Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts.

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression of IL6 mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10  µM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. The IL6 mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

Raloxifene enhances spontaneous microaggregation of platelets through upregulation of p44/p42 MAP kinase: a case report.

UNLABELLED: A 60-year-old postmenopausal woman diagnosed as primary osteoporosis began to take raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated after the treatment, concomitant with the significant upregulation of p44/p42 mitogen-activated protein (MAP) kinase induced by adenosine diphosphate (ADP). After the cessation of raloxifene, the spontaneous microaggregates of platelets and the acceleration of ADP-induced p44/p42 MAP kinase phosphorylation was diminished. We concluded that raloxifene caused platelet hyperaggregability to shear stress and p44/p42 MAP kinase was involved in the pathological state. INTRODUCTION: A 60-year-old postmenopausal woman suffering from severe lumbago was diagnosed as primary osteoporosis with combined vertebral fractures. After the acute phase, she began to take 60 mg daily of oral raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated significantly after 8 weeks from the beginning of raloxifene treatment and observed at 12 weeks. RESULTS: The platelet aggregation induced by ADP was little changed; however, low doses (0.3 and 1 microM) of ADP significantly induced the phosphorylation of p44/p42 MAP kinase in the platelets obtained at 12 weeks. Although there were few subjective complaints except for paroxysmal headache, the medication was stopped with her consent to avoid any adverse effects. The spontaneous microaggregates of platelets gradually decreased after the cessation of medication. At 12 weeks after the cessation, the phosphorylation of p44/p42 MAP kinase induced by low doses of ADP was no more observed. CONCLUSION: These results strongly suggest that raloxifene caused platelet hyperaggregability to shear stress and subclinical thrombus formation in this case and that p44/p42 MAP kinase was involved in the pathological state.

Glycyrrhizin and some analogues induce growth of primary cultured adult rat hepatocytes via epidermal growth factor receptors.

We investigated the effects of glycyrrhizin (GL-1) and some analogues on DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. The hepatocytes underwent DNA synthesis and proliferation in response to GL-1 and some analogues. The effects of these agents occurred in a time- and dose-dependent manner. The proliferative potency as judged by half-maximal effective concentrations was in the following order: 18-beta-H-glycyrrhetinic acid (GL-3; 4.5 x 10(-9) M)<18-beta-H-glycyrrhizin (GL-1; 4.4 x 10(-8) M)<18-alpha-H-glycyrrhetinic acid (GL-6; 6.0 x 10(-8) M). The analogue 18-alpha-H-glycyrrhetinic acid 3-O-beta-D-monoglucuronide (GL-5; 1.0 x 10(-7) M) weakly stimulated hepatocyte DNA synthesis and proliferation, whereas 18-alpha-H-glycyrrhizin (GL-4) and 18-beta-H-glycyrrhetinic acid 3-O-beta-D-monoglucuronide (GL-2) did not. The growth-promoting effects of GL-1, GL-3 and GL-6 were significantly inhibited at higher initial plating densities (7.0 x 10(4) and 10 x 10(4) cells/cm(2)). A monoclonal antibody against epidermal growth factor (EGF) receptor (1-100 ng/ml), but not that against EGF (1-100 ng/ml), dose-dependently inhibited glycyrrhizin- and analogue-induced hepatocyte DNA synthesis and proliferation. Specific inhibitors of growth-related signal transducers, such as genistein, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin, completely blocked glycyrrhizin- and analogue-induced hepatocyte DNA synthesis and proliferation. Treatment of hepatocytes with GL-1, GL-3 and GL-6 rapidly stimulated tyrosine phosphorylation of the EGF receptor and p42 MAP kinase, which were inhibited by genistein and PD98059, respectively. These results suggest that glycyrrhizin and some analogues are primary hepatocyte mitogens that bind to EGF receptors and subsequently stimulate the receptor tyrosine kinase/mitogen-activated protein kinase pathway to induce hepatocyte DNA synthesis and proliferation.

