Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons.

Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia and major depression, and a haplotype located in an intronic region of the L-type voltage-gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in iNs homozygous for the risk genotype, compared with non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human iNs, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease.

Antidepressant and anxiolytic profiles of newly synthesized arginine vasopressin V1B receptor antagonists: TASP0233278 and TASP0390325.

BACKGROUND AND PURPOSE: Vasopressin V1B receptor antagonists may be effective for the treatment of depression and anxiety and the objective of this study was to characterize the pharmacological profiles of two newly synthesized arginine vasopressin receptor 1B (V1B receptor) antagonists, TASP0233278 and TASP0390325. EXPERIMENTAL APPROACH: We investigated the in vitro profiles of TASP0233278 and TASP0390325. In addition, the effect of TASP0390325 on the increase in plasma adrenocorticotropic hormone (ACTH) levels induced by corticotropin-releasing factor (CRF)/desmopressin (dDAVP) was investigated. We also investigated the antidepressant and anxiolytic profiles of TASP0233278 and TASP0390325 in animal models. KEY RESULTS: Both TASP0233278 and TASP0390325 showed a high affinity and potent antagonist activity for V1B receptors. Oral administration of TASP0390325 antagonized the increase in plasma ACTH levels induced by CRF/dDAVP in rats, indicating that TASP0390325 blocks the anterior pituitary V1B receptor in vivo. Oral administration of TASP0233278 or TASP0390325 also exerted antidepressant effects in two models of depression (a forced swimming test and an olfactory bulbectomy model). Moreover, TASP0233278 improved depressive-like behaviour induced by repeated treatment with corticosterone, a model that has been shown to be resistant to treatment with currently prescribed antidepressants. In addition to depression models, TASP0233278 or TASP0390325 exerted anxiolytic effects in several anxiety models (social interaction, elevated plus-maze, stress-induced hyperthermia, separation-induced ultrasonic vocalization and sodium lactate-induced panic-like responses in panic-prone rats). CONCLUSION: TASP0233278 and TASP0390325 are potent and orally active V1B receptor antagonists with antidepressant and anxiolytic activities in rodents.

The H19 gene: regulation and function of a non-coding RNA.

The H19 gene encodes a 2.3-kb non-coding mRNA which is strongly expressed during embryogenesis. This gene belongs to an imprinted cluster, conserved on mouse chromosome 7 and human chromosome 11p15. H19 is maternally expressed and the neighbouring Igf2 gene is transcribed from the paternal allele. These two genes are co-expressed in endoderm- and mesoderm-derived tissues during embryonic development, which suggests a common mechanism of regulation. The regulatory elements (imprinted control region, CTCF insulation, different enhancer sequences, promoters of the two genes, matrix attachment regions) confer a differential chromatin architecture to the two parental alleles leading to reciprocal expression. The role of the H19 gene is unclear but different aspects have been proposed. H19 influences growth by way of a cis control on Igf2 expression. Although H19(-/-) mice are viable, a role for this gene during development has been suggested by viable H19(-/-) parthenogenetic mice. Finally it has been described as a putative tumour suppressor gene. H19 has been studied by numerous laboratories over the last fifteen years, nevertheless the function of this non-coding RNA remains to be elucidated.

The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H(+)-ATPase.

We cloned the genomic genes encoding the murine 16 kDa subunit (proteolipid, PL16) of vacuolar H(+)-ATPase (V-ATPase) and determined their nucleotide sequences. At least three independent genes were found in the murine genome. One gene consisted of three exons and was largely identical in sequence to that of PL16 cDNA reported previously (Hanada et al., Biochem. Biophys. Res. Commun. 176 (1991) 1062). In the 5'-flanking region of this gene, several possible transcriptional cis-elements were found. TATA and CAAT sequences were not found, which is characteristic for promoters of house-keeping genes. The other two genes identified did not contain introns. One of these genes had an open reading frame that potentially encoded PL16 but contained six amino acid substitutions and a frame-shift mutation that would result in a truncated protein unable to participate in V-ATPase activity. The other gene had the same sequence in the reading frame as that in the cDNA. However, this gene contained a polyA sequence at the same position where polyA is normally added to mRNA. The gene also had 15 bp repetitive sequences near the transcription initiation site and next to the polyA sequence. These observations suggest that this gene may have been generated by the insertion of reverse-transcribed double-stranded cDNA, as is usually observed for pseudogenes. In conclusion, there is a single functional PL16 gene and two pseudogenes in the murine genome. It is unlikely that PL16 isoforms contribute to variation in V-ATPase function.

