Rice NPH1 homologues, OsNPH1a and OsNPH1b, are differently photoregulated.

The members of nonphototropic hypocotyl 1 (NPH1) family of genes in plants are considered to be the blue-light photoreceptors for phototropism. We isolated and characterized two NPH1 homologues from rice named as OsNPH1a and OsNPH1b. The predicted proteins of both OsNPH1 genes include LOV (LOV1 and LOV2) and protein kinase domains which are typically conserved regions in all the members of NPH1 family. Comparison of OsNPH1 apoproteins with those from other plant species revealed a close homology of OsNPHla with Arabidopsis, maize and oat NPH1, while the OsNPH1b appeared to be more closer to the recently found Arabidopsis NPL1. These structural homologies indicate that NPH1 homologues can be grouped into two classes namely "NPH1 type" and "NPL1 type". Northern blot analysis showed that OsNPH1a was strongly expressed in coleoptiles, whereas OsNPH1b was highly expressed in leaves of dark-grown rice seedlings. When the dark-grown seedlings were transferred to the continuous white light, the abundance of the OsNPH1a transcript in coleoptiles rapidly declined to the minimum levels, whereas the OsNPH1b transcript level in leaves gradually increased. These results lead us to conclude that expression of OsNPH1a and OsNPH1b is differently photoregulated in different tissues of rice seedlings.

Cryptochrome nucleocytoplasmic distribution and gene expression are regulated by light quality in the fern Adiantum capillus-veneris.

Numerous cellular responses are reportedly regulated by blue light in gametophytes of lower plants; however, the molecular mechanisms of these responses are not known. Here, we report the isolation of two blue light photoreceptor genes, designated cryptochrome genes 4 and 5 (CRY4 and CRY5), from the fern Adiantum capillus-veneris. Because previously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largest identified to date in plants. The deduced amino acid sequences of the five genes show remarkable similarities with previously identified cryptochromes as well as class I photolyases. Like the other plant cryptochromes, none of the cryptochromes of this fern possesses photolyase activity. RNA gel blot analysis and competitive polymerase chain reaction analysis indicate that the expression of the newly identified CRY4 and CRY5 genes is regulated by light and is under phytochrome control. The intracellular distribution of reporter beta-glucuronidase (GUS)-CRY fusion proteins indicates that GUS-CRY3 and GUS-CRY4 localize in fern gametophyte nuclei. The nuclear localization of GUS-CRY3 is regulated in a light-dependent manner. Together with our physiological knowledge, these results suggest that CRY3, CRY4, or both might be the photoreceptor that mediates inhibition of spore germination by blue light.

A phytochrome from the fern Adiantum with features of the putative photoreceptor NPH1.

In plant photomorphogenesis, it is well accepted that the perception of red/far-red and blue light is mediated by distinct photoreceptor families, i.e., the phytochromes and blue-light photoreceptors, respectively. Here we describe the discovery of a photoreceptor gene from the fern Adiantum that encodes a protein with features of both phytochrome and NPH1, the putative blue-light receptor for second-positive phototropism in seed plants. The fusion of a functional photosensory domain of phytochrome with a nearly full-length NPH1 homolog suggests that this polypeptide could mediate both red/far-red and blue-light responses in Adiantum normally ascribed to distinct photoreceptors.

Isolation and characterization of homologues of plant blue-light photoreceptor (cryptochrome) genes from the fern Adiantum capillus-veneris.

Many blue-light mediated physiological responses have been studied in the fern Adiantum capillus-veneris. We have isolated genomic clones encoding sequences similar to those encoding blue-light photoreceptors (cryptochromes) in higher plants using the Arabidopsis CRY1 cDNA as a probe, and these positive clones fall into five independent groups. Using RACE procedures, we obtained full-length cDNA sequences for three of these five groups. The deduced amino acid sequences include the photolyase-homologous domain in the N-terminal half, and they also contain a C-terminal extension of about 200 amino acids in length. These structural features indicate that the genes indeed encode Adiantum cryptochromes and represent a small gene family having at least three members.

Determinations of free and bound ethanol, acetaldehyde, and acetate in human blood and urine by headspace gas chromatography.

The improved PCA method leads to accurate measurement of ethanol, acetaldehyde, and acetate in blood and urine by headspace gas chromatography. It is important to prevent the formation of artifactual acetaldehyde from coexistent ethanol. The column used for detection of alcohol metabolites was the fused silica glass capillary column bonded with PEG-20M or the fused silica glass capillary column of Pora PLOT Q. In bound alcohol metabolites, recent measurements of hemoglobin-associated acetaldehyde in blood, and ethanol conjugate and acetaldehyde conjugate in urine are reviewed and described as a marker of alcohol abuse.

