Analysis of Unique Motility of the Unicellular Green Alga Chlamydomonas reinhardtii at Low Temperatures down to -8 °C.

Previous studies of motility at low temperatures in Chlamydomonas reinhardtii have been conducted at temperatures of up to 15 °C. In this study, we report that C. reinhardtii exhibits unique motility at a lower temperature range (-8.7 to 1.7 °C). Cell motility was recorded using four low-cost, easy-to-operate observation systems. Fast Fourier transform (FFT) analysis at room temperature (20-27 °C) showed that the main peak frequency of oscillations ranged from 44 to 61 Hz, which is consistent with the 60 Hz beat frequency of flagella. At lower temperatures, swimming velocity decreased with decreasing temperature. The results of the FFT analysis showed that the major peak shifted to the 5-18 Hz range, suggesting that the flagellar beat frequency was decreasing. The FFT spectra had distinct major peaks in both temperature ranges, indicating that the oscillations were regular. This was not affected by the wavelength of the observation light source (white, red, green or blue LED) or the environmental spatial scale of the cells. In contrast, cells in a highly viscous (3.5 mPa·s) culture at room temperature showed numerous peaks in the 0-200 Hz frequency band, indicating that the oscillations were irregular. These findings contribute to a better understanding of motility under lower-temperature conditions in C. reinhardtii.

Spatial Distribution of Flagellated Microalgae Chlamydomonas reinhardtii in a Quasi-Two-Dimensional Space.

Although the phenomenon of collective order formation by cell-cell interactions in motile cells, microswimmers, has been a topic of interest, most studies have been conducted under conditions of high cell density, where the space occupancy of a cell population relative to the space size ϕ>0.1 (ϕ is the area fraction). We experimentally determined the spatial distribution (SD) of the flagellated unicellular green alga Chlamydomonas reinhardtii at a low cell density (ϕ≈0.01) in a quasi-two-dimensional (thickness equal to cell diameter) restricted space and used the variance-to-mean ratio to investigate the deviation from the random distribution of cells, that is, do cells tend to cluster together or avoid each other? The experimental SD is consistent with that obtained by Monte Carlo simulation, in which only the excluded volume effect (EV effect) due to the finite size of cells is taken into account, indicating that there is no interaction between cells other than the EV effect at a low cell density of ϕ≈0.01. A simple method for fabricating a quasi-two-dimensional space using shim rings was also proposed.

Collimated Microbeam Reveals that the Proportion of Non-Damaged Cells in Irradiated Blastoderm Determines the Success of Development in Medaka (Oryzias latipes) Embryos.

It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

Intermittent schedules of the oral RAF-MEK inhibitor CH5126766/VS-6766 in patients with RAS/RAF-mutant solid tumours and multiple myeloma: a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study.

