice.

METHODS: FPKO and wild-type (WT) mice were bred and acclimatized under a 12-hour light-dark cycle. IOP was measured under general anesthesia by a microneedle

METHOD: To evaluate the effects of each drug, a single drop (3 muL) of each drug solution was topically applied in a masked manner to a randomly selected eye. IOP reduction was evaluated by the difference in IOP between the treated eye and the untreated contralateral eye in the same mouse. First, the diurnal variation and baseline IOP in WT and FPKO mice were measured. Then, to determine the window feasible for demonstrating the most marked ocular hypotensive effect, 0.005% latanoprost was applied to WT mice during the day or at night. The time when the ocular hypotensive effect was larger was selected for further studies to evaluate the effects of latanoprost (0.005%), travoprost (0.004%), bimatoprost (0.03%), and unoprostone (0.12%). In addition, bunazosin (0.1%) was also applied to demonstrate functional uveoscleral outflow in FPKO mice. All experiments were conducted under a masked study design.

RESULTS: The baseline IOP (mean +/- SEM) in WT and FPKO mice was 15.0 +/- 0.2 and 15.0 +/- 0.3 mm Hg, respectively, during the day, and 18.9 +/- 0.4 and 19.2 +/- 0.4 mm Hg, respectively, at night. In WT mice, latanoprost significantly lowered IOP both during the day and at night, at 2 to 6 hours and 1 to 6 hours after application, respectively. Maximal IOP reduction was observed at 3 hours after drug instillation both during the day (10.9 +/- 1.8%) and at night (23.2 +/- 1.1%). At 3 hours after instillation, latanoprost (10.9 +/- 1.8% and 23.2 +/- 1.1%, daytime and nighttime, respectively), travoprost (15.9 +/- 1.4% and 26.1 +/- 1.2%) and bimatoprost (8.8 +/- 2.0 and 19.8 +/- 1.5%) significantly lowered IOP in WT mice both during the day and at night; isopropyl unoprostone significantly lowered IOP at night (13.7 +/- 1.9%) but not during the day (5.3 +/- 3.2%). In FPKO mice, latanoprost, travoprost, bimatoprost, and unoprostone showed no significant IOP-lowering effect. Bunazosin significantly lowered IOP in both WT (22.1 +/- 1.6%) and FPKO mice (22.2 +/- 2.1%).

CONCLUSIONS: A single application of latanoprost, travoprost, bimatoprost, or unoprostone had no effect on IOP in FPKO mice with presumed functional uveoscleral outflow pathways. The prostanoid FP receptor plays a crucial role in the mechanism of early IOP lowering of all commercially available prostaglandin analogues.

Abstract  Nitric oxide (NO) exerts multiple biologic roles in animal cells through differential regulation of three distinct forms of NO synthase encoded by separate genes. Macrophage-inducible nitric oxide synthase (iNOS) has been correlated with inhibition of viral growth, but little is known about the mechanism of this effect. To study the antiviral role of NO, we have generated a vaccinia virus (VV) recombinant expressing iNOS under the control of Escherichia coli LacI operator/repressor elements. When cultured cells of various origins are infected with this recombinant virus, there is inducible expression of iNOS in the presence of isopropylthio-beta-galactoside, as determined by Western blot and by detection of nitrite, a NO oxidation product. The levels of nitrite increase with time after infection, correlating with marked inhibition of VV DNA and late protein synthesis. Expression of VV early proteins is not affected by NO. Inhibition of VV DNA synthesis is likely to be in part a consequence of NO-mediated inhibition of viral ribonucleotide reductase, as this inhibition can be partially overcome by addition of deoxyribonucleosides. Inhibition of the essential viral functions by NO results in a reduction of virus yields by 50 to 90%, depending on the cell line. Thus, our results demonstrate a direct antiviral effect of NO, with inhibition of VV replication occurring at the level of DNA synthesis.

Abstract  Accurately predicting noise propagation in gene networks is crucial for understanding signal fidelity in natural networks and designing noise-tolerant gene circuits. To quantify how noise propagates through gene networks, we measured expression correlations between genes in single cells. We found that noise in a gene was determined by its intrinsic fluctuations, transmitted noise from upstream genes, and global noise affecting all genes. A model was developed that explains the complex behavior exhibited by the correlations and reveals the dominant noise sources. The model successfully predicts the correlations as the network is systematically perturbed. This approach provides a step toward understanding and manipulating noise propagation in more complex gene networks.

