Nucleotide sequence of the human 70 kDa peroxisomal membrane protein: a member of ATP-binding cassette transporters.

The cDNA sequence of human liver 70 kDa peroxisomal membrane protein (hPMP70) was determined. The nucleotide sequence contains an open reading frame of 1977 base pairs and encodes an amino acid sequence of 659 residues which exhibits 95.0% identity with that of rat liver PMP70. hPMP70 shares close similarity to the members of a superfamily of ATP-binding transport proteins.

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase.

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.

A possible role of immunoglobulin E in patients with hyperthyroid Graves' disease.

To investigate the possible participation of immunoglobulin E (IgE) in the autoimmune process of Graves' disease, incidence of elevation of serum IgE level, TSH receptor antibody (TRAb), and thyroid status were studied in 66 patients with hyperthyroid Graves' disease, 54 patients with Hashimoto's thyroiditis, 19 patients with bronchial asthma, and 15 patients with pollen allergy. In hyperthyroid Graves' patients, elevation of serum IgE levels (> or = 170 U/mL) was found in 19 of 66 patients (29%), 11 of whom had hereditary and/or allergic conditions. Elevations of serum IgE levels were found in 63% of patients with bronchial asthma and in 40% of patients with pollen allergy. Mean values of serum IgE were the same in patients with hyperthyroid Graves' disease and with bronchial asthma. During methimazole treatment TRAb decreased without fluctuation of IgE levels in both groups. The decrease in TRAb was significantly greater in patients with normal IgE than in patients with IgE elevation. After prednisone administration, reduction in TRAb was greater in patients with normal IgE than that in patients with IgE elevation. High incidence of IgE elevation in hyperthyroid Graves' disease and slower reduction in TRAb in association with IgE elevation suggest a difference in the autoimmune processes in Graves' disease with and without elevation of IgE.

Degradation of overexpressed wild-type and mutant uricase proteins in cultured cells.

Wild-type and mutated urate oxidase (UO) proteins were overexpressed in Cos-1 and HEK293 cells and were analyzed by Western blotting and several morphological methods. By immunoelectron microscopy, wild-type UO formed large aggregates in the cytoplasm and nucleoplasm and exhibited a crystalloid structure. Mutated UO (UOdC), from which 28 amino acids, including peroxisomal targeting signal at the C-terminus, were deleted, formed dispersed aggregates in the cytoplasm and nucleus. Chimeric UO (MUOdC), which was made by addition of the mitochondrial targeting signal of serine:pyruvate/glyoxylate aminotransferase to the N-terminus of UOdC, attached to ER to form a complicated MUOdC-ER complex. These three structures were immunostained for ubiquitin- and p32-subunits of proteasomes. Western blotting showed strong signal for UO and UOdC but very weak signal for MUOdC. The results suggest that overexpressed UO and UOdC accumulate in the cells because their synthesis rate is higher than the degradation rate, whereas MUOdC forming a complex with ER is degraded very rapidly. The ubiquitin-proteasome pathway may be involved in the degradation of these proteins.

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat mitochondrial acetoacetyl-CoA thiolase.

cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1,460 bases and contained a 1,272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44,694 and 41,364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.

Pharmacokinetics of amikacin and cephalothin in bedridden elderly patients.

The pharmacokinetics of amikacin (5.5 mg/kg intramuscularly) and cephalothin (1000 mg/body intravenously) in bedridden elderly patients were studied in comparison with those in healthy volunteers. The eliminations of amikacin and cephalothin from the plasma followed the course of a one-compartment open model. For amikacin, five healthy volunteers, elimination rate constant Kel was 0.396 hr-1, biologic half-life t1/2 was 1.80 hour, volume of distribution Vd was 0.201 l./kg; in five bedridden elderly patients, Kel was 0.208 hr-1, t1/2 was 3.55 hours, Vd was 0.376 l./kg. Cumulative renal excretion of amikacin in 8 hours was 44 per cent of the total dose in bedridden elderly patients and 69 per cent in healthy volunteers. For cephalothin, in seven healthy volunteers, Kel was 0.0353 min-1, t1/2 was 19.7 min, Vd was 0.176 l./kg; in four bedridden elderly patients, Kel was 0.0127 min-1, t1/2 was 56.4 min, Vd was 0.283 l./kg. Cumulative renal excretion of cephalothin reached a plateau by 4 hours of 40.8 per cent of the total dose in bedridden elderly patients and of 56.7 per cent in healthy volunteers. These results suggest that in bedridden elderly patients decreased renal excretion of amikacin and cephalothin is related to decreased renal function and an increased Vd.