a4, a unique kidney-specific isoform of mouse vacuolar H+-ATPase subunit a.

The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.

An auto-tuning method for focusing and astigmatism correction in HAADF-STEM, based on the image contrast transfer function.

An auto-tuning method for high-angle annular detector dark field scanning transmission electron microscopy (HAADF-STEM) is proposed which corrects the defocus to the optimum Scherzer focus and compensates the astigmatism. Because the method is based on the image contrast transfer function formulated for the HAADF-STEM, the defocus and the astigmatism are accurately measured from input of two different defocus images. The method is designed to work independent of object function in the linear imaging model by analysing the spectral ratio between two Fourier spectra of their images, which is useful for cases where the spectrum of object function is not uniformly spread out over the reciprocal space. The method was preliminarily tested in a Hitachi HD-2000 STEM, and successful results of the auto-tunings from the viewpoint of verification of the algorithm were obtained using general specimens of Au fine particles and a thin section of a semiconductor device.

Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.

Primordial germ cells (PGCs) in mice have been recognized histologically as alkaline phosphatase (AP) activity-positive cells at 7.2 days post coitum (dpc) in the extra-embryonic mesoderm. However, mechanisms regulating PGC formation are unknown, and an appropriate in vitro system to study the mechanisms has not been established. Therefore, we have developed a primary culture of explanted embryos at pre- and early-streak stages, and have studied roles of cell and/or tissue interactions in PGC formation. The emergence of PGCs from 5.5 dpc epiblasts was observed only when they were co-cultured with extra-embryonic ectoderm, which may induce the conditions required for PGC formation within epiblasts. From 6.0 dpc onwards, PGCs emerged from whole epiblasts as did a fragment of proximal epiblast that corresponds to the area containing presumptive PGC precursors without neighboring extra-embryonic ectoderm and visceral endoderm. Dissociated epiblasts at these stages, however, did not give rise to PGCs, indicating that interactions among a cluster of a specific number of proximal epiblast cells is needed for PGC differentiation. In contrast, we observed that dissociated epiblast cells from a 6.5-b (6.5+15-16 hours) to 6.75 dpc embryo that had undergone gastrulation gave rise to PGCs. Our results demonstrate that stage-dependent tissue and cell interactions play key roles in PGC determination.

Upstream regions directing heart-specific expression of the GATA6 gene during mouse early development.

The expression of murine transcription factor GATA6 is restricted to tissues including the heart and gastrointestinal systems during embryogenesis, and is maintained throughout postnatal life. We have characterized the 5' upstream region (6.4 kb) of the mouse GATA6 gene, and identified two closely spaced transcription initiation sites. The flanking sequence lacks a typical TATA-box, and is rich in guanine and cytosine. The role of the 5' upstream region was examined using the lacZ reporter gene in transgenic mice. A construct containing the 5' flanking sequence (4.9 kb), untranslated exon 1 and 1.3 kb intron 1 could drive the gene expression in the embryonic and adult heart regions. Weak expression was also observed in the stomach, liver, and bronchial arch in addition to the cardiac region. Deletion of the 5' upstream region ( approximately 1.2 kb) or intron 1 abolished all this expression, indicating that at least two cis-acting control elements are necessary for heart-specific expression of GATA6 in vivo.

Sensing of cadmium and copper ions by externally exposed ADL, ASE, and ASH neurons elicits avoidance response in Caenorhabditis elegans.