Abstract  We describe herein the properties at the air/water (A/W) interface of hydrophobically end-modified (HM) poly(2-isopropyl-2-oxazoline)s (PiPrOx) bearing an n-octadecyl chain on both termini (telechelic HM-PiPrOx) or on one chain end (semitelechelic HM-PiPrOx) for different subphase temperatures and spreading solvents using the Langmuir film balance technique. The polymer interfacial properties revealed by the pi-A isotherms depend markedly on the architecture and molecular weight of the polymer. On cold water subphases (14 degrees C), diffusion of PiPrOx chains onto water takes place for all polymers in the intermediate compressibility region (5mNm(-1)). At higher subphase temperatures (36 and 48 degrees C), the HM-PiPrOx film exhibited remarkable stability with time. Brewster angle microscopy (BAM) imaging of the A/W interface showed that the polymer assembly was not uniform and that large domains formed, either isolated grains or pearl necklaces, depending on the polymer structure, the concentration of the spreading solution and the subphase temperature. The Langmuir films were transferred onto hydrophilic substrates (silica) by the Langmuir-Blodgett (LB) technique and onto hydrophobic substrates (gold) by Langmuir-Schaefer (LS) film deposition, resulting in the formation of adsorbed particles ranging in size from 200 to 500nm, depending on the polymer architecture and the substrate temperature. The particles presented "Janus"-like hydrophilic/hydrophobic characteristics.

Abstract  A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.

Abstract  Affinity sites of N-isopropyl-[125I]p-iodoamphetamine (IMP) were studied in rat brain employing in vitro technique of receptor autoradiography. High degrees of IMP accumulation were observed in almost regions of white matter and some of gray matter. Phenethylamine, glutamic acid and serotonin suppressed IMP accumulation in all brain regions, in cerebral cortex and in hypothalamus, respectively. These results indicated that IMP was accumulated mainly due to its physicochemical property of lipophilicity and might have some interactions with neurotransmitter receptors.

Abstract  Allosteric modification of ligand binding is central to LacI transcription control. Recently, the conformational change between LacI operator- and inducer-bound states was simulated with targeted molecular dynamics (TMD) [Flynn, T. C., Swint-Kruse, L., Kong, Y., Booth, C., Matthews, K. S., and Ma, J. (2003) Protein Sci., 12, 2523-2541]. Atomic-level analyses of TMD results indicate the structural importance of the core pivot region that connects the N- and C-subdomains flanking the inducer-binding site. Further, a number of LacI mutations in the core pivot have been identified recently by their altered behaviors in phenotypic screens. Biochemical characterization of three of these variants-L148F, S151P, and P320A-provides an opportunity to directly explore the role of the core pivot in repressor function. For L148F, inducer IPTG binding affinity is strengthened, whereas O(1) operator DNA binding is diminished approximately 30-fold. In contrast, O(1) binding is increased for S151P, whereas IPTG binding is decreased. UV-difference spectroscopy and urea denaturation indicate long-range effects in both variants. Interestingly, P320A binds to DNA approximately 4-fold more tightly than wild-type, yet inducer binding is unaffected. To examine linkage between the core pivot and DNA binding domains, the L148F substitution was combined with Q60G, a previously known mutant with enhanced operator affinity. The double mutant exhibits the properties of both parent proteins, resulting in near wild-type DNA binding affinity and enhanced inducer sensitivity. These features may render Q60G/L148F more cost-effective in technological applications than wild-type repressor. As a group, the behaviors of the core pivot mutants are consistent with the allosteric structural role predicted for this region by TMD and reflect the significant long-range impact that single substitutions can elicit on protein function.