Systematic approach to dipole localization of interictal EEG spikes in children with extratemporal lobe epilepsies.

OBJECTIVES: To assess the reliability of dipole localization based on residual variances (RV), using equivalent current dipole analysis of interictal EEG spikes in children with extratemporal lobe epilepsy. METHODS: Four pediatric patients with extratemporal lobe epilepsy were studied. Digital EEG was recorded from 19 scalp electrodes. Computer programs for spike detection and clustering analysis were used to select spikes. Dipoles were calculated 5 times for each spike using different initial guesses by the moving dipole model. Standard deviation (SD) of the dipole positions was calculated at each time point in the 5 trials. RESULTS: We analyzed the dipoles at 1097 time points from 4 patients. Among 106 time points with RV < 2%, the SD was < 1 mm in 78 (74%), while in those with SD > 1 mm the dipole positions varied between 2.8 and 52.6 mm. Of dipoles with RV < 1%, 26 of 27 (96%) had an SD < 1 mm; the one dipole with SD > 1 mm varied within 2.5 mm. The dipole localizations with RV < 2% corresponded to the epileptogenic zones identified on intracranial invasive video EEG and intraoperative ECoG. CONCLUSIONS: The systematic approach of equivalent current dipole analysis using spike detection, clustering analysis, and an RV < 2% as a standard is useful for identifying extratemporal epileptic regions.

Development of transient thyroid disease and reaction during treatment of chronic hepatitis C with interferon.

Six of 50 (12%) patients with chronic hepatitis C who were treated with interferon developed thyroid disease or an autoimmune thyroid reaction while undergoing treatment. One patient developed silent thyroiditis, with an increase in serum triiodothyronine (T3), thyroxine (T4), free T3, free T4, and markedly suppressed thyroid-stimulating hormone (TSH) levels, accompanied by the appearance of both antithyroglobulin (TgAb) and antimicrosomal antibodies (McAb). One patient developed hypothyroidism in association with moderately elevated TSH levels and high titers of McAb. TSH, TgAb, and McAb levels returned to the initial values at least 4 months after the end of interferon treatment (9 months of follow up). Four patients whose TgAb and/or McAb levels were elevated during treatment with interferon had been diagnosed as having subclinical autoimmune thyroiditis; however, their thyroid function remained in the normal range. These results suggested that treatment with interferon can cause a transient autoimmune thyroid reaction and disease as a side effect.

A new type of mitochondrial monoamine oxidase distinct from type-A and type-B.

The characteristics of mitochondrial monoamine oxidase (MAO) in carp liver were studied with MAO inhibitors and substrates. This enzyme was thermolabile, but was stabilized in the presence of bovine serum albumin. With clorgyline and deprenyl, single-sigmoidal curves for inhibition of the activity towards tyramine or 5-hydroxytryptamine were obtained; the sensitivities to the two inhibitors were identical. The activity towards beta-phenylethylamine was not completely inhibited by clorgyline or deprenyl, but the remaining activity was inhibited by semicarbazide and the inhibition curves by either clorgyline or deprenyl and semicarbazide were also identical to the curves with the other two substrates. These results suggest that carp liver mitochondria contain "classical" MAO and a clorgyline- and deprenyl-resistant amine oxidase and that the classical MAO does not seem to be MAO-A or MAO-B, which are present in mitochondria of most mammalian tissues.