BACKGROUND: CH5126766 (also known as VS-6766, and previously named RO5126766), a novel MEK-pan-RAF inhibitor, has shown antitumour activity across various solid tumours; however, its initial development was limited by toxicity. We aimed to investigate the safety and toxicity profile of intermittent dosing schedules of CH5126766, and the antitumour activity of this drug in patients with solid tumours and multiple myeloma harbouring RAS-RAF-MEK pathway mutations. METHODS: We did a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study at the Royal Marsden National Health Service Foundation Trust (London, UK). Patients were eligible for the study if they were aged 18 years or older, had cancers that were refractory to conventional treatment or for which no conventional therapy existed, and if they had a WHO performance status score of 0 or 1. For the dose-escalation phase, eligible patients had histologically or cytologically confirmed advanced or metastatic solid tumours. For the basket dose-expansion phase, eligible patients had advanced or metastatic solid tumours or multiple myeloma harbouring RAS-RAF-MEK pathway mutations. During the dose-escalation phase, we evaluated three intermittent oral schedules (28-day cycles) in patients with solid tumours: (1) 4·0 mg or 3·2 mg CH5126766 three times per week; (2) 4·0 mg CH5126766 twice per week; and (3) toxicity-guided dose interruption schedule, in which treatment at the recommended phase 2 dose (4·0 mg CH5126766 twice per week) was de-escalated to 3 weeks on followed by 1 week off if patients had prespecified toxic effects (grade 2 or worse diarrhoea, rash, or creatinine phosphokinase elevation). In the basket dose-expansion phase, we evaluated antitumour activity at the recommended phase 2 dose, determined from the dose-escalation phase, in biomarker-selected patients. The primary endpoints were the recommended phase 2 dose at which no more than one out of six patients had a treatment-related dose-limiting toxicity, and the safety and toxicity profile of each dosing schedule. The key secondary endpoint was investigator-assessed response rate in the dose-expansion phase. Patients who received at least one dose of the study drug were evaluable for safety and patients who received one cycle of the study drug and underwent baseline disease assessment were evaluable for response. This trial is registered with ClinicalTrials.gov, NCT02407509. FINDINGS: Between June 5, 2013, and Jan 10, 2019, 58 eligible patients were enrolled to the study: 29 patients with solid tumours were included in the dose-escalation cohort and 29 patients with solid tumours or multiple myeloma were included in the basket dose-expansion cohort (12 non-small-cell lung cancer, five gynaecological malignancy, four colorectal cancer, one melanoma, and seven multiple myeloma). Median follow-up at the time of data cutoff was 2·3 months (IQR 1·6-3·5). Dose-limiting toxicities included grade 3 bilateral retinal pigment epithelial detachment in one patient who received 4·0 mg CH5126766 three times per week, and grade 3 rash (in two patients) and grade 3 creatinine phosphokinase elevation (in one patient) in those who received 3·2 mg CH5126766 three times per week. 4·0 mg CH5126766 twice per week (on Monday and Thursday or Tuesday and Friday) was established as the recommended phase 2 dose. The most common grade 3-4 treatment-related adverse events were rash (11 [19%] patients), creatinine phosphokinase elevation (six [11%]), hypoalbuminaemia (six [11%]), and fatigue (four [7%]). Five (9%) patients had serious treatment-related adverse events. There were no treatment-related deaths. Eight (14%) of 57 patients died during the trial due to disease progression. Seven (27% [95% CI 11·6-47·8]) of 26 response-evaluable patients in the basket expansion achieved objective responses. INTERPRETATION: To our knowledge, this is the first study to show that highly intermittent schedules of a RAF-MEK inhibitor has antitumour activity across various cancers with RAF-RAS-MEK pathway mutations, and that this inhibitor is tolerable. CH5126766 used as a monotherapy and in combination regimens warrants further evaluation. FUNDING: Chugai Pharmaceutical.

Discrepancy between musical ability and language skills in children with Williams syndrome.

BACKGROUND: Children with Williams syndrome (WS) show a marked interest in music, a characteristic often explored in clinical settings. However, the actual musical abilities of patients with WS remain debatable due to some of the relevant data being derived from experimental tasks that require a verbal response, despite the known language impairments in WS. The present study aimed to examine musical ability in children with WS using a newly invented pitch discrimination task with minimal involvement of language and clarify its relationship with language skill. METHODS: Eleven children with WS participated in the study. We used a novel pitch discrimination task that required minimal language use. Two piano tones were presented sequentially, and children were asked to give a non-verbal response as to whether the second tone was higher than, lower than, or the same as the first tone. RESULTS: Pitch discrimination performance in children with WS was lower than the level predicted for their chronological age (CA), even in the non-verbal task. Pitch discrimination ability and verbal mental age (VMA) were shown to be dissociated, such that children with WS with a lower skill level for language showed an unexpectedly higher level of pitch discrimination ability and vice versa. CONCLUSIONS: Our results indicated reduced musical ability with respect to CA in children with WS. The dissociation between musical ability and language skills may indicate unique developmental relationships that differ from those in normal children. These findings provide new evidence to support the importance of assessing actual musical ability in WS prior to implementing interventional music therapy.

Randomized, Controlled, Phase 3 Trials of Carteolol/Latanoprost Fixed Combination in Primary Open-Angle Glaucoma or Ocular Hypertension.