Estimation of absorption rate of alpha-human atrial natriuretic peptide from the plasma profile and diuretic effect after intranasal administration to rats.

The absorption rate of alpha-human atrial natriuretic peptide (alpha-hANP) after intranasal (i.n.) administration to rats was estimated from the plasma profile and pharmacological effect (diuretic effect) using a pharmacokinetic (PK) model and a PK-pharmacodynamic (PD) model involving data obtained after intravenous (i.v.) bolus injection. The plasma concentrations of alpha-hANP after i.v. administration at different doses were fitted to a two-compartment PK model with zero-order excretion and input of endogenous alpha-rat atrial natriuretic peptide (alpha-rANP) and two elimination processes represented by Michaelis-Menten and first-order kinetics. However, the saturable process was ignored at low doses. The plasma concentrations after low doses via the i.n. route could also be expressed by this model, but with first-order absorption, so that an absorption rate constant was calculated using a deconvolution method. In addition, the diuretic effect plotted against the i.v. dose was represented by the Hill equation and showed an anti-clockwise hysteresis loop versus the plasma concentration. These results suggest that the diuretic effect could be estimated by a PK-PD model having an 'effect' compartment or a homeostatic system. Such a PK-PD model accurately expressed the diuretic effect of alpha-hANP at all doses after i.v. and i.n. administrations. The resulting absorption rate constant calculated using the PK-PD model agreed closely with that obtained by the PK model alone. The absorption rate and simulated diuretic effect suggest that, for i.n. administration of alpha-hANP, a higher absorption rate constant causes a more potent diuretic effect (a dramatic effect over the early period), whereas greater bioavailability is associated with a better hypotensive effect (sustained effect).

Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats.

The effect of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of FITC-dextrans with a mean molecular weight ranging from 4.3 to 167 kDa and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats were studied. When FITC-dextrans were co-administered intranasally with 1.0 w/v% poly-L-Args of different molecular weight (MW, ca. 45.5 and 92 kDa, poly-L-Arg (50) and poly-L-Arg (100)), the bioavailability (F(infinity)) increased markedly compared with that after administration of FITC-dextran alone. However, the F(infinity) decreased exponentially with the increasing molecular weight of FITC-dextrans. There was no significant difference between the enhanced nasal absorption of FITC-dextrans achieved by the co-administration of poly-L-Arg (50) and poly-L-Arg (100). Moreover, the relationship between the F(infinity) and the molecular weight of FITC-dextrans indicated that the molecular weight of protein drugs, which exhibited efficient absorption with poly-L-Arg, was about 20 kDa, when the lower limit of bioavailability for developing a potent transnasal delivery system was assumed to be about 10%. Indeed, the nasal absorption of rhG-CSF, which has a molecular weight of 18.8 kDa, was also increased after co-administration of 1.0 w/v% poly-L-Arg (50) and the F(infinity) was about 11%. It seems likely that poly-L-Arg can be used to provide adequate nasal absorption of various protein drugs which have a molecular weight of about 20 kDa, thereby allowing the successful development of a variety of transnasal drug delivery systems.

Improved nasal absorption of drugs using poly-L-arginine: effects of concentration and molecular weight of poly-L-arginine on the nasal absorption of fluorescein isothiocyanate-dextran in rats.

