Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.

Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.

Prenatal one-carbon metabolism dysregulation programs schizophrenia-like deficits.

The methionine-folate cycle-dependent one-carbon metabolism is implicated in the pathophysiology of schizophrenia. Since schizophrenia is a developmental disorder, we examined the effects that perturbation of the one-carbon metabolism during gestation has on mice progeny. Pregnant mice were administered methionine equivalent to double their daily intake during the last week of gestation. Their progeny (MET mice) exhibited schizophrenia-like social deficits, cognitive impairments and elevated stereotypy, decreased neurogenesis and synaptic plasticity, and abnormally reduced local excitatory synaptic connections in CA1 neurons. Neural transcript expression of only one gene, encoding the Npas4 transcription factor, was >twofold altered (downregulated) in MET mice; strikingly, similar Npas4 downregulation occurred in the prefrontal cortex of human patients with schizophrenia. Finally, therapeutic actions of typical (haloperidol) and atypical (clozapine) antipsychotics in MET mice mimicked effects in human schizophrenia patients. Our data support the validity of MET mice as a model for schizophrenia, and uncover methionine metabolism as a potential preventive and/or therapeutic target.

A genome-wide association study identifies two novel susceptibility loci and trans population polygenicity associated with bipolar disorder.

Genome-wide association studies (GWASs) have identified several susceptibility loci for bipolar disorder (BD) and shown that the genetic architecture of BD can be explained by polygenicity, with numerous variants contributing to BD. In the present GWAS (Phase I/II), which included 2964 BD and 61 887 control subjects from the Japanese population, we detected a novel susceptibility locus at 11q12.2 (rs28456, P=6.4 × 10-9), a region known to contain regulatory genes for plasma lipid levels (FADS1/2/3). A subsequent meta-analysis of Phase I/II and the Psychiatric GWAS Consortium for BD (PGC-BD) identified another novel BD gene, NFIX (Pbest=5.8 × 10-10), and supported three regions previously implicated in BD susceptibility: MAD1L1 (Pbest=1.9 × 10-9), TRANK1 (Pbest=2.1 × 10-9) and ODZ4 (Pbest=3.3 × 10-9). Polygenicity of BD within Japanese and trans-European-Japanese populations was assessed with risk profile score analysis. We detected higher scores in BD cases both within (Phase I/II) and across populations (Phase I/II and PGC-BD). These were defined by (1) Phase II as discovery and Phase I as target, or vice versa (for 'within Japanese comparisons', Pbest~10-29, R2~2%), and (2) European PGC-BD as discovery and Japanese BD (Phase I/II) as target (for 'trans-European-Japanese comparison,' Pbest~10-13, R2~0.27%). This 'trans population' effect was supported by estimation of the genetic correlation using the effect size based on each population (liability estimates~0.7). These results indicate that (1) two novel and three previously implicated loci are significantly associated with BD and that (2) BD 'risk' effect are shared between Japanese and European populations.

Abnormal asymmetries in subcortical brain volume in schizophrenia.

Subcortical structures, which include the basal ganglia and parts of the limbic system, have key roles in learning, motor control and emotion, but also contribute to higher-order executive functions. Prior studies have reported volumetric alterations in subcortical regions in schizophrenia. Reported results have sometimes been heterogeneous, and few large-scale investigations have been conducted. Moreover, few large-scale studies have assessed asymmetries of subcortical volumes in schizophrenia. Here, as a work completely independent of a study performed by the ENIGMA consortium, we conducted a large-scale multisite study of subcortical volumetric differences between patients with schizophrenia and controls. We also explored the laterality of subcortical regions to identify characteristic similarities and differences between them. T1-weighted images from 1680 healthy individuals and 884 patients with schizophrenia, obtained with 15 imaging protocols at 11 sites, were processed with FreeSurfer. Group differences were calculated for each protocol and meta-analyzed. Compared with controls, patients with schizophrenia demonstrated smaller bilateral hippocampus, amygdala, thalamus and accumbens volumes as well as intracranial volume, but larger bilateral caudate, putamen, pallidum and lateral ventricle volumes. We replicated the rank order of effect sizes for subcortical volumetric changes in schizophrenia reported by the ENIGMA consortium. Further, we revealed leftward asymmetry for thalamus, lateral ventricle, caudate and putamen volumes, and rightward asymmetry for amygdala and hippocampal volumes in both controls and patients with schizophrenia. Also, we demonstrated a schizophrenia-specific leftward asymmetry for pallidum volume. These findings suggest the possibility of aberrant laterality in neural pathways and connectivity patterns related to the pallidum in schizophrenia.

