Influence of Drinking and/or Cooking with Jinzu River Water on the Development of Itai-Itai Disease

This study was undertaken to estimate the amount of cadmium (Cd) exposure needed for the development of Itai-itai disease and the influence of using Jinzu River water for drinking and/or cooking on its development. The subjects comprised 38 Itai-itai disease patients admitted both for questionnaire survey and medical testing. The period when the patients started to perceive leg/back pain was defined as ‘mild disease onset’ and that when they experienced the most severe manifestations such as ambulatory disturbance as ‘severe disease onset’. In a comparison of non-users/users of river water, mean age and lifetime Cd intake (LCd) estimated from the daily consumption of rice and other foods at the onset of mild or severe disease showed smaller values in the users than non-users. LCd in non-users/users of river water for drinking and/or cooking were estimated as 3.46–3.60 g/2.58–2.63 g at mild disease onset and 4.24–4.44 g/3.50–3.54 g at severe disease onset. A portion of the differences in LCd in non-users/users is attributable to the influence of Cd consumption from the use of river water, although it was suggested that LCd estimated from eating Cd-polluted rice exerted a greater influence on the development of Itai-itai disease than drinking and/or cooking with Cd-polluted river water.     

Pochonicine,a polyhydroxylated pyrrolizidine alkaloid from fungus Pochonia suchlasporia var. suchlasporia TAMA 87 as a potent β-N-acetylglucosaminidase inhibitor

A new polyhydroxylated pyrrolizidine alkaloid designated as pochonicine (1) was isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87. The structure of 1 was determined using NMR and MS techniques as (1R*, 3S*, 5S*, 6S*, 7R*, 7a S*)-5-acetamidomethyl-3-hydroxymethyl-1,6,7-trihydroxypyrrolizidine. Pochonicine (1) showed potent inhibition against β-N-acetylglucosaminidases (GlcNAcases) of various organisms including insects, fungi, mammals, and a plant but no inhibition against β-glucosidase of almond, α-glucosidase of yeast, or chitinase of Bacillus sp. The GlcNAcase inhibitory activity of pochonicine (1) was comparable to nagstatin, a potent GlcNAcase inhibitor of natural origin.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

Detrimental Effects of Microgravity on Mouse Preimplantation Development In Vitro

Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (µG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10^–3 G using 3D rotation. Fertilization occurred normally in vitro under µG. However, although we obtained 75 healthy offspring from µG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under µG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under µG environment in a mammal, but normal preimplantation embryo development might require 1G.     

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.     

Molecular identification of ghrelin receptor (GHS-R1a) and its functional role in the gastrointestinal tract of the guinea-pig

Ghrelin stimulates gastric motility in vivo in the guinea-pig through activation of growth hormone secretagogue receptor (GHS-R). In this study, we identified GHS-R1a in the guinea-pig, and examined its distribution and cellular function and compared them with those in the rat. Effects of ghrelin in different regions of gastrointestinal tract were also examined. GHS-R1a was identified in guinea-pig brain cDNA. Amino acid identities of guinea-pig GHS-R1a were 93% to horses and 85% to dogs. Expression levels of GHS-R1a mRNA were high in the pituitary and hypothalamus, moderate in the thalamus, cerebral cortex, pons, medulla oblongata and olfactory bulb, and low in the cerebellum and peripheral tissues including gastrointestinal tract. Comparison of GHS-R1a expression patterns showed that those in the brain were similar but the expression level in the gastrointestinal tract was higher in rats than in guinea-pigs. Guinea-pig GHS-R1a expressed in HEK 293 cells responded to rat ghrelin and GHS-R agonists. Rat ghrelin was ineffective in inducing mechanical changes in the stomach and colon but caused a slight contraction in the small intestine. 1,1-Dimethyl-4-phenylpiperazinium and electrical field stimulation (EFS) caused cholinergic contraction in the intestine, and these contractions were not affected by ghrelin. Ghrelin did not change spontaneous and EFS-evoked [³H]-efflux from [³H]-choline-loaded ileal strips. In summary, guinea-pig GHS-R1a was identified and its functions in isolated gastrointestinal strips were characterized. The distribution of GHS-R1a in peripheral tissues was different from that in rats, suggesting that the functional role of ghrelin in the guinea-pig is different from that in other animal species.     

Effects of various steroids on platelet-derived endothelial cell growth factor (PD-ECGF) and its mRNA expression in uterine endometrial cancer cells

Progestins diminish the estrogen-induced angiogenic potential related to basic fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in uterine endometrial cancer cells. This led us to study the effect of various steroids on the expression of platelet-derived endothelial cell growth factor (PD-ECGF) as the other pertinent angiogenic factor in well-differentiated uterine endometrial cancer cell line Ishikawa.In Ishikawa cells, estradiol induced the expression of PD-ECGF and its mRNA. The estrogen-induced expression was increased approximately two-fold by progesterone and by its metabolite, 17alpha-hydroxyprogesterone, but not by medroxyprogesterone acetate (MPA). Therefore, progesterone and 17alpha-hydroxyprogesterone as endogenous steroids might induce PD-ECGF-related angiogenic potential in uterine endometrial cancer cells, but not MPA as a synthetic steroid. In conclusion, the failure of PD-ECGF induction by MPA might be the great merit of anti-angiogenic treatment with MPA for uterine endometrial cancers.     

A novel transchromosomic system: stable maintenance of an engineered Mb-sized human genomic fragment translocated to a mouse chromosome terminal region

Transchromosomic (Tc) technology using human chromosome fragments (hCFs), or human artificial chromosomes (HACs), has been used for generating mice containing Mb-sized segments of the human genome. The most significant problem with freely segregating chromosomes with human centromeres has been mosaicism, possibly due to the instability of hCFs or HACs in mice. We report a system for the stable maintenance of Mb-sized human chromosomal fragments following translocation to mouse chromosome 10 (mChr.10). The approach utilizes microcell-mediated chromosome transfer and a combination of site-specific loxP insertion, telomere-directed chromosome truncation, and precise reciprocal translocation for the generation of Tc mice. Human chromosome 21 (hChr.21) was modified with a loxP site and truncated in homologous recombination-proficient chicken DT40 cells. Following transfer to mouse embryonic stem cells harboring a loxP site at the distal region of mChr.10, a ~4 Mb segment of hChr.21 was translocated to the distal region of mChr.10 by transient expression of Cre recombinase. The residual hChr.21/mChr.10ter fragment was reduced by antibiotic negative selection. Tc mice harboring the translocated ~4 Mb fragment were generated by chimera formation and germ line transmission. The hChr.21-derived Mb fragment was maintained stably in tissues in vivo and expression profiles of genes on hChr.21 were consistent with those seen in humans. Thus, Tc technology that enables translocation of human chromosomal regions onto host mouse chromosomes will be useful for studying in vivo functions of the human genome, and generating humanized model mice.