The effects of the concentration and molecular weight of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of fluorescein isothiocyanate-labeled dextran (MW, 4 kDa, FD-4) in rats were studied. When poly-L-Arg with a range of different molecular weights (MW, 8.9, 45.5 and 92.0 kDa) was applied intranasally at various concentrations, the bioavailability (F(0-9 h)) of FD-4 increased with the increasing concentration of poly-L-Arg. The enhanced absorption was also dependent on the molar concentration, in that the poly-L-Arg with a higher molecular weight increased F(0-9 h) at a lower molar concentration. In addition, for each applied concentration, the poly-L-Arg exhibited a molecular weight-dependence as far as the enhancement of FD-4 absorption was concerned. On the other hand, the maximum absorption rate (MAR) of FD-4, calculated by means of a deconvolution method, tended to reach a maximum plateau level at a lower applied concentration for the poly-L-Arg with the highest molecular weight, but this plateau level was almost the same for poly-L-Arg with molecular weights of 45.5 and 92.0 kDa. Moreover, the simulated absorption profiles of FD-4 indicate that the degree of enhancement (the level of MAR and the subsequent reduction in the absorption rate) was dependent on the molecular weight of poly-L-Arg, while the effect of poly-L-Arg was maintained for a longer period, depending on the applied concentration, although the MAR was relatively similar. These results indicate that the molecular weight of poly-L-Arg appears to affect both the enhancing efficiency (absorption rate) and the time-frame of this enhancing effect, whereas the concentrations of each poly-L-Arg system applied only have an effect on the time-frame. These effects may also be associated with the charge density of a poly-L-Arg molecule.

Malignant hyperthermia in a patient with Graves' disease during subtotal thyroidectomy.

We report the case of a 31-year-old man with Graves' disease who manifested malignant hyperthermia during subtotal thyroidectomy. His past medical history and family history were unremarkable. Before surgery, his condition was well controlled with propylthiouracil, beta-adrenergic blocker and iodine. During the operation, anesthesia was induced by intravenous injection of vecuronium and thiopental, followed by suxamethonium for endotracheal intubation. Anesthesia was maintained with nitrous oxide and sevoflurane. One hour after induction of anesthesia, his end tidal carbon dioxide concentration (ET(CO2)) increased from 40 to 50 mmHg, heart rate increased from 90 to 100 beats per min and body temperature began to rise at a rate of 0.3 degrees C per 15 min. Suspecting thyroid storm, propranolol 0.4 mg and methylprednisolone 1,500 mg were administered, which, however, had little effect. Despite the lack of muscular rigidity, the diagnosis of malignant hyperthermia was made based on respiratory acidosis. Sevoflurane was discontinued and dantrolene was given by intravenous bolus. Soon after the treatment, ET(CO2), heart rate and body temperature started to fall to normal levels. His laboratory findings showed abnormally elevated serum creatine phosphokinase and myoglobin but normal thyroid hormone levels. Since dantrolene is efficacious in thyrotoxic crisis and malignant hyperthermia, an immediate intravenous administration of dantrolene should be considered when a hypermetabolic state occurs during anesthesia in surgical treatment for a patient with Graves' disease.

[Pharmaceutical evaluation of fast-disintegrant tablet containing nicorandil-loaded particles].

AIM: To improve the bioavailability and taste of fast-disintegrating tablet (FD tablet) containing nicorandil-loaded particles. METHODS: A FD tablet containing nicorandil-loaded particles with 1%-4% croscarmellose sodium in addition of D-mannitol and lactose (9:1) was prepared and the dissolution and absorption characteristics were examined, in comparison with FD tablet and commercial tablets of nicorandil. In vivo absorption of nicorandil from FD tablet was evaluated in beagle dogs. RESULTS: The disintegration time of FD tablets containing 1% croscarmellose sodium with 6 mm and 10 mm in diameter were about 12 and 23 seconds, respectively. When nicorandil-loaded particles consist of myristyl alcohol and stearyl alcohol were put in FD tablet, nicorandil release from FD tablet continued until 6 h while nicorandil release from Sigmart and FD tablet containing nicorandil crystals finished within 5 min. In vivo absorption of nicorandil from Sigmart and FD tablet containing nicorandil crystals was very similar after oral administration in beagle dogs and no statistic difference in AUC, Tmax, Cmax was observed between these tablets. However pharmacokinetics parameters of nicorandil after oral administration of FD tablet containing nicorandil-loaded particles showed that nicorandil was delivered into the body at a suitable absorption rate with similar AUC, delayed Tmax and lower Cmax. CONCLUSION: The reports suggest that the modification of properties of myristyl alcohol and stearyl alcohol released from the drug-loaded particles system would lead to more acceptable bioavailability of the system. However, The formulation of particles and may have a masking effect against the bitter taste and irritation of the drug.