Possible association of CUX1 gene polymorphisms with antidepressant response in major depressive disorder.

Association between response to antidepressant treatment and genetic polymorphisms was examined in two independent Japanese samples of patients with major depressive disorder (MDD). Genome-wide approach using the Illumina Human CNV370-quad Bead Chip was utilized in the analysis of the 92 MDD patients in the first sample. In all, 11 non-intergenic single-nucleotide polymorphisms with uncorrected allelic P-value <0.0001 were selected for the subsequent association analyses in the second sample of 136 MDD patients. Difference in allele distribution between responders and nonresponders were found in the second-stage sample for rs365836 and rs201522 of the CUX1 gene (P=0.005 and 0.004, respectively). The allelic P-values for rs365836 and rs201522 in both samples combined were 0.0000023 and 0.0000040, respectively. Our results provide the first evidence that polymorphisms of the CUX1 gene may be associated with response to antidepressant treatment in Japanese patients with MDD.

Allosteric modulators of the δ GABAA receptor subtype demonstrate a therapeutic effect in morphine-antinociceptive tolerance and withdrawal in mice.

The present study evaluated the effects of compounds targeting extrasynaptic δ subunit-containing γ-aminobutyric acid type A receptors (δ*-GABAARs) to interrogate the role of tonic inhibition in the development of antinociceptive tolerance caused by repeated morphine administration. We investigated the effect of subchronic or acute treatment with non-steroidal positive allosteric modulators (PAMs) of δ*-GABAARs, such as 2-261, on the morphine-antinociceptive tolerance. Mice were treated twice daily with morphine for 9 days and antinociception was measured using the hot water tail immersion test. Co-treatment with 2-261 and morphine prevented morphine-antinociceptive tolerance and acute administration of 2-261 on day 9 was sufficient to reverse the tolerance. Other compounds with activity at δ*-GABAARs also reversed morphine tolerance, whereas an enaminone that lacked activity at δ*-GABAARs did not. Acute administration of 2-261 did not cause an additive or synergistic antinociceptive effect when combined with an acute submaximal dose of morphine. We then used Cre/LoxP recombination to generate GABAA δ-subunit knockout mice to corroborate the pharmacological results. Observations of male δ-knockout mice demonstrated that the δ*-GABAARs was necessary for 2-261 modulation of both analgesic tolerance and somatic withdrawal symptoms produced by subchronic morphine. While female mice still benefited from the positive effects of 2-261, the δ-subunit was not necessary for these effects, highlighting a distinction of the different pathways that could have implications for some of the sex-related differences seen in human opioid-induced outcomes. Consequently, subtype-specific allosteric modulators of GABAARs may warrant further investigation as pharmacological targets to manage tolerance and withdrawal from opioids.

How many well vs. unwell days can you expect over 10 years, once you become depressed?

OBJECTIVE: Prognostic studies of major depression have mainly focused on episode remission and relapse, and only a limited number of studies have examined long-term course of depressive symptomatology at threshold and subthreshold levels. METHOD: The Group for Longitudinal Affective Disorders Study has conducted prospective serial assessments of a cohort of heretofore untreated major depressive episodes for 10 years under naturalistic conditions. RESULTS: Of the 94 patients in the cohort, the follow-up rate was 70% of the 11,280 person-months. Around 77% of the follow-up months were spent in euthymia, 16% in subthreshold depression and 7% in major depression. Duration of the index episode before reaching recovery was the only significant predictor of the ensuing well time. CONCLUSION: On average, patients with major depression starting treatment today may expect to spend three quarters of the next decade in euthymia but the remaining one quarter in subthreshold or threshold depression.

