Yasuhiro Sumino

A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

Tissue distribution of prolactin-releasing peptide (PrRP) and its receptor.

Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.

Purification and characterization of acid urease from Lactobacillus fermentum

SummaryAcid urease was purified to an electrophoretically homogeneous state, and the molecular weight was estimated to be 220 000. The enzyme consisted of three kinds of subunits, designated α, β and λ, with molecular weights of 67 000, 16 800 and 8600, respectively, in a (α1 \2λ1)2 structure. The isoelectric point of the enzyme was 4.8. The nickel content was found to be 1.9 atoms of nickel per α1β2λ1 unit. The amino acid profile was different from those of known bacterial neutral ureases. The enzyme was most active at pH 2 and around 65° C. It was stable between pH 3 and 9, and below 50° C. The Km for urea was 2.7 mM at pH 2. The enzyme activity was inhibited by Ag+, Hg2+, Cu2+, p-chloromercuribenzoate and acetohydroxamate. The enzyme was separated into three subunits by reverse phase HPLC. The amino terminal amino acid sequences of the subunits α, β and λ were Ser-Phe-Asp-Met-, Met-Val-Pro-Gly- and Met-Arg-Leu-Thr-, respectively.

Organization and expression inPseudomonas putida of the gene cluster involved in cephalosporin biosynthesis fromLysobacter lactamgenus YK90

TheLysobacter lactamgenus YK90pcbAB gene encoding δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is located immediately upstream of thepcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. ThepcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411 593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within thepcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. The domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthatase 2 ofBacillus brevis. ThepcbAB gene was expressed under the control of thelac promoter inPseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis inP. putida led to the accumulation of β-lactam antibiotics. Deletion analysis of an open-reading frame located between thecefE andcefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis inL. lactamgenus appear to be clustered in the orderpcb AB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.

Purification and characterization of acid urease from Lactobacillus reuteri

Acid urease was purified from Lactobacillus reuteri and characterized. The enzyme preparation was electrophoretically homogeneous and the molecular weight of the enzyme was estimated to be 220,000. The enzyme consisted of three kinds of polypeptides, designated as α, s and γ, with molecular weights of 68,000, 16,100 and 8,800, respectively, in a (α1β2γ1)2 structure. The isoelectric point of the enzyme was 4.7. The nickel content was found to be 1.8 atoms of nickel per α1β2γ1 unit. The amino acid profile was different from those of known bacterial neutral ureases. The enzyme was most active at pH 2 and at around 65°C. It was stable between pH 3 and 8, and below 50°C. The Km for urea was 2.8 mM at pH 2. The enzyme activity was inhibited by Ag+, Hg2+, Cu2+, p-chloromercuribenzoate and acetohydroxamate. The acid urease eliminated urea in alcoholic beverages that are acidic in general.

Molecular analysis of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90

Determination of the nucleotide sequence downstream from the Lysobacter lactamgenus pcbC gene encoding isopenicillin N synthase revealed that five open-reading frames (ORF) including the pcbC gene were tightly linked in the same orientation. Each ORF has the remarkable feature of the protein-coding frame in the DNA sequence with a high G + C content. Expression in Escherichia coli and a comparison of the deduced amino acid sequences with published sequences showed that the gene cluster contained a deacetoxycephalosporin C synthetase (DAOCS) gene (cefE), an ORF having homology with the Cephalosporium acremonium DAOCS/deacetylcephalosporin C synthetase gene (cefEF), an isopenicillin N epimerase gene(cefD), and a β-lactamase gene. The gene order was pcbC-cefE-ORF3-cefD-β-lactamase.

Scale-up of purine nucleoside fermentation from a shaking flask to a stirred-tank fermentor

Scaling-up purine nucleoside fermentation by a mutant strain of Bacillus subtilis from a shaking flask to a stirred-tank fermentor was attempted. The dimensions and the operating conditions of the stirred tank were determined in order to satisfy the optimum conditions of O2 transfer and power consumption per unit volume for the shaking flask. When the purine nucleoside fermentation was carried out in the stirred-tank fermentor under these conditions, in which the temperature simulated that in the shaking flask, the total amount of purine nucleosides produced was almost the same as that in the shaking flask, but the accumulation ratio of guanosine to total nucleotides was different from that in the flask. Since urea could not be utilized so efficiently in the stirred-tank fermentor, the NHp+f4 concentration and the pH of the culture broth were lower than those in the shaking-flask culture during fermentation. The activity of inosine monosphosphate dehydrogenase and the accumulation ratio were significantly affected by the NHp+f4 concentration. When the pH of the stirred-tank culture was maintained at 6.9 by ammonia water to keep the NHp+f4 level higher, the ratio was improved to the same level as that observed in the shaking-flask culture. The fermentation heat calculated from the shaking-flask data and its pattern of change were similar to those in the stirred-tank fermentor.

Measurement of the overall volumetric coefficient of heat transfer of a shaking flask

Abstract The overall volumetric coefficient of heat transfer ( Ua ) of a shaking flask was measured under various shaking conditions using three types of flask. Ua was significantly affected by flask weight ( W F ), which could be attributed to the thickness of the flask wall, the rotational speed of the shaker ( N ), wind velocity ( V W ), and the liquid volume in the flask ( V L ). The limiting step of heat transfer seemed to be the heat radiation process from the surface of the flask to the surroundings. To predict the value of Ua , the following empirical equations were obtained for each type of flask by the least squares method: 1. (1) for deformed creased flask (M-type flask), Ua =17.4 W F −0.43 V L −0.61 N 0.12 V W 0.36 2. (2) for creased flask (N-type flask), Ua =9.2 W F −0.36 V L −0.57 N 0.17 V W 0.27 3. (3) for smooth flask (S-type flask), Ua =5.3 W F −0.26 V L −0.62 N 0.19 V W 0.25 .

Cloning and expression of the isopenicillin N synthase gene from Lysobacter lactamgenus YK90

The Lysobacter lactamgenus YK90 pcbC gene encoding isopenicillin N synthase (IPNS) was cloned using the corresponding Acremonium chrysogenum pcbC gene as a probe. The cloned pcbC gene encoded a polypeptide composed of 326 amino acid residues with a molecular weight of 36,562. The predicted amino acid sequence had a high similarity with those from fungus A. chrysogenum and actinomycetes Streptomyces clavuligerus. The pcbC gene was expressed in E. coli and in A. chrysogenum under control of the tac and GAP promoters, respectively.

Isolation and Taxonomie Characterization of Acid Urease-producing Bacteria

About 16,000 isolates from animal feces and intestines were assayed for the production of acid urease, and the 700 strains selected as producers were examined as to their taxonomic properties. Of these 700 strains, 370 belonged to the genus Streptococcus, 312 to the genus Lactobacillus, 9 to the genus Escherichia, 6 to the genus Staphylococcus, 2 to the genus Morganella and 1 to the genus Bifidobacterium. The majority of the streptococci were identified as Streptococcus mitior, the remainder being Streptococcus salivarius, Streptococcus faecalis, Streptococcus faecium, Streptococcus avium and Streptococcus gallinarum. The majority of the lactobacilli were considered to be Lactobacillus reuteri or Lactobacillus fermentum and Lactobacillus animalis or Lactobacillus salivarius, the remainder being Lactobacillus ruminis, Lactobacillus viridescens, Lactobacillus vac- cinostercus and strains considered to be Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus or Lactobacillus gasseri.