Comparison of electroencephalographic dipoles of interictal spikes from prolonged scalp video-electroencephalography and magnetoencephalographic dipoles from short-term recording in children with extratemporal lobe epilepsy.

We retrospectively compared electroencephalographic (EEG) dipoles of interictal spikes from prolonged video-EEG monitoring with magnetoencephalographic dipoles from short-term recording in four children with extratemporal lobe epilepsy. We analyzed both sets of dipoles using individual interictal spikes and single moving dipole modeling and evaluated the profiles of spike appearance, dipole position, and orientation in EEG and magnetoencephalography. We obtained more than 100 magnetoencephalographic spikes in two patients who manifested frequent interictal EEG spikes throughout both day and night but fewer than 40 magnetoencephalographic spikes in two patients who had interictal EEG spikes mainly during sleep. The dipole positions of EEG and magnetoencephalography were in close proximity and included in the surgical resection area. Most of the dipoles between EEG and magnetoencephalography were oriented perpendicularly. A combination of EEG dipole analysis from prolonged video-EEG monitoring and magnetoencephalographic dipole analysis provides complementary information for presurgical evaluation in children with intractable extratemporal lobe epilepsy.

Hypothalamic hormones: clinical aspects.

Clinical aspects of TRH, GHRH, Somatostatin (SS) and CRH as well as an effect of glucocorticoid on GH secretion are reported.

Clinical diagnosis of pituitary adenoma.

Clinical symptoms, signs, endocrine examinations, image diagnosis of GH cell adenoma, prolactin cell adenoma, TSH cell adenoma and nonsecreting pituitary adenoma are discussed.

Effects of bromocriptine on experimental GH3 cell tumors.

Bromocriptine in concentrations up to 10(-4) M was studied for morphological and endocrinological effects upon the GH3 cell line as well as the GH1 and AtT-20 cell lines. The cells (10(5)/ml) were incubated with RPMI 1640 or in some experiments Dulbecco's Modified Eagle's Medium supplemented with 10% FCS. Bromocriptine was added in concentrations of 10(-4) to 10(-8) mol/L and aliquots of medium were obtained at 2 and 24 hs for the determination of growth hormone and prolactin. Significant reductions in concentrations of growth hormone and prolactin as well as cell number were observed with a concentration of bromocriptine of 10(-4) M at 24 hs. The electron microscopic appearance of GH3 cells treated with 10(-4) mol/L concentrations of bromocriptine for 24 hs demonstrated extensive and marked vacuolization in the cytoplasm which had already appeared 2 hs after treatment with bromocriptine. In bromocriptine-treated (10(-4) mol/L for 24 hs) GH1 cells and AtT-20 cells, the morphologic features were essentially unchanged, compared to the untreated group. Since many previous reports demonstrated a defective dopamine receptor system in GH3 cells, it must be concluded that bromocriptine has an extradopaminergic action which is selectively observed in GH3 cell.

An ACTH and FSH producing invasive pituitary adenoma with Crooke's hyalinization.

We report on a patient with ACTH and FSH producing invasive pituitary adenoma complaining of cutaneous pigmentation. Elevations in plasma ACTH, beta-endorphin and cortisol levels as well as urinary 17-OHCS and cortisol excretion were found. Serum FSH concentration was just within the upper limit of the normal range, whereas serum LH level was reduced and alpha-subunit level was normal. Roentogenographic examination showed an almost complete loss of sellar floor and destruction of the posterior clinoids and dorsum sella. CT scan and MRI demonstrated an enlarged tumor invasion of the clivus and its extension to the sphenoid sinus. After subtotal removal of the large pituitary tumor, serum cortisol and plasma beta-endorphin levels as well as plasma ACTH concentrations returned to normal and serum FSH levels also remarkably decreased. Histologically, the tumor corresponded to a chromophobe, slightly PAS positive adenoma. These tumor cells exhibited positive immunostaining with antibody to ACTH (1-24), beta-LPH, beta-endorphin and FSH, while immunostaining of the adenoma cells was negative for LH, TSH, GH and prolactin. The immunogold technique also demonstrated ACTH and FSH particles in the secretory granules in the cytoplasm of the adenoma cells. Some of the tumor cells disclosed Crooke's hyalinization and type I microfilament occupied most of the cytoplasm. In the present study, a very rare case of ACTH and FSH producing invasive pituitary adenoma is reported.

