The Piccolo Intronic Single Nucleotide Polymorphism rs13438494 Regulates Dopamine and Serotonin Uptake and Shows Associations with Dependence-Like Behavior in Genomic Association Study.

Piccolo (PCLO) inhibits methamphetamine-induced neuropharmacological effects via modulation of dopamine (DA) uptake and regulation of the transport of synaptic vesicles in neuronal cells. Clinical studies have recently suggested that the single nucleotide polymorphism (SNP) rs13438494 in the intron 24 of the PCLO gene is associated with psychiatric disorder, in the meta-analysis of GWAS. Therefore, in this study, we attempted to evaluate the possible role of the PCLO SNP in the mechanisms of uptake of monoamines. To characterize rs13438494 in the PCLO gene, we constructed plasmids carrying either the C or A allele of the SNP and transiently transfected them into SH-SY5Y cells to analyze genetic effects on the splicing of PCLO mRNA. The C and A allele constructs produced different composition of the transcripts, indicating that the intronic SNP does affect the splicing pattern. We also transfected DA and serotonin (5-hydroxytryptamine; 5- HT) transporters into cells and analyzed their uptakes to elucidate the association to psychiatric disorders. In the cells transfected with the C allele, both the DA and 5-HT uptake were enhanced compared to the A allele. We also conducted a clinical study, in order to clarify the genetic associations. PCLO rs13438494 exhibits a relationship with the symptoms of drug dependence or related parameters, such as the age of first exposure to methamphetamine, eating disorders, tobacco dependence and fentanyl requirement. Our findings suggest that rs13438494 is associated with drug abuse and contributes to the pathogenesis of psychiatric disorders via modulation of neurotransmitter turnover.

Ethnic differences in GRHPR mutations in patients with primary hyperoxaluria type 2.

The objective of this study was to investigate ethnic differences in the glyoxylate reductase/hydroxypyruvate reductase (GRHPR) gene in patients with primary hyperoxaluria type 2 (PH2). GRHPR was genotyped in Japanese patients with PH2 and all GRHPR mutations described to date were reviewed in terms of geographic and ethnic association. We identified a novel mutation, a two-nucleotide deletion (c.248_249delTG) in exon 3 creating a premature 'stop' at codon 91. Also, we found that the c.864_865delTG mutation was associated with the rs35891798 single-nucleotide polymorphism. The allelic frequencies of the c.103delG, c.494G>A, c.403_404+2 delAAGT, and c.864_865delTG mutations in PH2 patients were 37.8%, 15.6%, 10.0%, and 10.0%, respectively. All patients with the c.103delG mutation were Caucasian. Patients with the c.494G>A mutation and 78% (7/9) of those with the c.403_404+2 delAAGT mutation were from the Indian subcontinent, whereas those with the c.864_865delTG mutation were Chinese or Japanese. Molecular analysis of GRHPR of four Japanese PH2 patients identified a novel mutation (c.248_249delTG in exon 3). Caucasians with PH2 should be screened for the c.103delG mutation; patients from the Indian subcontinent for c.494G>A; and patients of East Asian origin (particularly) for c.864_865delTG. The prevalence of the latter mutation in PH2 patients from East Asia was 75.0%.

[Lung cancer with a cystic lesion formed by the check-valve mechanism].

A 55-year-old man was admitted to our hospital with a growing cystic lesion in the left middle field of the lung. After we diagnosed it as non-small cell lung cancer, we performed left upper lobectomy. A series of chest X-ray revealed that the cyst was formed by the check-valve mechanism due to the lung cancer, retrospectively. We should keep in mind the existence of lung cancer and other malignant tumors adjacent to cystic lesions.

Ewing's sarcoma/peripheral primitive neuroectodermal tumor (pPNET) arising in the omentum as a multilocular cyst with intracystic hemorrhage.

