Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp. strain NS671.

DNA fragments containing the genes involved in the conversion of 5-substituted hydantoins to their corresponding L-amino acids have been cloned from the 172-kb native plasmid (pHN671) of Pseudomonas sp. strain NS671. The largest recombinant plasmid, designated pHPB14, encoded the ability to convert D-5-substituted hydantoins to the corresponding L-amino acids, whereas the smallest one, designated pHPB12, encoded the ability to convert them to their corresponding N-carbamyl-D-amino acids. Restriction analysis suggested that the inserts of both recombinant plasmids are derived from the identical portion in pHN671 and that the insert of pHPB14, compared with that of pHPB12, has an extra 5.3 kb in length. DNA sequencing revealed that pHPB14 contains two additional complete open reading frames, designated ORF5 and hyuE. Analysis of deletion derivatives of pHPB14 indicated that hyuE is required for the ability to produce L-amino acids from the corresponding D-5-substituted hydantoins, but ORF5 is not. Cells of Escherichia coli transformed with a plasmid containing hyuE were capable of racemizing different 5-substituted hydantoins, indicating that hyuE is a gene encoding a hydantoin racemase.     

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging

Marine Biotechnology - Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study...     

Characterization of the carp myosin heavy chain multigene family

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Role of c-Myc in nitric oxide-mediated suppression of cytochrome P450 3A4

Cytochrome P450 (CYP) 3A4, which is abundant in human liver and small intestine and participates in the metabolism of various drugs and xenochemicals, is known to be induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the colon carcinoma cell line Caco-2 cells. Nitric oxide (NO) is able to inhibit CYP3A4 expression and catalytic activity. In this study, we investigated the mechanism of suppression by NO of 1,25(OH)2D3-induced CYP3A4 expression in Caco-2 cells. Caco-2 cells were exposed for 36 h to 400 nM 1,25(OH)2D3, and the induction of CYP3A4 mRNA expression was detected by real-time PCR. Because c-Myc regulates the expression of several genes, we examined its effect on the CYP3A4 expression induced by 1,25(OH)2D3. The expression of c-myc mRNA was increased in the early stage but decreased 36 h after the treatment of Caco-2 cells with 1,25(OH)2D3. The NO donor NOR-4 suppressed CYP3A4 expression induced by 1,25(OH)2D3 in Caco-2 cells in contrast, it significantly induced c-myc gene expression. Treatment of Caco-2 cells with the c-myc antisense oligonucleotide reversed the inhibitory effect of NOR-4 on CYP3A4 expression induced by 1,25(OH)2D3. These results suggest that the suppression of 1,25(OH)2D3-induced CYP3A4 expression by NO is due to c-myc expression.