DISC1 causes associative memory and neurodevelopmental defects in fruit flies.

Originally found in a Scottish family with diverse mental disorders, the DISC1 protein has been characterized as an intracellular scaffold protein that associates with diverse binding partners in neural development. To explore its functions in a genetically tractable system, we expressed the human DISC1 in fruit flies (Drosophila melanogaster). As in mammalian neurons, DISC1 is localized to diverse subcellular domains of developing fly neurons including the nuclei, axons and dendrites. Overexpression of DISC1 impairs associative memory. Experiments with deletion/mutation constructs have revealed the importance of amino-terminal domain (46-290) for memory suppression whereas carboxyl domain (598-854) and the amino-terminal residues (1-45) including the nuclear localization signal (NLS1) are dispensable. DISC1 overexpression also causes suppression of axonal and dendritic branching of mushroom body neurons, which mediate a variety of cognitive functions in the fly brain. Analyses with deletion/mutation constructs reveal that protein domains 598-854 and 349-402 are both required for the suppression of axonal branching, while amino-terminal domains including NLS1 are dispensable. In contrast, NLS1 was required for the suppression of dendritic branching, suggesting a mechanism involving gene expression. Moreover, domain 403-596 is also required for the suppression of dendritic branching. We also show that overexpression of DISC1 suppresses glutamatergic synaptogenesis in developing neuromuscular junctions. Deletion/mutation experiments have revealed the importance of protein domains 403-596 and 349-402 for synaptic suppression, while amino-terminal domains including NLS1 are dispensable. Finally, we show that DISC1 functionally interacts with the fly homolog of Dysbindin (DTNBP1) via direct protein-protein interaction in developing synapses.

Emerging roles for the FCRL family members in lymphocyte biology and disease.

Members of the extended Fc receptor-like (FCRL) family in humans and mice are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. Although the majority of these proteins repress B cell receptor-mediated activation, there is an emerging evidence for their bifunctionality and capacity to counter-regulate adaptive and innate signaling pathways. In light of these findings, the recent discovery of ligands for several of these molecules has begun to reveal exciting potential for them in normal lymphocyte biology and is launching a new phase of FCRL investigation. Importantly, these fundamental developments are also setting the stage for defining their altered roles in the pathogenesis of a growing number of immune-mediated diseases. Here we review recent advances in the FCRL field and highlight the significance of these intriguing receptors in normal and perturbed immunobiology.

Intraarticular administration of platelet-rich plasma with biodegradable gelatin hydrogel microspheres prevents osteoarthritis progression in the rabbit knee.

OBJECTIVE: To investigate the therapeutic potential of administration of gelatin hydrogel microspheres containing platelet-rich plasma (PRP), by examining its effects on progression of osteoarthritis (OA) in a rabbit model. METHODS: PRP and platelet-poor plasma (PPP) were prepared from rabbit blood. Adult rabbit chondrocytes were cultured in the alginate beads with the presence of 3% PRP or 3% PPP. Glycosaminoglycan (GAG) synthesis was quantified using dimethylmethylene blue assay. To confirm the anabolic effect of PRP in vivo, cartilage matrix gene expression was examined after intraarticular administration of PRP contained in gelatin hydrogel microspheres. The PRP contained in gelatin hydrogel microspheres was administered into the rabbit knee joint twice with an interval of 3 weeks, beginning 4 weeks after anterior cruciate ligament transection (ACLT). Ten weeks after ACLT, gross morphological and histological examinations were performed. RESULTS: PRP significantly stimulated chondrocyte GAG synthesis in vitro. In the knee joint, expression of proteoglycan core protein mRNA in the articular cartilage increased after administration of PRP contained in microspheres. Intraarticular injections of PRP in gelatin hydrogel microspheres significantly suppressed progression of OA in the ACLT rabbit model morphologically and histologically. CONCLUSION: The present findings indicate that sustained release of growth factors contained in PRP has preventive effects against OA progression. These preventive effects appear to be due to stimulation of cartilage matrix metabolism, caused by the growth factors contained in PRP.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

Perceived importance of being thin and smoking initiation among young girls.

