Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin.

AIMS/HYPOTHESIS: The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function. METHODS: Isolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1(-/-)) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells. RESULTS: BetaRac1(-/-) mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1(-/-) mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion. CONCLUSIONS/INTERPRETATION: In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.

N = 16 spherical shell closure in 24O.

The unbound excited states of the neutron drip-line isotope 24O have been investigated via the 24O(p,p')23O + n reaction in inverse kinematics at a beam energy of 62 MeV/nucleon. The decay energy spectrum of 24O* was reconstructed from the momenta of 23O and the neutron. The spin parity of the first excited state, observed at E(x) = 4.65±0.14 MeV, was determined to be J(π) = 2+ from the angular distribution of the cross section. Higher-lying states were also observed. The quadrupole transition parameter ß2 of the 2(1)+ state was deduced, for the first time, to be 0.15±0.04. The relatively high excitation energy and small ß2 value are indicative of the N = 16 shell closure in 24O.

Structural evolution in the neutron-rich nuclei ¹°6Zr and ¹°8Zr.

The low-lying states in ¹°6Zr and ¹°8Zr have been investigated by means of ß-γ and isomer spectroscopy at the radioactive isotope beam factory (RIBF), respectively. A new isomer with a half-life of 620 ± 150 ns has been identified in ¹°8Zr. For the sequence of even-even Zr isotopes, the excitation energies of the first 2⁺ states reach a minimum at N = 64 and gradually increase as the neutron number increases up to N = 68, suggesting a deformed subshell closure at N = 64. The deformed ground state of ¹°8Zr indicates that a spherical subshell gap predicted at N = 70 is not large enough to change the ground state of ¹°8Zr to the spherical shape. The possibility of a tetrahedral shape isomer in ¹°8Zr is also discussed.

ß-decay half-lives of very neutron-rich Kr to Tc isotopes on the boundary of the r-process path: an indication of fast r-matter flow.

The ß-decay half-lives of 38 neutron-rich isotopes from (36)Kr to (43)Tc have been measured; the half-lives of (100)Kr, (103-105)Sr, (106-108)Y, (108-110)Zr, (111,112)Nb, (112-115)Mo, and (116,117)Tc are reported here. The results when compared with previous standard models indicate an overestimation in the predicted half-lives by a factor of 2 or more in the A≈110 region. A revised model based on the second generation gross theory of ß decay better predicts the measured half-lives and suggests a more rapid flow of the rapid neutron-capture process (r-matter flow) through this region than previously predicted.

Halo structure of the island of inversion nucleus 31Ne.

The cross sections for single-neutron removal from the very neutron-rich nucleus 31Ne on Pb and C targets have been measured at 230 MeV/nucleon using the RIBF facility at RIKEN. The deduced large Coulomb breakup cross section of 540(70) mb is indicative of a soft E1 excitation. Comparison with direct-breakup model calculations suggests that the valence neutron of 31Ne occupies a low-l orbital (most probably 2p(3/2)) with a small separation energy (S(n) approximately < 0.8 MeV), instead of being predominantly in the 1f(7/2) orbital as expected from the conventional shell ordering. These findings suggest that 31Ne is the heaviest halo system known.

Standardization of (18)F using the 4pi(beta+gamma) integral counting technique.

Alpha 4pi(beta+gamma) integral counting technique using a 4pibeta-4pigamma detector configuration was adopted for the standardization of (18)F. In this technique, the beta-detector is composed of two thin plastic scintillators sandwiching the source, coupled with a slender photomultiplier tube. The beta-detector part with the source was inserted into a large well-type NaI(Tl) scintillation detector for gamma-ray detection, making a 4pibeta-4pigamma coincidence counting system. In this work, positron particles were detected with high efficiency in the beta-channel and annihilation quanta were also detected with high efficiency in the 4pigamma channel. The very small inefficiency of the 4pi(beta+gamma) integral counter for the beta-plus branch has been confirmed by EGS5 Monte Carlo simulation. The result using this technique agreed within the uncertainties with the result obtained by the conventional 4pibeta-gamma coincidence counting with the efficiency extrapolation technique using the same detector configuration and a conventional 4pibeta-gamma coincidence counter.