The role of cysteinyl-LT(1)receptor (CysLT(1)R) in renal ischemia-reperfusion injury.

The pathogenesis of ischemia-reperfusion (I/R) injury is known to involve cytokines and particularly surface adhesion molecules, the expression of which initiates the attachment of inflammatory cells. Renal I/R injury, a clinically important problem, is an invariable consequence of renal transplantation. The problem begins at the onset of acute tubular necrosis (ATN), when the transplantation includes a long ischemic interval or by use of a cardiac arrest donor's kidney. The cysteinyl leukotriene-1 (CysLT(1)), a potent lipid mediator in allergic disease, acts through the CysLT(1)R receptor. We researched the expression of CysLT(1)R in rat renal I/R injury as well as correlations with the degree of ATN. The right kidney was harvested and the left renal artery and vein were clamped at laparotomy. The kidney was reperfused after 90 minutes of ischemia; rats were sacrificed at 0, 3, 5, 12, and 24 hours after reperfusion. CysLT(1)R expression was analyzed by immunohistochemistry. CysLT(1)R expression was observed only in endothelial cells of a normal kidney. CysLT(1)R expression was most intense on endothelial cells at 3 hours after reperfusion, and CysLT(1)R expression on endothelial cells gradually became weaker. Twelve hours after reperfusion, ATN extended throughout the ischemic kidney. Renal I/R injury gradually progressed at time after reperfusion. Several hours after the maximal CysLT(1)R expression, we observed the maximum renal I/R injury.

Definitions of recovery and outcomes of major depression: results from a 10-year follow-up.

OBJECTIVE: Consensus operational definitions for symptomatic remission and recovery of a major depressive episode have been proposed but only irregularly followed. METHOD: We examined the predictive validity of different definitions of recovery in a multi-center 10-year follow-up study of an inception cohort of untreated unipolar major depressive episodes (n = 95). Time to recovery and time to recurrence after recovery were estimated by Kaplan-Meier survival analyses for alternative definitions requiring 2, 4, 6 or 12 months of remission to declare recovery. RESULTS: The median time to recovery was 3.0, 4.0, 4.0 and 12.0 months respectively. The index episode lasted longer than 24 months in 9.4%, 9.2%, 12.6% and 24.5%. The median time to subthreshold recurrence was 16.0, 32.0, 42.0 and 74.0 months. CONCLUSION: Either 4- or 6-month duration of remission defined a change point before which the episode was continuous and after which the recurrence was reasonably unlikely.

Study of cysteinyl leukotriene-1 receptor in rat renal ischemia-reperfusion injury.

Renal ischemia-reperfusion (I/R) injury is a major cause of renal transplant dysfunction. Recent studies of I/R injury have focused on the function of neutrophils, the mechanisms of action of inflammatory cytokines, and oxygen free radicals, as well as other mediators. However, few reports address the cysteinyl leukotriene-1 receptor (CysLT1R), an important mediator of bronchial asthma in human beings. We examined the expression of CysLT1R in rat renal I/R injury. At laparotomy, the right kidney was harvested and the left renal artery and vein were clamped. The kidney was reperfused after 90 minutes of ischemia, and the rats were killed after 0, 3, 5, 12, or 24 hours. Expression of CysLT1R analyzed at immunohistochemistry was observed only in endothelial cells in nonischemic kidney. At 0 to 3 hours after reperfusion, CysLT1R expression on endothelial cells gradually became stronger, being most intense at 3 hours after reperfusion. Twelve hours after reperfusion, necrosis extended throughout the ischemic kidney; nearly all of the tubular epithelial cells were destroyed. At 3 to 12 hours after reperfusion, CysLT1R expression gradually became weaker on endothelial cells. At 24 hours after reperfusion, CysLT1R expression was almost at the level of that in nonischemic kidney. Expression of CysLT1R was noted in a rat model of renal I/R injury. Several hours after the maximal CysLT1R expression, we observed the maximum renal I/R injury. These results may suggest a relationship between the CysLT1R and renal I/R injury.