Histological changes of thyroid tissues in patients with liver cirrhosis.

Alterations in thyroid hormone regulation and metabolism, such as low serum T3 and T4 with normal TSH, are frequently noted in liver cirrhosis, but few morphological studies have ever been done on thyroid tissue in liver cirrhosis. In this study we analyzed the histological changes of thyroid tissue in the patients with liver cirrhosis. Specimens of thyroid gland obtained from autopsies in 16 cirrhotic patients were examined, and compared with those of two control groups. Control group I consisted of 7 patients with diabetes mellitus and control group II, 12 patients who died from sudden onset of cardiovascular problems. We measured follicular diameter and epithelial width on light micrographs of the central portion of thyroid specimens. We graded the degree of colloid vacuole, lipofuscin deposition in follicular epithelia, regenerative reaction and perivascular fibrosis in 10 consecutive light microscopic fields of the specimen. In the cirrhotic group, mean follicular diameter and epithelial width were significantly shorter and thinner, respectively, than those in the two control groups. Perivascular fibrosis was more prominent in the cirrhosis group than in the controls. These findings suggest that thyroid glands in patients with liver cirrhosis have the characteristic features of hypoactivity.

[Transdermal microparticle delivery by a supersonic-Helios gun system].

AIM: To investigate the effect of particle size and high speed flow of helium gas on the systemic absorption of indomethacin using a needle-less injection system. METHODS: Poly-L-lactic acid microspheres containing indomethacin was prepared by the o/w solvent evaporation technique. After anesthetizing the male hairless rat, microspheres filled in the tube cartridge was accelerated by a stream of helium gas at various velocity in the Helios gun system, and then was introduced to the abdominal skin. RESULTS: Introduction of indomethacin to the hairless rat skin was proportionally increased with enhancing the helium pressure (supersonic flow). Bioavailability and Cmax were also dependent on the helium pressure. CONCLUSION: This method can be used to deliver the powered drug and/or microparticulate systems into the skin tissues and the systemic circulation.

Very strong correlation between dominant negative activities of mutant thyroid hormone receptors and their binding avidity for corepressor SMRT.

The syndrome of resistance to thyroid hormone (RTH) is an inherited disorder involving a mutation of the thyroid hormone receptor (TR) gene. Mutant (m) TR inhibits wild-type (wt) TR functions in a dominant negative manner, and this dominant negative effect (DNE) is a crucial factor in RTH pathogenesis. The molecular mechanism of the DNE is still unclear, although several possibilities (including competition between wt- and mTRs at the T(3) response element (TRE), sequestration of TR-associated protein(s) and titration out of functional TR) have been considered. Here we report that the DNE of mTRs is strongly correlated with their binding avidity for the retinoid X receptor (RXR), and especially for corepressor SMRT (silencing mediator for retinoid and thyroid hormone receptor), but not for the nuclear receptor corepressor, NCoR. The DNE of six natural TRs and four artificially constructed mTRs was assayed using a TR reporter gene containing TRE-DR4 (DR=direct repeat), TRE-pal (pal=palindrome) or TRE-lap (lap=inverted palindrome) in CV1 cells treated with 10 nM T(3). Of the mTRs examined, F451X (with a carboxy-terminal 11-amino-acid truncation) identified in a patient with RTH exhibited the strongest DNE on all TREs. The binding affinities between mTRs and corepressors SMRT or NCoR were quantified using a two-hybrid interference assay system consisting of VP16-TR(LBD) (LBD=ligand binding domain) and Gal4(DBD)-SMRT (DBD=DNA binding domain), or Gal4(DBD)-NCoR respectively, together with the Gal4 reporter gene. In this assay, VP16-TR(LBD) and Gal4(DBD)-SMRT (or Gal4 (DBD)-NCoR) interact with each other and trans-activate the Gal4 reporter gene. When an equal amount of mTR is coexpressed, it reduces the transcriptional activity of the reporter gene, depending on its binding avidity for a corepressor. A very strong correlation was observed between the SMRT-binding activity and the potency of the DNE among six natural mTRs and also among all mTRs, including four artificially constructed ones. The relationship between NCoR and DNE, however, was not significant. When we assayed the binding avidity of mTRs for RXR by using a two-hybrid assay system consisting of Gal4(DBD)-RXR(LBD) and VP16-TR(LBD), a significant correlation between DNE and binding avidity for the RXR was also observed. These results suggest that a corepressor plays an important role in DNE pathogenesis.