[Effect of long-term ethanol administration (2). Free type and bound type ethanol and related substances contents of the urine from ethanol administrated rats, indices in the serum, and renal tissues].

Histological effects of ethanol on the kidney were published in our previous report. In the present paper, results of the following measurement will be reported: contents of ethanol and related substances in the urine, both free and bound types, collected during the periods from 30 minutes to 11 hours after ethanol administration to rats, and ACE, alpha-GST, LPO, 25(OH)-D3, 1 alpha-25(OH)2-D3, 24, 25(OH)2-D3 in the serum of rats which had ethanol every day for a month. These will be reported together with histological observation of the kidney excised immediately after the blood sample was collected. The measurement of free and bound types ethanol, acetaldehyde, acetone and methanol in the urine was made up to 11 hours after administration of 4 g/kg b.w./day, p.o. and its results showed the highest contents at 9 hours after the administration. Bound type acetic acid showed the high contents at both 90 minutes and 9 hours after the administration. In 11 hours free type ethanol and acetaldehyde recovered their pre-administration value but as to the bound type only acetic acid recovered it. In the serum of the rats which were ethanol 4 g/kg b.w./day, oral administrated for a mouth, ACE showed significantly high value and 1 alpha, 25(OH)2-D3 and 24, 25(OH)2-D3 showed significantly low value relative to the control. Also alpha-GST showed a low value. In the kidney of the same rats the following changes were observed: swelling of glomerulus, thickening of basement membrane of glomerulus, PAS positive deposits in glomerulus, proliferation of mesangial cell, proliferation of juxtaglomenular cell, dilation of tubular lumen, swelling of tubular epithelial cell, its falling, hyaline droplet in tubular epithelial cell, cell infiltration to interstitial tissue, and basophilic tubule. There was not only difference between findings in the control and those in the liver and the brain of the rats which showed changes above-mentioned. As described above, changes were seen in the renal tissue caused by ethanol administration and in this connection changes in indices related to renal function were observed, too. Furthermore, urinary ethanol and related substances, not only free type but also bound type, that went through the kidney were observed for a long period time. The bound type, in particular, was observed for longer duration and hence effects of ethanol on the kidney were surely assumed. Presently longer term experiments are proceeding and other indices connected with renal functions are being studied.

Effects of bezafibrate on ethanol oxidation in rats.

Bezafibrate is used to lower serum lipid levels in humans. Fibrate derivatives induce an enzyme participating in the beta-oxidation by peroxisomes. We gave ethanol (2 g/kg) orally to bezafibrate-treated (300 mg/kg) male rats of the Wistar strain. Blood ethanol levels were remarkably lower and ethanol elimination stood at 432.6 mg/kg/hr (control, 336.6 mg/kg/hr) in the bezafibrate group (p < 0.01). Blood acetate levels were conversely higher in the bezafibrate group. The fatty acid beta-oxidation activity of liver peroxisome in bezafibrate-treated, clofibrate-treated, or gamma-linolenic acid-treated rats for 4 days was assayed. The activity was 5.8-fold higher in rats given bezafibrate, 5.4-fold in the clofibrate (p < 0.01), and 2.0-fold in the gamma-linolenic acid (p < 0.05). Alcohol dehydrogenase and aldehyde dehydrogenase activity of cytosol in the liver was not induced by the hypolipidemic drugs, but aldehyde dehydrogenase activity in the liver homogenate was induced. From foregoing results, bezafibrate induced in the organism beta-oxidation by peroxisomes and increased H2O2 production, which led to augmented ethanol metabolism by catalase.

Effects of tea (Camellia sinensis) chemical compounds on ethanol metabolism in ICR mice.

The effects of a green tea (Camellia sinensis) extract on ethanol metabolism in ICR male mice were studied. A crude green tea extract (GTE) and the tea components as (-)-epigallocatechin gallate (EGCg), (-)-epigallocatechin (EGC), and caffeine were administered before the tests. One hour later, the mice were orally given 2g/kg body weight (b.w.) of ethanol (20% ethanol w/v). The results show that the levels in the blood and liver of ethanol and acetaldehyde were lower, and that the levels of acetate and acetone were higher than in the controls orally given 500 mg/kg b.w. of GTE. After the administration of 75 mg/kg b.w. and 225 mg/kg b.w. of EGCg, the acetate and acetone concentrations in the blood and liver were lower than in the controls. The mice given caffeine at the same dose as that in GTE showed almost the same effects as the group treated with GTE. This suggests that EGCg and caffeine, the principal components of GTE, both had an effect on ethanol metabolism.

[An experiment on drinking using breath alcohol monitor (Alcomed 3010) by an electrochemical method].