A novel approach to successful ABO-incompatible high-titer renal transplantation.

BACKGROUND: Currently the long-term outcome among recipients of ABO-incompatible renal transplantations is excellent in Japan. However, previous reports have documented poor outcomes in patients with high (> 1:256) anti-A/B antibody titers pretreatment. The immunosuppressive protocol for ABO-incompatible high-titer renal transplantation has remained a medical challenge. METHODS: We treated 3 patients with high (> 1:512) anti-A/B antibody titers prior to ABO-incompatible renal transplantation. Our immunosuppressive protocol was initiated 1 month prior to surgery and included mycophenolate mofetil (1 g/d) and low-dose steroid (methylprednisolone [8 mg/d]). Two doses of the anti-CD20 antibody rituximab, (150 mg/m2) were administered 2 weeks before and on the day of transplantation. We performed antibody removal with 6 to 8 sessions of plasmapheresis (plasma exchange or double-filtration plasmapheresis) before transplantation. Splenectomy was also performed on the day of transplantation. Postoperative immunosuppression followed the same regimen as ABO-compatible cases, in which calcineurin inhibitors were initiated 3 days before transplantation combined with 2 doses of basiliximab. RESULT: With this protocol, the anti-A/B antibody was reduced to below 1:16 in all cases. All 3 patients underwent successful transplantation with a mean current serum creatinine of 1.32 mg/dL (range, 1.22-1.50 mg/dL). There were no episodes of antibody-mediated rejection. No serious complications or side effects were encountered. CONCLUSIONS: A preconditioning protocol consisting of rituximab infusions, splenectomy, plasmapheresis, and pharmacologic immunosuppression enabled ABO-incompatible renal transplantation in patients with high (> 1:512) anti-A/B antibody titer.

A carboxy-terminal truncation of human alpha-galactosidase A in a heterozygous female with Fabry disease and modification of the enzymatic activity by the carboxy-terminal domain. Increased, reduced, or absent enzyme activity depending on number of amino acid residues deleted.

Fabry disease is an X-linked disorder of glycosphingolipid metabolism caused by a deficiency of alpha-galactosidase A (alpha-Gal A). We identified a novel mutation of alpha-Gal A gene in a family with Fabry disease, which converted a tyrosine at codon 365 to a stop and resulted in a truncation of the carboxy (C) terminus by 65 amino acid (AA) residues. In a heterozygote of this family, although the mutant and normal alleles were equally transcribed in cultured fibroblasts, lymphocyte alpha-Gal A activity was approximately 30% of the normal control and severe clinical symptoms were apparent. COS-1 cells transfected with this mutant cDNA showed a complete loss of its enzymatic activity. Furthermore, those cotransfected with mutant and wildtype cDNAs showed a lower alpha-Gal A activity than those with wild type alone (approximately 30% of wild type alone), which suggested the dominant negative effect of this mutation and implied the importance of the C terminus for its activity. Thus, we generated mutant cDNAs with various deletion of the C terminus, and analyzed. Unexpectedly, alpha-Gal A activity was enhanced by up to sixfold compared with wild-type when from 2 to 10 AA residues were deleted. In contrast, deletion of 12 or more AA acid residues resulted in a complete loss of enzyme activity. Our data suggest that the C-terminal region of alpha-Gal A plays an important role in the regulation of its enzyme activity.

Fluctuating serotonergic function in premenstrual dysphoric disorder and premenstrual syndrome: findings from neuroendocrine challenge tests.