We developed a quantitative assay for Caenorhabditis elegans avoidance behavior. This was then used to demonstrate that the worm moved away from toxic concentrations of Cd2+ and Cu2+, but not Ni2+, all ions that prevented development from larval to adult stages. Mutants that have structural defects in ciliated neurons (che-2 and osm-3) as well as worms with three laser-operated neurons (ADL, ASE, and ASH), showed no avoidance behavior from Cd2+ and Cu2+. These results suggest that the avoidance from Cd2+ and Cu2+ are mediated through multiple neural pathways including ADL, ASE, and ASH neurons. We hypothesize that the three sensing neurons provide increased accuracy of the sensory response and a survival advantage in the natural environment of the worm.

Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice.

The Pic-1, Oct-1,2, Unc-86 (POU) transcription factor Oct-4 is specifically expressed in the germ cell line, and a previous study has indicated that the expression of the lacZ gene inserted into an 18 kb genomic fragment encompassing the Oct-4 gene can come close to mimicking the endogenous embryonic expression pattern of Oct-4 in transgenic mice. In the present study transgenic mice expressing green fluorescent protein (GFP) in the germ cell line were generated using the same Oct-4 genomic fragments and the expression pattern was analyzed in detail through all stages of germ cell development. The GFP expressing primordial germ cells were first detected as early as 8.0 days post-coitum (d.p.c.; early head fold stage) at the base of the allantois in living embryos. The GFP expression was thereafter found in both male and female germ cells at all developmental stages except in male germ cells after differentiating into type A spermatogonia in the postnatal testis. There was also a lower level of expression in female germ cells in the prophase of the first meiotic division. These transgenic mice therefore proved to be powerful tools for isolating living germ cells at various developmental stages to study their nature and to isolate new genes.

Essential Cys-Pro-Cys motif of Caenorhabditis elegans copper transport ATPase.

Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast delta ccc2 mutant (copper ATPase gene disruptant). We found that CUA-1 with Cys-Pro-Cys to Cys-Pro-Ala mutation could not rescue the yeast delta ccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.

[Combination of transcatheter arterial infusion of SMANCS and embolization (SMANCS-TAE) for hepatocellular carcinoma--second report].

Patients with unresectable hepatocellular carcinoma (hepatoma) with hypervascularity were treated by SMANCS-TAE. A superselective catheterization technique was used to inject gelatin sponge particles after administration of SMANCS. In 30 patients of first hepatoma treated by SMANCS-TAE. Grade 4 was obtained after 1.7(1-3) courses. The 2-year survival rate was 22%. Some of the 24 patients of second hepatoma treated by SMANCS-TAE have survived over 2 years. Sixteen patients with advanced hepatoma (Vp2-3 or T4) were treated only by SMANCS injection, but none survived over 1 year. SMANCS-TAE appears to have the same potential and safety as L-TAE, when used selectively. Moreover, we can reduce the course of treatment and obtain good QOL for hepatoma patients except in advanced cases.

Caenorhabditis elegans cDNA for a Menkes/Wilson disease gene homologue and its function in a yeast CCC2 gene deletion mutant.

The full-length cDNA coding for a putative copper transporting P-type ATPase (Cu2+-ATPase) was cloned from Caenorhabditis elegans. The putative Cu2+-ATPase is a 1,238-amino acid protein, and highly homologous to the Menkes and Wilson disease gene products mutations of which are responsible for human defects of copper metabolism. The Saccharomyces cerevisiae mutant with a disrupted CCC2 gene (yeast Menkes/Wilson disease gene homologue) was rescued by the cDNA for the C. elegans Cu2+-ATPase but not by the cDNA with an Asp-786 (an invariant phosphorylation site) to Asn mutation, suggesting that the C. elegans Cu2+-ATPase functions as a copper transporter in yeast. The expressed C. elegans protein was detected in yeast vacuolar membranes by immunofluorescence microscopy. The yeast expression system may facilitate further studies on copper transporting P-type ATPases.

[Abnormal chest shadow on CT in immunosuppressed patients].