A drinking experiment was performed to evaluate the efficiency of a breath alcohol monitor, Alcomed 3010. The ethanol concentrations in blood and breath were determined by gas chromatography, and in particular the breath ethanol concentration was determined with the breath alcohol monitor and by gas chromatography. The results obtained by two methods were compared. Based on the blood and breath ethanol concentrations, the following conclusions were drawn reading the breath alcohol monitor. The monitor has practical merit for determination of the breath ethanol level. It is small, usable anywhere, with little error in determination. In measuring principle, tobacco and acetone did not affected levels with the meter, but methanol, n-propanol and n-butanol affected determinations with the alcohol monitor. The breath (AM)/blood (GC) ethanol ratio was 1:2555. Comparison of the values determined with the alcohol monitor and gas chromatography yielded the equation: y = 0.998 x +/- 0.012 (r = 0.994). When determinations were made on the pure ethanol gas by the meter and gas chromatograph, the equation was: y = 0.974 x +/- 0.021 (r = 0.994). It may be said therefore that the alcohol monitor is both practically and functionally excellent.

Simultaneous measurement of alcohols and hydrogen cyanide in biological specimens using headspace gas chromatography.

We attempted to analyze biological specimens simultaneously for alcohols and hydrogen cyanide. A headspace gas chromatographic method with thick film wide bore column (PEG 20M) for the simultaneous determinations of methanol, ethanol, n-propanol and hydrogen cyanide in blood has been developed. This method was applied for the determinations of methanol, ethanol and hydrogen cyanide in a forensic autopsy case and animal experiments.

Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.

[Concentrations of ethanol and ethanol metabolites and symptoms of acute alcohol-intoxicated patients].

Five patients who presented to an emergency room and did not have other injury and disease with acute alcohol intoxication were analyzed about blood and urine ethanol, acetaldehyde, acetate and acetone levels. The average concentrations of ethanol, acetaldehyde, acetate and acetone in blood were 37.0 mM (1.7 mg/ml), 18 microM, 1.00 mM and 18 microM, respectively and the concentrations in urine were 50.8 mM (2.3 mg/ml), 37 microM, 0.79 mM and 47 microM, respectively. Clinical symptoms were concerned with both ethanol concentration and concentrations of ethanol metabolites. Their symptoms of acute alcohol-intoxicated patients were caused by the ethanol concentrations which was less than the levels reported in early studies.

Changes in free and bound alcohol metabolites in the urine during ethanol oxidation.

Free and bound ethanol, acetaldehyde, acetate, acetone and methanol in urine during alcohol oxidation were analyzed by means of a head space gas chromatography. Four healthy male volunteers drank beer for 20 min with 16 ml/kg for non-flushers (A, B) and 8 ml/kg for flushers (C, D). In the urine, the highest bound ethanol levels were between 0.5-1.1 mM for the non-flushers (NF) and 0.2-0.3 mM for the flushers (F). The urine free ethanol levels were 23-70 times as high as bound ethanol levels. The maximum free acetaldehyde in urine was 11-13 microM for the NF and 26-55 microM for the F. The urine bound acetaldehyde levels were 4-5 microM for the NF and 7-15 microM for the F. Urine acetaldehyde existed in free forms at 2.4-3.6 times as high concentrations as in bound forms during ethanol oxidation. The urine free acetate ranged between 0.3-2.0 mM. The bound acetate varied between 0.7-1.1 mM. The urine free methanol at 70-110 microM before the intake increased to 104-180 microM. The bound methanol reached to 78-126 microM from 48-97 microM before the intake. Ethanol levels in the urine were ethanol dose-dependent, whereas it was thought that free and bound acetaldehyde or acetate reflected individual metabolic abilities and not the amount of ethanol consumed.

Effects of seed saponins of Thea sinensis L. (Ryokucha saponin) on alcohol absorption and metabolism.

We evaluated the effects of the seed saponins of Thea sinensis L. on alcohol absorption and metabolism in rats and mice. An ethanolic extract from the seeds of T. sinensis was orally administered to the rats 1 hr before or 0.5 hr after administration of ethanol (2 g/kg), and the blood ethanol assayed 0.5, 1, 2, 3, and 4 hr after ethanol administration. The ethanol level decreased after both pre- and post-administration of the extract. The extract was further purified to obtain a saponin fraction which was orally administered to mice 1 hr before ethanol administration. Blood, liver, and stomach were obtained 0, 1, 3, and 6 hr after ethanol administration, and the ethanol, acetaldehyde, acetate, and acetone concentrations in each specimen were measured by head space gas chromatography. The saponin fraction decreased the ethanol levels in the blood and liver but increased that in the stomach five-fold over the control level, suggesting inhibition of alcohol absorption. The ethanol disappearance time from the blood was shortened, suggesting the promotion of alcohol disappearance. The acetate and acetone levels were unaffected. However, the acetaldehyde level decreased in the blood, liver, and stomach. The decreases in the ethanol and acetaldehyde levels in the liver suggested the protective effects of the seed saponins on the liver. The saponins did not directly inhibit hepatic alcohol dehydrogenase activity. The seed saponins of T. sinensis seem to suppress alcohol absorption by slowing gastric emptying and by inhibiting absorption across the cell membranes of the digestive tract.