Abstract  SsrA RNA of Escherichia coli, also known as 10Sa RNA or tmRNA, acts both as tRNA and mRNA when ribosomes are paused at the 3' end of an mRNA lacking a stop codon. This process, referred to as trans-translation, leads to the addition of a short peptide tag to the C-terminus of the incomplete nascent polypeptide. The tagged polypeptide is then degraded by C-terminal-specific proteases. Here, we focused on endogenous targets for the SsrA system and on a potential regulatory role of SsrA RNA. First, we show that trans-translation events occur frequently in normally growing E. COLI: cells. More specifically, we report that the lacI mRNA encoding Lac repressor (LacI) is a specific natural target for trans-translation. The binding of LacI to the lac operators results in truncated lacI mRNAs that are, in turn, recognized by the SsrA system. The SsrA-mediated tagging and proteolysis of LacI appears to play a role in cellular adaptation to lactose availability by supporting a rapid induction of lac operon expression.

Abstract  The porphyrinogenicity of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is related to the process of mechanism-based destruction of cytochrome P450 (P450) heme, accompanied by conversion of heme to N-alkylprotoporphyrins (N-alkylPPs). Certain DDC analogues (4-isopropyl, 4-isobutyl, 4-hexyl) are weakly porphyrinogenic in comparison to the potent porphyrinogen, 4-ethyl DDC. We have examined the abilities of these DDC analogues to promote irreversible binding of radiolabeled heme to protein in rat liver microsomal preparations. The goals of this study were to determine whether DDC analogues with different porphyrinogenicities differ in the extents to which they cause heme adduct formation, and whether P450 isozymes differ in their capacities to catalyze heme covalent binding. Incubation of microsomes with NADPH alone promoted heme covalent binding, while loss of spectral P450 heme was minimal or absent. In microsomal incubations containing NADPH, the 4-ethyl, 4-isopropyl, and 4-isobutyl analogues caused heme covalent binding to extents which paralleled their P450 destructive activities. In contrast, 4-hexyl DDC caused less heme covalent binding as a function of P450 loss than the other analogues in microsomes from untreated and beta-naphthoflavone (beta NF)-treated rats. Thus, the weakly porphyrinogenic DDC analogues do not cause greater heme covalent binding than 4-ethyl DDC. Weak porphyrinogenicity, therefore, cannot be explained by diversion of the heme moiety of P450 from conversion to N-alkylPPs towards utilization for formation of heme-derived protein adducts. Treatment of rats with P450 inducing agents altered the degree to which DDC analogues caused heme covalent binding. The greatest heme adduct formation occurred in microsomes from untreated and dexamethasone (DEX)-treated rats, whereas treatment with phenobarbital and especially beta NF reduced heme covalent binding as a function of P450 loss. Thus, these microsomal studies suggest that constitutive P450 isozymes and members of the DEX-inducible P450IIIA subfamily appear to catalyze heme covalent binding, while beta NF-inducible forms such as P450IA1 (P450c) seem to be relatively inactive in this regard.

Abstract  The purpose of this research was to evaluate the effect of penetration enhancers on permeation kinetics of nitrendipine (NTP) through two different skin models. The permeation profile and related kinetics parameters such as activity parameter, diffusion parameter, lag time, relative activity parameter and relative diffusion parameter of NTP was determined in presence of some novel and widely accepted permeation enhancers. Among all the more pronounced enhancing effect was obtained with oleic acid (OA) as it presented the highest permeability coefficient. The enhancement was found to be increased in the following order: Dimethyl sulphoxide (DMSO)<Isopropyl myristate (IPM)<Sodium lauryl sulphate (SLS)<Tween 20 (T20)<Myristic acid (MA)<Lauric acid (LA)<Capric acid (CA)<Tween 80 (T80)<Span 80 (S80)<Thyme oil (TO)<Palmarosa oil (PA)<Petitlgrain oil (PT)<Basil oil (BO)<Oleic acid (OA). The increase in drug permeation was attributed mainly to the decrease in diffusion path length. Essential oils and surfactant (span 80) have shown relative activity parameter (Kr value) well above unity and relative diffusion parameter (Dr value) almost equal or less than unity, which would be attributed to gradual increase in membrane permeability leading to higher permeation. Oleic acid is effective for augmenting skin permeation of NTP, with potential application in transdermal delivery of the drug.