PURPOSE: To assess the intraocular pressure (IOP)-lowering effects and safety of a carteolol/latanoprost fixed combination drug (OPC-1085EL) vs latanoprost (Study 1) and carteolol (Study 2) in patients with primary open-angle glaucoma (POAG) or ocular hypertension (OH). DESIGN: Multicenter, randomized, evaluator-masked (Study 1)/double-masked (Study 2), parallel-group studies. METHODS: Setting: Twenty-eight clinical sites (Study 1) and 19 clinical sites (Study 2) in Japan. STUDY POPULATION: Outpatients with bilateral POAG or OH whose predose IOP was 18 to <35 mm Hg in the study eye after 4 weeks' treatment with latanoprost (Study 1) or carteolol (Study 2) (defined as baseline). INTERVENTION: In Study 1, 237 patients applied OPC-1085EL (n = 118) or latanoprost (n = 119) for 8 weeks. In Study 2, 193 patients applied OPC-1085EL (n = 78), carteolol (n = 78), or carteolol/latanoprost concomitant therapy (n = 37) for 8 weeks. MAIN OUTCOME MEASURE: Adjusted mean IOP reduction at predose from baseline to week 8. RESULTS: In Study 1, the adjusted mean IOP reductions (95% confidence interval [CI]) were 2.9 (2.5-3.3) mm Hg and 1.6 (1.2-2.0) mm Hg in the OPC-1085EL and latanoprost groups, respectively (P < .0001). In Study 2, the adjusted mean IOP reductions (95% CI) were 3.5 (3.1-3.9) mm Hg and 1.6 (1.2-2.0) mm Hg in the OPC-1085EL and carteolol groups, respectively (P < .0001). All adverse drug reactions of OPC-1085EL observed in both studies were mild in severity and only 1 patient in each study discontinued because of an adverse drug reaction. CONCLUSIONS: OPC-1085EL is superior to latanoprost or carteolol alone in terms of lowering IOP, and was well tolerated.

Evolutionary pathway of pseudogenization of globin genes, α5 and ß5, in genus Oryzias.

Hemoglobin transports oxygen in many organisms and consists of α- and ß-globin chains. Previously, using molecular phylogenetic analysis, we proposed that both α- and ß-globins of teleost could be classified into four groups. We also showed that the Hd-rR strain of medaka (Oryzias latipes) inhabiting southern Japan had all four groups of globin genes but that the α- and ß-globin genes of group III were pseudogenized (α5(ψα), ß5(ψß)). Based on the small degree of nucleotide variations, the pseudogenization of ß5 was assumed to have occurred at a relatively late stage of evolution. Here, we compared the α5(ψα)-ß5(ψß) of two other strains of O. latipes and found that both α5(ψα) and ß5(ψß) of the northern Japanese and Korean strains were pseudogenized similar to those of Hd-rR. In a Philippine population (Oryzias luzonensis), α5(ψα) was also pseudogenized, but the structure was different from that of O. latipes, and ß5(ψß) was almost deleted. Interestingly, an Indonesian population (Oryzias celebensis) had α5 and ß5 genes that were deduced to be functional. Indeed, they were expressed from the young to adult development stages, and this expression pattern was consistent with the expression of α2 and ad.α1 in Hd-rR. Because α2 and ad.α1 in Hd-rR were assigned to groups I and II, respectively, we speculate that their expression patterns might be altered by pseudogenization of group III genes. These results provide a basis for further investigations of recruiting and changing expression patterns of one globin gene after pseudogenization of other globin genes during evolution.

The Parapineal Is Incorporated into the Habenula during Ontogenesis in the Medaka Fish.