A rare case of Ewing's sarcoma/peripheral primitive neuroectodermal tumor arising in the greater omentum in a 41-year-old man is reported. The patient presented with a hemorrhagic mesenteric cyst that was disclosed by the results of an abdominal echogram, a computed tomography scan, and magnetic resonance imaging. A laparotomy showed a multilocular cyst with intra-cystic hemorrhage. Histologically, the tumor wall consisted of sheets of small round cells separated by thick desmoplastic stroma. Rosette formations or ribbon-like cell arrangements were absent. Further pathological examination revealed that the membrane of the tumor cells was positive for MIC-2, and negative for epithelial membrane antigen, cytokeratin, and desmin, which are usually positive in intra-abdominal desmoplastic small round-cell tumors. An EWS/FLI1 fused transcript was detected by reverse transcription-polymerase chain reaction. These findings confirmed the diagnosis of Ewing's sarcoma/peripheral primitive neuroectodermal tumor. The patient died of tumor recurrence 4 months after his first admission. The autopsied tumor tissue exhibited neural differentiation in certain regions. To our knowledge, this is the first case to be reported of Ewing's sarcoma/peripheral primitive neuroectodermal tumor arising in the omentum with unique pathological features and the occurrence of partial neural differentiation during the clinical course. This case pointed out to us, as gastroenterologists, that only thorough examination confirms a definitive diagnosis of small round-cell tumor of the abdomen, it also shows that Ewing's sarcoma/peripheral primitive neuroectodermal tumor should be included in the differential diagnosis of cystic lesions in the omentum.

Interaction of EphB2-tyrosine kinase receptor and its ligand conveys dorsalization signal in Xenopus laevis development.

The Eph class is the largest family of receptor tyrosine kinases and has been shown to play various roles in neural development including axon pathfinding and neural crest migration. EphB2 associates with transmembrane ligands Ephrin-B1 and -B2, which leads to tyrosine phosphorylation of both the ligands and receptor and is presumed to regulate cell-to-cell interactions by bidirectional signaling. We have investigated the biological effects of the EphB2-induced signal in the early stage of Xenopus laevis development. Xenopus EphB2 transcripts were detected maternally and were expressed at equal levels between the ventral and dorsal halves of the gastrulae, with expression increasing after the late gastrula stage. EphB2 mRNA expression in dorsal marginal zone explants from gastrulae increases during later development while that in ventral explants does not. We show here that microinjection of RNA encoding EphB2 into a ventral blastomere of embryos induced a partial secondary dorsal axis which consisted of neural tissues, notochord and somites. Analysis with molecular markers verified that the microinjected EphB2 dorsalized the mesoderm of ventral marginal zone explants. These dorsalizing effects of EphB2 in both the whole embryo and ventral explants were inhibited by the coinjection of RNA encoding the soluble form of Ephrin-B1. Furthermore, co-injection of EphB2 and Ephrin-B1 RNAs synergistically enhanced the dorsalization effect. These data show that the interaction between EphB2 and its ligands including Ephrin-B1 causes signaling events which lead to dorsal development, and strongly suggests the existence of proteins which mediate the dorsalization induced by EphB2 in early stage embryos of Xenopus laevis.

Proliferative activity in breast carcinoma evaluated by BrdU and PCNA. Correlation with expression of p53, c-erbB-2, estrogen receptor and P-glycoprotein.

The proliferative activity in 35 cases of breast carcinoma was evaluated by bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA) and was compared with benign breast lesion. Overexpression of p53 and c-erbB-2 oncoprotein, presence of estrogen receptor (ER) and cellular localization of multidrug resistance gene product P-glycoprotein (P-gp) were immunohistochemically examined to investigate the relation with the proliferative activity and clinicopathologic characteristics. The mean BrdU labeling index (LI) was 12.6% and PCNA labeling rate (LR) was 33.5% in breast carcinomas, and good correlation was found between them. The proliferative activity of breast carcinomas was significantly higher than that of benign lesions. The BrdU LI correlated positively with tumor size, histologic grade, TNM stage and p53 immunoreactivity, and negatively with the presence of ER. PCNA LR correlated with histologic grade and expression of p53. p53 protein was demonstrated in 43% of the breast carcinomas and correlated with proliferative activity. The extent of p53 immunoreactivity on carcinoma cells was also related to BrdU LI. c-erbB-2 oncoprotein was demonstrated in 51% of the breast carcinomas and correlated with histologic grade. ER was found in 34% of the breast carcinomas and correlated negatively with histologic grade, lymph node metastasis and TNM stage. P-gp was observed in 49% of the breast carcinomas and no correlation was found with clinicopathologic characteristics. None of the benign lesions expressed p53 protein, c-erbB-2 oncoprotein and P-gp. BrdU is a reliable standard and a more useful tool for the evaluation of proliferative activity of breast tumors. High proliferative activity, overexpression of p53 protein and the absence of ER are considered as a high grade malignancy of breast carcinoma. Expression of c-erbB-2 oncoprotein and P-gp may be related to malignant transformation of breast tumors.