BACKGROUND: Smoking among adolescents remains unacceptably high and the difference in potential risk factors for smoking initiation between male and female adolescents has been explored. Although the association between smoking initiation and dieting behaviour has been observed among girls, the mechanism of the association is unknown. OBJECTIVE: To examine prospectively the association between perceived importance of being thin at baseline and smoking initiation among girls. DESIGN: A four year prospective cohort survey including perceived importance of being thin at baseline and smoking behaviour, conducted in 1993 and 1996. SETTING AND PARTICIPANTS: 273 Massachusetts female adolescents aged 12-15 years at baseline who reported having smoked no more than one cigarette by the time of the baseline survey, drawn from households sampled by random digit dialing. MAIN OUTCOME MEASURE: Progression to established smoking, defined as having smoked 100 or more cigarettes in their lifetime. RESULTS: After adjusting for age, smoking status at baseline, and race/ethnicity, girls who valued thinness most strongly and somewhat strongly were both more likely to have become established smokers, compared to the girls who valued thinness least strongly. The odds ratios are 4.5 (95% confidence interval (CI), 1.4 to 16.7) and 3.4 (95% CI 1.04 to 10.9), respectively. CONCLUSIONS: The level of perceived importance of being thin among young female adolescents predicts future smoking initiation. Smoking prevention programmes designed for female adolescents may therefore benefit from the inclusion of content related to importance of being thin.

Hepatic parenchymal enhancement in the cirrhotic liver: evaluation by triple-phase dynamic MRI.

BACKGROUND: To evaluate the changes of liver parenchymal enhancement in the cirrhotic liver by means of triple-phase dynamic magnetic resonance (MR) imaging. METHODS: Triple-phase multisection dynamic MR imaging was performed in 32 patients with liver cirrhosis. The control group consisted of 19 patients without liver cirrhosis. After precontrast images were obtained, arterial phase images were acquired 20 s after the start of intravenous bolus administration of 0.10 mmol/kg of gadopentetate dimeglumine. Portal and delayed phase images were then acquired 1 and 3 min, respectively, after the injection of contrast material. On each phase image, the signal-to-noise ratio (S/N) from the liver parenchyma was measured by operator-defined regions of interest (ROIs). The contrast-enhanced ratio (CER) on each phase was then obtained according to the following formula: [S/N(arterial or portal or delayed phase image) - S/N(precontrast image)] / S/N(precontrast image). The portal perfusion index (PPI) also was obtained according to the following formula: [S/N(portal phase image - S/N(arterial phase image)] / S/N(arterial phase image). The results were expressed as mean +/- SD. RESULTS: The CERs of arterial, portal, and delayed phase images in patients with and without liver cirrhosis were 0.256 +/- 0.211, 0.640 +/- 0.384, and 0.554 +/- 0.318 and 0.132 +/- 0.094, 0.404 +/- 0.204, and 0.324 +/- 0.144, respectively. The CERs were highest in the portal phase and lowest in the arterial phase in patients with and without liver cirrhosis. The CER of the cirrhotic liver was significantly higher than that of the normal liver in every phase (p < 0.05). PPIs with and without liver cirrhosis were 2.90 +/- 4.03 and 3.86 +/- 3.89, respectively. The PPI with liver cirrhosis was significantly lower than that without liver cirrhosis (p < 0.05). CONCLUSION: The enhancement of cirrhotic liver parenchyma is greater than that of the normal liver parenchyma at every phase of triple-phase dynamic MR imaging.

Sequence heterogeneity of the ten rRNA operons in Clostridium perfringens.

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA(Ile) genes between the 16S and 23S rDNAs, and the other had a tRNA(Ile) genes between the 16S and 23S rDNAs and a tRNA(Asn) gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.

Four subunit a isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific expression during development.

We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717-6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M. (2000) J. Biol. Chem. 275, 29556-29561). The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.

Inferior phrenic arteries: depiction with thin-section three-dimensional contrast-enhanced dynamic MR imaging with fat suppression.