The parapineal is present in many teleost families, while it is absent in several others. To find out why the parapineal is absent at adult stages in the latter families, the development of the epithalamus was examined in the medaka fish (Oryzias latipes). For this purpose, a green fluorescent protein-transgenic medaka line, in which the pineal complex (pineal and parapineal) is visible fluorescently, was used. We found that a distinct parapineal was present in the roof plate at early developmental stages. Subsequently, however, the parapineal and the associated roof plate began to be incorporated into the habenula between embryonic stages 28 and 29. Between embryonic stages 29 and 30, the entire parapineal was incorporated into the habenula. That is, the parapineal became a small caudomedial region (termed the 'parapineal domain') within the left habenula in the majority of embryos, resulting in the left-sided asymmetry of the epithalamus. Thereby the left habenula became larger and more complex than its right counterpart. In the minority of embryos, the parapineal was incorporated into the right habenula or into the habenulae on both sides. In the majority of embryos, the parapineal domain projected a fiber bundle to a subnucleus (termed the 'rostromedial subnucleus') in the left habenula. The rostromedial subnucleus sent axons, through the left fasciculus retroflexus, to the rostral region of the left half of the interpeduncular nucleus. We further found that the ratio of the left-sided phenotype was temperature dependent and decreased in embryos raised at a high temperature. The present study is the first demonstration that the supposed lack of a distinct parapineal in adult teleost fishes is due to ontogenetic incorporation into the habenula.

Phase III clinical trials comparing the immunogenicity and safety of the vero cell-derived Japanese encephalitis vaccine Encevac with those of mouse brain-derived vaccine by using the Beijing-1 strain.

The immunogenicity and safety of an inactivated cell culture Japanese encephalitis vaccine (CC-JEV) were compared with those of an inactivated mouse brain-derived Japanese encephalitis vaccine (MB-JEV) in phase III clinical multicenter trials conducted in children. The vaccines contain the same Japanese encephalitis virus strain, the Beijing-1 strain. Two independent clinical trials (trials 1 and 2) were conducted. Trial 1 was conducted in 468 healthy children. Each subject was injected with 17 µg per dose of either CC-JEV or MB-JEV, and the immunogenicity and safety of the vaccines were investigated. Trial 1 showed that CC-JEV was more immunogenic and reactive than MB-JEV at the same dose. Therefore, to adjust the immunogenicity of CC-JEV to that of MB-JEV, a vaccine that has had a good track record regarding its efficacy for a long time, trial 2 was conducted in 484 healthy children. To improve the stability, CC-JEV was converted from a liquid type to a freeze-dried type of vaccine. Each subject was injected subcutaneously with either 4 µg per dose of CC-JEV, 8 µg per dose of CC-JEV, or 17 µg per dose of MB-JEV twice, at an interval of 2 to 4 weeks, followed by an additional booster immunization 1 to 15 months after the primary immunization. Based on the results of trial 2, 4 µg per dose of the freeze-dried CC-JEV (under the label Encevac) was selected as a substitute for the MB-JEV. Encevac was approved and launched in 2011 and has since been in use as a 2nd-generation Japanese encephalitis vaccine in Japan. (These studies have been registered at the JapicCTI under registration no. JapicCTI-132063 and JapicCTI-080586 for trials 1 and 2, respectively).

Immunodiagnosis of alveolar echinococcosis using urine samples.

Alveolar echinococcosis (AE) is one of the most lethal zoonotic parasitic infections. The diagnosis is based on the combination of the abdominal imaging including CT, MRI and PET, and serology. To develop a new diagnostic tool for AE with urine as samples, mouse-Echinococcus multilocularis (Em) model and then human cases were studied. The antibody levels of urine and serum samples from the infected mice and AE cases were well correlated with each other. The sensitivity and specificity of the method with urine were 91% and 98%, respectively, when IgG4 to crude Em was examined. Comparing with serum samples, the collection of urine is easier and safer and the urine diagnostic tool makes surveys of this silent disease easier.

[Factors associated with cardiovascular death and events in patients with end stage renal disease].

