Enzymatic transesterification of purine nucleoside having a low solubility in organic medium

Enzymatic transesterification of guanosine having low solubility against organic solvent was examined. For the transesterification between guanosine and divinyl adipate catalyzed by alkaline protease from Bacillus (Bioprase), DMSO was added to DMF to increase the solublility of the nucleoside, and the conversion rate of guanosine to the vinyl guanosine ester was less than 30%. To overcome the reversible inactivation of enzyme by hydrophilic organic solvents, the reaction was carried out with 10% (v/v) water. The transesterification reaction was effectively catalyzed in DMF/DMSO in the presence of water and the conversion rate increased ca. 70% after 7 d reaction. The result shows that the water effect of Bioprase would be a useful method for the synthesis of low solublility nucleoside esters.     

Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound

The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of Candida macedoniensis was cloned and sequenced. A 1212bp nucleotide fragment (oye) was confirmed to be the gene encoding OYE based on the agreement of internal amino acid sequences. Oye encodes a total 403 amino acid residues, and the deduced amino acid sequence shows a high degree of similarity to those of other microbial OYE family proteins. An expression vector, pETOYE, that contains the full length of oye was constructed. Escherichia coli harboring pETOYE exhibited an about six-fold increase in specific KIP-reducing activity under the control of the T7 promoter as compared with that of C. macedoniensis. (6R)-Levodione formed with washed cells of the transformant and a cofactor regeneration system amounted to 638 mM (98.2 mg ml(-1)), the a molar yield being 96.9%. The asymmetric reduction of KIP to (6R)-levodione with E. coli cells, which co-expressed both oye and the glucose dehydrogenase gene (gdh), as a catalyst was investigated. The (6R)-levodione formed amounted to 627 mM (96.6 mg ml(-1)), the a molar yield being 95.4%. Since the use of E. coli BL21 (DE3) cells co-expressing oye and gdh as a catalyst is simple and does not require the addition of glucose dehydrogenase, it is highly advantageous for the practical synthesis of (6R)-levodione.     

Characterization of the splice sites in GT-AG and GC-AG introns in higher eukaryotes using full-length cDNAs

For the purpose of analyzing the relation between the splice sites and the order of introns, we conducted the following analysis for the GT-AG and GC-AG splice site groups. First, the pre-mRNAs of H. sapiens, M. musculus, D. melanogaster, A. thaliana and O. sativa were sampled by mapping the full-length cDNA to the genomes. Next, the consensus sequences at different regions of pre-mRNAs were analyzed in the five species. We also investigated the mononucleotide and dinucleotide frequencies in the extensive regions around the 5' splice sites (5'ss) and 3' splice sites (3'ss). As a result, differential frequencies of nucleotides at the first 5'ss in both the GT-AG and GC-AG splice site groups were observed in A. thaliana and O. sativa pre-mRNAs. The trend, which indicates that GC 5'ss possess strong consensus sequences, was observed not only in mammalian pre-mRNAs but also in the pre-mRNAs of D. melanogaster, A. thaliana and O. sativa. Furthermore, we examined the consensus sequences of the constitutive and alternative splice sites. It was suggested that in the case of the alternative GC-AG introns, the tendency to have a weak consensus sequence at 5'ss is different between H. sapiens and M. musculus pre-mRNAs.     

Fission yeast Dna2 is required for generation of the telomeric single-strand overhang

It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells. However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand. Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature. The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature. Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay. Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature. These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells. The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant. Our results reveal that DSB ends and telomere ends are processed by different mechanisms.     

Core 2 GlcNAc transferase and kidney tubular cell-specific expression

The expression of glycan chains is precisely regulated in a time- and space-dependent manner. We summarize here our recent work on the kidney tubular cell-specific regulation of core 2 beta-1,6-GlcNAc transferase. Gsl5 gene was first identified by genetic analysis on the basis of polymorphic expression of kidney glycolipids among inbred strains of mice and turned out to be a regulatory gene controlling the level of mRNA of kidney-specific core 2 beta-1,6-GlcNAc transferase. This kidney-specific core 2 GlcNAc transferase takes glycolipids having Gal beta 1-3GalNAc at their termini, Gal beta 1-3GalNAc alpha 1- and beta 1-oligosaccharide derivatives, and glycoproteins having core 1 structure, as substrates. Immunohistochemistry with anti-core 2-Le( x ) monoclonal antibody demonstrated that vesicles located just below the microvillous membrane of proximal tubule cells were clearly stained in a Gsl5 -wild type mouse. Western blotting with the monoclonal antibody detected a major glycoprotein with a molecular mass of 500 kDa in the microsomal fraction of the wild type mouse kidney. In situ hybridization with anti-sense cDNA of kidney-specific core 2 GlcNAc transferase confirmed that Gsl5 gene controls the expression of the core 2 beta-1,6-GlcNAc transferase mRNA in a proximal tubular cell-specific manner. The 5' upstream sequences of the kidney-specific core 2 GlcNAc transferase gene in inbred and wild-derived strains of mice were analyzed, and the phylogenetic analysis of these sequences suggests that functional Gsl5 gene might be produced by the time of subspeciation of M. musculus, about one million years ago.     

Novel algorithm for automated genotyping of microsatellites

Microsatellites or short tandem repeats (STRs) are abundant in the human genome with easily assayed polymorphisms, providing powerful genetic tools for mapping both Mendelian and complex traits. Microsatellite genotyping requires detection of the products of polymerase chain reaction (PCR) amplification by electrophoresis, and analysis of the peak data for discrimination of the true allele. A high-throughput genotyping approach requires computer-based automation at both the detection and analysis phases. In order to achieve this, complicated peak patterns from individual alleles must be interpreted in order to assign alleles. Previous methods consider limited types of noise peaks and cannot provide enough accuracy. By pattern recognition of various types of noise peaks, such as stutter peaks and additional peaks, we have achieved an overall average accuracy of 94% for allele calling in our actual data. Our algorithm is crucial for a high-throughput genotyping system for microsatellite markers by reducing manual editing and human errors.     

