Novel expression of equivocal messages containing both regions of choline/ethanolamine kinase and muscle type carnitine palmitoyltransferase I.

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure.

To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I.

We isolated a human muscle type of carnitine palmitoyltransferase I (CPTI-M) genomic clone and determined its entire nucleotide sequence. By comparison of the nucleotide sequence of the genomic clone with that of cDNA, we determined the intron/exon junctions. For detection of the exon(s) in the 5'-region of the CPTI-M gene, we isolated cDNA clones corresponding to the 5'-region of its transcript by 5'-rapid amplification of cDNA ends (5'-RACE method). Results showed two alternative exons, 1A and 1B, that do not encode amino acids in the 5'-region of the human CPTI-M gene. The gene encoding human CPTI-M was found to consist of two 5'-non-coding exons, 18 coding exons and one 3'-non-coding exon spanning approximately 10 kbp. Furthermore, on analysis of the 5'-flanking region, a putative gene encoding a 'choline kinase homologue' was found to be located only about 300 bp upstream from exon 1A of the human CPTI-M gene. Comparison of the gene structure of human CPTI-M with the reported partial gene structure of human liver type CPTI (CPTI-L) showed that the intron insertion sites were completely conserved in these two genes.

Rectal submucosal tumor-like lesion originating from intestinal tuberculosis.

We present the case of a 55-year-old man who underwent transsacral local excision for a rectal submucosal tumor-like lesion suspected to originate from tuberculosis. The lesion, 2 cm in size, was found incidentally in the posterior wall of the lower rectum during anal fistulectomy. The lesion was apart from the primary crypt of the anal fistula. Barium enema and colonoscopy revealed a protuberant submucosal growth with a shallow depression of the overlying mucosa. Although computed tomography and magnetic resonance imaging showed a well defined round mass within the rectal wall, digital rectal examination suggested extramural origin. Since repeated endoscopic biopsies were negative, we selected the transsacral approach for excisional biopsy to achieve histological diagnosis. The lesion was confined to the rectal wall and the full-thickness rectal wall was excised. Histologically, a foreign-body granuloma with acute inflammation was the main component of the lesion. Caseating granulomas and Langhans' giant cells, consistent with tuberculosis, were also found.

Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I.

With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.

High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone.

To characterize energy metabolism in rat brown adipose tissue (BAT), we carried out differential screening of a cDNA library of BAT with a cDNA probe of white adipose tissue (WAT) and isolated one cDNA clone. It contained a single open reading frame of 2,316 bases which encodes a protein of 88.2 kDa. The predicted amino acid sequence showed the highest homology (62.6%) with that of rat carnitine palmitoyltransferase I (CPTI). The transcript corresponding to this cDNA was found to be abundantly expressed in BAT and heart. Therefore, the isolated clone is concluded to encode a CPTI like protein expressed in BAT and heart.

Studies on inhibitors of skin tumor promotion, XII. Rotenoids from Amorpha fruticosa.

As a part of screening studies for chemopreventive agents (anti-tumor-promoters), six North American plants belonging to the Amorpha genus were tested using an in vitro assay system. Of these plants, Amorpha fruticosa exhibited strong inhibitory effects on Epstein-Barr virus early antigen (EBA-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Also six rotenoids, amorphispironone [1], tephrosin [2], amorphigenin [3], 12a-hydroxyamorphigenin [4], 12a-hydroxydalpanol [5], and 6'-O-D-glucopyranosyldalpanol [6], were isolated from the leaves of A. fruticosa. Among these retenoids, 1 and 2 exhibited remarkable inhibitory effects of EBV-EA activation induced by TPA. Further, 1 and 2 exhibited significant anti-tumor-promotion effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These investigations suggested that these rotenoids might be valuable anti-tumor-promoters.

Antitumor agents, 138. Rotenoids and isoflavones as cytotoxic constitutents from Amorpha fruticosa.