Abstract  The aim of this work was to investigate the effect of enhancers and organic amines on the in-vitro percutaneous absorption of the major pharmacologically active compound, scutellarin, obtained from breviscapine extract. The donor vehicle consisted of isopropyl myristate-ethanol in a ratio of 4:1. Percutaneous absorption across full thickness rat skin was investigated in-vitro using 2-chamber diffusion cells, with reverse-phase HPLC for quantification of the permeating scutellarin. Organic amines increased scutellarin permeation by ion-pair formation. We also found that the cumulative amount of scutellarin over a period of 12 h of scutellarin was inversely related to the molecular weight of organic amines (r = 0.9134), as well as the logarithm of scutellarin permeability coefficient inversely related to the partition coefficient of organic amines (r = 0.8929). All the permeation enhancers tested increased the cumulative amount of scutellarin over a period of 12 h, and the order of this increase was n-methyl-2-pyrrolidone, oleic acid, menthol or Azone. Drug solubility in donor phase was markedly increased by Azone and n-methyl-2-pyrrolidone, and reduced by menthol and oleic acid. The combined effects of ethanolamine plus Azone, ethanolamine plus menthol, and Azone plus menthol were also investigated. Azone plus menthol had a synergistic effect on the cumulative amount of scutellarin over a period of 12 h.

Abstract  1. Organophosphorus compounds that produce a delayed neurotoxic effect in hens phosphorylate a specific site in the brain soon after administration. 2. Phosphorylation of the specific site by di-isopropyl [(32)P]phosphorofluoridate in vitro is blocked by the prior addition of phenyl phenylacetate. 3. A small proportion of the total activity of hen brain hydrolysing phenyl phenylacetate in vitro was shown to be due to an enzyme different from two others previously described. 4. This enzyme is only slightly inhibited in vitro by concentrations of tetraethyl pyrophosphate and paraoxon (diethyl 4-nitrophenyl phosphate) up to 64mum and is completely inhibited by 6mum-di-isopropyl phosphorofluoridate and 128mum-mipafox. 5. It is also inhibited in vivo by effective doses of neurotoxic organophosphorus compounds but not by high doses of non-neurotoxic analogues. 6. It is deduced that the active site of this enzyme is the phosphorylation site associated with the genesis of delayed neurotoxicity.

Abstract  This paper examines the effects of phosphate pool and expression of polyphosphate kinase on glucose uptake by expressing the polyphosphate kinase under the control of lac promoter. The E. coli transformant of pL1, containing an IPTG controllable element for polyphosphate kinase expression, showed that the total intracellular phosphate significantly increased. However, the rate of glucose uptake by the resting plasmid-bearing cells with IPTG induction significantly decreased. These findings suggest that the polyphosphate can not directly function as an energy source in E. coli or at least not as a good energy supplier.

Abstract  Recent advances in genomics and molecular pathogenesis studies have determined that many diseases are caused by a multiplicity of factors. New drug regimens may consist of multiple biologically active agents designed to act synergistically on multiple biochemical targets. Live cell assays are becoming a standard for identifying new drug candidates with an emphasis on "homogeneous" living cell assays in which multiple cell lines are mixed and monitored simultaneously. In this study, we used a high-density single living cell array, based on an optical imaging fiber bundle microwell array, to simultaneously monitor "promiscuous" drug effects on single cells of multiple cell types. Such a capability allows for a more comprehensive understanding of how cells dynamically respond to combinatorial drug libraries or how different cellular pathways and regulation circuits respond cooperatively to drugs in individual cells.

Abstract  Five members (6-10) of an homologous series of 7-alkyloxycarbonyltheophylline (7-AOC-Th) and four members (2-5) of a homologous series of 7-alkylcarbonyltheophylline (7-AC-Th) prodrugs have been synthesized by known methods and characterized. All of the members in both series were much more soluble in isopropyl myristate (S(IMP)) (10-200 times) and in each series, at least one member was more soluble in pH 4.0 buffer (S(AQ)) than Th. However, in the 7-AC-Th series, only the acetyl member, 2, which exhibited about 90% of the S(AQ) of Th, was sufficiently stable to be evaluated - it gave four times the flux of Th/IPM (isopropyl myristate). In the 7-AOC-Th series, all the members were sufficiently stable to be evaluated but the member which exhibited the greatest S(AQ), 6 (methyloxycarbonyl), did not exhibit the greatest flux. Instead, 8 (propyloxycarbonyl), which exhibited the second greatest S(AQ) (about 80% of the S(AQ) of Th), but exhibited over ten times the S(IPM) of 6 gave the greatest flux - two times the flux of Th/IPM. Thus, good biphasic solubility was the best predictor of increased flux. All of the prodrugs delivered only Th through the mouse skin. Only 2/IPM actually delivered more Th into the skin than Th/IPM which correlated with its ability to increase the flux of Th through the skin.