Differences between the silencing-related properties of the extreme carboxyl-terminal regions of thyroid hormone receptors alpha 1 and beta 1.

Human thyroid hormone receptor (TR) is encoded by two distinct genes, TR alpha and TR beta. TR heterodimerizes with retinoid X receptor (RXR) and binds efficiently to the thyroid hormone (T(3)) response element (TRE) of target genes. In the absence of T(3), unliganded TR suppresses the basal promoter activity of positively regulated genes (silencing). Silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR) interact with unliganded TR and function as corepressor proteins. Previously, we found beta F451X with carboxyl (C)-terminal 11-amino acid deletion had stronger silencing potency than wild-type TR beta 1 and beta E449X with C-terminal 13-amino acid deletion on a subset of TREs. In the present study, to assess the isoform-specific effects of the C-terminal truncations on TR silencing, we constructed two mutant TR alpha 1s (alpha F397X and alpha E395X) with the same respective C-terminal truncations as beta F451X and beta E449X and analysed their silencing activities. Unlike beta F451X and beta E449X, alpha F397X and alpha E395X showed similarly stronger silencing potency than wild-type TR alpha 1. We further studied the abilities of wild-type and the mutant TR beta 1s and alpha 1s on RXR and co-repressor binding by a two-hybrid interference assay. beta F451X had significantly stronger abilities to bind to RXR and SMRT than did wild-type TR beta 1 and beta E449X. In contrast, wild-type TR alpha 1, alpha F397X and alpha E395X showed similar abilities to bind to RXR and SMRT. beta E449X and alpha E395X, which have identical C-terminal truncation, showed less ability to bind to N-CoR than did wild-type TR beta 1 and beta F451X and wild-type TR alpha 1 and alpha F397X respectively. These results indicate that an identical C-terminal truncation gives rise to different effects on TR beta 1 and alpha1 with respect to silencing potency, RXR binding and SMRT binding. The difference in the silencing potency among wild-type TR beta 1, beta F451X and beta E449X correlated well with the difference in the ability to bind co-repressor SMRT.

Evaluation of bile acids and fusidate derivative as nasal absorption enhancers using an electrophysiological technique.

The present study was carried out to investigate the reversibility of the action of two nasal absorption enhancers, bile acids and fusidate derivative, on nasal membrane resistance. The nasal mucosa was isolated from rabbit nasal septum and mounted in a Ussing-type chamber to allow the monitoring of the membrane resistance and flux of fluorescein isothiocyanate-labeled dextran (FD10, M.W. 9400). Membrane resistance was reduced by 46% following treatment with 0.5% (w/v) sodium taurodihydrofusidate (STDHF) for 10 min and then gradually returned to the control level after being wash. The resistance was restored to 76% of the control level following a 30 min treatment with 0.5% (w/v) STDHF. However, there was no recovery of resistance following treatment with 0.5% (w/v) STDHF for 120 min or 1% (w/v) STDHF for 10in. Concurrently, FD10 transport was enhanced while membrane resistance was reduced. Treatment with 0.5% (w/v) sodium deoxycholate (DC) for more than 10 min showed no reversible action and marked FD10 transport enhancement, whereas a 10-30 min treatment with 0.5% (w/v) sodium glycocholate (GC) or sodium taurocholate (TC) resulted in the rapid recovery of membrane resistance without any enhancement of FD10 permeation. STDHF transport across the nasal mucosa was approximately 2-fold faster than that of DC, GC, and TC. The accumulation of STDHF in the nasal mucosa was 2-fold lower than that of DC and 1.7-fold higher than that of GC and TC after a 30 min treatment. The rank order of hydrophobicity determined by reverse-phase HPLC was: DC>STDHF>GC>TC. These results suggest that the reduction in membrane resistance and its reversibility appear to be due to a balance between the accumulation and clearance of STDHF.

