Copy number variation at 22q11.2: from rare variants to common mechanisms of developmental neuropsychiatric disorders.

Recently discovered genome-wide rare copy number variants (CNVs) have unprecedented levels of statistical association with many developmental neuropsychiatric disorders, including schizophrenia, autism spectrum disorders, intellectual disability and attention deficit hyperactivity disorder. However, as CNVs often include multiple genes, causal genes responsible for CNV-associated diagnoses and traits are still poorly understood. Mouse models of CNVs are in use to delve into the precise mechanisms through which CNVs contribute to disorders and associated traits. Based on human and mouse model studies on rare CNVs within human chromosome 22q11.2, we propose that alterations of a distinct set of multiple, noncontiguous genes encoded in this chromosomal region, in concert with modulatory impacts of genetic background and environmental factors, variably shift the probabilities of phenotypes along a predetermined developmental trajectory. This model can be further extended to the study of other CNVs and may serve as a guide to help characterize the impact of genes in developmental neuropsychiatric disorders.

Commensal microbiota modulate murine behaviors in a strictly contamination-free environment confirmed by culture-based methods.

BACKGROUND: There is increasing evidence suggesting the existence of an interaction between commensal microbiota, the gut and the brain. The aim of this study was to examine the influence of commensal microbiota on the host behaviors in a contamination-free environment, which was verified by culture-based methods. METHODS: Open-field and marble-burying tests were used to analyze anxiety-like behaviors and locomotor activity in gnotobiotic BALB/c mice with a common genetic background in a sterile isolator. The monoamine levels in several regions of the brain were measured in germfree (GF) mice and commensal fecal microbiota-associated mice (EX-GF). KEY RESULTS: A 24-h exposure to the environment outside the sterile isolators rendered GF mice less anxious than those not contaminated, while there was no change in the locomotion. EX-GF mice, the gnotobiotic mice with normal specific pathogen-free microbiota, were less anxious and active than GF mice using open-field and marble-burying tests. The norepinephrine, dopamine, and serotonin turnover rates were higher in the EX-GF mice than in the GF mice in most regions of the brain, suggesting that monoaminergic neurotransmission might increase in the EX-GF mice comparing the GF mice. Monoassociation with Brautia coccoides reduced the anxiety level, but it did not affect the locomotor activity. In contrast, colonization with Bifidobacterium infantis decreased the locomotor activity, while having little effect on the anxiety level. CONCLUSIONS & INFERENCES: These results strongly support the current view that gut microorganisms modulate brain development and behavior.

Gramine increase associated with rapid and transient systemic resistance in barley seedlings induced by mechanical and biological stresses.

Systemic acquired resistance (SAR) is one of the intriguing issues for studying the mechanism in signal transduction system in a whole plant. We found that SAR and increase of an antifungal compound were induced rapidly and transiently in barley (Hordeum vulgare L. cv. Goseshikoku) by mechanical and biological stresses. One of the major antifungal compounds was identified as an indole alkaloid, gramine (N,N-dimethyl-3-aminomethylindole), by mass spectrum and NMR analyses. Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) and wheat (B. graminis f.sp. tritici). However, in the leaf detached from unwounded seedlings or in the leaf inoculated with the barley powdery mildew fungus, gramine did not increase at all. In the water droplets contacted with barley leaves, the amount of leaked gramine increased dependently upon the time after the seedling was injured mechanically. We also found a tight correlation between gramine increase and enhancement of resistance to the barley powdery mildew fungus in barley leaves treated with an endogenous elicitor. Furthermore, such a systemic resistance was not observed in a barley cultivar Morex that lacks the biosynthetic pathway of gramine. From these results, we conclude that gramine is the excellent marker in rapid and transient systemic acquired resistance in barley.

A novel human rad54 homologue, Rad54B, associates with Rad51.

Members of the SNF2/SWI2 family, characterized with sequence motifs similar to those found in DNA and RNA helicases, play roles in various aspects of cellular fundamental processes such as transcriptional regulation, chromosome stability, nucleotide excision repair, and recombination. We have isolated a novel member of the human SNF2/SWI2 family, RAD54B, which is highly homologous to mammalian RAD54. The RAD54 gene is a member of the RAD52 epistasis group which is involved in the recombinational repair of DNA damage. Here we demonstrate that human Rad54B (hRad54B), like human Rad54 (hRad54), associates with human Rad51 (hRad51). Both hRad54B and hRad54 associate with hRad51 through their NH(2)-terminal domains, but there are differences in their ways of association with hRad51. In contrast to Rad54, whose association with Rad51 is induced by ionizing radiation, Rad54B associates with Rad51 constitutively in immunoprecipitation experiments. Also, the failure to detect the interaction between hRad54B and hRad51 in the yeast two-hybrid assay suggests that their interaction, unlike that between hRad54 and hRad51, may be indirect. Immunofluorescence microscopy revealed that hRad54B formed nuclear foci that colocalized with hRad51, hRad54, and BRCA1. These findings suggest that Rad54B may be functionally distinct from Rad54, although it may play an active role in recombination processes in concert with other members of the RAD52 epistasis group.