BACKGROUND: It is very important to evaluate atherosclerosis at an early stage since cardiovascular disease is the main cause of death in patients with end stage renal disease. The purpose of our study was to examine which of the following parameters of atherosclerosis is the best index for the prediction of cardiovascular death or events in hemodialysis patients: intima media thickness (IMT), ankle-brachial index (ABI) and cardio-ankle vascular index (CAVI), and whether visceral fat area (VFA) and subcutaneous fat area (SFA), also predict those events. METHODS: VFA, SFA, IMT, ABI and CAVI were measured using CT or a dedicated device in 270 hemodialysis patients(age: 63.3 +/- 12.3 years, male 56.3%). RESULTS: During a median follow-up period of 54 months, cardiovascular deaths or events occurred in 92 (34.1%) patients. Seventy (25.9%) patients died, 27 (38.6%) of them due to cardiovascular events. Whereas several baseline clinical covariates showed an associated risk for composite cardiovascular events in a univariate Cox proportional hazards analysis, almost all of them became insignificant when analyzed together. Only age, SFA, and a prevalence of diabetes remained significant in multivariate analysis. When both IMT and ABI were included in this model, all other covariates became insignificant, while ABI, but not IMT, was also related to the prediction of cardiovascular death on top of age and SFA. CONCLUSIONS: Both ABI and IMT were useful predictors for composite cardiovascular events, with ABI being also associated with a risk for cardiovascular deaths. In addition, SFA was a useful predictor for both cardiovascular events and cardiovascular deaths.

Propagation of Caenorhabditis japonica in the nest of its carrier insect, Parastrachia japonensis.

We demonstrated the disembarkation of the bacterial-feeding nematode Caenorhabditis japonica dauer larvae (DL) from adult Parastrachia japonensis female insects and observed the propagation of nematodes in artificial insect nests. Our results clarify the process of propagation in this nematode species and provide insights into the nematode-insect relationship. Quiescent C. japonica DL resumed their mobility only at > 99.9% relative humidity (RH) at 25°C in the presence or absence of the carrier insect. In artificial nests with > 99.9% RH, DL resumed their mobility and the number of DL on female insects decreased gradually after oviposition, although numerous DL remained on the insects. Very few DL were detected on mother insects after hatching. Nematode propagation was observed on the egg mass after hatching and on nymphal carcasses; the total number of nematodes in the nest increased dramatically after this point. These results indicate that humidity is an important factor for disembarkation of C. japonica DL and that C. japonica propagates in the nest of P. japonensis where it feeds on the remains of eggs and nymph carcasses, indicating that C. japonica and P. japonensis have a unique phoretic and necromenic association.

Species-specific and female host-biased ectophoresy in the roundworm Caenorhabditis japonica.

Caenorhabditis japonica is a bacteriophagous nematode species that was discovered on the semi-social burrower bug, Parastrachia japonensis, which demonstrates egg-guarding and provisioning behaviors. To understand the life history of C. japonica in relation to P. japonensis, we demonstrated the specificity of this association and fluctuations in nematode number on the insect throughout the year. C. japonica dauer larvae (DL), larvae in a nonfeeding diapause stage, were predominantly found as clumps on the adult female insects but rarely found on the male insects in all populations examined. This female-biased association was consistent throughout the year, but after the nymphs hatched, nematodes were not detected on the mother insects showing provisioning behavior. DL appeared on the nymphs, and the number of DL on the newly emerged female insects gradually increased thereafter. C. japonica has never been detected on other invertebrates collected from the P. japonensis habitat thus far. Our data suggest that the life cycles of C. japonica and P. japonensis are synchronized.

Transgenic medaka fish which mimic the endogenous expression of neuronal kinesin, KIF5A.

Intracellular transport is spatiotemporally controlled by microtubule-dependent motor proteins, including kinesins. In order to elucidate the mechanisms controlling kinesin expression, it is important to analyze their genomic regulatory regions. In this study, we cloned the neuronal tissue-specific kinesin in medaka fish and generated transgenic fish which mimic endogenous neuronal kinesin expression in order to elucidate the mechanisms which regulate kinesin expression. Searches for medaka neuronal orthologues by RT-PCR identified a candidate gene expressed only in neuronal tissues. Using BAC clones, we determined the cDNA sequence and the gene structure of the candidate neuronal kinesin. Evolutionary analysis indicated that the candidate gene encoded medaka KIF5Aa. The endogenous medaka orthologue was found to be expressed only in the nervous system, including the brain and spinal cord, while expression of KIF5Ab was not exclusive to neuronal tissues. Transgenic (Tg) medaka that expressed EGFP under the control of the 6.9 kbp 5' and 1.9kbp 3' flanking regions of the KIF5Aa gene showed characteristic expression throughout the nervous system, including the brain, spinal cord, olfactory pit, eye and cranial nerve. Immunohistological analysis showed that EGFP expression in Tg fish co-localized with expression of HuC/D, a neuronal marker. These results demonstrate that the 6.9 kbp 5' and 1.9 kbp 3' flanking regions of medaka KIF5Aa have neuronal-specific promoter activity mimicking endogenous expression of medaka KIF5Ab. This transgenic fish strain will be useful for further functional analysis of the effects of these regulatory regions on gene expression.