Screening of cationic compounds as an absorption enhancer for nasal drug delivery.

Several cationic compounds were screened as potential nasal absorption enhancers to increase intranasal absorption of a model drug, fluorescein isothiocyanate labeled dextran (MW 4.4 kDa, FD-4), without nasal membrane damage in rats. Their effects were compared with those of classical enhancers. Various cationic compounds (poly-L-arginines with different molecular weights (MW 8.9, 45.5 and 92.0 kDa, poly-L-Arg (10), (50) and (100), respectively), L-arginine (L-Arg), L-lysine (L-Lys), and cetylpyridinium chloride (CPCL) were evaluated. Of the cationic compounds, poly-L-Arg and CPCL greatly enhanced the intranasal absorption of FD-4, as did chitosan, a cationic polysaccharide which has been reported to show a great effect on the transnasal delivery of peptide and protein drugs. The enhancing intensity by poly-L-Arg was dependent on its molecular weight. Rank order of the enhancing ratio, calculated from the AUC ratio for the enhancer treatment against the untreatment, was 0.5% poly-L-Arg (100) congruent with0.5% sodium dodecylsulfate congruent with0.5% CPCL?0.5% poly-L-Arg (50)?0.5% sodium deoxycholate congruent with0.5% sodium taurodihydrofusidate?0.5% polyoxyethylene-9-lauryl ether congruent with0.5% lysophosphatidylcholine?0.5% chitosan congruent with0.5% poly-L-Arg (10)>/=10% L-Arg congruent with10% L-Lys?0.5% sodium glycocholate congruent with0.5% sodium taurocholate congruent with0.5% EDTA. Only the poly-L-Args represented almost the same degree of hemolysis of cationic compounds compared with pH 7.0 phosphate buffered saline in the rat erythrocyte lysis experiment. The enhancing ratio by classical enhancers correlated with leaching of protein, phospholipids and LDH from isolated rabbit nasal mucosa. CPCL also fell on the regression lines between the enhancing ratio and their degree of leaching from classical enhancers. In contrast, the enhancing intensities by poly-L-Arg (10), (50) and (100) were greatly shifted from the regression line: the amount of leaching was markedly low in spite of a great enhancement of FD-4 absorption. These findings suggest that of the assessed enhancers only the poly-L-Args enhance the transnasal delivery of high molecular substances without severe damage to the nasal mucosal membrane. Poly-L-Arg is therefore a promising candidate having a good balance between enhancing activity and safety for nasal peptide and protein delivery.

Alterations in the enzyme activity and protein contents of protein disulfide isomerase in rat tissues during fasting and refeeding.

Protein disulfide isomerase (PDI) is an enzyme that participates in the formation of disulfide bonds. It is also known to be the subunits of some enzymes and the membrane-associated thyroid hormone-binding protein. In this study, we measured the quantitative distribution of PDI protein in rat tissues and examined the relationship between protein level and enzyme activity in PDI during fasting and refeeding. Western blotting with specific anti-PDI antiserum detected the PDI protein band of 55 kd. Among several tissues, liver contained the largest amount of PDI protein, followed by kidney and fat, in which one-third to one-fourth of the hepatic PDI protein existed. The PDI protein band was also detected in heart and muscle. Fasting for 3 days decreased PDI protein levels in rat liver by 40%; control levels were recovered after 3 days of refeeding. The same change was observed in kidney. PDI activity, measured by the scrambled ribonuclease method, did not show the parallel alteration to PDI protein level in liver and kidney. Isomerase activity decreased to 50% of control values during fasting, but did not recover by refeeding. Thyroidal status did not affect either PDI protein level or isomerase activity. These findings show that fasting and refeeding affect PDI protein and enzyme activity, and that PDI protein level does not always reflect PDI activity.