Abstract  The influence of some process variables on the productivity of the fractions (liquid yield times fraction per cent) obtained from SCFE of a Brazilian mineral coal using isopropanol and ethanol as primary solvents is analyzed using statistical techniques. A full factorial 2(3) experimental design was adopted to investigate the effects of process variables (temperature, pressure and cosolvent concentration) on the extraction products. The extracts were analyzed by the Preparative Liquid Chromatography-8 fractions method (PLC-8), a reliable, non-destructive solvent fractionation method, especially developed for coal-derived liquids. Empirical statistical modeling was carried out in order to reproduce the experimental data. Correlations obtained were always greater than 0.98. Four specific process criteria were used to allow process optimization. Results obtained show that it is not possible to maximize both extract productivity and purity (through the minimization of heavy fraction content) simultaneously by manipulating the mentioned process variables. (C) 1998 Elsevier Science B.V.

Abstract  In the picolinic acid (PA) promoted Cr-VI oxidation of dimethyl sulfoxide (DMSO), the Cr-VI-PA complex formed at the pre-equilibrium step undergoes nucleophilic attack by the S of DMSO to form a positively charged reactive intermediate which experiences an oxygen transfer or a ligand coupling to give the products; the anionic surfactant (SDS) accelerates the process while the cationic surfactant (CPC) retards the reaction.

Abstract  A series of ZrO2-TiO2 mixed oxides was prepared varying mole fraction of titanium from 0 to 1, and characterized by XRD, FT-IR, TG/DTA, UV-VIS-DRS, surface area, surface acidity, and sulphur content. The UV-VIS-DRS and pH of point of zero charge (pH(ZPC)) data for the mixed oxide samples determined by acid-base titration method revealed that a different surface was formed when zirconium and titanium were co-precipitated. XRD pattern of 700degreesC calcined samples showed the formation of ZrTiO4 phase. The optimum concentration of zirconium in the zirconia-titania mixed oxide in the precipitation solution was 60 mol%, for obtaining high surface area and highly acidic mixed oxide system. Sulphur analysis of the sulphate promoted samples showed that the sample containing Ti mole fraction 0.4 retained the highest amount of sulphur. The surface acidity determined by irreversible adsorption of organic bases, such as pyridine, piperidine and 2,6-dimethyl pyridine showed the highest acidity for the above sample. This catalyst also showed the highest catalytic activity towards isopropanol dehydration. (C) 2002 Elsevier Science B.V. All fights reserved.

Abstract  Crosslinked poly (vinyl alcohol) (PVA) membranes were Prepared using maleic acid (MA) and citric acid (CA) as crosslinking agents. First the water separation ability of the crosslinked membranes from aqueous acetic acid (10-90% acetic acid) and ethanol (20-80% ethanol) solutions at 30 to 90 degrees C was tested by pervaporation. The effects of temperature, feed concentration, crosslinker type on pervaporation characteristics were investigated. The PVAMA membrane showed separation factors of 1.9-16.2 and fluxes of 4.26-38.48 kg/m(2) h and PVACA membrane showed separation Factors of 3.8-33 and fluxes of 2.17-480.8 kg/m(2) h for acetic acid-water separation. In case of ethanol-water separation. the PVAMA membrane showed separation factors of 0.7-17.9 and fluxes of 2.5-36.2 kg/m(2) h and PVACA membrane showed separation factors of 1.4-41.9 and fluxes of 3.8-359.7 kg/m(2) h. PVACA membrane has shown high separation efficiency of water than that of PVAMA membrane. Having high selectivity and good permeating ability for water both the membranes were then used to remove water of esterification from reaction of acetic acid with ethanol by a batch pervaporation. Compared to around 60% conversion in conventional esterification, LIP to 98% conversion to ethyl acetate was achieved from ethanol and acetic acid when water by product was removed by pervaporation using the Prepared membranes at same temperature.

