Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor

The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS–PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 h. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression.     

Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role

Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age‐dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives.     

ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.     

Increased Activity of Liver Xanthine Oxidase in Rats from Increasing Dietary Calcium

A new binary vector, pZT4B, containing the UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence.     

Determination of 2-Aminoethylphosphonic Acid and Its Y-iM ethyl Derivative in Animal Tissues by Gas Chromatography with Flame Photometric Detection

A sensitive and selective method was developed for the determination of 2-aminoethylphosphonic acid (AEP) and W-methyl AEP in animal tissues by gas chromatography (GC). These compounds were converted into their A-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection (FPd-GC), using 0.5 % FFAP on Uniport HP as the GC column packing. The calibration curves for AEP and A-methyl AEP in the range of 0.02 ~ 2 μg were linear, and the detection limit was about 20 pg as an injection amount. AEP and A^-methyl AEP in animal tissues were found in the free form and bound form with lipid and other biological macromolecules, and they could be measured without any influence from coexistent substances by FPd-GC. The recoveries of AEP and A'-methyl AEP added to the tissue samples were 92 —105 %, and their reproducibility was found to be satisfactory. The distribution of these compounds in various animals was also studied by using this new method.     

Stability and bifurcation analysis of a ratio-dependent community dynamics model on Batesian mimicry

Journal of Mathematical Biology - Batesian mimicry is the similarity of coloration and patterns in an unpalatable species (the “model-species”) and a palatable species (the...