Extracellular ATP inhibits starvation-induced apoptosis via P2X2 receptors in differentiated rat pheochromocytoma PC12 cells.

Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explored the effects of extracellular ATP on starvation-induced apoptosis in rat pheochromocytoma PC12 cells. Incubation of differentiated PC12 cells with ATP for 6h suppressed apoptosis. 2-Methylthio-ATP, a P2 purinoceptor agonist, was as potent as ATP in suppressing apoptosis, whereas adenosine, ADP, alpha,betamethylene-ATP or UTP was totally ineffective. The suppressive action of ATP was dependent upon the presence of extracellular Ca2+ and blocked by co-incubation with the P2 antagonist, suramin. DNA ladder formation, a typical symptom of apoptosis in starved cells, was inhibited by ATP, 2-methylthio-ATP but not by UTP. These results suggest that the inhibitory action of extracellular ATP on apoptotic cell death is mediated via the activation of P2X2 receptors in differentiated PC12 cells.

[Inflammatory pseudotumor of the lung with infiltration identified radiographically and hemoptysis].

The patient was a 53-year-old man whose chief complaint was a dry cough and hemoptysis. Chest X-ray films and computed tomographic scans revealed a hazy shadow with unclear margins in the left middle lobe. A diagnosis of inflammatory pseudotumor was made because transbronchial lung biopsy specimens showed proliferation of plasma cells and lymphocytes. Initially, in response to steroid therapy, his clinical condition and pulmonary infiltration improved, but about 30 days after the start of steroid therapy, hemoptysis occurred and the shadow increased, and therefore left segmentectomy was performed. Histology revealed not only proliferation of acute and chronic inflammatory cells but also changes in the vessels. These pathologic findings were considered to be related to the infiltration shadows and the hemoptysis.

The role of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 genotypes in alcohol-induced vasospastic angina.

Alcohol ingestion often provokes attacks in patients with vasospastic angina. Type 2 aldehyde dehydrogenase (ALDH2) deficiency, which is based on a single point mutation (Glu487Lys) of the ALDH2 gene, is common in the Japanese population, but rare among the Caucasian population. We investigated how the genotype of ALDH2 affects the characteristics of alcohol-induced vasospastic angina. Ninety-one patients with vasospastic angina who had ingested alcohol daily or occasionally were studied. Patients had been diagnosed as vasospastic angina by a provocation test with an intracoronary injection of ergonovine or acetylcholine during coronary angiography. The Glu487Lys mutation was detected by allele specific PCR. We interviewed the patients to obtain information concerning the relationship between alcohol ingestion and anginal attacks. Alcohol ingestion induced attacks in 16 of 66 patients without the Glu487Lys mutation, 8 of 22 in heterozygotes, and 1 of 3 in mutant homozygotes. The intervals between alcohol ingestion and the onset of anginal attacks were shorter in homozygotes (0.17 hours) and heterozygotes (1.5+/-0.6 hours) for ALDH2*2 than in normal homozygotes for ALDH2*1 (5.4+/-0.6 hours). The amount of ethanol which induced attacks was significantly greater in normal homozygotes than in homozygotes (11 ml) and heterozygotes (42.5+/-7.1 ml) for ALDH2*2 (96.1+/-13.4 ml in normal patients). The frequency of anginal attacks induced by alcohol ingestion did not differ between ALDH deficient and normal homozygotes. In ALDH deficient patients, however, anginal attacks were induced by a smaller amount of alcohol immediately after its ingestion. Thus, the ALDH2 genotype modifies the characteristics of the anginal attacks as a co-factor for the induction of vasospastic angina after alcohol ingestion.

[Radiotherapy alone for elderly patients with stage III non-small cell lung cancer].

