Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture.

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.

IGF-1 regulate the expression of uncoupling protein 2 via FOXO1.

Mitochondria uncoupling protein2 (UCP2) expressed ubiquitously is a key molecule of energy metabolism. Insulin-like growth factor-1 (IGF-1) is a hormone, a target molecule of growth hormone (GH) signal pathway, which is also known as the drug "mecasermin" for clinical usages. IGF-1 is seemed to be closely related to metabolic diseases, such as adult GH deficiency. However, there has not been reports depicted possible relationship with each other. So, we sought to elucidate the mechanisms by which expression of UCP2 is regulated by IGF-1 via FOXO1. The findings suggested that three sequences in the consensus UCP2 promoter play complementary functional roles in the functional expression of FOXO1. So, we found that FOXO1 is involved in IGF-1-mediated energy metabolism greater than that of direct action of GH via STAT5. Our findings suggested that IGF-1 was involved in energy metabolism by regulating the expression of UCP2 via the PI3K/Akt/FOXO1 pathway.

Cigarette Smoke Extract Modulates Functions of Peroxisome Proliferator-Activated Receptors.

Cigarette smoke extract (CSE) contains many toxicants and may derange the physiological processes, such as cholesterol metabolism. We examined the impact of CSE on transcriptional regulation mediated peroxisome proliferator-activated receptors (PPARs) and its interaction with cofactors to elucidate differences in the molecular mechanism between CSE and other agonists of PPARs. We constructed several mutant PPARs (mPPARs) with amino acid substitution in the ligand-binding domain, which according to the molecular modeling, may affect the binding of agonists. In transient expression assays, each wild-type peroxisome proliferator-activated receptor (PPAR) mediated transcription stimulated by CSE was faintly yet significantly elevated compared to the control. The CSE-induced transcriptional activation was abolished in the H323A, H323Y, S342A, and H449A mPPARγs, although the activation elevated by pioglitazone was reserved. In the mPPARγ with Y473A and mPPARß/δs with H286Y and Y436A, the pioglitazone-induced or L165041-activated transcriptional elevations were decreased and were lower than that of CSE-induced stimulation. These results suggested that CSE activated both mutant PPARs to be selectively different from those ligands. Mammalian two-hybrid assay illustrated that CSE could mildly recruit SRC1 or GRIP1 to the wild-type PPARγ. Representative ingredients, such as acrolein and crotonaldehyde present in CSE, could stimulate PPAR isoforms even at the toxicological concentrations and might possibly contribute to stimulatory effects. CSE mildly regulates the cholesterol metabolism-related genes, such as low density lipoprotein (LDL) receptor and Liver X receptor (LXR)ß. In conclusion, these CSE effects the nuclear hormone receptors and their cofactors thereby disturbing metabolic phenomena. Therefore, CSE might be involved in cholesterol metabolism.

Cigarette Smoke Extract Disrupts Transcriptional Activities Mediated by Thyroid Hormones and Its Receptors.

Cigarette smoke contains over 4800 compounds, including at least 200 toxicants or endocrine disruptors. Currently, effects of cigarette smoke on thyroid hormone (TH) levels remains to be clarified. Here, we demonstrate that cigarette smoke extract (CSE) possesses thyroid hormone properties and acts synergistically as a partial agonist for thyroid hormone receptors (TRs) in the presence of TH. In transient gene expression experiments, CSE stimulated transcriptional activity with TH in a dose-dependent manner. Stimulatory effects were observed with physiological TH concentrations, although CSE did not activate TRs without TH. CSE (5%) dissolved in phosphate-buffered saline (PBS) supplemented with 1 nM TH was approximately comparable to 3.2±0.1 and 2.3±0.2 nM of TRα1 and TRß1, respectively. To illustrate probable mechanisms of the CSE agonistic activity, effects on TR mediated transcriptional functions with cofactors were investigated. With a mammalian two-hybrid assay, CSE recruited the nuclear coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC1) to the TR. Unsaturated carbonyl compounds, acrolein, crotonaldehyde, and methyl vinyl ketone, representative constituents of CSE, retained such agonistic properties and possibly contributed to stimulatory effects. The results suggest that CSE recruits a transcriptional activator and may reinforce TH binding to the TR additively, resulting in gene expression. CSE partially agonizes TH action and may disturb the function of various nuclear hormone receptor types and their cofactors to disrupt the physiological processes.