Observation of beta-ray spectra after penetrating absorbing materials.

beta-Ray spectra after penetrating absorbing materials of various thicknesses were observed by the use of a scintillation-type beta-ray spectrometer equipped with a flat NE-102 plastic scintillator of 5mm thickness for sources of (60)Co, (90)Sr-(90)Y, (137)Cs, (147)Pm and (204)Tl. Although the spectra changed rapidly with increasing absorber thickness, the average beta-ray energy was kept nearly constant for a wider range. These results are consistent in that the beta-ray absorption curve becomes quasi-linear in a semi-logarithmic plot. Spectra including scattered beta-rays from several materials placed behind the source were also measured for (137)Cs and (204)Tl. It may be concluded that mean energy measurements by the use of beta-ray spectrometer of this kind is useful for the identification of nuclides in radiation protection purposes even in worse source-conditions.

[Long term outcome of arterial switch surgery for transposition of the great arteries: evaluation of the reconstruction of the pulmonary artery].

We assessed the effect of reconstructing the pulmonary artery during arterial switch surgery for transposition of the great arteries on late pulmonary stenosis. Sixty-five patients who underwent Lecompte procedure between September 1991 and December 2006 were divided, by the procedure used chronologically to reconstruct the pulmonary artery, into group XP (single pantaloon patch with equine pericardium, n = 11), group P (direct reconstruction, n = 47), and group AP (single pantaloon patch with fresh autopericardium, n = 7). Outcome and pulmonary stenosis on the most recent ultrasound cardiography (UCG) were compared in the 3 groups. The median follow-up was 13, 7.5, and 1.3 years, respectively. Both early and late mortalities were 1.5% (1/65). Although percutaneous trans-pulmonary angioplasty was necessary in 1, 13, and 3 patients, there was 1, 1, and 0 reoperation for pulmonary stenosis in the 3 groups, respectively. Pulmonary stenosis (pulmonary arterial maximum flow velocity > 3 m/sec on UCG) was present in 4 (40%). 14 (30%). and 3 patients (43%). Although there was no significant difference among the 3 procedures in preventing pulmonary stenosis 10 years after arterial switch surgery, direct reconstruction of the pulmonary artery may show a superior outcome, in particular, over 10 years after arterial switch surgery.

4πß(PS)-4πγ(GE) list-mode coincidence counter and its applications.

We developed a 4πß-4πγ counter composed of a 4π plastic scintillation detector and a well-type Ge detector, employing digital coincidence counting and data storage in list-mode. In both of the ß- and γ-channels, the amplified pulses from a linear amplifier feed the input channel of the digitizer directly via delay circuits. A signal from the peak-hold of each channel is fed to a sliding scale ADC (14bits, 200MHz clock) after peak detection and converted into 13bit digital data, registered along with a time stamp and event channel allowing various data analysis to be implemented offline. When employing multiple gamma window settings, a weighted average of each apparent efficiency might be introduced to improve the efficiency functions. This idea was investigated along with reasonable estimates of the weighing factors, and activity measurements of (59)Fe using this system are presented.

Activity measurement of (68)Ge-(68)Ga by use of 4π(ß(+)+γ) integral counting method.

A 4π(ß(+)+γ) integral counting method using 4πß-4πγ detector configuration composed of a large well type NaI(Tl) scintillation detector and stacked plastic scintillators positioned in the center of the well and coupled with a slender PMT was adopted for activity measurement of (68)Ge-(68)Ga. Several source preparation schemes were studied to reduce the activity loss due to volatility. The possible contribution of EC events were rejected with pulse-height discrimination. Owing to the high counting efficiencies in both channels and the multiplicity of photons and ß-particles emitted, the 4π(ß(+)+γ) integral counting system gives a count rate very nearly equal to the positron emission rate. The activity can be determined simply from this value divided by the positron emission branching ratio. The remaining overall inefficiency was evaluated by the EGS5 code.