Proliferating cell nuclear antigen (PCNA), immunostaining and flow cytometric DNA analysis of renal cell carcinoma.

Nuclear PCNA immunoreactivity is found in the proliferating compartment of normal and tumor cells. The purpose of the present study was to examine immunohistochemical staining of PCNA in renal cell carcinoma and its relationship to tumor grading, flow cytometric DNA analysis and prognosis. The resected kidneys from 81 patients with renal cell carcinoma were used. Anti-PCNA monoclonal antibody was applied to paraffin sections to identify proliferating cells by the SAB technique. The proportion of PCNA-positive cells to negative cells was determined by counting the tumor cells stained by PCNA at 400x magnification and expressed as percentage of positive cells. In parallel with the PCNA immunostaining DNA flow cytometry was performed, using the cell suspension prepared from paraffin sections and stained with propidium iodide. 79 tumors were evaluable for PCNA immunohistochemistry and 54 tumors for DNA flow cytometry. Immunohistochemically, there was no significant difference in percentage of PCNA-positive cells between tumor grades G 1 and G 2 (1.4 +/- 1.3% in G 1 and 1.6 +/- 1.4% in G 2), while G 3 tumors contained high percentage of PCNA positive cells, the mean value being 7.6 +/- 5.3%. The percentage of PCNA-positive tumor cells was found to be correlated with histological grading of tumor and also with the S+G2+M phase fraction and the S phase fraction measured by flow cytometry (p < 0.01). The mean percentage of PCNA-positive tumor cells was higher in aneuploid tumors than in diploid tumors. There was no significant relationship between the percentage of PCNA positive cells and tumor staging. Sixty-one cases were available for survival analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascertainment of IgA1 (kappa)-transferrin complex in a case of multiple myeloma associated with hypersiderinemia.

IgA1(kappa)-transferrin complex was ascertained in a case of multiple myeloma with hypersiderinemia. On gel filtration, this complex disclosed an orange-colored protein with two peaks between the 19S and 7S fractions. On polyacrylamide gel electrophoresis and Western blotting analysis, the complex migrated at molecular weights of 430 and 700 kD in the non-reducing condition. A small amount of free transferrin was detected simultaneously. The complex was further separated into monoclonal IgA1(kappa) and transferrin at pH 4.5; these recombined at pH 7.2 after incubation (37 degrees C, 2 h). We also showed that the pepsin-digested F(ab')2 fragment of IgA1(kappa) combined with the transferrin. From these results it can be suggested that monoclonal IgA1(kappa) has an antibody activity for the transferrin. Clinically, this complex distributed the iron transport to erythroid cells in the bone marrow, resulting in hypersiderinemia, anemia, and iron deposits in the liver.

[Endocrine and hemodynamic responses to total body hyperthermia in humans].

We investigated effects of total body hyperthermia (TBH) on endocrine and hemodynamic responses. A total of five treatments were performed in five patients with gastric cancer under neuroleptanesthesia with morphine followed by 0.2 to 0.4% enflurane. TBH was extracorporeally induced with veno-venous shunt incorporating with heat exchanger to keep their temperature between 41.5 degrees C and 42.0 degrees C for three hours. The patients were administered angiotensin to maintain tumor blood flow. Lactated Ringer's solution was administered at the rate of 10 to 15 ml.kg-1.hr-1 for five hours. Plasma cortisol levels decreased significantly to about one third of the control value after heating and the levels recovered to the control value after cooling. Plasma norepinephrine level increased significantly to about 7 to 9 times the control value following TBH, but this hormonal response was insufficient to reveal marked direct hemodynamic effects. The magnitude of fall in SVR was more significant in spite of the administration of angiotensin. Cardiac index increased significantly to about 2.0 to 2.6 fold of control value, but mean arterial blood pressure (MAP) decreased significantly to about two thirds to four fifths of the control value. Morphine relieved the hormonal response in ACTH and cortisol strongly, but morphine suppressed hemodynamics by decreasing SVR. Neither norepinephrine released from sympathetic nerve endings nor even 50 to 200 ng.kg-1.min-1 of angiotensin administered failed to restore SVR or MAP during hyperthermia.