Interaction of Rif1 Protein with G-Quadruplex in Control of Chromosome Transactions.

Recent studies on G-quadruplex (G4) revealed crucial and conserved functions of G4 in various biological systems. We recently showed that Rif1, a conserved nuclear factor, binds to G4 present in the intergenic regions and plays a major role in spatiotemporal regulation of DNA replication. Rif1 may tether chromatin fibers through binding to G4, generating specific chromatin domains that dictate the replication timing. G4 and its various binding partners are now implicated in many other chromosome regulations, including transcription, replication initiation, recombination, gene rearrangement, and transposition.

Vitamin D3/VDR resists diet-induced obesity by modulating UCP3 expression in muscles.

BACKGROUND: The impact of vitamin D3 (VD3) on obesity has been reported in the past. Our study was aimed at investigating the possible mechanisms by which VD3 affects obesity induced by a high fat diet. METHODS: Eight-week-old C57BL/6 J male mice were fed a normal- or high-fat diet for 9 weeks and were treated with a gavage of vehicle (corn oil) or cholecalciferol (50 µg/kg, daily). Body weight, white adipose tissue weight, blood lipid and glucose levels were measured. In addition, we investigated the expression of 1,25(OH)2D3 (calcitriol)/VDR-regulated genes involved in energy and lipid metabolism, such as of uncoupling protein 3 (UCP3), by using qRT-PCR in the liver, adipose tissue, skeletal muscle and C2C12, L6, and H-EMC-SS cells. We also measured UCP3 promoter transcription in the same cell lines using a Dual Luciferase Assay. Furthermore, we analyzed the binding site consensus sequences of VDR on the UCP3 promoter. RESULTS: Mice consuming a high-fat diet treated with cholecalciferol had lower body weight and adipose tissue weight and higher expression of UCP3 compared to the other treatment groups. Changes in the expression of genes correlated with calcitriol/VDR. Luciferase activity was dose-dependently associated with calcitriol/VDR levels. We confirmed the functional VDR binding site consensus sequences at -2200, -1561, -634, and +314 bp in the UCP3 promoter region. CONCLUSION: We suggest that VD3/VDR inhibits weight gain by activating UCP3 in the muscles.

Molecular characterization of human thyroid hormone receptor ß isoform 4.

Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor ß isoform (TRß4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRß4 from a molecular basis. Temporal expression of TRß4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRß1 is potentially stable. TRß1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRß4 in a dose-dependent manner. Thus, TRß4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRß1 and TRß4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRß4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRß1. In contrast, TRß4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRß1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRß4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRßs, we found that both TRß1 and TRß4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRßs within the nucleus.

Growth hormone directly stimulates GATA2 expression.

OBJECTIVE: GATA2 is a key transcription factor involved in the differentiation and determination of thyrotrophs and gonadotrophs in pituitary and hematopoietic development. However, studies on the upstream ligands of the GATA2 signal transduction pathway have been limited. To identify upstream ligands, we examined growth hormone (GH) as a plausible stimulator. DESIGN: We evaluated GH-induced GATA2 expression in murine TtT/GF thyrotrophic pituitary tumor cells and its direct impact on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. RESULTS: GATA2 expression increased with activated STAT5B in a dose-dependent manner and was inhibited by a STAT5 specific inhibitor. Moreover, we found functional STAT5B binding site consensus sequences at -359 bp in the GATA2 promoter region. CONCLUSION: These findings suggest that GH directly stimulates GATA2 via the GHR/JAK/STAT pathway and participates in various developmental phenomena mediated by GATA2.