A novel compound heterozygous mutation in the steroidogenic acute regulatory protein gene in a patient with congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomal recessive disorder characterized by impaired synthesis of all adrenal and gonadal steroid hormones. Recently, it was reported that mutations in the steroidogenic acute regulatory protein (StAR) gene cause CLAH. In the present study, we have analyzed the StAR gene of a Japanese patient with CLAH. PCR amplification and subsequent nucleotide sequencing of the StAR gene and those of her parents revealed that the patient has a compound heterozygous mutation of this gene. In one allele, an undescribed G to C transversion in codon 217, which occurred at the last base of exon 5 and thus altered the splice donor site sequence, apparently resulted in a substitution of Arg to Thr (AGG to ACG: R217T), and in the other allele, a C to T transition in codon 218 caused a substitution of Ala to Val (GCG to GTG: A218V), which has been previously shown to abolish StAR activity. In vitro expression analysis of an allelic minigene that consists of exons 4-6 of the R217T mutant StAR gene showed that the G to C transversion in the splice donor site of exon 5 caused by the R217T mutation disrupts normal splicing, resulting in the complete skipping of exon 5, which alters the translation reading frame of exon 6, introduces a stop codon at amino acid position 174, and thus impairs the activity. A functional expression study of the R217T replacement mutant revealed that the mutant has no steroidogenesis-enhancing activity if the transcript of the R217T mutant allele is ever spliced normally and translated into the protein. From the genetic analysis of 50 healthy subjects, the novel R217T mutation was unlikely to be due to polymorphism. Together, these results indicate that this patient is a compound heterozygote for the mutation in the StAR gene (T217R and A218V) and that these mutations inactivate the StAR function and give rise to clinically manifest CLAH.

Increased plasma cholesteryl ester transfer activity in obese children.

To determine whether enhanced activity of cholesteryl ester transfer protein (CETP) contributes to the development of atherogenic lipoprotein profiles in obese children, plasma CETP activity was assayed according to a micro-method, by co-incubating lipoprotein-deficient samples with exogenous donor and acceptor lipoproteins. The study subjects were 31 obese children (14 males and 17 females). Serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), TC:high-density lipoprotein (HDL)-C, LDL-C:HDL-C, apolipoprotein (apo) B, and apo B:apo Al were increased in obese children. Thus they appeared to exhibit an atherogenic lipoprotein profile, with a relative decrease in cholesterol carried by HDL compared with the cholesterol in the other lipoprotein fractions. The mean fasting plasma insulin level was also increased. CETP activity was significantly higher in the obese children than in nonobese control children, and was correlated with LDL-C, TC:HDL-C, LDL-C:HDL-C, and apo B:apo Al. These results suggest that an increase in plasma CETP activity results in atherogenic change in lipoprotein metabolism in obese children. The increase in CETP may be due to the adiposity or insulin resistance. Alternatively, dyslipidemia per se, physical inactivity or excessive fat intake, that are commonly found in obese children, may contribute to the increase in CETP activity.

Cellular basis of skin allograft rejection in mice: specific lysis of allogeneic skin components by non-T cells.

BACKGROUND: It has been generally assumed that CD8+ T cells mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4+ T cells. However, both the generation of CD4- or CD8-deficient mice and adoptive transfer experiments with CD4+ T cells from CD8-deficient mice demonstrate that noncytotoxic CD4+ T cells alone are sufficient to induce skin or organ allograft rejection. Furthermore, we have reported that the major effector cells responsible for allografted-tumor (e.g., Meth A) rejection are allograft-induced macrophages (AIM) with MHC haplotype specificity. METHODS: We characterized the macrophages migrating into the rejection site of allografted skin by immunohistochemical and in situ hybridization analyses using an antibody (K16.5) specific for AIM and a cDNA (pK30) encoding the antigen. To determine the in situ effector cells responsible for the rejection, we prepared both effector cells and target cells from the graft-graft bed border. RESULTS: The macrophages seemed to be morphologically (monocytic), phenotypically (K16.5+/pK30+), and functionally (cytotoxic against Meth A cells) AIM. The AIM population in bulk infiltrates taken from the rejection site was cytotoxic against allografted, but not self, skin components (e.g., fibroblasts, myocytes, endothelial cells, and epithelial cells). In contrast, other types of infiltrating cells including lymphocytes and granulocytes were virtually inactive toward these targets, and NK-1.1+ cells hardly infiltrated into the rejection site. CONCLUSIONS: These data suggest that the major effector cells mediating allografted skin rejection are AIM and not T cells.

Association study of structural mutations of the tyrosine hydroxylase gene with schizophrenia and Parkinson's disease.