The purpose of this study was to evaluate visibility of the inferior phrenic arteries in normal subjects at thin-section, multiphasic, three-dimensional (3D) contrast-enhanced dynamic magnetic resonance (MR) imaging with fat suppression, and to compare the appearances and frequencies of MR visualization of these vessels between normal and cirrhotic patients. This study included 95 patients (44 normal and 51 cirrhotic patients) who underwent 3D contrast-enhanced dynamic imaging on a high-performance gradient (25 mT/m) system as a part of abdominal MR examinations. The right and left inferior phrenic arteries were visible in 84% and 73% of the normal subjects, respectively. The averaged rating for visibility in the right inferior phrenic artery was significantly greater in the cirrhotic patients than in the normal subjects (2.1 +/- 0.1 vs. 1.7 +/- 0.2; P = 0.040). Mean diameters of the right inferior phrenic artery in the cirrhotic patients (1.7 +/- 0.1 mm) were significantly larger (P = 0.002) than those in the normal subjects (1.3 +/- 0.1 mm). No significant difference was noted in the mean diameters and the visibility of the left inferior phrenic artery between the two groups. The inferior phrenic arteries can frequently be identified on thin-section, 3D contrast-enhanced arterial-phase dynamic MR images with fat-suppression techniques. Dilatation of the right inferior phrenic artery depicted by this technique may be a nonspecific but an additional secondary finding suggestive of cirrhosis.

Heterogeneity of T cell clones specific for a single indirect alloantigenic epitope (I-Ab/H-2Kd54-68) that mediate transplant rejection.

BACKGROUND: One of the complexities of solid organ allograft rejection is the inherent diversity of the specific T cell antigenic epitopes that participate in this response, including the role of direct alloantigen recognition and indirect recognition of donor-derived peptides in recipient antigen-presenting cells. To probe the role of distinct T cell receptor (TCR) avidity differences and the role of cytokine expression patterns of different effector T cells that may participate in allograft rejections, we have identified a dominant allopeptide derived from the H-2Kd molecule, recognized by H-2b CD4 T cells in the context of syngeneic I-Ab. METHODS: To identify a stimulatory peptide derived from the H-2Kd molecule, a panel of synthetic overlapping peptides was screened for immunogenicity and a panel of T cell clones established. These clones were characterized for TCR Vbeta usage by mAb staining and/or reverse transcribed-polymerase chain reaction analysis, peptide dose sensitivity as a marker of TCR avidity, cytokine expression phenotype in vitro, and their ability to mediate rejection of a vascularized cardiac allograft after adoptive transfer to immunodeficient mice. RESULTS: The H-2Kd54-68 peptide was identified as a dominant stimulatory peptide by the ability of T cells from C57BL/6 (H-2b) mice primed by a combination of allogeneic spleen cell injection and mixed peptide immunization to mount an in vitro proliferative response and interferon-gamma production by peptide stimulation. Furthermore, direct immunization with synthetic H-2Kd54-68 peptide of normal C57BL/6 mice resulted in accelerated rejection of both skin and cardiac allografts from B10.D2 (H-2d) mice, but not 3rd party B10.BR (H-2k) grafts. A panel of 15 distinct CD4+ clones specific for H-2Kd54-68 peptide were established and shown to utilize a variety of TCR Vbeta and different apparent TCR avidities to H-2Kd54-68 peptide when stimulated in vitro. To characterize these clones further, two clones were chosen based on the difference of avidity to H-2Kd54-68 peptide. The cytokine expression pattern was determined and indirect alloantigen specificity confirmed by analysis of responses to purified peptide and B10.D2 spleen cells using normal H.2b and I-Abeta chain knockout mice as APC donors. Both of these T cell clones were able to mediate rejection of B10.D2, but not B10.BR hearts, in immunodeficient mice, but the morphological pattern of T cell infiltration was distinct. CONCLUSIONS: These results demonstrate the potential importance of fine dissection of the alloantigeneic response to solid organ transplants and provide unique insights into the role of TCR avidity and cytokine expression patterns in different morphological patterns of transplant rejection.

Oddi sphincter and common channel: evaluation with pharmacodynamic MR cholangiopancreatography using fatty meal and secretin stimulation.