Eight cytotoxic compounds have been isolated from the CHCl3 extract of Amorpha fruticosa. One compound, 6'-O-D-beta-glucopyranosyldalpanol [10], is a new cytotoxic rotenoid. Another known rotenoid, 12 alpha beta-hydroxyamorphigenin [6], was first shown to exhibit extremely potent cytotoxicity (ED50 < 0.001 microgram/ml) in six neoplastic cell lines. In addition to these compounds, three isoflavones (afrormosin [1], 7,2',4', 5'-tetramethoxyisoflavone [2], 8-methylretusin [3]) and five rotenoids (amorphispironone [4], amorphigenin [5], dalpanol [7], 12a beta-hydroxydalpanol [8], and tephrosin [9]) were isolated. Compound 8 was isolated for the first time as a natural product from this plant. The structures of these compounds were established on the basis of spectral data; some were further confirmed by X-ray crystallographic analysis.

[Experience of predeposit autologous blood transfusion and medication of recombinant human erythropoietin in pediatric open heart surgery].

To avoid using the homologous blood, 11 children between the age of 5 and 15 years donated autologous blood of 10 ml/kg of body weight (upper limit 400 ml) once a week for two weeks prior to elective open heart surgery. Five of 11 children received erythropoietin (100 U/kg of body weight) intravenously three times a week for two weeks. Only one patient experienced a mild donor reaction but no adverse effects occurred in erythropoietin therapy. In all the patients cardiac operations were able to be completed without homologous blood transfusion. Patients treated with erythropoietin were not anemic despite of preoperative donation although without erythropoietin therapy patients were mildly anemic. Our experience documents safety and effectiveness of predeposit autologous blood transfusion and erythropoietin therapy in pediatrics.

[A case of delirium which occurred during otorhinolaryngological treatment under local anesthesia--the importance of close communication between practitioners and local general hospitals].

We experienced a case of delirium which occurred during treatment of chronic sinusitis under local anesthesia in a ENT practitioner, which took an unique course. We were able to treat the patient immediately after occurrence of the complication because we have emergency hot-line between anesthesiologists in our hospital and the practitioners in our area. Fortunately the patient recovered without severe sequelae due to immediate therapy. We consider that in emergency cases, a close communication between practitioners and local general hospitals is important for exact diagnosis and suitable treatments as well as for life-saving procedures.

Possible involvement of lignin structure in anti-influenza virus activity.

Commercial lignins suppressed the growth of influenza A virus infecting MDCK cells, and the RNA-dependent RNA synthesis, as efficiently as the high-molecular weight fractions extracted from pine cone of Pinus parviflora Sieb. et Zucc. The anti-influenza A virus activity of both pine cone extract and commercial alkali-lignin was considerably reduced by treatment with sodium chlorite, but was not affected by sulfuric acid or trifluoroacetic acid. The degraded components of lignin, various synthesized polyphenols unrelated to lignin, and natural and chemically modified glucans, were not appreciably inhibitory. The data suggest that the polymerized phenolic structure of lignified materials is responsible for the anti-influenza A virus activity.

Effect of lard and corn oil intake on serum lipids in young men.

An experimental diet with lard (30 g/day for 7 days) and corn oil (30 g/day for 7 days) on high carbohydrate (basal diet) was given to four healthy Japanese young men and the effect of diets containing different fat on serum lipids was examined. Serum total cholesterol was increased significantly from a basal diet of 106 +/- 23 to 141 +/- 26 mg/dl on lard diet, and then decreased significantly (p less than 0.05) to 111 +/- 22 mg/dl on corn oil diet. Serum triglycerides increased significantly (p less than 0.01) from 66 +/- 38 to 173 +/- 32 mg/dl on basal diet. Serum HDL-cholesterol was decreased significantly (p less than 0.01) from 41.9 +/- 1.6 to 31.2 +/- 3.8 mg/dl on lard diet and increased significantly (p less than 0.05) to 41.9 +/- 4.6 mg/dl on corn oil diet. Serum HDL-cholesterol fraction was decreased significantly (p less than 0.01) from 41.6 +/- 4.9 to 28.1 +/- 3.2% on basal diets, but increased significantly (p less than 0.05) to 44.3 +/- 3.1% on lard diet, and then decreased to 36.3 +/- 2.5% on corn oil diet. Serum HDL phospholipid fraction decreased significantly (p less than 0.05) from 62.5 +/- 6.7 to 50.7 +/- 1.8% on basal diet and increased significantly (p less than 0.05) to 60.4 +/- 1.0% on lard and corn oil diet. Serum phospholipids did not change by experimental diets. It is concluded that lard and corn oil have different and specific roles in lipid metabolism.

Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products.

A liquid chromatographic method for the determination of ochratoxin A in coffee beans (green and roast), instant coffee, and coffee drink is described. The sample is subjected to extraction with methanol-1% aqueous sodium bicarbonate (1 + 1) and C18 cartridge cleanup. The extract is chromatographed on a Nucleosil 5C18 column with a mobile solvent of acetonitrile-water-0.2M phosphate buffer pH 7.5 (50 + 47 + 3) containing 3 mM cetyltrimethylammonium bromide as an ion-pair reagent. Ochratoxin A is detected with a fluorometer (excitation 365 nm, emission 450 nm). The sensitivity was increased 20-fold by using ion-pair resolution. The detection limits corresponded to 2 micrograms/kg for coffee beans, 5 micrograms/kg for instant coffee, and 0.2 microgram/kg for coffee drink. The recoveries from coffee products were generally better than 80.7% and the relative standard deviations were 3.43-5.93%. The peak coinciding with ochratoxin A can be confirmed by treatment using alcohol (methanol, ethanol, or n-propanol) and H2SO4.

Studies on the analysis of food additives by high-performance liquid chromatography. V. Simultaneous determination of preservatives and saccharin in foods by ion-pair chromatography.

A high-performance liquid chromatographic method for the simultaneous determination of sorbic acid, benzoic acid, p-hydroxybenzoic acid and its methyl, ethyl, isopropyl, n-propyl, isobutyl, n-butyl esters and saccharin in foodstuffs is described. For good separations of these compounds, acetonitrile-water-0.2 M phosphate buffer pH 3.6 (7:12:1) containing 2 mM cetyltrimethylammonium bromide as an ion-pair reagent and a Nucleosil 5C18 column are required. A steam distillation method and a Sep-Pak C18 cartridge method for the sample preparation are compared. The recoveries from a coffee drink were generally better than 93.8% and the relative standard deviations were 0.85-2.15% for the Sep-Pak C18 cartridge method.

Caffeine degradation and increased ochratoxin A production by toxigenic strains of Aspergillus ochraceus isolated from green coffee beans.

The growth and ochratoxin A production of Aspergillus ochraceus strains S-235-100 and IFM 0458, which were isolated from green coffee beans and glutinous rice, respectively, were examined in yeast extract-sucrose (YES) medium containing 0.1 to 1.0% caffeine. The mycelial growth and ochratoxin A formation of strain IFM 0458 was inhibited by caffeine at concentrations over 0.1%, and ochratoxin A was not produced at caffeine levels of 0.5% and 1.0%. Contrary to this, A. ochraceus strain S-235-100 produced a larger amount of ochratoxin A in the presence of 0.5% and 1.0% caffeine when grown on YES medium, reaching a maximum after 15 to 20 days of incubation. The formation of ochratoxin A by nine additional strains of A. ochraceus, three strains of A. elegans and one strain of A. sclerotiorum isolated from green coffee beans was determined on rice and ground green coffee media. A significant degree of degradation of caffeine in the green coffee medium was demonstrated with cultures of nine A. ochraceus isolates from green coffee beans. Most of these isolates showed the potential to grow on moist green coffee beans and to produce a significant amount of ochratoxins.