An abnormal chest shadow was observed on CT scans in 25 cases of 23 immunosuppressed patients. Pulmonary disease was pathologically confirmed to be pneumocystis carinii pneumonia (PC pneumonia) in four patients, cytomegalovirus pneumonia (CMV pneumonia) in one, bacterial pneumonia in seven, fungal infection in three, miliary tuberculosis in one, leukemic infiltration in two, lymphangitis carcinomatosa in three, drug-induced pneumonitis in three, and ARDS in one. In almost all patients, especially those with infectious diseases such as PC pneumonia, CMV pneumonia, and bacterial pneumonia, the abnormal shadow was wide and visible in the bilateral lung fields. We presumed that such findings as lobular shadow, centrilobular shadow, and mosaic pattern reflected the extension of disease via the respiratory tract, and that those findings are typical of infectious diseases. Because such findings as abnormal linear shadow and swelling of a broncho-vascular bundle were very frequently recognized in patients with lymphangitis carcinomatosa and frequently recognized in those with drug-induced pneumonitis, these diseases may be distinguished from other diseases. An area of slightly increased density was frequently recognized in patients with PC pneumonia, bacterial pneumonia, and drug-induced pneumonitis. Such lesions were pathologically confirmed to be located in the interstitium and/or alveolus. CT was extremely useful in comprehending the character and extension of particular diseases among various diseases. As the number of patients studied was small, the utility of CT in immunosuppressed patients requires further investigation in a larger number of patients.

Altered regional clearance of 99Tcm-DTPA in radiation pneumonitis.

99Tcm-diethylene triaminepenta-acetic acid (99Tcm-DTPA) lung scintigrams were investigated in 11 patients with radiation pneumonitis to assess the alteration of clearance of this small solute. Thirty minutes after the injection of 99Tcm-DTPA, all 11 patients showed abnormal accumulation. This uptake was seen in the areas with reduced 99Tcm-macroaggregated albumin activity. The curves of the lung/heart ratios obtained after 1 min after injection of 99Tcm-DTPA in areas showing abnormal accumulation were of three types. Comparison of the slope for the initial 5 min of the lung/heart curves between the abnormal accumulation sites and the contralateral non-irradiated lung revealed a significant difference. The uptake ratio (the mean counts of the last frame divided by initial peak count) was related to the degree of abnormal accumulation in the radiation pneumonitis. Moreover, study of rabbits with radiation pneumonitis revealed similar behaviour by 99Tcm-DTPA as observed clinically. This simple and rapid method may be useful in detecting and monitoring the altered regional kinetics of 99Tcm-DTPA in radiation pneumonitis.

Scintigraphic analysis of hemodynamics in a patient with a single large pulmonary arteriovenous fistula.

A pulmonary perfusion study was performed on a 54-year-old man with a single large pulmonary arteriovenous fistula (aneurysmal type) using Tc-99m MAA. On the static imaging, a defect corresponding to the location of the mass and right-to-left shunt was clearly documented. Decreased uptake of Tc-99m MAA in the lung field surrounding the lesion was observed and was thought to reflect a "steal" of blood flow by the fistula. One advantage of using radionuclide angiography with Tc-99m MAA rather than with Tc-99m pertechnetate was that it more easily provided a time-activity curve that added useful quantitative information about the altered pulmonary hemodynamics.

[Demonstration of the architecture of the tracheobronchial wall by MR imaging: an experimental study].

To study the potential of MRI to demonstrate the architecture of the tracheobronchial wall, resected tracheobronchi of rabbits were examined with MRI. As a result, epithelium and cartilage were demonstrated as relatively low intensity bands on T1- and T2-weighted images (T1WI and T2WI). The subepithelium was of intermediate-high intensity on T1WI and of markedly high intensity on T2WI and Gd-DTPA enhanced T1WI. Peritracheobronchial fat was of markedly high intensity on T1WI and of relatively low intensity on T2WI. In conclusion, MRI was found to have the potential to demonstrate the architecture of the tracheobronchial wall as 4 layers on each sequence.