We undertook a retrospective study of elderly patients with stage III non-small cell lung cancer who had been treated solely with radiotherapy during the period 1986 to 1995. Our study was designed to assess the influence of age on survival and malnutrition in patients aged 75 years or older (elderly group) and patients aged 74 years or younger (younger group). Radiotherapy alone resulted in a median survival period of 11.5 months in the younger group and 6.3 months in the elderly group (p = 0.0043). With the Cox multivariate model, good performance status, age less than 75 years, and good response were significant favorable independent predictors. Furthermore, the elderly group patients more frequently died of respiratory infections and had lower prognostic nutritional indexes than the younger group patients before and after radiotherapy. These findings suggested elderly patients with stage III non-small cell lung cancer who had been treated with radiotherapy alone had a poor prognosis and that malnutrition caused by radiotherapy was a factor contributing to the risk of death from respiratory infection in such patients.

Mutations of a novel human RAD54 homologue, RAD54B, in primary cancer.

Association of breast tumor susceptibility gene products BRCA1 and BRCA2 with the RAD51 recombination protein suggested that cancer could arise through defects in recombination. The identification of NBS1, responsible for Nijmegen breakage syndrome, from the MRE11/RAD50 recombination protein complex also supports this hypothesis. However, our mutation analysis revealed that known members of the RAD52 epistasis group are rarely mutated in human primary cancer. Here we describe the isolation of a novel member of the SNF2 superfamily, characterized with sequence motifs similar to those in DNA and RNA helicases. The gene, designated RAD54B, is significantly homologous to the RAD54 recombination gene. The expression of RAD54B was high in testis and spleen, which are active in meiotic and mitotic recombination. These findings suggest that RAD54B may play an active role in recombination processes in concert with other members of the RAD52 epistasis group. RAD54B maps to human chromosome 8q21.3-q22 in a region associated with cancer-related chromosomal abnormalities. Homozygous mutations at highly conserved positions of RAD54B were observed in human primary lymphoma and colon cancer. These findings suggest that some cancers arise through alterations of the RAD54B function.

[So-called benign metastasizing leiomyoma of the lung presenting with bone metastases].

The patient, a 42-year-old woman, was admitted to our hospital because of abnormal shadows on chest X-ray films obtained during a routine medical check-up. Her medical history included a uterine myomectomy at the age of 21, and thereafter, periodic lumbago and back pain for which she had not sought any medical treatment. Chest computed tomography detected a number of pulmonary nodules in both whole lung fields, and magnetic resonance imaging revealed many spherical metastatic lesions in the thoracic and lumbar vertebrae. Although we initially suspected lung cancer, no primary lesion was found. A thoracoscopic lung biopsy revealed leiomyomatous tumors that were histologically similar to the uterine myoma removed 21 years previously. The final diagnosis was so-called benign metastasizing leiomyoma (BML). Because the removed tumor contained a high concentration of progesterone receptors (240 fmol/mg), a gonadotropin-releasing hormone analogue was administered, and proved effective in relieving the patient's periodic lumbago and back pain. The findings in this case suggested that the so-called BML was in fact a metastasis of a low-grade uterine leiomyosarcoma.

[Concurrent high-dose thoracic irradiation plus daily low-dose cisplatin and vindesine in locally advanced unresectable stage III non-small cell lung cancer].

Patients with unresectable non small-cell lung cancer were treated to evaluate the toxicity and efficacy of high-dose thoracic irradiation (RT) combined with concurrent daily cisplatin plus vindesine. Fourteen evaluable patients with unresectable stage III non small-cell lung cancer treated with continuous-course RT (70 Gy in 35 fractions of 2 Gy once daily) and concurrent daily intravenous cisplatin (6 mg/m2) plus vindesine (3 mg/m2 on day 1 and day 8). The objective response rate was 86%, and two patients achieved a radiographic complete response. Leukocytopenia was the severe toxicity, but there were no episodes of discontinuation of treatment. Only one patient had grade 3 acute radiation esophagitis. Ten patients experienced late radiation pneumonitis and nine of those had grade 1 or grade 2. There was only one life-threatening case of toxicity (grade 5 pneumonitis). We concluded that the regimen of high-dose thoracic RT combined with concurrent daily cisplatin plus vindesine obtained a high response rate. Further testing on late toxicities and survival time is required.

Colocalization of scavenger receptor in CD68 positive foam cells in verruciform xanthoma.