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle.

The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.

Concentrations of blood and urine ethanol, acetaldehyde, acetate and acetone during experimental hangover in volunteers.

Blood and urine samples were analyzed for ethanol, acetaldehyde, acetate and acetone during experimental hangover in 6 healthy male volunteers (A, B, C, D, E, F). They drank freely for some 4 hr. In flushers (A, F) at 9 hr after ingestion (ethanol: 92 g, 1.2 g/kg and 1.3 g/kg), acetaldehyde levels were low in the blood, but high in the urine (37 microM, 45 microM). Heavy drinkers, non-flushers of B (ethanol: 176 g, 2.5 g/kg), C (157 g, 2.4 g/kg) and E (182 g, 2.9 g/kg) had a slightly high [lactate]/[pyruvate] ratio and 3-hydroxybutyrate in the blood at 11 hr after alcohol ingestion. Blood ethanol levels were dose dependent and blood acetaldehyde in B and C had a slightly high 6.3 microM and 8.0 microM 9 hr later, respectively. B, C and E had a high urine acetone concentration (100 microM over) in hangover. In C, in particular, urine acetate and acetone levels were unusually high. The ratio in blood (urine) among alcohol metabolites at 9 hr after drinking was approximately ethanol 1000 (1000): acetaldehyde 0.2-1.0 (0.1-5.9): acetate 36-163 (22-1554): acetone 1-11 (3-47).

Effects of amino acids on acute alcohol intoxication in mice--concentrations of ethanol, acetaldehyde, acetate and acetone in blood and tissues.

Condensation reactions between some SH-amino acids (L-and D-cysteine 1%) and acetaldehyde (50 microM) were studied in vitro experiment. In the aqueous solution, free acetaldehyde was reduced to 41.3% by L-cysteine and to 36.4% by D-cysteine. In the reaction with human blood medium, after the medium was deproteinized with perchloric acid reagent, acetaldehyde was reduced to 47.0% by L-cysteine and to 43.8% by D-cysteine. D-Cysteine appears to have great stability of reacting acetaldehyde. In vitro experiment reactability for D-cysteine exhibited 3-8% higher than that for L-cysteine. Next, effects of some amino acids on alcohol metabolism were studied in male ICR mice. The animals were given ethanol through a gastric catheter at a dose of 2 g/kg and they were intraperitoneally injected L-cysteine (300 mg/kg), D-cysteine (300 mg/kg), L-alanine (300 mg/kg) and control (saline), respectively in the period of one hour before the injection of ethanol. Blood and tissues samples were analyzed for ethanol, acetaldehyde, acetate and acetone during alcohol intoxication in mice by head space gas chromatography. In the groups administered D-cysteine and L-cysteine, the mice showed a definitely faster oxidation and disappearance of ethanol. Especially in the D-cysteine group, ethanol levels in blood, liver and brain remained lower than that in the other groups (p less than 0.01). Acetaldehyde levels in blood, liver and brain remained low by L-cysteine. Ethanol metabolites during alcohol oxidation by chemical reactabilities of L- and D-cysteine showed different distribution in the mice, respectively. In the mice received L-alanine, acetate and acetone levels in blood, liver and brain were distinctly reduced (p less than 0.01). L-Alanine is reported to supply an abundance of pyruvic acid that performs the NAD-generating system. NAD produced is introduced to alcohol metabolism and the TCA cycle. It was thus presumed that the L- or/and D-cysteine, and L-alanine was effective in acute alcohol intoxication by heavy drinking.

Liquid protein diets and torsade de pointes.

Three women, aged 27, 33, and 35 years, experienced recurrent syncope five months after losing 36 to 41 kg using liquid protein diets. No abnormalities were noted during physical examination except in one who was hypothyroid. Serum potassium levels varied between 2.9 and 3.9 mEq/liter. The ECGs demonstrated prominent U waves, QUc prolongation, and ST and T wave abnormalities, with left axis deviation in two patients. Syncopal episodes were due to ventricular tachycardia and fibrillation, which were not responsive to conventional antiarrhythmic agents used in two patients. Patients using liquid protein diets may thus experience reversible QUc prolongation giving rise to serious arrhythmias that are probably best treated with drugs that shorten the QTc interval. Caution should be exercised in the use of liquid protein diets for weight reduction in obesity.