Predicting rTMS effect for deciding stimulation parameters.

Repetitive transcranial magnetic stimulation (rTMS) is used in the medical field to modulate cortical excitability. However, when applied in this setting, rTMS stimulation parameters are not usually decided objectively. The aim of this study is to make a model that predicts the rTMS effect, allowing stimulation parameters (intensity and pulse number) to be easily determined before use. First, we investigated the relationship between stimulation condition and rTMS outcome. rTMS delivered at 1 Hz was applied with stimulation intensities of 85%, 100%, or 115% resting motor threshold (RMT) over the primary motor cortex in the left hemisphere. Motor-evoked potentials (MEPs) were measured before rTMS and after every 200 rTMS pulses. Eighteen hundred pulses were applied for each stimulation condition. Results showed that more pulses and stronger intensities lead to a larger decrease in MEP amplitude. An initial prediction model was then made by applying multiple regression analysis over the experimental data. We then adjusted the model depending on the size of the initial MEP amplitude before rTMS, and confirmed the improvement.

Relationship between pulse number of rTMS and inter reversal time of perceptual reversal.

The aim of this study is to investigate the stimulus parameter which affects the repetitive Transcranial Magnetic Stimulation (rTMS) effect. It is said that the condition under 1Hz rTMS induces the inhibition effect. On the other hand, the condition over 1 Hz rTMS induces the facilitation effect. However the number of pulses of rTMS is also important factor. In this study, we focused on the number of pulses. We used the cognitive task of perceptual reversal and compared the rTMS effects of different condition under 1 Hz which is the inhibition condition. It has been known that the right superior parietal lobule (SPL) has a role in perceptual reversal. We applied rTMS over the SPL and measured the inter-reversal time (IRT) of perceptual reversal. The results showed that when 0.25 Hz 60 pulses, 0.5 Hz 60 pulses and 1 Hz 60 pulses of rTMS was applied over the right SPL, the IRT was significantly smaller. On the other hand, when 1 Hz 240 pulses of rTMS was applied over the right SPL, the IRT was significantly longer. When 0.25 Hz 12 0 pulses, 0.5 Hz 120 pulses and 1Hz 120 pulses of rTMS was applied over the right SPL, there were no significant differences. Furthermore, to investigate the rTMS effects, when rTMS are applied over the motor area, we measured the motor evoked potential (MEP). The more pulses of rTMS was applied, the smaller the amplitude of MEP became. From these results, it was found that the IRT of perceptual reversal and the amplitude of MEP primarily affected by the number of pulses of rTMS.

Time change of perceptual reversal of ambiguous figures by rTMS.

The aim of this study was to investigate the effect of stimulus frequency and number of pulses during rTMS (repetitive transcranial magnetic stimulation) on the phenomenon of perceptual reversal. Particularly, we focused on the temporal dynamics of perceptual reversal in the right SPL (superior parietal lobule), using the spinning wheel illusion. We measured the IRT (inter-reversal time) of perceptual reversal. To investigate whether stimulus frequency or the number of pulses is critical for the rTMS effect, we applied the following schedules over the right SPL and the right PTL (posterior temporal lobe): 0.25Hz 60 pulses, 0.25Hz 120pulses, 0.5Hz 120 pulses, and 1Hz 120 pulses biphasic rTMS at 90% of the resting motor threshold. As a control, we included a No-TMS condition. The results showed that rTMS with 0.25Hz 60 pulses over the right SPL caused shorter IRT. There were no significant differences between IRTs for rTMS with 0.25Hz 120 pulses, 0.5Hz 120 pulses or 1Hz 120 pulses over the right SPL. Comparing these results with those of a previous study, we found that an rTMS condition with 60 pulses causes shorter IRT; 240 pulses causes longer IRT; and 120 pulses does not change IRT. Therefore, when applying rTMS over the right SPL, the IRT of perceptual reversal is primarily affected by the number of pulses.

Direct evidence for susceptibility genes for type 2 diabetes on mouse chromosomes 11 and 14.