Verruciform xanthoma is a rare variant of xanthoma that appears predominantly on oral mucosa and the anogenital area. The histologic features are characterized by marked accumulation of foam cells in the papillary dermis with verrucous epidermal acanthosis and hyperkeratosis. However, little is known of the nature or origin of foam cells. Recent studies have emphasized the crucial role of macrophage scavenger receptors in the formation of foam cells in atherogenesis. We examined the immunohistologic localization of scavenger receptors in genital verruciform xanthoma. We found that the massively infiltrated foam cells in the papillary dermis were CD68+ monocyte-macrophage lineage cells, and that the majority of CD68+ cells coexpressed scavenger receptors. The in situ staining pattern of scavenger receptor in foam cells was mainly of an intracytoplasmic vacuolar pattern similar to that of dermal resident macrophages found in normal skin. Furthermore, CD1a+ Langerhans cells, completely negative for scavenger receptor, were markedly decreased in number in verruciform xanthoma. These results indicate that scavenger receptor-bearing CD68+ cells are also actively involved in the development of cutaneous verruciform xanthoma, as has been shown to be the case in atherogenesis.

Abnormalities of pulmonary function in patients with non-insulin-dependent diabetes mellitus.

To examine the possible association between the vascular complications of diabetes and changes in pulmonary function, we performed pulmonary function tests including assessment of the diffusing capacity (%DLco) in 80 patients with non-insulin-dependent diabetes mellitus (45 males and 35 females) without overt lung or heart disease. The mean age of the subjects was 57.9 years and the mean duration of diabetes was 10.8 years. The %DLco decreased significantly as the duration of diabetes increased (r = -0.38, p less than 0.01), and the same relationship was also observed in non-smoking subjects (N = 37). The reduction in %DLco was greater in patients with diabetic microangiopathy (especially nephropathy) and in those treated with insulin. Other pulmonary function tests (%VC, FEV1.0, PaO2 and PaCO2) showed no relationship to the duration of diabetes, the degree of microangiopathy or the type of treatment. These results suggest that diabetic microangiopathy may play an important role in the decrease of %DLco.

Responses of atrium and ventricle to sustained sympathetic nerve stimulation.

To determine whether chronotropic and atrial inotropic responses to sympathetic nerve stimulation are maintained longer than ventricular inotropic response, the present study was performed with control and acute reserpinized dogs. We stimulated the right stellate ganglion of both groups supramaximally for 60 min and compared right atrial responses (chronotropism and inotropism) with left ventricular (LV) dP/dtmax. In the control group, heart rate (HR) immediately increased and was only slightly attenuated with 60 min of stimulation, and right atrial (RA) inotropic response was less attenuated than was LV response (7% in HR, 33% in RA dP/dtmax, 50% in LV dP/dtmax, P less than 0.01, from the peak value of each response). RA and LV norepinephrine (NE) content was decreased by the stimulation but remained higher than the LV control value. In the reserpinized group, NE content in the RA was low before the stimulation and was further decreased by the stimulation. In this group, HR response was attenuated (27% in HR, P less than 0.01) as was LV dP/dtmax, and the difference in contractile responsiveness between atrium and ventricle disappeared (58% in RA dP/dtmax vs. 61% in LV dP/dtmax, NS). The results indicate that the chronotropic response was only slightly attenuated and that the atrial contractile response was attenuated less than the ventricular response, with sustained sympathetic nerve stimulation in the normal heart. This can be ascribed to the much higher NE content in the RA than that in the LV.

Influence of reduced presynaptic myocardial norepinephrine stores on left ventricular contractility.

Many investigators have reported that myocardial norepinephrine content is decreased in congestive heart failure. However there have been no studies of how decrease in myocardial norepinephrine might influence myocardial contraction. To clarify whether decreased myocardial norepinephrine per se affects myocardial contraction, we observed the change in left ventricular contractility during 30 min of left stellate ganglion stimulation in control and acutely reserpinized dogs. We obtained left ventricular max dp/dt and left ventricular end-systolic pressure-segment length relationships as indicators of left ventricular contractility. Both parameters decreased after left stellate ganglion stimulation in reserpinized dogs (left ventricular max dp/dt: 2064 +/- 200 to 1608 +/- 168 mmHg/s, left ventricular end-systolic pressure-segment length slope 117 +/- 22 to 79 +/- 14 mmHg/mm, n = 8, P less than 0.05), while they did not change in controls. In reserpinized dogs, left ventricular norepinephrine content decreased to one-third that of controls before the stimulation, and further decreased after stimulation. These data indicate that lowered myocardial norepinephrine itself may be responsible for the negative effect on left ventricular contractility in congestive heart failure.

Myocardial alpha 1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolism. Species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium.