RATIONALE: Premenstrual dysphoric disorder (PMDD) has been assumed to be a subtype of premenstrual syndrome (PMS) with depressive symptoms, such as depressive mood, tension, anxiety, and mood liability during luteal phase. At present, no conclusion has been established about serotonergic function in PMDD. OBJECTIVE: The purpose of this study was to investigate the serotonergic function of PMDD subjects in comparison to PMS without PMDD subjects and normal controls via neuroendocrine challenge tests. SUBJECTS AND METHODS: Twenty-four women (seven with PMDD, eight with PMS without PMDD, and nine normal controls) were tested on three occasions (follicular phase, early luteal phase, and late luteal phase) receiving paroxetine 20 mg orally as a serotonergic probe at 8:00 A: .M: . Plasma ACTH and cortisol were measured prior to the administration and every hour for 6 h thereafter. RESULTS: As a whole, there were significant differences in serotonergic function measured by ACTH and cortisol responses to paroxetine challenge across these three groups. PMDD subjects showed higher serotonergic function in follicular phase but lower serotonergic function in luteal phase, compared with women with PMS without PMDD and normal controls. CONCLUSION: The present findings suggest that PMDD women have fluctuating serotonergic function across their menstrual cycles and that the pattern may be different from PMS without PMDD.

Estimation of whole-body radiation exposure from brachytherapy for oral cancer using a Monte Carlo simulation.

Early stage oral cancer can be cured with oral brachytherapy, but whole-body radiation exposure status has not been previously studied. Recently, the International Commission on Radiological Protection Committee (ICRP) recommended the use of ICRP phantoms to estimate radiation exposure from external and internal radiation sources. In this study, we used a Monte Carlo simulation with ICRP phantoms to estimate whole-body exposure from oral brachytherapy. We used a Particle and Heavy Ion Transport code System (PHITS) to model oral brachytherapy with 192Ir hairpins and 198Au grains and to perform a Monte Carlo simulation on the ICRP adult reference computational phantoms. To confirm the simulations, we also computed local dose distributions from these small sources, and compared them with the results from Oncentra manual Low Dose Rate Treatment Planning (mLDR) software which is used in day-to-day clinical practice. We successfully obtained data on absorbed dose for each organ in males and females. Sex-averaged equivalent doses were 0.547 and 0.710 Sv with 192Ir hairpins and 198Au grains, respectively. Simulation with PHITS was reliable when compared with an alternative computational technique using mLDR software. We concluded that the absorbed dose for each organ and whole-body exposure from oral brachytherapy can be estimated with Monte Carlo simulation using PHITS on ICRP reference phantoms. Effective doses for patients with oral cancer were obtained.

PCLO rs2522833-mediated gray matter volume reduction in patients with drug-naive, first-episode major depressive disorder.

Major depressive disorder (MDD) has been linked to differences in the volume of certain areas of the brain and to variants in the piccolo presynaptic cytomatrix protein (PCLO), but the relationship between PCLO and brain morphology has not been studied. A single-nucleotide polymorphism (SNP) in PCLO, rs2522833, is thought to affect protein stability and the activity of the hypothalamic-pituitary-adrenal axis. We investigated the relationship between cortical volume and this SNP in first-episode, drug-naive patients with MDD or healthy control subjects. Seventy-eight participants, including 30 patients with MDD and 48 healthy control subjects, were recruited via interview. PCLO rs2522833 genotyping and plasma cortisol assays were performed, and gray matter volume was estimated using structural magnetic resonance images. Among the individuals carrying the C-allele of PCLO rs2522833, the volume of the left temporal pole was significantly smaller in those with MDD than in healthy controls (family-wise error-corrected, P=0.003). No differences were detected in other brain regions. In addition, the C-carriers showed a larger volume reduction in the left temporal pole than those in the individuals with A/A genotype (P=0.0099). Plasma cortisol levels were significantly higher in MDD-affected C-carriers than in the healthy control C-carriers (12.76±6.10 vs 9.31±3.60 nm, P=0.045). We conclude that PCLO SNP rs2522833 is associated with a gray matter volume reduction in the left temporal pole in drug-naive, first-episode patients with MDD carrying the C-allele.