Polymyositis associated with thymoma and the subsequent development of pure red cell aplasia.

A 53-year-old woman with polymyositis associated with thymoma subsequently developed pure red cell aplasia (PRCA). She was hospitalized because of fever and muscle weakness, and diagnosed as having polymyositis by muscle biopsy. Remarkable clinical improvement followed administration of prednisolone. Progressive anemia became evident, however, while prednisolone was being tapered. Erythroid aplasia and the presence of thymoma confirmed the diagnosis of PRCA. Further examinations revealed that cytotoxic T cells may play an important role in the pathogenesis of this case.

Ultrasonic imaging of the temporomandibular joint: a clinical trial for diagnosis of internal derangement.

Kinematic imaging of the temporomandibular joint (TMJ) was applied for diagnosis of TMJ disorders using an ultrasonic diagnostic imaging system. Patients with a normal TMJ (male, 24 y 1 mon) and a symptomatic TMJ (female, 20 y 2 mon) were selected for imaging. The transducer must be placed in a specific location in order to propagate ultrasound through soft tissue because it is difficult for ultrasound to penetrate bone such as the condyle and the eminence. Therefore the ultrasonic images were not taken in sagittal cross-section, as is the case with magnetic resonance images. The ultrasonic diagnostic imaging system showed a transverse cross-section and no hard tissue images. It was difficult to become accustomed to these images, thus making it difficult to find differences between the normal TMJ and the symptomatic TMJ on the basis of static ultrasonic images alone. However a difference between the kinematic images of the normal and symptomatic TMJ was observed during jaw opening. Irregularity in the striated pattern of the soft tissue surrounding the condyle was observed in the image of the symptomatic TMJ. In order to make a precise diagnosis using ultrasonic imaging, it may be useful to understand the kinematics of the soft tissue surrounding the TMJ during jaw opening and closing.

Carboxyphenyl chloroanthranilic acid (CCA) in the modulation of immune response in mice.

A study to elucidate the immunopharmacological mechanisms of carboxyphenyl chloroanthranilic acid (CCA) was carried out, employing as a parameter the rosette formation of mouse spleen and thymus cells with red blood cells of sheep. The immunopharmacological properties of CCA and levamisole (LMS) were compared. In C57BL/6 mice, the immune response was either suppressed or enhanced by LMS, whereas CCA caused a suppression of rosette formation. Both drugs enhanced response in adult thymectomized C57BL/6 mice. LMS induced Thy-1 positive rosette-forming cells after adult thymectomy in C57BL/6 mice, whereas CCA did not. CCA was significantly effective in restoring the impaired immune response in C57BL/6 mice pretreated with immunosuppressants. The effect of LMS or CCA on the immune response varied among six different mouse strains (C57BL/6, BALB/c, C3H/He, DBA/2, (NZB X NZW) F1 and BXSB). The results suggest that both drugs have immunomodulation or immunonormalization properties.

[Effects of cysteine compounds on antibiotics I. Effects of L-cysteine and its derivatives on the potency of antibiotics (author's transl)].