Species-dependent variations of myocardial alpha 1-adrenoceptor-mediated positive inotropic effects of epinephrine were assessed in relation to characteristics of alpha 1-receptor bindings and acceleration of phosphatidylinositol metabolism in the isolated rat, rabbit, and dog ventricular myocardium. Epinephrine in the presence of the beta-adrenoceptor antagonist bupranolol (10(-6) M) elicited a positive inotropic effect through activation of alpha 1-adrenoceptors in rat and rabbit, whereas in dog ventricular myocardium, bupranolol abolished the positive inotropic effect of epinephrine. [3H]Prazosin bound to membrane fractions derived from rat, rabbit, and dog ventricular muscle with high affinities in a saturable and reversible manner. In dog, Bmax and Kd values of alpha 1-adrenoceptor binding sites were identical to those in rabbit ventricular muscle. The Bmax value of alpha 1-adrenoceptors in rat ventricle was the highest, amounting to two to four times those in rabbit and dog. Epinephrine displacement curves for the specific binding of [3H]prazosin in the membrane fraction of these species showed high and low affinity sites with slope factors significantly less than unity, which were shifted to single low affinity sites with slope factors close to unity by addition of 5'-guanylylimidodiphosphate. Accumulation of [3H]inositol 1-phosphate [( 3H]IP1) in ventricular slices prelabeled with [3H]myo-inositol was increased by epinephrine in a time- and concentration-dependent manner in rat ventricular slices. [3H]IP1 accumulation likewise was facilitated by alpha 1-adrenoceptor stimulation in rabbit ventricular slices, whereas the extent of [3H]IP1 accumulation was much less than that in rat. In dog ventricular slices, [3H]IP1 was not accumulated by epinephrine. In rabbit papillary muscle, the time course of increase in contractile force induced by alpha-adrenoceptors coincided with the prolongation of the action potential duration with a similar time course, which is in strong contrast to previous findings in rat that the contractile response was dissociated from the electrophysiological response to alpha-adrenoceptor stimulation. The present results indicate that a wide range of variation of alpha 1-adrenoceptor-mediated regulation of myocardial contractility may be ascribed to different contributions of facilitatory as well as inhibitory regulatory processes that lead to intracellular Ca2+ mobilization subsequent to myocardial alpha 1-adrenoceptor activation among mammalian species.

Pulmonary lymphangiomyomatosis.

Two cases of pulmonary lymphangiomyomatosis are reported and findings of high resolution computed tomography (CT) are described. CT reveals that most lesions appearing reticular or emphysematous on radiographs are actually cysts, and accurately displays the extent and distribution of cystic change of the lung. On high resolution CT, individual cystic walls are much better displayed than on routine 10 mm section CT. Further, it is possible to detect even trivial pleural effusion and mediastinal lymph node swelling by CT.

The preferential inhibition of alpha 1- over beta-adrenoceptor-mediated positive inotropic effect by organic calcium antagonists in the rabbit papillary muscle.

Experiments were carried out to elucidate the mechanism that the positive inotropic effect mediated by alpha 1-adrenoceptors is more susceptible to organic calcium antagonists than the beta-adrenoceptor-mediated effect. Verapamil and diltiazem displaced the specific binding of [3H]prazosin to the membrane fraction derived from the rabbit ventricular myocardium, verapamil being about 70 times more potent than diltiazem. Nifedipine did not displace the binding. While these compounds suppressed the positive inotropic effect mediated via alpha 1-adrenoceptors in a concentration-dependent manner, there was no correlation between the potency of the compounds to displace the [3H]prazosin binding and to inhibit the alpha-mediated positive inotropic effect. The relative potency of three calcium antagonists to decrease the basal force of contraction and the alpha 1-mediated effect (of the same extent as compared to basal force of contraction) was consistent to each other. The positive inotropic effect mediated by beta-adrenoceptors was inhibited much less, and was enhanced by low concentrations of organic calcium antagonists. The differential action of calcium antagonists on the alpha- and beta-mediated positive inotropic effect was mimicked by lowering the extracellular calcium concentration to 1/2, 1/4 and 1/8 of that in normal Krebs-Henseleit solution (2.5 mmol/l). These results indicate that the alpha 1-adrenoceptor blocking activity does not play an essential role for the preferential inhibition of alpha-mediated positive inotropic effect by organic calcium antagonists. Difference in the subcellular mechanism involved in mobilization of intracellular Ca2+ subsequent to alpha 1- and beta-adrenoceptor activation may be responsible for the differential inhibitory action of calcium antagonists in the rabbit heart.