Diverse coordination modes in tin analogues of a cyclopentadienyl anion depending on the substituents on the tin atom.

Reactions of an anionic heavy ruthenocene with CCl4, MeI, EtBr and Me3SiCl afforded the first stannole monoanion complexes. Surprisingly, coordination modes of the stannole rings are highly dependent on the substituents on the tin atom. The chloro derivative exhibits a η(4)-fashion-like coordination mode with a bent stannole ring, whereas the trimethylsilyl derivative adopts the conventional η(5)-coordination mode. Coordination modes of the alkyl derivatives are in between the two types. Cyclic voltammograms for these complexes reveal that the electron-donating character of the stannole ligand becomes stronger as the stannole ring becomes planar. Theoretical calculations elucidate that the different coordination modes originate from both electronegativity of an adjacent atom to the tin atom and bulkiness of a substituent on the tin atom.

Thy28 protects against anti-CD3-mediated thymic cell death in vivo.

Apoptotic cell death plays a pivotal role in the development and/or maintenance of several tissues including thymus. Deregulated thymic cell death is associated with autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), a prototype murine model for analysis of human multiple sclerosis. Because Thy28 expression is modulated during thymocyte development, we tested whether Thy28 affects induction of EAE as effectively as antigen-induced thymocyte deletion using Thy28 transgenic (TG) mice. Thy28 TG mice showed partial resistance to anti-CD3 monoclonal antibody (mAb)-induced thymic cell death in vivo, as assessed by annexin V-expression and loss of mitochondrial membrane potential. The resistance to anti-CD3 mAb-induced cell death in Thy28 TG mice appeared to correlate with a decreased c-Jun N-terminal kinase phosphorylation and reduced down-regulation of Bcl-xL. Moreover, thymic hyperplasia was detected in Thy28 TG mice, although thymocyte development was unaltered. Development of peripheral lymphoid tissues including spleen and lymph nodes was also unaltered. Thy28 TG spleen T cells showed an increased production of IFN-γ, but not IL-17, in response to both anti-CD3 and anti-CD28 mAbs. Finally, Thy28 TG mice displayed accelerated induction of EAE as assessed by disease incidence, clinical score, and pathology following immunization with myelin oligodendrocyte glycoprotein compared with control WT mice. These findings suggest that modulation of Thy28 expression plays a crucial role in the determination of thymic cell fate, which may contribute to the development of EAE through proinflammatory cytokine production.

Air shower simulation for WASAVIES: warning system for aviation exposure to solar energetic particles.

WASAVIES, a warning system for aviation exposure to solar energetic particles (SEPs), is under development by collaboration between several institutes in Japan and the USA. It is designed to deterministically forecast the SEP fluxes incident on the atmosphere within 6 h after flare onset using the latest space weather research. To immediately estimate the aircrew doses from the obtained SEP fluxes, the response functions of the particle fluxes generated by the incidence of monoenergetic protons into the atmosphere were developed by performing air shower simulations using the Particle and Heavy Ion Transport code system. The accuracy of the simulation was well verified by calculating the increase count rates of a neutron monitor during a ground-level enhancement, combining the response function with the SEP fluxes measured by the PAMELA spectrometer. The response function will be implemented in WASAVIES and used to protect aircrews from additional SEP exposure.

Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by Toll-like receptor 4 in mice.

AIMS/HYPOTHESIS: Hyperlipidaemia is an independent risk factor for the progression of diabetic nephropathy, but its molecular mechanism remains elusive. We investigated in mice how diabetes and hyperlipidaemia cause renal lesions separately and in combination, and the involvement of Toll-like receptor 4 (TLR4) in the process. METHODS: Diabetes was induced in wild-type (WT) and Tlr4 knockout (KO) mice by intraperitoneal injection of streptozotocin (STZ). At 2 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Functional and histological analyses were carried out 6 weeks later. RESULTS: Compared with treatment with STZ or HFD alone, treatment of WT mice with both STZ and HFD markedly aggravated nephropathy, as indicated by an increase in albuminuria, mesangial expansion, infiltration of macrophages and upregulation of pro-inflammatory and extracellular-matrix-associated gene expression in glomeruli. In Tlr4 KO mice, the addition of an HFD to STZ had almost no effects on the variables measured. Production of protein S100 calcium binding protein A8 (calgranulin A; S100A8), a potent ligand for TLR4, was observed in abundance in macrophages infiltrating STZ-HFD WT glomeruli and in glomeruli of diabetic nephropathy patients. High-glucose and fatty acid treatment synergistically upregulated S100a8 gene expression in macrophages from WT mice, but not from KO mice. As putative downstream targets of TLR4, phosphorylation of interferon regulatory factor 3 (IRF3) was enhanced in kidneys of WT mice co-treated with STZ and HFD. CONCLUSIONS/INTERPRETATION: Activation of S100A8/TLR4 signalling was elucidated in an animal model of diabetic glomerular injury accompanied with hyperlipidaemia, which may provide novel therapeutic targets in progressive diabetic nephropathy.

Pharmacokinetics and metabolism of KW-4490, a selective phosphodiesterase 4 inhibitor: difference in excretion of KW-4490 and acylglucuronide metabolites between rats and cynomolgus monkeys.

1. The pharmacokinetics and metabolism of KW-4490, a selective phosphodiesterase 4 inhibitor, were investigated in rats and monkeys. After oral administration, KW-4490 was rapidly absorbed, and then its plasma concentrations apparently declined with half-lives of approximately 5 h in rats and 3.5 h in cynomolgus monkeys; however, a number of secondary peaks were apparent in the profiles for both species. The plasma pharmacokinetics of KW-4490 were comparable between rats and monkeys. 2. After oral administration, KW-4490 was mainly eliminated by metabolism to acylglucuronides and renal excretion in the unchanged form. KW-4490 acylglucuronides were found in monkey but not rat urine. In rats, KW-4490 acylglucuronides were excreted only in bile. Although the pathway of excretion of acylglucuronides differed between rats and monkeys, cumulative excretion in the two animals was very similar, as expected from comparable hepatic clearance for glucuronidation in rat and monkey liver microsomes. 3. The glomerular filtration rate of unbound KW-4490 indicated that renal tubular secretion was significant in monkeys, whereas reabsorption was significant in rats. These species differences in urinary excretion of KW-4490 and its acylglucuronide metabolites are most likely due to substrate specificity of active transporters in rat and monkey kidney.

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.

Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-beta-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation.

Overexpression of connective tissue growth factor in podocytes worsens diabetic nephropathy in mice.

Connective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes.

LPS-induced apoptosis is dependent upon mitochondrial dysfunction.

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.

Allosterically controlled single-chained maxizymes with extremely high and specific activity.

For the treatment of chronic myelogenous leukemia (CML), attempts have been made to design various ribozyme motifs that can specifically recognize and cleave BCR-ABL fusion mRNAs. In the case of L6 BCR-ABL b2a2 mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure [Kuwabara, T.; et al. Mol. Cell 1998, 2, 617-627; Tanabe, T.; et al. Nature 2000, 406, 473-474]. Specifically, we tailored the maxizyme with molecular switching function: the maxizyme splices a cleavable GUC site, but only when it appears within a strand of mRNA that possesses the abnormal splice junction. We demonstrated that this approach is generalizable [Tanabe, T.; et al. Biomacromolecules 2000, 1, 108-117]. All the maxizymes designed in the past functioned as a result of the formation of a dimeric structure. Questions have been asked whether a similar molecular switching might be possible within a single molecule when two monomer units of the maxizyme were connected via a linker sequence. We found that an analogous conformational change could not be induced within a single molecule when two maxizyme units were simply connected via a nonregulatable linker sequence. However, an active conformation was achieved by the introduction of an antisense modulator within the linker sequence that adjusted the overall structure to the correct form. Results of studies in cultured cells suggested that the desired conformational change could indeed be induced within the modified single-chained maxizyme and such a construct caused apoptosis only in leukemic cells with the Philadelphia chromosome.

Transport of intracellularly active ribozymes to the cytoplasm.

Ribozymes are RNA molecules with enzymatic activity which selectively bind and cleave specific target RNAs. To date, numerous studies directed toward the application of ribozymes in vivo have been performed and many successful experiments have been reported. However, to induce high-level activities of ribozymes in vivo, several factors must be considered. Here we report that the cytoplasmic localization of ribozymes is important for their intracellular activity in mammalian cells. Northern blot analysis revealed that a tRNA(Val) ribozyme, which can assume a cloverleaf structure similar to that of a native tRNA, is efficiently transported to the cytoplasm. In contrast, the tRNAiMet-driven ribozyme, which does not maintain the cloverleaf structure, remained predominantly in the nucleus. In correlation with the localization, the activity of the exported ribozyme was higher than that of the ribozyme retained in the nucleus. These results should provide insight into the design of ribozymes that have high-level activity in mammalian cells.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

RNA-protein hybrid ribozymes that efficiently cleave any mRNA independently of the structure of the target RNA.