AIMS/HYPOTHESIS: Diabetogenic loci for type 2 diabetes have been mapped to mouse chromosome (Chr) 11 and 14 in the Nagoya-Shibata-Yasuda (NSY) mouse, an animal model of type 2 diabetes. We aimed to obtain direct evidence of these genes on each chromosome and to clarify their function and interaction in conferring susceptibility to type 2 diabetes. METHODS: We established three consomic strains homozygous for diabetogenic NSY-Chr11, NSY-Chr14 or both on the control C3H background (C3H-11(NSY), C3H-14(NSY) and C3H-11(NSY)14(NSY), respectively), and monitored diabetes-related phenotypes longitudinally. The glucokinase gene was sequenced as a positional candidate gene on Chr11. RESULTS: C3H-11(NSY) mice showed hyperglycaemia associated with impaired insulin secretion and age-dependent insulin resistance without obesity. C3H-14(NSY) mice exhibited hyperglycaemia mainly due to insulin resistance, with a slight increase in percentage body fat. C3H-11(NSY)14(NSY) double consomic mice showed marked hyperglycaemia and obesity, which was not observed in single consomic strains. Sequences of the glucokinase gene were allelically variant between NSY and C3H mice. CONCLUSIONS/INTERPRETATION: These data provide direct evidence that Chr11 and Chr14 harbour major susceptibility genes for type 2 diabetes. These two chromosomes interact to cause more severe hyperglycaemia and obesity, which was not observed with the presence of either single chromosome, indicating different modes of gene-gene interaction depending on the phenotype. Marked changes in the phenotypes retained in the consomic strains will facilitate fine mapping and the identification of the responsible genes and their interaction with each other, other genes and environmental factors.

Inactivation of hepatitis A virus by heat and high hydrostatic pressure: variation among laboratory strains.

BACKGROUND AND OBJECTIVES: Hepatitis A virus (HAV) transmission via contaminated blood products has been reported. Cell-adapted HAV strains are generally used to confirm virus inactivation in manufacturing blood products, but the strains may differ in their sensitivity to inactivation treatment. To select an appropriate cell-adapted HAV strain for virus validation, we compared the inactivation efficiency among four strains under two different physical inactivation treatments: heat and high hydrostatic pressure. MATERIALS AND METHODS: The cell-adapted HAV strains used here were KRM238, KRM003 (subgenotype IIIB), KRM031 (IA), and TKM005 (IB). The strains were treated at 60 degrees C for up to 10 h or under high hydrostatic pressure (up to 420 MPa). The reduction in HAV infectivity was measured by an immunofocus-staining method. RESULTS: The heat treatment at 60 degrees C for 10 h reduced HAV infectivity in the range of 3 to 5 log(10) among the strains; KRM238 and TKM005 were harder to inactivate than the other two. The high hydrostatic pressure treatment at 420 MPa also reduced infectivity in the range of 3 to 5 log(10) among the strains, and KRM031 was easier to inactivate than the other strains. CONCLUSION: Heat treatment and high hydrostatic pressure treatment revealed differences in inactivation efficiencies among cell-adapted HAV strains, and each strain reacted differently depending on the treatment. KRM238 may be the best candidate for virus validation to ensure the safety of blood products against viral contamination, as it is harder to inactivate and it replicates better in cell culture than the other strains.

Fatty liver and obesity: phenotypically correlated but genetically distinct traits in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Obesity and fatty liver are commonly associated with type 2 diabetes, but the genetic and functional bases linking fatty liver with obesity and diabetes are largely unknown. Our aim was to investigate the association of fatty liver with obesity and other diabetes-related phenotypes and to define the genetic control of obesity and fatty liver. MATERIALS AND METHODS: We established 306 F2 mice by crossing Nagoya-Shibata-Yasuda (NSY) mice, an animal model of type 2 diabetes, with control C3H mice, and analysed their phenotypes. Whole-genome screening of F2 mice was performed to identify the loci responsible for fatty liver and obesity. RESULTS: A strong association of fatty liver with obesity, hyperinsulinaemia and hyperglycaemia was observed in F2 mice. Using whole-genome screening in 306 F2 mice, we mapped a new locus for fatty liver (Fl1n) on chromosome 6 (maximum logarithm of odds score [MLS] 10.0) and one for body weight (Bw1n) on chromosome 7 (MLS 5.1). Fl1n was linked to epididymal fat weight as well as fatty liver, but its effects were opposite in the two tissues in that the NSY allele increased liver fat but decreased epididymal fat, suggesting a role of Fl1n in partitioning of fat mass. The sequence of peroxisome proliferator-activated receptor gamma (Pparg), a candidate for Fl1n, showed allelic variation between NSY and C3H mice. CONCLUSIONS/INTERPRETATION: These data suggest that fatty liver and obesity are phenotypically related but genetically independent. Loci homologous to Fl1n and Bw1n are good candidate genes for susceptibility to fatty liver and obesity in humans.