Various derivatives of L-cysteine obtained by conversion to an -S-S- bond in the mucoprotein by means of -SH in the chemical structure are widely used as expectorants because they show mucous dissolving action. Recently, there have been reports that L-cysteine derivatives lower the potencies of various antibiotics. Various types of antibiotics and cysteine-type expectorants are often used concomitantly for the treatment of bacterial infections in respiratory tract diseases, and any decrease in the antibiotic potency presents a major therapeutic problem. We investigated the effects of four cysteine derivatives on 12 antibiotics, ampicillin (ABPC), amoxicillin (AMPC), sulbenicillin (SBPC), cefazolin (CEZ), cephalexin (CEX), cephalothin (CET), oxytetracycline (OTC), doxycycline (DOTC), minocycline (MINO), erythromycin (EM), ribostamycin (VSM) and lincomycin (LCM), widely used clinically in vitro with the minimum inhibitory concentration (MIc) obtained by the liquid dilution method as an index. L-Cysteine, acetylcysteine, ethylcysteine and mecysteine lowered the potencies of almost all of the antibiotics at high concentrations (500 mcg/ml), but at low concentrations (12.5 mcg/ml), mecysteine lowered the potencies of only three antibiotics and L-cysteine those of only four antibiotics, while acetylcysteine decreased the potencies of six and ethylcysteine those of seven antibiotics.

[Changes in endocrine status in cancer patients and the effect of chemotherapy on endocrine functions].

Changes in serum growth hormone (GH) concentrations before and after TRH injection, oral glucose tolerance test (OGTT), L-dopa, CB-154 and LHRH administration were investigated in 78 gastric, 32 hepatocellular, 23 colonic, 20 pulmonary, 11 esophageal, 9 pancreatic and 9 other types of cancer patients. In addition, the effect of 5-FU or tegafur on thyroid function was studied in 39 gastric, 12 colonic, 8 hepatocellular, 5 pancreatic and 8 other types of cancer patients. The results obtained were as follows. 1) Paradoxical responses of growth hormones to TRH and OGTT were demonstrated in cancer patients, although normal increases in GH concentrations after L-dopa and CB-154 administration were shown. Incidences of GH response to TRH were significantly higher in female cancer patients than in male cancer patients, although there was no sex difference in the paradoxical responses of serum GH concentrations to OGTT. 2) After curative surgery, paradoxical responses of serum GH levels to TRH and OGTT were gradually diminished. Immunostaining of tumors using GH Ab was negative. Anti-tumor agents showed a stimulatory effect on the paradoxical response to OGTT. 4) More than 3 months after 5-FU or tegafur administration, significant elevations of serum T3, T4 and TBG concentrations were demonstrated in cancer patients, compared to pre-treatment values or those within 2 months after therapy, although there were no significant changes in serum thyroglobulin and TSH levels before and after therapy. In conclusion, tumor-dependent hypothalamic-pituitary changes were recognized in cancer patients. Long-term administration of 5-FU or tegafur caused elevations of serum T3 and T4 through high TBG concentrations.

[Fundamental and clinical studies of changes in endocrine conditions in cancer patients].

The aim of the present study was to investigate hypothalamic-pituitary function, pituitary-thyroid function as well as pituitary-adrenal function in cancer patients. 1. Hypothalamic-pituitary function: We already reported a tumor-related paradoxical increase of serum growth hormone (GH) in oral glucose tolerance test (OGTT) and TRH test, which seemed to be due to changes in pituitary GH cell, not hypothalamus. The high incidence of the paradoxical increase of GH in OGTT was found in patients with hepatocellular cancer and pancreatic cancer, whereas in TRH test it was observed in patients with pancreatic, colonic and gastric cancer. The highly sensitive GH enzyme immunoassay demonstrated in 12 non-responder patients in OGTT a significant reduction of serum GH at 30 minutes compared to basal values. 2. Pituitary-thyroid function: We already demonstrated that a high incidence of low T3 syndrome in elderly cancer patients compared to younger patients and metabolic changes in glucose must be involved in the pathogenesis. In addition, this syndrome is significantly related to prognosis. There were no significant changes in thyroid function before and after curative operation for tumors except in patients with poor prognosis. 3. Pituitary-adrenal function: It was shown that cancer cachexia is characterized by hypercortisolemia, which must be due to a defect of negative feedback control.