Epstein-Barr nuclear antigen 1 (EBNA1)-dependent recruitment of origin recognition complex (Orc) on oriP of Epstein-Barr virus with purified proteins: stimulation by Cdc6 through its direct interaction with EBNA1.

Origin recognition complex (Orc) plays an essential role in directing assembly of prereplicative complex at selective sites on chromosomes. However, Orc from vertebrates is reported to bind to DNA in a sequence-nonspecific manner, and it is still unclear how it selects specific genomic loci and how Cdc6, another conserved AAA(+) factor known to interact with Orc, participates in this process. Replication from oriP, the latent origin of Epstein-Barr virus, provides an excellent model system for the study of initiation on the host chromosomes because it is known to depend on prereplicative complex factors, including Orc and Mcm. Here, we show that Orc is recruited selectively at the essential dyad symmetry element in nuclear extracts in a manner dependent on EBNA1, which specifically binds to dyad symmetry. With purified proteins, EBNA1 can recruit both Cdc6 and Orc independently on a DNA containing EBNA1 binding sites, and Cdc6 facilitates the Orc recruitment by EBNA1. Purified Cdc6 directly binds to EBNA1, whereas association of Orc with EBNA1 requires the presence of the oriP DNA. Nuclease protection assays suggest that Orc associates with DNA segments on both sides adjacent to the EBNA1 binding sites and that this process is stimulated by the presence of Cdc6. Thus, EBNA1 can direct localized assembly of Orc in a process that is facilitated by Cdc6. The possibility of similar modes of recruitment of Orc/Cdc6 at the human chromosomal origins will be discussed.

Characterization of Apparently Paradoxical Thyrotropin Binding Inhibitory Immunoglobulins With Neutral Bioactivity.

Context: The thyrotropin (TSH) receptor (TSH-R) autoantibody activity is clinically measured by inhibition of labeled ligand (TSH or M22) binding to the TSH-R (TSH-binding inhibitory immunoglobulin [TBII]) or by stimulation (TSH-R stimulating antibody [TSAb]) or inhibition (TSH-R blocking antibody [TSBAb]) of 3',5'-cyclic adenosine 5'-monophosphate (cAMP) production in isolated cells. Objective: We experienced a patient with hypothyroid Graves disease (GD) having strong positive TBII but with almost neutral bioactivities on the TSH-R. The aim of this study is the characterization of this apparently paradoxical TBII (serum sample S). Methods: We first compared the TBII, TSAb, and TSBAb activities of serum sample S with mixtures of stimulating (S-mAb) and blocking monoclonal Ab (B-mAb). Next, we serially measured cAMPs stimulated by various serum samples in the presence or absence of TSH. Results: Mixtures of S-mAb and B-mAb did not reproduce the characteristics of serum sample S. Instead, serum sample S had a unique feature that blocked the TSH-stimulated cAMP initially but disappeared the blocking activity thereafter to reach the control level. Conclusion: We present here the TBIIs with neutral bioactivities found in the patient with autoimmune thyroid disease, which strongly inhibit TSH binding to the TSH-R but exerts neither TSAb nor TSBAb activity. Differences in the methods of detecting TRAb between TBII in vitro and bioassay may cause the discrepancy. Although serum sample S may be an extreme example, a variety of TRAb that not only stimulates or blocks but also interferes with TSH-R binding for only a short time may exist in the serum samples of GD patients.

Kbus/Idr, a mutant mouse strain with skeletal abnormalities and hypophosphatemia: identification as an allele of 'Hyp'.