Treatment with edaravone improves the survival rate in renal warm ischemia-reperfusion injury using rat model.

Renal ischemia-reperfusion (I/R) injury during renal transplantation is a significant cause of renal dysfunction. The pathological role of free radicals in this process is a major concern. We investigated the effect of a free radical scavenger, edaravone (MCI-186), in renal I/R injury. Male Lewis rats (270 to 320 g) were used for the model. The right kidney was harvested and left renal artery and vein were clamped as laparotomy. The kidney was reperfused after 90 minutes of ischemia. Edaravone (10 mg/kg) was delivered intravenously before ischemia and after reperfusion to prevent the neutrophil activation. In the nontreatment I/R group, no rat survived beyond 4 days. However, in the edaravone I/R treatment group, one among five rats survived more than 7 days. These results suggested that treatment with edaravone ameliorated renal I/R injury, and that the agent has the potential to ameliorate preservation injury in renal transplantation.

Benefical effect of neutrophil elastase inhibitor on renal warm ischemia-reperfusion injury in the rat.

Renal ischemia-reperfusion (I/R) injury is a significant problem in renal transplantation. Neutrophils play an important role in renal I/R injury. Several reports have demonstrated that neutrophil elastase derived from the activated neutrophils might play an important role in this injury. We investigated the effect of a neutrophil elastase inhibitor in renal I/R injury. Male Lewis rats (270-320 g) were used in the model. The right kidney was harvested and the left renal artery and vein were clamped at laparotomy. The kidney was reperfused after 90 minutes of ischemia. Neutrophil elastase inhibitor (ONO-5046: 30 mg/kg) was delivered intravenously before ischemia and after reperfusion to prevent neutrophil activation. In the nontreatment I/R group, no hosts survived 4 days. However, after treatment with neutrophil elastase inhibitor, 3 of 10 rats in the I/R group, survived more than 7 days. These results demonstrated that treatment with neutrophil elastase inhibitor ameliorated renal I/R injury.

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells.

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region a fragment from -1645 to -1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from -1605 to -1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.

Impact of natural IRS-1 mutations on insulin signals: mutations of IRS-1 in the PTB domain and near SH2 protein binding sites result in impaired function at different steps of IRS-1 signaling.

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and M209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and M209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in [3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and M209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and M209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and M209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.

The risk of radiation-induced cancer in patients with squamous cell carcinoma of the head and neck and its results of treatment.

The purpose of this study was to determine the incidence and the results of treatment of cancer induced by radiotherapy for early stage (stage I and II) squamous cell carcinoma of the head and neck (SCH). The clinical records of 355 patients with early stage malignant lymphoma of the head and neck region treated by radiotherapy were reviewed, and then the records of 1358 patients with early stage SCH (oral cavity, 956; larynx, 154; oropharynx, 110; maxillary sinus, 86; lip, 20; epipharynx, 17; hypopharynx, 15) who underwent radiotherapy were reviewed. The disease-specific 10-year survival rate of the patients with 355 malignant lymphoma was 61%, and 5 cases of radiation-induced cancer occurred more than 8 years after irradiation. The crude incidence of radiation-induced cancer in the malignant lymphoma patients was 1.4%, and the 10-year probability by the actuarial life table method was 0.8%. The 10-year survival rate of the early stage SCH patients was 71%. The crude incidence of a second cancer in a previously irradiated field after an 8-year latent period (SCI) in the SCH patients was 1.8% (25/1358), and the 10-year probability was 1.6%. 12 SCIs were treated by surgery and 8 of those 12 patients (67%) resulted in success, whereas treatment by radiation resulted in failure in every other case. The risk of SCIs in the SCH group was higher than in the early stage malignant lymphoma group, although the difference was not statistically significant. The possibility of radiation-induced cancer in SCH is small, and the advantage of radiation therapy compares favourably with the risks of other treatments.