Effect of exercise on kidney function, oxidative stress, and inflammation in type 2 diabetic KK-A(y) mice.

Exercise is recommended for the management of type 2 diabetes, but its effects on diabetic nephropathy (DN) are still unknown. We hypothesized that appropriate exercise improves early DN via attenuation of inflammation and oxidative damage. Type 2 diabetic KK-A(y) mice, a spontaneous DN model, underwent two different kinds of exercise (i.e., moderate and low intensity). Sedentary mice or those undergoing an exercise regimen causing no significant body weight loss were used. We examined the urinary excretion of albumin, number of podocytes and macrophages, renal expressions of HIF-1α and MCP-1, and biomarkers of oxidative stress such as urinary 8-OHdG and serum SOD. Exercise reduced urinary levels of albumin and also maintained the number of podocytes in the exercised KK-A(y) mice independently of improvements of overweight and hyperglycemia, although moderate-intensity exercise increased expression of HIF-1α. Sedentary KK-A(y) mice showed increased expression of MCP-1 and infiltration of macrophage, increased urinary 8-OhdG, and decreased serum SOD levels compared with exercised KK-A(y) mice. On the whole, low-intensity exercise attenuates progression of early DN without affecting marked renal ischemia. Reduction rates of urinary albumin and maintained podocyte numbers, with parallel improvements in oxidative damage and inflammation, are related to beneficial effects of exercise in diabetic kidney disease.

The primary brain vesicles revisited: are the three primary vesicles (forebrain/midbrain/hindbrain) universal in vertebrates?

It is widely held that three primary brain vesicles (forebrain, midbrain, and hindbrain vesicles) develop into five secondary brain vesicles in all vertebrates (von Baer's scheme). We reviewed previous studies in various vertebrates to see if this currently accepted scheme of brain morphogenesis is a rule applicable to vertebrates in general. Classical morphological studies on lamprey, shark, zebrafish, frog, chick, Chinese hamster, and human embryos provide only partial evidence to support the existence of von Baer's primary vesicles at early stages. Rather, they suggest that early brain morphogenesis is diverse among vertebrates. Gene expression and fate map studies on medaka, chick, and mouse embryos show that the fates of initial brain vesicles do not accord with von Baer's scheme, at least in medaka and chick brains. The currently accepted von Baer's scheme of brain morphogenesis, therefore, is not a universal rule throughout vertebrates. We propose here a developmental hourglass model as an alternative general rule: Brain morphogenesis is highly conserved at the five-brain vesicle stage but diverges more extensively at earlier and later stages. This hypothesis does not preclude the existence of deep similarities in molecular prepatterns at early stages.

Identification of quantitative trait loci for diabetic nephropathy in KK-Ay/Ta mice.