[Respiratory gas exchange during total body hyperthermia in man].

Respiratory gas exchange was evaluated using indirect calorimetry during total body hyperthermia (TBH) in 7 postoperative patients with disseminated gastric cancer to the peritoneal cavity. TBH was induced using veno-venous bypass and extracorporeal circuit incorporating a heat exchanger to keep pulmonary arterial temperature 42 degrees C. The high temperature was maintained for 3 hours under general anesthesia with droperidol (0.15 mg.kg-1), morphine (1 mg.kg-1) and enflurane (less than 0.5%). Oxygen consumption (VO2) was also calculated using the Fick equation and thermodilution cardiac output. Hyperthermia for three hours increased both VO2 and carbon dioxide output (VCO2) values to 1.5 times as compared to the control values before heating, respectively. VO2 values measured by indirect calorimetry correlated well with the values calculated from the Fick equation when respiratory and cardiovascular system were relatively stable during the procedure. Gradual but statistically insignificant increase in respiratory quotient was observed after recooling started. These results suggest that indirect calorimetry was valuable to measure respiratory gas exchange continuously during hyperthermic state as well as normothermic state. However, RQ value observed in the present study may be modified by several factors including lactate accumulation in the blood, and may not reflect substrate utilization during the hyperthermic state.

[Chronic myelomonocytic leukemia (CMMoL) with systemic lymph node swelling and paroxysmal nocturnal hemoglobinuria (PNH)-like complication].

A 37-year-old male was diagnosed as having chronic myelomonocytic leukemia (CMMoL) with chief complaint of systemic lymph node swelling. On admission, his peripheral blood revealed mild anemia and mild thrombocytopenia with giant platelets, and monocytosis (1480/microliters). NAP score was low. Serum lysozyme increased. The bone marrow showed normal cellularity consisting of 4% myeloblasts and 14.4% promyelocytes, and a few myeloid cells were positive for double staining by alpha-naphthyl butyrate and naphthol ASD chloroacetate esterase. Biopsied specimens of the cervical lymph node showed infiltration of monocytoid cells, which were positive for lysozyme staining, into interfollicular tissue. As for chromosome variation, 21 large satellite was observed in all dividing cells from his bone marrow and peripheral blood. Furthermore, hemolytic anemia with hemoglobinuria developed during his course. Sugar water test was positive, but Ham test negative. Coombs test and Donath-Landsteiner reaction were negative. Abnormal hemoglobin, spherocyte and fragmentation were not found. Hemolysis disappeared about two months later. However, blastic crisis appeared and he died. We showed a case of CMMoL with 21 large satellite and paroxysmal nocturnal hemoglobinuria (PNH)-like complication. Satellite have usually been reported as asymptomatic, and thus this chromosome variant and CMMoL may have been coincidentally observed.

Cardiac distribution of alpha-hANP in an infant with total anomalous pulmonary venous return.

Atrial natriuretic polypeptide like immunoreactivity (irANP) was searched in the right and left atrial and ventricles following necropsy of a 2-month-old boy who underwent anesthesia for radical surgery to total anomalous pulmonary venous return (TAPVR), who could not be removed from a cardiopulmonary bypass. The irANP concentration was 630 micrograms.g-1 in the right atrium, 3.2 micrograms.g-1 in the right ventricule in 1 100 micrograms.g-1 in the left atrium and 1.7 micrograms.g-1 in the left ventricle, respectively in spite of numerous studies reported that ir ANP cannot be detected in adult ventricles.