BACKGROUND: The endopeptidase encoded by Phex (phosphate-regulating gene with homologies to endopeptidases linked to the X chromosome) is critical for regulation of bone matrix mineralization and phosphate homeostasis. PHEX has been identified from analyses of human X-linked hypophosphatemic rickets and Hyp mutant mouse models. We here demonstrated a newly established dwarfism-like Kbus/Idr mouse line to be a novel Hyp model. METHODS: Histopathological and X-ray examination with cross experiments were performed to characterize Kbus/Idr. RT-PCR-based and exon-directed PCR screening performed to identify the presence of genetic alteration. Biochemical assays were also performed to evaluate activity of alkaline phosphatase. RESULTS: Kbus/Idr, characterized by bone mineralization defects, was found to be inherited in an X chromosome-linked dominant manner. RT-PCR experiments showed that a novel mutation spanning exon 16 and 18 causing hypophosphatemic rickets. Alkaline phosphatase activity, as an osteoblast marker, demonstrated raised levels in the bone marrow of Kbus/Idr independent of the age. CONCLUSIONS: Kbus mice should serve as a useful research tool exploring molecular mechanisms underlying aberrant Phex-associated pathophysiological phenomena.

[Diamine oxidase as blood biomarker in rats and humans to GI tract toxicity of fluorouracil anti-cancer drugs].

Diarrhea is a side effect of a 5-fluorouracil (5-FU) anti-cancer drug-induced intestinal mucosal disorder, which sometimes becomes more severe. Blood diamine oxidase (DAO; EC1. 4. 3. 6) activity is reported to be significantly correlated with activity in the small intestinal mucosal tissue, and to be a reliable indicator of small intestinal mucosal integrity and maturity. Here, we investigated whether blood DAO activity can be a biomarker for the gastrointestinal (GI) mucosal disorder caused by 5-FU anti-cancer drugs, both in rats and humans. From results of the rat study, the degree of jejunal mucosal disorder caused by the 5-FU anti-cancer drug was well correlated with a decrease in blood DAO activity. Clinically, 12 out of 28 patients (43%) administered 5-FU anti-cancer drug suffered from diarrhea. The plasma DAO activity within one week of the onset of diarrhea significantly decreased compared with that before the administration. Furthermore, before drug administration, plasma DAO activity in patients suffering from diarrhea was higher than those in patients without diarrhea. Although DAO activity differs by the individual, it is a useful biomarker for estimating the degree of intestinal mucosal disorder, and possibly for estimating manifestations of diarrhea induced by 5-FU anti-cancer drug administration.

G-quadruplex binding protein Rif1, a key regulator of replication timing.

DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing and locations of replication. Among them, Rif1 plays a major role to shape the 'replication domains' that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.

Identification of a novel human thyroid hormone receptor beta isoform as a transcriptional modulator.

Thyroid hormone exerts a pleiotropic effect on development and homeostasis. A novel thyroid hormone receptor beta isoform (hereafter referred to as TR beta 4) was cloned using PCR from a human pituitary cDNA library as a template. Analysis of the PCR products revealed a 137-bp insertion, which contains a stop codon in the middle, between the 5th and 6th exons that encode the ligand-binding domain of TR beta. The corresponding sequence of this insertion exists within the 5th intron of the human TR beta gene and consensus splice sequences were found at the junction sites. RACE analysis revealed that TR beta 4 is a carboxyl-terminal splicing variant of TR beta 1. RT-PCR and Northern blot analyses indicate that TR beta 4 mRNA is expressed in various human tissues, and especially abundant in testis and skeletal muscle. The TR beta 4 protein was unable to bind thyroid hormone (T3) and transient transfection assays demonstrate that TR beta 4 construct does not mediate T3-dependent gene regulation. TR beta 4 weakly but significantly inhibited transcription mediated by functional TR. Thus, this novel isoform may modulate hormone action as an endogenous antagonist in the tissue or cellular context.

A case of central nervous system lymphomatoid granulomatosis; characteristics of PET imaging and pathological findings.