Tyrosine hydroxylase (TH) gene is the rate-limiting enzyme in the synthesis of catecholamines. Functional polymorphisms of the TH gene may be involved in the pathogenesis of neuropsychiatric diseases such as schizophrenia, affective disorders, and Parkinsonism. This study examined a possible association of two polymorphisms, both of which result in an amino acid change of the TH protein, with schizophrenia and Parkinson's disease (PD). The Val81Met polymorphism is a common variation, although its effect on the enzyme expression is unclear. Leu205Pro polymorphism is a rare mutation that is reported to cause Parkinsonism in infancy for individuals who are homozygous for the mutated type. We genotyped a Japanese sample of 194 schizophrenics, 99 patients with PD, and 161 controls for the Val81Met polymorphism by using mis-match PCR and digestion by the restriction enzyme BalI. There was no significant allelic or genotypic association of the Val81Met polymorphism with schizophrenia or PD. The Leu205Pro polymorphism was examined by using PCR and digestion by AluI; however, there was no individual who carried the mutated type of Pro205 among 50 schizophrenics or 50 patients with PD. Thus we obtained no evidence for the involvement of the two structural mutations of the TH gene in the pathogenesis of schizophrenia or PD.

Significance of AgNOR counts for distinguishing carcinoma from adenoma and hyperplasia in parathyroid gland.

Nucleolar organizer region proteins, which can be stained and visualized by an argyrophil technique (AgNORs), are markers of cell activities, such as DNA transcription and proliferation, and they are useful for differential diagnosis between benign and malignant tumors. We counted both AgNOR numbers in 25 parathyroid lesions (three carcinomas, 11 adenomas, 10 hyperplasias, and one hyperplasia with carcinoma) to determine if the AgNOR number could be useful as a diagnostic aid in parathyroid neoplasms and hyperplasias, because it is often difficult to histopathologically distinguish among these lesions. The AgNOR numbers were significantly higher in carcinomas (3.18 +/- 0.05) than in adenomas (1.67 +/- 0.30, P < .001) or hyperplasias (1.85 +/- 0.16, P < .001), but there was no significant difference between adenomas and hyperplasias. These results suggest that AgNORs may be useful as an adjunct to discriminating carcinomas from adenomas or hyperplasias in the parathyroid gland.

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii.

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.

Neuropeptide levels in discrete brain regions in the iminodipropionitrile-induced persistent dyskinesia rat model.

To clarify the role of neuropeptides in dyskinesia induced by iminodipropionitrile (IDPN), the levels of five representative neuropeptides were examined in discrete regions of the rat brain 4 weeks after intraperitoneal injection of IDPN. The five neuropeptides examined were methionine-enkephalin (Met-Enk), substance P (SP) and somatostatin, which are closely related to extrapyramidal function, and thyrotropin-releasing hormone (TRH) and cholecystokinin octapeptide (CCK-8), which are closely related to the neural mechanism of the dopamine system. IDPN pretreatment significantly increased Met-Enk in the basal ganglia but not SP or somatostatin; however, all three neuropeptide levels were increased in the hindbrain. In IDPN-treated rats, TRH and CCK-8 levels were increased in the nucleus accumbens, and the frontal cortical CCK-8 level was extremely increased. These findings, together with previous reports, suggest that neuropeptides in the basal ganglia, hindbrain and cerebral cortex play important roles in the manifestation of dyskinetic symptoms.

Endoscopic incision of persistent ureteral infoldings.

We report 2 cases of persistent ureteral infolding in a four-month-old infant and an eight-year-old boy, both presenting with hydronephrosis. Initial diagnostic evaluation showed multiple pleats in the upper ureter. Endoscopic incision of the pleats relieved hydronephrosis. The concept of persistent ureteral infolding seems to apply to these cases.

HLA-A and B antigens in patients with Peyronie disease.

Using microdroplet lymphocyte cytotoxicity test, as a method of HLA typing, 9 patients with Peyronie disease were studied from a viewpoint of relationship between this disease and HLA-A and B antigens. The frequencies of each antigen in the patients were compared with those of healthy controls. No significant difference of distribution in HLA antigens was observed between the patients and the control group.

Müllerian duct cyst extending into the abdomen.

Müllerian duct cysts occupying the pelvic/abdominal region are rare. We describe a müllerian duct cyst extending into the lower abdomen in a 47-year-old man complaining of urinary retention. We removed the cyst through a suprapubic retrovesical approach. No malignancy was found in the surgical specimen.