Ribozyme activity in vivo depends on achieving high-level expression, intracellular stability, target colocalization, and cleavage site access. At present, target site selection is problematic because of unforeseeable secondary and tertiary RNA structures that prevent cleavage. To overcome this design obstacle, we wished to engineer a ribozyme that could access any chosen site. To create this ribozyme, the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, was attached to our ribozymes so that the helicase-bound, hybrid ribozymes would be produced in cells. This modification significantly enhanced ribozyme activity in vivo, permitting cleavage of sites previously found to be inaccessible. To confer cleavage enhancement, the CTE must retain helicase-binding activity. Binding experiments demonstrated the likely involvement of RNA helicase(s). We found that attachment of the RNA motif to our tRNA ribozymes leads to cleavage in vivo at the chosen target site regardless of the local RNA secondary or tertiary structure.

Photochromism of viologens included in crown ether cavity.

The effect of a pi-electron-donating macrocyclic molecule on the photochromic behavior of viologen derivatives was investigated in a thin polymer film. The intermolecular interactions between the viologens and the macrocyclic molecule were investigated in a solution before photoirradiation. In acetone, benzylviologens, N,N'-dibenzyl-4,4'-bipyridinium hexafluorophosphate (1) and N,N'-dibenzyl-trans-1,2-bis(4-pyridinium)ethylene hexafluorophosphate (2) each derived from 4,4'-bipiyridine and trans-1,2-bis(4-pyridyl)ethylene, respectively, form an inclusion complex with p-benzocrown ether (3) with binding constants of ca 200 M-1, which was driven by a charge transfer interaction. The peak wavelength of the charge transfer absorption band was at 453 and 421 nm for the inclusion complexes of 1 and 2 with 3, respectively. Upon photoirradiation to the polymer film containing 1, the film changed color from colorless to blue, associated with the reduction of 1 from the dication to the radical cation. The original dication was recovered after 120 min. The addition of 3 into the film containing 1 caused not only the color change from colorless to yellow, associated with the charge transfer interaction between 1 and 3 before photoirradiation, but also an acceleration in the bleaching rate of the photoreduced 1. When p-dimethoxybenzene (4) was used as an acyclic analog of 3, a negligible change in the photochromic behavior of 1 was observed. Similar effect of 3 on the photochromic behavior of 2 was observed. These results imply that the pi-electron-donating macrocyclic molecule causes a faster bleaching of photoreduced viologens by forming the inclusion complex.

Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms.

PURPOSE: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

A possible relationship between abortions and placental embolism in pregnant rabbits given human granulocyte colony-stimulating factor.

Developmental toxicity studies were conducted in rats and rabbits with a human G-CSF derivative (NTG). As reported for G-CSF, increases in abortions and fetal mortality were observed in rabbits, but not in rats given NTG. Histopathological examination of the rabbit placenta revealed accumulation of neutrophils in vessels and necrosis of the tissues surrounding these vessels. To assess the mechanism of abortion and fetal death in rabbits given G-CSF, 125I-labeled NTG was given intravenously on Day 18 of pregnancy after repeated administration of cold NTG on Days 6 through 17 of pregnancy, and the feto-maternal distribution of radioactivity was examined. In a rabbit given 20 micrograms/kg, high radioactivity was observed in the endometrium, placenta, and some parts of the decidua at 6 hr when the concentration of radioactivity in maternal blood had already decreased. At 24 hr after administration of 200 micrograms/kg NTG, high radioactivity was still detected in parts of the maternal placenta. These patterns of distribution suggest that embolism occurred in parts of the uterus and placenta which might have caused congestion. Radioactivity in the TCA precipitates in the fetus was low, suggesting that NTG does not readily transfer to the fetus. These results strongly suggest that neutrophils accumulated in the vessels of placenta and induced embolism leading to abortions and fetal mortality in the rabbits given G-CSF.

Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells.

Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting step in vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules.

Pharmacokinetics, distribution, metabolism and excretion of.

PURPOSE: To evaluate the metabolic fate of UCN-01, a signal transduction inhibitor, blood and plasma concentrations, distribution, metabolism and excretion were investigated in rats and dogs after intravenous administration of [3H]UCN-01. METHODS: The radioactivity in plasma, blood and tissues was measured after intravenous administration of UCN-01. In addition, the radioactivity excreted in bile, urine and feces was also determined. RESULTS: The radioactivity in rat and dog plasma disappeared triphasically with terminal half-lives of 21.3 and 27.2 h, respectively. The ratios of the blood-to-plasma concentrations ranged from 0.82 to 1.13 in rats and 0.81 to 1.73 in dogs. From 0.5 to 4 h after giving [3H]UCN-01 to rats, the radioactivity in all tissues except the brain and testes was higher than in plasma. The highest concentration was observed in the lungs followed by the liver and kidneys. The radioactivity was mainly excreted in feces, reaching 96.0% of the radioactivity dose in rats and 78.4% in dogs up to 168 h after injection. Since the biliary excreted radioactivity was 67.2% over 48 h in bile duct-cannulated rats, most of the radioactivity excreted in feces was from biliary radioactivity. There were several metabolites in bile samples, but little UCN-01. CONCLUSIONS: UCN-01 is mainly eliminated by the liver, and there are high concentrations of radioactivity derived from [3H]UCN-01 in all tissues except the brain and testes.