Allelic variation in class I K gene as candidate for a second component of MHC-linked susceptibility to type 1 diabetes in non-obese diabetic mice.

AIMS/HYPOTHESIS: Recent studies have revealed that MHC-linked susceptibility to Type 1 diabetes is determined by multiple components. In the non-obese diabetic (NOD) mouse, a second component (Idd16) has been mapped to a region adjacent to, but distinct from Idd1 in the class II region. In this study, we investigated the class I K gene as a candidate gene for Idd16. METHODS: We determined the genomic sequences of the class I K gene as well as the reactivity of K molecules with monoclonal antibodies in the NOD mouse, the Cataract Shionogi (CTS) mouse, and the NOD.CTS-H-2 congenic strain, which possesses a resistance allele to Type 1 diabetes at the Idd16 on the NOD genetic background genes. RESULTS: While the K sequence of the NOD mouse was identical to that of Kd type, ten nucleotide substitutions were identified in the CTS mouse compared with the NOD mouse. Of these, three were in exon 4, giving two amino acid substitutions, which were identical to those seen in KK type. These characteristics were retained in the NOD.CTS-H-2 congenic strain, which had a lower incidence and delayed onset of Type 1 diabetes owing to a resistance allele at Idd16. Lymphocytes from NOD.CTS-H2 congenic mice reacted with anti-Kd and anti-Kk monoclonal antibodies, reflecting the unique sequence of the K gene. The nucleotide sequence of the K gene in the non-obese non-diabetic (NON) mouse was also unique, consisting of a combination of Kk- and Kb-like sequences. CONCLUSIONS/INTERPRETATION: These data suggest that H2-K is unique in CTS and NON mice, and that allelic variation of the class I K gene may be responsible for Idd16.

Significance of fractionated irradiation for the biological therapeutic gain of carbon ions.

It is well established that the RBE (relative biological effectiveness) for cell killing depends on LET (linear energy transfer), and that a maximum RBE is observed at approximately 150 keV.micron-1. However, the therapeutic gain depends on the ratio of the RBEs for the effects on the cancer cell population and the effects on normal tissues. The RBE of a given radiation quality depends not only on LET but also on dose, biological system and effect, and irradiation conditions. There is no data available to answer the question: which LET is suitable to improve the biological therapeutic gain of carbon ions? Here, three different LET values of 290 MeV/u carbon ions were selected, and the relative biological effectiveness was compared between tumour-growth retardation and skin damage using a murine transplantable tumour. Larger RBE values for tumours after than the skin type were obtained when carbon ions of intermediate LET were delivered daily for 2 to 5 fractions. The biological therapeutic gain would be high for the carbon ion SOBP if the number of fractions were correctly selected in clinical trials.

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Lack of association between hepatocyte nuclear factor-1beta gene and common forms of type 2 diabetes in the Japanese population.

Mutations in the hepatocyte nuclear factor-1beta (HNF-1beta) gene have been shown to be a cause of maturity-onset diabetes of the young (MODY). We studied the contribution of the HNF-1beta gene to susceptibility to common forms of Type 2 diabetes in the genetically homogeneous Japanese population, by investigating the allelic association of Type 2 diabetes with two markers in the HNF-1beta region. The frequency of a nonsense mutation, R177X, which was previously reported in a Japanese family, was also studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using a mismatch primer. A total of 200 subjects were studied. There was no significant difference in allele frequencies of either of the two polymorphisms studied between patients with Type 2 diabetes and control subjects, or between subgroups of patients subdivided by the presence of mild or severe diabetic nephropathy. None of the subjects studied had R177X mutation, giving a frequency of less than 1.1% in common forms of Type 2 diabetes in Japan. These results suggest that mutations in the HNF-1beta gene derived from a limited number of founders are not a major cause of common forms of Type 2 diabetes, even in the genetically homogeneous Japanese population.