Lymphomatoid granulomatosis (LYG) in the central nervous system (CNS) is an uncommon lymphoproliferative disorder with low grade malignant potential. Here we report a case of CNS-LYG, in particular, its characteristics of radioisotope imaging and pathological findings. A 65-year-old man complained of visual disturbance and homonymous hemianopsia was designated. CT and MRI revealed an edematous, enhanced irregular and nodular lesion in the right occipital and parietal lobes. Although 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan showed low uptake in the lesion, Methionine (MET)-PET scan indicated high uptake. Proton magnetic resonance spectroscopy ((1)H-MRS) at 3T revealed a decrease of the peak of the N-acetylaspartate (NAA), suggesting a possible neoplastic lesion. The patient was diagnosed with CNS-LYG based on the surgically removed material showing perivascular infiltration of CD3-positive small T-lymphocytes with granulomatous lesions. The post-operative steroid therapy was effective and the recurrence or exacerbation has not been observed by radiological imaging until now.

Experimental Reproduction of Dynamic Fluctuation of TSH Receptor-Binding Antibodies Between Stimulation and Inhibition.

CONTEXT: Hyperthyroidism in Graves disease (GD) is caused by autoantibody stimulation of the TSH receptor (TSHR). TSHR autoantibody (TSHR-Ab) activity is measured routinely by inhibition of labeled ligand (TSH or M22) binding to the TSHR [TSH-binding inhibitory immunoglobulins (TBIIs)] or by stimulation of cAMP production in isolated cells [TSH receptor-stimulating antibodies (TSAbs)]. Usually, measurements of TSHR-Abs by TBIIs agree reasonably well with TSAb values at least in the setting of hyperthyroidism, and both measurements tend to change in parallel during treatment with some exceptions. In this study, we describe three unusual cases, which illustrate nearly pure stimulating, blocking, or neutral properties of TSHR-Abs. OBJECTIVE: Whether patient serum TSHR-Abs can be reproduced by mixtures of human monoclonal autoantibodies to the TSHR was studied because the sera in most patients show moderate properties having both of TBII and TSAb activities. DESIGN: We compared the TBII and TSAb activities of serum from four unusual patients in detail with mixtures of human monoclonal TSHR-Abs (mAbs) M22 (stimulating), K1-18 (stimulating), and K1-70 (blocking). RESULTS: Characteristic of a patient's serum was similar to M22 or K1-18, another was similar to K1-70, whereas another was similar to a mixture of K1-70 and M22 (or K1-18). Additionally, some patients seemed to have neutral TSHR-Abs in their sera. CONCLUSIONS: Our studies suggest that the characteristics of TSHR-Abs in the patient serum can be mimicked by mixtures of human mAbs to the TSHR, stimulating, blocking, and neutral if any.

Rif1 promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization activities.

Rif1 is a conserved protein regulating replication timing and binds preferentially to the vicinity of late-firing/dormant origins in fission yeast. The Rif1 binding sites on the fission yeast genome have an intrinsic potential to generate G-quadruplex (G4) structures to which purified Rif1 preferentially binds. We previously proposed that Rif1 generates chromatin architecture that may determine replication timing by facilitating the chromatin loop formation. Here, we conducted detailed biochemical analyses on Rif1 and its G4 binding. Rif1 prefers sequences containing long stretches of guanines and binds preferentially to the multimeric G4 of parallel or hybrid/mix topology. Rif1 forms oligomers and binds simultaneously to multiple G4. We present a model on how Rif1 may facilitate the formation of chromatin architecture through its G4 binding and oligomerization properties.

Insulin-like growth factor-1 directly mediates expression of mitochondrial uncoupling protein 3 via forkhead box O4.