[A case of atopic myelitis in the area other than Kyushu Island].

A case of acute myelitis associated with atopic dermatitis, hyperIgEemia and mite antigen specific IgE was reported. A 22-year old woman with a history of atopic dermatitis since her childhood began to have numbness in her legs, which extended upward to her upper part of chest in four days. Neurological examination revealed Lhermitte's sign, moderate disturbance of superficial and deep sensation below the Th4 level, and sensory ataxia. She also showed exaggerated deep tendon reflexes in the upper extremities and urinary difficulty. MRI studies revealed an intramedullary T1-low, T2-high lesions without Gd-DTPA contrast enhancement in the posterior part of the cervical spinal cord at C3 level. The laboratory examination revealed normal CSF, and the elevated level of serum IgE and mite-antigen-specific IgE in sera. We conclude that this case is thought to be atopic myelitis, which was reported as acute myelitis associated with hyperIgEemia and atopic dermatitis proposed by Kira et al. This is the first report of a case outside of Kyushu area in Japan.

Allosterically controllable maxizyme-mediated suppression of progression of leukemia in mice.

Chronic myelogenous leukemia (CML) is a hematopoietic malignant disease associated with expression of a chimeric BCR-ABL gene. We recently succeeded in designing a novel allosterically controllable ribozyme, the maxizyme (Tanabe et al. Biomacromolecules 2000, 1, 108-117; Kuwabara et al. Biomacromolecules 2001, 2, 788-799), that not only specifically cleaves BCR-ABL mRNA and induces apoptosis in cultured CML cells but also shows significant inhibition against the growth of an established BV173 cell line in a mouse model (Tanabe et al. Nature 2000, 406, 473-474). As an extension of our studies, we tested the maxizyme against primary CML cells in the same mouse model. The maxizyme under the control of a tRNA(Val) promoter showed significant inhibition against the growth of the primary bone marrow cells from a Japanese patient with CML. Specifically, to examine the applicability of the maxizyme in the treatment of CML, we assessed the antitumor effect of the maxizyme in murine models of CML. Fourteen weeks after the injection of primary CML cells into a NOD-SCID mouse, the bone marrow of the mouse was filled with primary CML cells as a result of diffuse leukemia. In marked contrast, when maxizyme-expressing primary CML cells were injected, the mouse remained disease-free. These results further strengthen our earlier suggestion that the maxizyme technology might provide a useful approach to the treatment of CML.

Recognition of engineered tRNAs with an extended 3' end by Exportin-t (Xpo-t) and transport of tRNA-attached ribozymes to the cytoplasm in somatic cells.

Our recent analysis indicates that the cytoplasmic localization of tRNA-attached ribozymes (tRNA-Rz) is critical for its high-level intracellular activity, suggesting that mature mRNAs in the cytoplasm are more accessible to ribozymes than pre-mRNAs in the nucleus (Kato et al. J. Biol. Chem. 2001, 276, 15378-15385; Kuwabara et al. Nucleic Acids Res. 2001, 29, 2780-2788). Although studies in Xenopus oocytes led to the proposal that only correctly processed mature tRNAs are exported from nuclei in a RanGTP-dependent manner (Lund and Dahlberg Science 1998, 282, 2082-2085), our tRNA-Rz with an extended 3' end can also be exported to the cytoplasm in somatic cells. Xpo-t/RanGTP bound to tRNA-attached ribozymes in vitro and in somatic cells, with recognition basically resembling the recognition of mature tRNAs. In contrast, no binding to tRNA-attached ribozymes occurred in Xenopus oocytes. The injection of a nuclear extract of Xenopus oocytes together with tRNA-attached ribozymes inhibited the export of tRNA-attached ribozymes but not mature tRNAs in somatic cells, suggesting the existence of an inhibitor(s) of the Xpo-t-dependent export pathway. Moreover, the inhibitor(s) appears responsible for a proofreading mechanism that operates in oocytes.