Effects of neuronal norepinephrine uptake blockade on baroreflex neural and peripheral arc transfer characteristics

Neuronal uptake is the most important mechanism by which norepinephrine (NE) is removed from the synaptic clefts at sympathetic nerve terminals. We examined the effects of neuronal NE uptake blockade on the dynamic sympathetic regulation of the arterial baroreflex because dynamic characteristics are important for understanding the system behavior in response to exogenous disturbance. We perturbed intracarotid sinus pressure (CSP) according to a binary white noise sequence in anesthetized rabbits, while recording cardiac sympathetic nerve activity (SNA), arterial pressure (AP), and heart rate (HR). Intravenous administration of desipramine (1 mg/kg) decreased the normalized gain of the neural arc transfer function from CSP to SNA relative to untreated control (1.03 +/- 0.09 vs. 0.60 +/- 0.08 AU/mmHg, mean +/- SE, P < 0.01) but did not affect that of the peripheral arc transfer function from SNA to AP (1.10 +/- 0.05 vs. 1.08 +/- 0.10 mmHg/AU). The normalized gain of the transfer function from SNA to HR was unaffected (1.01 +/- 0.04 vs. 1.09 +/- 0.12 beats.min(-1).AU(-1)). Desipramine decreased the natural frequency of the transfer function from SNA to AP by 28.7 +/- 7.0% (0.046 +/- 0.007 vs. 0.031 +/- 0.002 Hz, P < 0.05) and that of the transfer function from SNA to HR by 64.4 +/- 2.2% (0.071 +/- 0.003 vs. 0.025 +/- 0.002 Hz, P < 0.01). In conclusion, neuronal NE uptake blockade by intravenous desipramine administration reduced the total buffering capacity of the arterial baroreflex mainly through its action on the neural arc. The differential effects of neuronal NE uptake blockade on the dynamic AP and HR responses to SNA may provide clues for understanding the complex pathophysiology of cardiovascular diseases associated with neuronal NE uptake deficiency.     

Muscle mechanoreflex induces the pressor response by resetting the arterial baroreflex neural arc

The effects of the muscle mechanoreflex on the arterial baroreflex neural control have not previously been analyzed over the entire operating range of the arterial baroreflex. In anesthetized, vagotomized, and aortic-denervated rabbits (n = 8), we isolated carotid sinuses and changed intracarotid sinus pressure (CSP) from 40 to 160 mmHg in increments of 20 mmHg every minute while recording renal sympathetic nerve activity (SNA) and arterial pressure (AP). Muscle mechanoreflex was induced by passive muscle stretch (5 kg of tension) of the hindlimb. Muscle stretch shifted the CSP-SNA relationship (neural arc) to a higher SNA, whereas it did not affect the SNA-AP relationship (peripheral arc). SNA was almost doubled [from 63 +/- 15 to 118 +/- 14 arbitrary units (au), P < 0.05] at the CSP level of 93 +/- 8 mmHg, and AP was increased approximately 50% by muscle stretch. When the baroreflex negative feedback loop was closed, muscle stretch increased SNA from 63 +/- 15 to 81 +/- 21 au (P < 0.05) and AP from 93 +/- 8 to 109 +/- 12 mmHg (P < 0.05). In conclusion, the muscle mechanoreflex resets the neural arc to a higher SNA, which moves the operating point towards a higher SNA and AP under baroreflex closed-loop conditions. Analysis of the baroreflex equilibrium diagram indicated that changes in the neural arc induced by the muscle mechanoreflex might compensate for pressure falls resulting from exercise-induced vasodilatation.     

Cardiac sympathetic nerve stimulation does not attenuate dynamic vagal control of heart rate via alpha-adrenergic mechanism

Complex sympathovagal interactions govern heart rate (HR). Activation of the postjunctional beta-adrenergic receptors on the sinus nodal cells augments the HR response to vagal stimulation, whereas exogenous activation of the presynaptic alpha-adrenergic receptors on the vagal nerve terminals attenuates vagal control of HR. Whether the alpha-adrenergic mechanism associated with cardiac postganglionic sympathetic nerve activation plays a significant role in modulation of the dynamic vagal control of HR remains unknown. The right vagal nerve was stimulated in seven anesthetized rabbits that had undergone sinoaortic denervation and vagotomy according to a binary white-noise signal (0-10 Hz) for 10 min; subsequently, the transfer function from vagal stimulation to HR was estimated. The effects of beta-adrenergic blockade with propranolol (1 mg/kg i.v.) and the combined effects of beta-adrenergic blockade and tonic cardiac sympathetic nerve stimulation at 5 Hz were examined. The transfer function from vagal stimulation to HR approximated a first-order, low-pass filter with pure delay. beta-Adrenergic blockade decreased the dynamic gain from 6.0 +/- 0.4 to 3.7 +/- 0.6 beats x min(-1) x Hz(-1) (P < 0.01) with no alteration of the corner frequency or pure delay. Under beta-adrenergic blockade conditions, tonic sympathetic stimulation did not further change the dynamic gain (3.8 +/- 0.5 beats x min(-1) x Hz(-1)). In conclusion, cardiac postganglionic sympathetic nerve stimulation did not affect the dynamic HR response to vagal stimulation via the alpha-adrenergic mechanism.