Insulin secretion to glucose as well as nonglucose stimuli is impaired in spontaneously diabetic Nagoya-Shibata-Yasuda mice.

To clarify the mechanisms of impaired insulin secretion in Nagoya-Shibata-Yasuda (NSY) mice, an inbred strain of mice with spontaneous development of type 2 (non-insulin-dependent) diabetes mellitus, the insulin response to glucose (5.5 to 27.8 mmol/L) and nonglucose stimuli (glibenclamide, arginine, and BayK8644, a Ca-channel opener) was studied in vitro using isolated islets from male NSY and control C3H/He mice at 36 weeks of age by the batch incubation method. Insulin response to 5.5 mmol/L glucose was not significantly different between NSY and C3H/He mice, but insulin response to a high concentration of glucose (> or = 11.1 mmol/L) was significantly smaller in NSY mice than in control C3H/He mice. The dose-response curve of insulin secretion showed a markedly reduced maximum response, but almost normal glucose sensitivity in NSY islets. Insulin responses to glibenclamide (1 mmol/L), arginine (20 mmol/L), and BayK8644 (0.1 mmol/L) were also significantly smaller in NSY mice than in C3H/He mice. Insulin content of islets, in contrast, was significantly higher in NSY mice than in C3H/He mice. The impaired insulin response to glucose and nonglucose stimuli together with higher insulin content in islets in the NSY mouse suggest that a defect in voltage-dependent Ca(2+)-channel or thereafter in the cascade of insulin secretion may be responsible for impaired insulin secretion in NSY mice. NSY mice, therefore, could be a novel animal model of type 2 diabetes with a defect in insulin secretion at a different site from that in previously known animal models.

Time course of reoxygenation in experimental murine tumors after carbon-beam and X-ray irradiation.

We compared the tumor reoxygenation patterns in three different murine tumor cell lines after X-irradiation with those after carbon-beam irradiation using a heavy-ion medical accelerator (HIMAC) system. The tumors of the cell lines SCCVII, SCCVII-variant-1 and EMT6 on the hind legs of mice received local priming irradiation with a carbon-beam (8 Gy, 73 keV/microm in LET, 290 MeV/u, 6 cm SOBP) or X-rays (13 Gy, 250 kVp). After various intervals, the mice were given whole-body test irradiation (16 Gy. 250 kVp X-ray) either in air or after they were killed. The hypoxic fractions were estimated as the proportions of the surviving fractions of the tumors in killed mice to those in air-breathing mice. In the SCCVII tumors, the hypoxic fractions at 0.5 h were 50% and 21% (p < 0.05) after the priming X-irradiation and carbon-beam irradiation, respectively. In the SCCVII-variant-1 tumors, the hypoxic fractions were 85% and 82% at 0.5 h, 84% and 20% at 12 h (p < 0.01), and 21% and 31% at 24 h after X-ray and after carbon-beam irradiation, respectively. In the EMT6 tumors, the reoxygenation patterns after X-irradiation and carbon-beam irradiation were quite similar. We concluded that the reoxygenation pattern differed among the three tumor cell lines, and that reoxygenation tended to occur more rapidly after carbon-beam irradiation than after X-irradiation for SCCVII and SCCVII-variant-1 tumors.

Sequence analysis of candidate genes for common susceptibility to type 1 and type 2 diabetes in mice.