BACKGROUND: The pathogenesis and development of human diabetic nephropathy involves genetic factors. Since human diabetic nephropathy is a heterogeneous disorder, identification of responsible gene loci is difficult. We studied candidate gene loci for diabetic nephropathy, using quantitative trait locus (QTL) analysis of a spontaneous animal model for diabetic nephropathy: KK-Ay/Ta × normal BALB/cA F2 intercross mice. METHODS: We examined 270 (KK-Ay/Ta × BALB/cA) F2 intercross mice for their urinary albumin to creatinine ratios (ACRs), HbA1c and fasting body weights (FBW) at 8, 12, 16 and 20 weeks. Genotypes were investigated using 86 microsatellite markers with QTL analysis. RESULTS: ACR in mice at 20 weeks and ACR gain showed a suggestive linkage to chromosome 9 (log of the odds [LOD] scores: 3.8 and 3.4, respectively; designated ACR-1). Gene loci contributing to HbA1c indicated a significant linkage to chromosome 7 (LOD: 5.8 and 8.9) in mice at 8 and 20 weeks (designated HbA1c-1), and FBW indicated a significant linkage to chromosome 1 (LOD: 5.5 and 5.2) in mice at 8 and 12 weeks (designated Fbw-1). At 20 weeks, glomerular to Bowman's capsule volume (G/B) ratio of F2 mice homozygous BB for D9Mit66 was significantly higher than that in homozygous KK and heterozygous KB F2 progeny. The sizes of pancreatic islets in F2 progeny homozygous KK and heterozygous KB for D7Mit100 were larger than those in homozygous BB F2 progeny. CONCLUSION: QTL analysis of KK-Ay/Ta mice revealed several new loci contributing to diabetic nephropathy and related phenotypes. Thus, it appears that type 2 diabetes and nephropathy of KK-Ay/Ta mice have different genetic factors.

1kbp 5' upstream sequence enables developmental stage-specific expressions of globin genes in the fish, medaka Oryzias latipes.

Hemoglobin of bony fish and higher vertebrates is a tetrameric protein constructed by 2 α- and 2 ß-globins, which are expressed in a developmental stage-specific manner. The genomic organization of genes for embryonic and adult α- and ß-globin varies from species to species. In fish, it is known that there is a unique genomic organization of globin genes, that is, α- and ß-globin genes are arranged in a bi-directional and head-to-head orientation with respect to transcription start sites. In medaka, we have demonstrated that 14 globin genes are located in 2 different clusters, and 5 pairs of the α- and ß-globin genes were found to be organized in a head-to-head orientation. The developmental expression patterns of the 11 globin genes were classified into 4 types. To clarify how their developmental stage-specific expressions are regulated, we produced 4 types of GFP- or RFP-transgenic medaka. Such transgenic medaka revealed that each of the 1-1.7 kbp 5' upstream sequences from respective globin genes possesses the ability to regulate the developmental stage-specific globin gene expression. In particular, the intervals between head-to-head α3 and ß3, and α4 and ß4 genes controlled the synchronized expression of the globin genes located at both sides of the intervals, which is significant to understand the mechanism by which equal amounts of α- and ß-globins are expressed in erythroid cells. We also demonstrated that the head-to-head intervals can control the expression of the globin genes located at both sides. These findings are significant to understand the mechanism by which α- and ß-globins are equally expressed in erythroid cells.

Environmental protection: researches in National Institute of Radiological Sciences.

Some studies for radiological protection of the environment have been made at the National Institute of Radiological Sciences (NIRS). Transfer of radionuclides and related elements has been investigated for dose estimation of non-human biota. A parameter database and radionuclide transfer models have been also developed for the Japanese environments. Dose (rate)-effect relationships for survival, growth and reproduction have been investigated in conifers, Arabidopsis, fungi, earthworms, springtails, algae, duckweeds, daphnia and medaka. Also genome-wide gene expression analysis has been carried out by high coverage expression profiling (HiCEP). Effects on aquatic microbial communities have been studied in experimental ecosystem models, i.e., microcosms. Some effects were detected at a dose rate of 1 Gy day(-1) and were likely to arise from interspecies interactions. The results obtained at NIRS have been used in development of frameworks for environmental protection by some international bodies, and will contribute to environmental protection in Japan and other Asian countries.

Attenuating effect of angiotensin-(1-7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-A(y)/Ta mice.

ANG-(1-7) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(1-7) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(1-7) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: 1) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(1-7) coinfusion group; and 4) ANG II+ANG-(1-7)+d-Ala(7)-ANG-(1-7) (A779) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(1-7), and A779. The ANG II+ANG-(1-7) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(1-7) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-κB and MAPK signaling activation was also attenuated by ANG-(1-7) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-ß1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(1-7)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy.