Exome sequencing for bipolar disorder points to roles of de novo loss-of-function and protein-altering mutations.

Although numerous genetic studies have been conducted for bipolar disorder (BD), its genetic architecture remains elusive. Here we perform, to the best of our knowledge, the first trio-based exome sequencing study for BD to investigate potential roles of de novo mutations in the disease etiology. We identified 71 de novo point mutations and one de novo copy-number mutation in 79 BD probands. Among the genes hit by de novo loss-of-function (LOF; nonsense, splice site or frameshift) or protein-altering (LOF, missense and inframe indel) mutations, we found significant enrichment of genes highly intolerant (first percentile of intolerant genes assessed by Residual Variation Intolerance Score) to protein-altering variants in general population, an observation that is also reported in autism and schizophrenia. When we performed a joint analysis using the data of schizoaffective disorder in published studies, we found global enrichment of de novo LOF and protein-altering mutations in the combined group of bipolar I and schizoaffective disorders. Considering relationship between de novo mutations and clinical phenotypes, we observed significantly earlier disease onset among the BD probands with de novo protein-altering mutations when compared with non-carriers. Gene ontology enrichment analysis of genes hit by de novo protein-altering mutations in bipolar I and schizoaffective disorders did not identify any significant enrichment. These results of exploratory analyses collectively point to the roles of de novo LOF and protein-altering mutations in the etiology of bipolar disorder and warrant further large-scale studies.

Oral administration of a recombinant cholera toxin B subunit promotes mucosal healing in the colon.

Cholera toxin B subunit (CTB) is a component of a licensed oral cholera vaccine. However, CTB has pleiotropic immunomodulatory effects whose impacts on the gut are not fully understood. Here, we found that oral administration in mice of a plant-made recombinant CTB (CTBp) significantly increased several immune cell populations in the colon lamina propria. Global gene expression analysis revealed that CTBp had more pronounced impacts on the colon than the small intestine, with significant activation of TGFß-mediated pathways in the colon epithelium. The clinical relevance of CTBp-induced impacts on colonic mucosa was examined. In a human colon epithelial model using Caco2 cells, CTBp, but not the non-GM1-binding mutant G33D-CTBp, induced TGFß-mediated wound healing. In a dextran sodium sulfate (DSS) acute colitis mouse model, oral administration of CTBp protected against colon mucosal damage as manifested by mitigated body weight loss, decreased histopathological scores, and blunted escalation of inflammatory cytokine levels while inducing wound healing-related genes. Furthermore, biweekly oral administration of CTBp significantly reduced disease severity and tumorigenesis in the azoxymethane/DSS model of ulcerative colitis and colon cancer. Altogether, these results demonstrate CTBp's ability to enhance mucosal healing in the colon, highlighting its potential application in ulcerative colitis therapy besides cholera vaccination.

Effects of calcium and bile acid feeding on colon tumors in the rat.

The hypothesis that dietary calcium alters the incidence of colorectal neoplasms was examined in an established model of carcinogenesis. Male Fischer 344 rats (28 days old) were quarantined for 2 weeks. All animals were fed the basal diet (AIN-76) supplemented with cholic acid (0.2%) and/or calcium (1.6%, corresponding to a 3-fold increase above that of the basal diet). N-Methyl-N-nitrosourea (MNU) (2 mg/dose) or saline (control) was given intrarectally to all animals on days 1 and 4 of the experiment. Groups 1-8 were fed the experimental diets concurrently with the first dose of MNU, while groups 9 and 10 were fed the diets 2 weeks prior to MNU (or saline). All animals were sacrificed after 28 weeks. No tumors were observed in the groups given saline (groups 1, 3, 5, 7, and 9). In groups receiving MNU, the addition of cholic acid to the diet (group 4) caused a significant increase in tumors (80% versus 55%), tumors/animal ratio (2.24 versus 0.75), and tumors/tumor-bearing animal ratio (2.80 versus 1.36), group 4 versus group 2, respectively. Increased dietary calcium did not inhibit tumor formation; 68% of animals in groups 6 and 10 had tumors. The combination of dietary cholic acid and calcium (group 8) gave a tumor incidence similar to cholic acid (group 4) alone (72% versus 80%, 2.00 versus 2.24 tumors/animal; 2.77 versus 2.80 tumors/tumor-bearing animal). Most tumors were adenomatous polyps but carcinomas in situ and invasive carcinomas were also present; dietary calcium reduced the number of invasive carcinomas (group 6 versus group 2, P less than 0.04).

A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery.

In this report, we deal with the isolation of a novel vasorelaxing peptide from a chymotryptic digest of ovalbumin and its vasorelaxing activities. This peptide is composed of Arg-Ala-Asp-His-Pro-Phe (RADHPF) in its sequence, corresponding to residues 359-364 of ovalbumin. This peptide (30-300 microM) exerted a dose-dependent vasodilation in an isolated mesenteric artery from a spontaneously hypertensive rat which was pre-constricted by phenylephrine, besides the relaxation being endothelium-dependent. It is noteworthy that the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester inhibited this relaxation, implying involvement of nitric oxide in its mechanism of action. Following oral administration of RADHPF at a dose of 10 mg/kg, the systolic blood pressure in a spontaneously hypertensive rat was significantly lowered.

Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin(2-7).

The potent anti-hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2-7). The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean beta-conglycinin alpha' subunit. The native alpha' subunit as well as the modified, RPLKPW-containing alpha' subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion-exchange chromatography. The RPLKPW peptide was released from recombinant RPLKPW-containing alpha' subunit after in vitro digestion by trypsin and chymotrypsin. Moreover, the undigested RPLKPW-containing alpha' subunit given orally at a dose of 10 mg/kg exerted an anti-hypertensive effect in spontaneously hypertensive rats, unlike the native alpha' subunit. These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site-directed mutagenesis could practically function in vivo even at a low dose.

SPECT images of technetium-99m-ethyl cysteinate dimer in cerebrovascular diseases: comparison with other cerebral perfusion tracers and PET.

UNLABELLED: To assess the clinical role of 99mTc-ethyl cysteinate dimer (ECD) as a cerebral perfusion tracer, 10 patients with unilateral cerebral infarction were studied. METHODS: ECD SPECT images were compared with IMP and/or HMPAO SPECT in nine patients, seven in chronic phases and two in subacute phases. Five of these patients and one additional patient with chronic infarction received PET imaging in order to compare ECD distribution with quantitative regional cerebral blood flow (rCBF) and oxygen metabolism (rCMRO2) images. RESULTS: In patients with chronic cerebral infarction, regression analysis showed excellent correlation between ECD and IMP in the uptake ratio of lesions-to-nonaffected cortices (r = 0.91). In two cases of subacute infarction, decreased uptake of ECD was observed in the area of "luxury perfusion," which showed elevated or preserved rCBF with diminished rCMRO2. On visual analysis, ECD image contrast was less prominent compared to that of IMP, but contrast was better than that of HMPAO. CONCLUSIONS: ECD uptake showed a curvilinear relationship against rCBF, suggesting flow-limited uptake in a high flow range. ECD is a clinically useful cerebral perfusion tracer with distinct characteristics when compared with other available agents.

PET and the autoradiographic method with continuous inhalation of oxygen-15-gas: theoretical analysis and comparison with conventional steady-state methods.

The steady-state method using 15O gas inhalation and positron emission tomography (PET) is a simple and practical way of imaging cerebral blood flow (CBF) and oxygen metabolism. Several disadvantages do exist, however, including prolonged examination time, requirement of steady-state and a large tissue heterogeneity effect. To avoid the drawbacks of the steady-state method but to preserve its simplicity, we applied the PET/autoradiographic method to the build-up phase during the continuous inhalation of 15O-gas with intermittent arterial sampling. A simulation study was performed to determine the optimal scanning period, evaluate the delay and dispersion effect of the input function and estimate the tissue heterogeneity effect. To assess the clinical feasibility of the proposed technique for the study of oxygen metabolism, sequential measurements with this method and the conventional steady-state method were performed in eight patients. The simulation study showed that a 5-min scan started 3 min after the commencement of 15O-gas inhalation was optimal. With this method, the delay and dispersion effect on CBF was the same as that of the conventional steady-state method, but the tissue heterogeneity effect was reduced. In eight patients, CBF values calculated by this method showed time dependency and were slightly higher than those obtained by the steady-state method. The oxygen extraction fraction showed no significant time dependency and was well correlated with that obtained by the steady-state method. We conclude that the proposed method is a simple and acceptable alternative to the conventional steady-state method.

Malignant mesenchymoma arising in an incisional scar of the abdominal wall.

A case of malignant mesenchymoma of the anterior abdominal wall, composed of leiomyosarcoma, chondrosarcoma and osteosarcoma is described. The tumour, arising in the old scar of a previous operation, was completely removed surgically. Eleven months after the operation the patient is well with no evidence of recurrence. There have been no previous reports of malignant mesenchymoma developing in operation scars. We believe that this traumatic factor played a significant role in the aetiology of this rare soft-tissue neoplasm.

Skull metastasis of thyroid carcinoma. Study of 12 cases.

Over the past 33 years the authors have treated 12 cases of skull metastasis from thyroid carcinoma, accounting for 2.5% of a total of 473 cases of thyroid cancer. A study of these 12 cases revealed the following characteristics. The mean age of the patients was 60.4 years and the predominant incidence was in the seventh decade of life. The incidence of skull metastasis from thyroid cancer was higher among women than among men. The primary sign was a soft hemispheric tumor resting on the skull. The tumors were rich in vascularity with osteolytic changes in the skull. The average period from diagnosis of the thyroid tumor until thyroidectomy was 14.3 years, and until discovery of the skull metastasis was 23.3 years. The clinical course was thus very long. The most frequent histopathological presentation was follicular adenocarcinoma. Such lesions were morphologically well differentiated, with little pleomorphism and atypism, but detailed examination showed infiltration into the vascular lumen or capsule. The mean survival time in these patients was 4.5 years from the time of diagnosis. The prognosis in this lesion was relatively poor, considering its long clinical course.

Noninvasive measurement of regional cerebral blood flow change with H(2)(15)O and positron emission tomography using a mechanical injector and a standard arterial input function.

To estimate changes in regional cerebral blood flow (rCBF) without arterial sampling in the study of functional-anatomical correlations in the human brain, using (15)O-labeled water and PET, a standard arterial input function was generated from the input function in 10 normal volunteers with dose calibration and peak time normalization. The speed and volume of injection were precisely controlled with a mechanical injector. After global normalization of each tissue activity image, the standard arterial input function was applied to obtain estimated CBF images. Relative changes in estimated rCBF to whole brain mean CBF(DeltaFest) and those in regional tissue activity (DeltaC) were compared with true relative rCBF changes (DeltaF) in 40 pairs of images obtained from 6 normal volunteers. DeltaFest correlated well with DeltaF, whereas DeltaC consistently underestimated DeltaF. This noninvasive method simplifies the activation studies and provides the accurate estimation of relative flow changes.

Dietary induction of cholesterol gallstones in hamsters from three different sources.

Cholesterol gallstones were produced in young male, golden Syrian hamsters, obtained from three different suppliers, by administering a nutritionally adequate, semipurified diet for periods of either 5 or 10 weeks. The major components of the lithogenic diet were casein, cornstarch, butterfat, corn oil and 0.3% cholesterol. The hamsters were obtained from Sesco, Harlan Sprague-Dawley (Engle hamster) and Charles River (Lakeview hamster). There were profound differences among the three groups with respect to gallstone formation and cholesterol metabolism: The highest incidence of gallstones occurred in Sesco hamsters, 44.4% and 63.6% after 5 and 10 weeks on the lithogenic diet, respectively. In the Engle hamster, after a 5-week feeding, cholesterol crystals and gallstones were absent. When the feeding period was extended to 10 weeks, cholesterol gallstones were present in 45.5% of the animals. In the Lakeview hamsters, neither gallstones nor cholesterol crystals were found in the gallbladder after a 5-week period. After 10 weeks, cholesterol gallstones were found in only a single hamster. In all groups, the lithogenic diet produced large increases of liver, serum and biliary cholesterol concentrations and increased liver weights. When the animals were fed for 5 weeks, only the bile of Sesco hamsters became supersaturated. Supersaturated bile was induced in all groups after a 10-week feeding of the lithogenic diet with cholesterol saturation ranging from 1.47 to 1.97. These data indicate that it is possible to induce cholesterol gallstones in hamsters by means of a nutritionally adequate, semipurified diet of moderate cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of alfalfa-corn diets on cholesterol metabolism and gallstones in prairie dogs.

Cholesterol gallstones were present in prairie dogs fed alfalfa plus corn with and without exogenous cholesterol (0.4%). The diets fed to the animals for eight weeks contained alfalfa plus corn in fixed proportions of 50:50, 85:15 and 15:85 (w/w). At sacrifice, all animals were healthy but had not gained weight; no deaths occurred during the experiment. Cholesterol gallstones were present in all groups. In the absence of exogenous cholesterol, the highest stone incidence was found in the animals which received the lowest fiber (highest corn) diets (alfalfa plus corn, 50:50, 67%; alfalfa plus corn, 15:85, 83%). Cholesterol gallstone incidence was 100% when exogenous cholesterol was added to the alfalfa plus corn diets (50:50 and 15:85). No pigment gallstones were detected in any animal. Liver and plasma cholesterol concentrations were highest in the animals receiving alfalfa plus corn (15:85) plus 0.4% cholesterol (4.29 mg/g, and 356 mg/dl, respectively). These values were lowest in animals receiving 85% alfalfa plus 15% corn without cholesterol (2.19 mg/g and 88 mg/dl, respectively). Lithogenic indices were below 1.00 in all groups. Biliary bile acids were mainly amidates of cholic acid and chenodeoxycholic acid, with the former predominating. Thus, gallstones can be formed in prairie dogs in the absence of exogenous cholesterol; gallstone incidence is reduced by dietary fiber.

Effect of 7-methylated bile acids and bile alcohols on cholesterol metabolism in hamsters.

The effect of 7-methyl substituted bile acid and bile alcohol analogues on cholesterol metabolism was studied in the hamster. Animals were fed chow plus 0.1% cholesterol supplemented with 0.1% of one of the following steroids: chenodeoxycholic acid, 7-methyl-chenodeoxycholic acid, 7 beta-methyl-24-nor-5 beta-cholestane-3 alpha, 7 alpha, 25-triol, cholic acid, 7-methyl-cholic acid, or 7 beta-methyl-24-nor-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25-tetrol. Cholesterol absorption was determined from fecal analysis after feeding of radiolabeled cholesterol and beta-sitosterol. Of the six compounds studied, chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid decreased intestinal cholesterol absorption (17% and 31% decrease, respectively). Only 7-methyl-chenodeoxycholic acid decreased serum cholesterol concentration (29% decrease), but there were no analogous changes of liver and biliary cholesterol concentration and cholesterol saturation of bile. Total fecal neutral sterol excretion was increased in the groups fed chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid. In addition, the production of coprostanol was increased in both groups. These data suggest that 7-methyl-chenodeoxycholic acid resembles chenodeoxycholic acid in its effect on cholesterol metabolism and may be a potential candidate for further studies of its gallstone-dissolving properties.

Cholelithiasis in hamsters: effects of cholic acid and calcium on gallstone formation.

Dietary cholic acid (0.1%) and/or calcium (2.6% as calcium carbonate) were added to a semipurified diet containing cholesterol and ethynyl estradiol to determine whether the incidence of pigment and/or cholesterol gallstones would be changed. Male golden Syrian hamsters were fed the experimental diets for 96 days (Group 1, control; Group 3, cholic acid plus calcium) or only an average of 60 days (Group 2, 0.1% cholic acid). Animals in Group 2 became ill (weight loss, low food intake, diarrhea) possibly due to cholic acid (or deoxycholic acid) toxicity. Cholesterol gallstones and crystals were absent in all experimental groups. The incidence of pigment gallstones was: control, Group 1, 12/16; 0.1% cholic acid, Group 2, 3/13; and 0.1% cholic acid plus calcium, Group 3, 11/22. Cholic acid with or without calcium produced an elevation of both liver and plasma cholesterol: Group 2, 80.1 mg/g and 501 mg/dl; Group 3, 103.7 mg/g and 475 mg/dl vs Group 1, 65 mg/g and 209 mg/dl, respectively. The lithogenic indices of the bile were lower in Groups 2 and 3 compared to Group 1, controls, 0.45 and 0.58 vs 1.16, respectively. The extent of the portal tract pathology could not be correlated with the presence or absence of pigment gallstones or with the levels of lithocholic acid in the hamster bile. In summary, when semipurified diets were supplemented with ethynyl estradiol and cholic acid, with and without calcium supplementation, no cholesterol gallstones formed and the incidence of pigment gallstones was not altered.

Bioactive peptides derived from food proteins preventing lifestyle-related diseases.

Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy.

Gastroduodenal intussusception secondary to a gastric carcinoma.

A case of gastroduodenal intussusception secondary to transpyloric prolapse of a primary gastric carcinoma is reported. Both the condition itself and the leading tumor, gastric carcinoma, are extremely exceptional. A diagnosis of this rare entity was established pre-operatively by endoscopy that demonstrated spontaneous reduction of the intussusception. This may be the first documentation of spontaneous reduction of the gastroduodenal intussusception during endoscopy.

[Simple quantification of brain perfusion SPECT with IMP using a large field gamma camera].

We have developed a simple method to quantitate brain perfusion SPECT with N-isopropyl-p-[123I]iodoamphetamine (IMP). After an intravenous bolus injection of IMP, serial dynamic images of the brain and the lung were obtained every 5 sec for 2 min, followed by a SPECT scan at 20 min. Based on the assumption of IMP as chemical microspheres, fractional uptake (FU) of IMP in the brain was calculated from the time-activity curves of the total brain and the total lungs. FU showed a strong positive correlation with inverse of the body surface area which was calculated from the height and weight of the individual subject, suggesting that FU is influenced by cardiac output. Quantification of IMP brain perfusion SPECT was attempted with FU and estimated cardiac output in each subject, providing an index of regional cerebral blood flow. Compared with PET measurement of regional cerebral blood flow, the present method tended to overestimate the flow values. This method has a great advantage that it can be applied in many institutions having large field gamma cameras. However, the accuracy of the measurement may be limited in the tall subjects and in the cases with cardiopulmonary diseases, and further investigation is required to clarify the usefulness and limitation of this approach.

[99mTc-HMPAO SPECT in the brain death--a case report].

Brain single photon emission computed tomography (SPECT) with 99mTc-d,l-hexamethyl-propyleneamine oxime (HMPAO) was performed twice in a 78-year-old man clinically diagnosed as brain death according to the standard criteria of the Japanese Ministry of Welfare. The first brain SPECT demonstrated the tracer accumulation in the brain, indicating preserved cerebral blood flow. The second brain SPECT performed 3 days later revealed cessation of the blood flow. In patients with preserved cerebral blood flow, the diagnosis of brain death cannot be made, even if they meet the existing criteria, because previous report noted the recovery in some of those patients. Brain perfusion SPECT plays an important role as a confirmatory test for the diagnosis of the brain death.

[A radioimmunometric assay for circulating tumor-associated glycoprotein (TAG-72) using the antigen determinant CA 72-4].

Tumor-associated glycoprotein (TAG-72) is an antigen recognized by monoclonal antibody (MAb) B 72.3 which was generated against a membrane enriched fraction of human mammary carcinoma cells. CA 72-4 is a novel antigen determinant on TAG-72 and is a quantitative radioimmunometric assay system utilizing two MAbs (CC-49, B 72.3) which react with circulating TAG-72 expressed by human carcinomas. We have employed the CA 72-4 RIA system to measure the antigen in sera. The optimum condition for this assay was found to be a 4 hour incubation at 37 degrees C for the first reaction and a 20 hour incubation at 4 degrees C for the second reaction. Under these conditions, intra-assay variation of the control sera was C.V. 3.0-5.2% and inter-assay variation was 5.6-8.3%. The mean +2 SD of CA 72-4 concentration in 468 healthy persons was 3.9 U/ml. Therefore, less than 4.0 U/ml was taken as the cut off level for the CA 72-4 serum assay. The largest population in healthy persons was at the range of 1.5-2.0 U/ml. Only 15 of 468 persons (3.2%) demonstrated serum CA 72-4 levels more than 4.0 U/ml. These data thus indicate the efficacy of CA 72-4 RIA system for the serum assay as a novel tumor marker. The clinical evaluation of CA 72-4 in patients with epithelial malignancies is now underway.

[A case of advanced gastric cancer with liver metastasis disappeared by oral administration of 5'-deoxy-5-fluorouridine after absolute non-curative operation].

A 64-year-old male was found to have a gastric cancer (Borrmann type 3) on the posterior wall of the stomach. Histological diagnosis was poorly differentiated adenocarcinoma. Ultrasonography revealed a hypoechoic lesion (18 x 14 mm) in the anteroinferior segment (S5) of the liver. The biopsy specimens from those lesions proved to be poorly differentiated adenocarcinoma. Total gastrectomy and distal pancreatosplenectomy were performed. During the operation, other liver metastases were found in the anterosuperior segment (S8) and inferolateral segment (S3). 5'-deoxy-5-fluorouridine (800 mg/day) was administered per os from the 33 rd postoperative day. The hypoechoic lesion of S5 in liver disappeared in the 17th postoperative month. The man is healthy with no evidence of recurrence 47 months after the operation. This case is very important because of the long-term CR.