Abstract  Reduction reactions of 3,5-diiodotyrosine (DTR) are reported in phosphate-buffered aqueous medium. Steady state gamma-radiolysis and electron pulse irradiation have been used to study reduction mechanisms of DTR in the presence of primary radicals, e(aq)(-) and H-., and secondary radicals derived from oxygen, HO2. and O-2(-), in acidic, neutral, and alkaline media. The rate constant for H-. addition to the DTR phenolic chromophore is estimated to be k = 2.4 x 10(9) at pH 1.8 and 1.6 x 10(9) dm(3) mol(-1) s(-1) at pH 5; a cyclohexadienyl type transient with peak absorption at 385 nm and epsilon(385) = 900 +/- 200 dm(3) mol(-1) cm(-1) is produced in both cases. Reactions of e(aq)- on the other hand show a marked pH dependence. Electron addition to the phenolic chromophore between pH 4 and 12 produces a transient species that absorbs below 300 nm with lambda(max) at 280 nm and epsilon(280) = 9300 +/- 700 dm(3) mol(-1) cm(-1) with concurrent liberation Of I-. G(I-) is constant over the pH range and is estimated to be 0.11 +/- 0.01 mu M Gy(-1). In addition, in acidic and neutral media, ammonia elimination occurs with G(NH3) = 0.19 at pH 4 and 0.12 mu M Gy(-1) at pH 7. The rate constants for these processes are intermediate to the rate, 8.0 x 10(9) dm(3) mol(-1) s(-1) for e(aq)- addition to the -NH3+ group (subsequently leading to deamination) and 1.55 x 10(10) dm(3) mol(-1) s(-1) for addition to the phenolic chromophore. DTR does not react with HO2. and O-2(-) radicals. The reducing a-hydroxy radical derived from isopropyl alcohol, (H3C)(2)(COH)-O-., and carboxyl radical, CO2-, do not react with DTR by either electron transfer, H-abstraction, or addition to the phenolic ring, indicating a reduction potential value of the DTR(-)/DTR couple more negative than -2.0 V vs NHE.

Abstract  Chemical kinetics of alkaline fading of crystal violent has been studied in aqueous mixtures of methanol, ethanol, isopropanol and t-butanol by measuring the specific rate constant of the reactions spectrophotometrically in these solvent systems. The kinetic solvent effect has been analyzed in the light of Gibb's free energies of transfer of reactants and transition states from water to aqueous mixtures of alcohols, derived thereof. The observed variation of transfer free energy parameters, characteristic of the S-N(2) reaction, with cosolvent compositions have been interpreted in terms of various specific and non-specific interactions of media with the reacting species and the transitions state involved in this reaction.

Abstract  The phosphorylation of cyclin D1 at threonine 286 by glycogen synthase kinase 3beta (GSK3beta) has been shown to be required for the ubiquitination and nuclear export of cyclin D1 and its subsequent degradation in the proteasome. The mutation of the nearby residue, threonine 288, to nonphosphorylatable alanine has also been shown to reduce the ubiquitination of cyclin D1, suggesting that phosphorylation at threonine 288 may also lead to degradation of cyclin D1. We now demonstrate that the G(0)/G(1)-active arginine-directed protein kinase Mirk/dyrk1B binds to cyclin D1 and phosphorylates cyclin D1 at threonine 288 in vivo and that the cyclin D1-T288A construct is more stable than wild-type cyclin D1. Transient overexpression of Mirk in nontransformed Mv1Lu lung epithelial cells blocked cells in G(0)/G(1). Depletion of endogenous Mirk by RNA interference increased cyclin D1 protein levels but not mRNA levels, indicating that Mirk destabilizes cyclin D1 protein. Destabilization was confirmed by induction of a stable Mirk transfectant of Mv1Lu cells, which blocked cell migration (Zou, Y., Lim, S., Lee, K., Deng, X., and Friedman, E. (2003) J. Biol. Chem. 278, 49573-49581), and caused a decrease in the half-life of endogenous cyclin D1, concomitant with an increase in Mirk expression. In vitro cyclin D1 was phosphorylated in an additive fashion by Mirk and GSK3beta. Mirk-phosphorylated cyclin D1 mutated at the GSK3beta phosphorylation site and was capable of phosphorylating cyclin D1 in the presence of the GSK3beta inhibitor LiCl. Mirk may function together with GSK3beta to assist cell arrest in G(0)/G(1) by destabilizing cyclin D1.