Difference in dominant negative activities between mutant thyroid hormone receptors alpha1 and beta1 with an identical truncation in the extreme carboxyl-terminal tau4 domain.

Although different expression patterns of thyroid hormone receptor (TR) alpha1 and beta1 have been reported, no essential distinction has been established in their functions. Unlike the TR beta gene, a mutation in the TR alpha1 gene has never been found in patients with resistance to thyroid hormone (RTH). Previously we found a mutant TR beta with an 11-carboxyl (C)-terminal amino acid truncation (betaF451X) in a girl with severe RTH. BetaF451X is a natural mutant with disruption of the transactivation domain, tau4, and it had very strong dominant negative activities. Based on the fact that the 46 amino acid sequence in the extreme C-terminal region is identical in TR alpha1 and TR beta, except for a C-terminal three amino acid extension of TR alpha1, we constructed a mutant TR alpha1 (alphaF397X) with the identical C-terminal truncation to betaF451X, to study functional differences between TR alpha1 and beta1. Both betaF451X and alphaF397X had negligible T3 binding and transcriptional activities even with 1 microM T3. The dominant negative activities of the mutant TRs were remarkable and T3 response element (TRE)-dependent. Co-expression of betaF451X decreased the CAT activity of either wild-type TR alpha1 or beta1 at 100 nM T3 by approximately 90% on the TRE-pal2 and 70% on DR4. AlphaF397X inhibited the transcriptional activities of both wild-type TR alpha1 and beta1 by approximately 50% on TRE-pal2 and by 60% on DR4. The dominant negative potency of betaF451X was significantly stronger than that of alphaF397X on the TRE-pal2, -DR4 and chicken lysozyme silencer F2, but similar on TRE-myosin heavy chain alpha and malic enzyme. No partiality for the TR subtypes was found in the dominant negative effects of betaF451X and alphaF397X. Co-expression with RXR enhanced the dominant negative effects of alphaF397X, but not of betaF451X. The results indicate that there are different dominant negative properties between alphaF397X and betaF451X, which are TRE-dependent, despite their identical C-terminal truncation. Deletion in the tau4 domain might affect the receptor structures of TR alpha1 and beta1 differently.

[Hypogonadism and osteoporosis].

Hypogonadism, whether caused by primary gonadal failure or secondary gonadotropin deficiency, is associated with osteoporosis. Even gonadotropin-releasing hormone therapy for endometriosis causes a decrease in bone mineral density by reducing serum estradiol level. Recent interesting studies of a male patient with a nonsense mutation in the estrogen receptor gene, a girl with missense mutation in the aromatase gene, and knock-out mice targeting estrogen receptor gene suggest that estrogen is very important to maintain normal bone density even in a male. Androgen deficiency increase risk for osteoporosis, but estrogen may compensate for it, since osteoporosis is not remarkable in patients with androgen resistance. Growth hormone deficiency and hyperprolactinemia are also related to gonadal function and thus bone metabolism.

[Targeting technology utilizing magnetic microparticulate system for cancer therapy].

Organ level-targeting of anticancer drugs or other relating stimuli to tumor tissues and their circumstances is important in cancer therapy. The successful targeting results in the greater antitumor effect and the lower numerous side effects. Application of a magnet from the outside of body is a useful tool to increase the targeting efficacy. In this review paper, targeting technology utilizing magnetic microparticulate system for cancer therapy was introduced and the future on the magnetic targeting system was augured.

A case of an iliopsoas abscess in a neonate.

The present study describes a male neonate with an iliopsoas abscess. He was born by Caesarean section at 35 weeks and 3 days gestation. At 24-days-old he had a fever and localized swelling of the groin to the femur. By ultrasonography (US) and computed tomography (CT), the swelling was diagnosed as iliopsoas abscess. We treated him through percutaneous needle drainage and antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in the culture of the fluid from the abscess and the urine. US and CT were useful for the diagnosis and provided guidance for the needle puncture and follow-up of the iliopsoas abscess.