Although type 1 and type 2 diabetes are regarded as clinically distinct diseases, several lines of evidence have suggested common genetic factors between the two types of diabetes. The non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, and the Nagoya-Shibata-Yasuda (NSY) mouse, a model of type 2 diabetes, are derived from the same outbred colony, Jcl:HCR, suggesting a shared susceptibility between the two types of diabetes in mice. Genetic as well as functional studies have supported the possibility that Tcf2, which encodes the transcription factor, hepatocyte nuclear factor 1beta (HNF-1beta), is a candidate gene for the common susceptibility between NSY and NOD mice. Txn, encoding thioredoxin which is a redox (reduction/oxidation)-active protein, is also a positional and functional candidate for a common susceptibility gene. To investigate whether either of these two genes is a common susceptibility gene, the coding nucleotide sequences of these two genes were compared among the NSY, NOD and control C3H strains. The coding sequence of Tcf2 of the NOD mouse was identical to that of the C3H mouse, but was different from that of the NSY mouse. The coding sequence of Txn was identical in the three strains. These data suggest that neither of the two genes is a common susceptiblity gene between type 1 and type 2 diabetes in mice.

A comparative study of Caucasian and Japanese mandibular clinical arch forms.

The purpose of this study was to clarify morphological differences between Caucasian and Japanese mandibular clinical arch forms in Class I, II, and III malocclusions. The study included 60 Class I, 50 Class II, and 50 Class III cases from each ethnic group. The most facial portion of 13 proximal contact areas was digitized from photocopied images of the mandibular dental arches. Clinical bracket points were calculated for each tooth based on mandibular tooth thickness data. Four linear and 2 proportional measurements were taken. The dental arches were classified into square, ovoid, and tapered forms to determine and compare the frequency distributions between the 2 ethnic groups. The Caucasian population had a statistically significant decreased arch width and increased arch depth compared with the Japanese population. When the subjects were regrouped by arch form, no statistically significant difference in arch dimension was observed between the 2 ethnic groups in any of the arch form samples. Our results suggest that there is no single arch form specific to any of the Angle classifications or ethnic groups. It appears to be the frequency of a particular arch form that varies among Angle classifications or ethnic groups.

Relative biological effectiveness of the 235 MeV proton beams at the National Cancer Center Hospital East.

A therapy-dedicated cyclotron was installed in the National Cancer Center Hospital East (NCCHE) at Kashiwa in 1997. Prior to the start of clinical use, we investigated the biological effectiveness of therapeutic proton beams for cell lethality. The proton beams accelerated up to 235 MeV were horizontally extracted from the cyclotron, and scattered by a bar-ridge filter to produce a Spread-Out-Bragg-Peak (SOBP) of 10-cm width. The biological systems used here were mouse intestinal crypt cells and three in vitro cell lines, including SCC61 human squamous cell carcinoma, NB1RGB human fibroblasts and V79 Chinese hamster cells. The dose responses after irradiation at either the entrance plateau or the middle portion of SOBP were compared with those after linac 6 MV X-ray irradiation. The fit of a linear quadratic model to survival curves showed that proton irradiation increased the alpha value of SCC61 and the beta value of V79 cells with a least change for alpha/beta ratio of NB1RGB cells. The isoeffect dose that reduces either cell survivals to 10% or mouse jejunum crypts to 10 per circumference was termed D10. The relative biological effectiveness (RBE) of protons obtained by comparing the D10 values between protons and X-rays ranged from 0.9 to 1.2. The depth distribution of cell lethality was measured by replating V79 cells after irradiation from a "cell stack chamber" that received a single dose of 7 Gy at the middle position of SOBP. The thus-obtained cell survivals at various depths coincided well with the estimated survivals, but tended to decrease at the distal end of SOBP. We conclude that an RBE of 1.1 would be appropriate for 235 MeV proton beams at the NCCHE.

Mapping and promoter sequencing of HNF-1beta gene in diabetes-prone and -resistant mice.

By using a novel single nucleotide polymorphism (SNP) in the coding sequence, the chromosomal location of Tcf2, encoding hepatic nuclear factor (HNF)-1beta, was determined in F2 intercrosses between Nagoya-Shibata-Yasuda (NSY) mice, an animal model of type 2 diabetes, and control C3H/He mice. The promoter region of Tcf2 gene was sequenced in NSY, non-obese diabetic (NOD) and control C3H/He mice. Tcf2 was mapped between genetic markers D11MIT320 and D11MIT195 with the following distances: D11MIT320-(7.3 cM)-Tcf2-(0.5 cM)-D11MIT195. A variant with insertion of C between -205 and -204 in the promoter region of Tcf2 was identified in NSY mice, but not NOD and C3H/He mice.