OBJECTIVE: The objective of our study was to examine the direct action of insulin-like growth factor-1(IGF-1) signaling on energy homeostasis in myocytes. DESIGN: We studied the IGF-1 stimulation of mitochondrial uncoupling protein 3 (UCP3) expression in the HEK 293 derived cell line TSA201, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal myoblasts. We also investigated the direct effect of IGF-1 on the Insulin/IGF-1 receptor (IGF-1R)/phosphatidylinositol 3 (PI3)-Akt/forkhead box O4 (FOXO4) pathway using a combination of a reporter assay, semi-quantitative polymerase chain reaction, western blotting, and animal experiments. RESULTS: We demonstrated that IGF-1 regulates UCP3 expression via phosphorylation of FOXO4, which is a downstream signal transducer of IGF-1. UCP3 expression increased with activated FOXO4 in a dose-dependent manner. We also examined the functional FOXO4 binding site consensus sequences and identified it as the -1922 bp site in the UCP3 promoter region. UCP3 was also found to be concomitantly expressed with IGF-1 during differentiation of C2C12 myoblasts. Our animal experiments showed that high fat diet induced IGF-1 levels which likely influenced UCP3 expression in the skeletal muscle. CONCLUSION: Our findings demonstrate that that IGF-1 directly stimulates UCP3 expression via the IGF-1/IGF-1R/PI3-Akt/FOXO4 pathway.

Sonodynamic Therapy for Malignant Glioma Using 220-kHz Transcranial Magnetic Resonance Imaging-Guided Focused Ultrasound and 5-Aminolevulinic acid.

Sonodynamic therapy (SDT) is used to treat various malignancies and can be applied to brain tumors using a transcranial magnetic resonance imaging-guided focused ultrasound (TcMRgFUS) device. This study investigated the efficacy of 220-kHz TcMRgFUS combined with 5-aminolevulinic acid (5-ALA) on malignant glioma in vitro and in vivo. F98 cells were irradiated with focused ultrasound (FUS) (4000 J, 20 W, 240 s, 100% duty cycle, target medium temperature <40°C) after treatment with 200 µg/mL 5-ALA, and cell viability and apoptosis were evaluated with the water-soluble tetrazolium-1 assay, triple fluorescent staining and Western blot analysis 20 h later. The anti-tumor effects of 5-ALA combined with FUS (500 J, 18 W, 30 s, 100% duty cycle, 10 repeats, target tissue temperature ≤42°C) were assessed on the basis of changes in tumor volume determined by MRI and histopathological analysis before and after treatment. The FUS/5-ALA combination reduced cell viability by inducing apoptosis and suppressed tumor proliferation and invasion as well as angiogenesis in vivo, while causing minimal damage to normal brain tissue. SDT with 220-kHz TcMRgFUS and 5-ALA can be safely used for the treatment of malignant glioma.

A case of cortisol producing adrenal adenoma without phenotype of Cushing's syndrome due to impaired 11beta-hydroxysteroid dehydrogenase 1 activity.

This report concerns a case of cortisol-producing adrenocortical adenoma without the phenotype of Cushing's syndrome. A left adrenal tumor was incidentally detected in this patient. A diagnosis of adrenal Cushing's syndrome was based on the results of endocrinological and radiological examinations, although she showed none of the physical signs of Cushing's syndrome, glucose intolerance, hypertension or dyslipidermia. After a successful laparoscopic left adrenalectomy, the pathological diagnosis was adrenocortical adenoma. Slow tapering of glucocorticoids was needed to prevent adrenal insufficiency after surgery, and the plasma ACTH level remained high even though the serum cortisol level had reached the upper limit of the normal range. Further examination showed a urinary THF + allo-THF/THE ratio of 0.63, which was lower than that of control (0.90 +/- 0.13, mean +/- SD). Serum cortisol/cortisone ratios after the cortisone acetate administration were also decreased, and the serum half-life of cortisol was shorter than the normal range which has been reported. These findings indicated a partial defect in 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity, which converts cortisone to cortisol. Our case suggests that a change in 11beta-HSD1 activity results in inter-individual differences in glucocorticoid efficacy.