PURPOSE: This study was performed to assess the usefulness of pharmacodynamic MR cholangiopancreatography (MRCP) in depicting the segment covered by the Oddi sphincter. MATERIALS AND METHODS: Twelve volunteers were studied by pharmacodynamic MRCP. After stimulation by the oral intake of a fatty meal and an intravenous injection of secretin, a single-shot rapid acquisition relaxation enhancement (RARE) sequence was used to obtain consecutive images of the segment covered by the Oddi sphincter. The contraction range of the Oddi sphincter and the lengths of the common channel were measured on the MR console by comparing the most contracted image of the sphincter with the most relaxed image. RESULTS: Pharmacodynamic MRCP showed periodic contraction of the Oddi sphincter in all cases. The range of sphincteric contraction over the biliary duct was 8-19 mm (11.8+/-3.2 mm, mean +/- standard deviation) and over the pancreatic duct 8-23 mm (10.0+/-1.5 mm). In 11 of the 12 cases, the common channel was depicted and its length ranged from 3-8 mm (5.2+/-1.3 mm). CONCLUSION: Pharmacodynamic MRCP clearly depicted the range of contraction of the Oddi sphincter and the common channel, which are not usually revealed by conventional MRCP.

Effects of market liberalisation on smoking in Japan.

OBJECTIVE: To document the effect of the liberalisation of the Japanese tobacco market on Japanese smoking rates and on Japanese tobacco industry practices. DATA SOURCE: Asahi Shimbun (major daily newspaper) from 1980 to 1996. STUDY SELECTION: Review of media coverage on the effects of market liberalisation following the imposition of the USA's section 301 trade sanction. DATA SYNTHESIS: The opening of Japan's tobacco market to foreign cigarette companies stalled a decline in smoking prevalence. Smoking rates among young women increased significantly, and also appear to be on the rise among adolescents. Aggressive marketing and promotional activities by US and Japanese tobacco companies in response to trade liberalisation appear responsible for these adverse trends. Steep increases in sales through vending machines were also possible contributors to the rising smoking prevalence among adolescents. On the positive side, market liberalisation indirectly promoted smoking control efforts in Japan, by causing an anti-smoking movement to coalesce. CONCLUSION: Market liberalisation in Japan played a significant role in increasing smoking prevalence among young women and adolescents while helping to transform the issue of smoking in Japan from a matter of individual choice to a public health problem.

A preliminary study of discrimination among the components of small pulmonary nodules by MR imaging: correlation between MR images and histologic appearance.

PURPOSE: To investigate whether magnetic resonance (MR) imaging depicts the internal characteristics of small pulmonary nodules. METHODS: We reviewed MR images of 39 surgically resected pulmonary nodules 3 cm or less and compared the components within the nodules. In 22 malignant nodules, eight histologic components were characterized by signal and enhancement patterns on MR images. RESULTS: MR images obtained from any single sequence discriminated all components in 26 (67%) nodules, whereas the combination of images from various sequences allowed discrimination in 35 (90%). Fourteen of 16 components of aggregated tumor cells showed marked early enhancement. Although fibrotic and necrotic components showed no or slight early enhancement, nine of 10 fibrotic components showed hypointensity and six of seven necrotic components showed hyperintensity on T2-weighted images. Component characterization in eight histologies by MR imaging was possible in 71-100%. CONCLUSION: Our study demonstrated that MR imaging offers the possibility of high tissue-contrast resolution in small pulmonary nodules.

Foreign serum-induced bile duct lesion (BDL) in athymic BALB/c nude mice.

To investigate a role of cellular immunity in foreign serum-induced bile duct lesion (BDL) in mice, athymic BALB/c nude (nu/nu) mice were intraperitoneally injected with swine serum (SS) twice a week up to 8 weeks and were compared with euthymic BALB/c heterozygote (nu/+) and wild-type (+/+) mice treated with SS in the same way for 4 weeks. All immunized nu/+ and +/+ mice developed marked BDL, and their sera showed high anti-SS IgE and IgG1 antibody titers, whereas no immunized nu/nu mice developed lesions, and their sera showed no elevation of antibody titers. Next, nu/nu mice were reconstituted with splenocytes derived from nu/+ mice, and then were intraperitoneally injected with SS twice a week for 3 weeks. Most of the reconstituted nu/nu mice developed BDL, and their sera showed the elevation of anti-SS IgE and IgG antibody titers. These results suggest that cellular immunity may play a pivotal role in the pathogenesis of swine serum-induced BDL.

Immunopathological study on the development of swine serum-induced bile duct lesions in BALB/c and DBA/2 mice.

To compare the difference in the development of swine serum (SS)-induced bile duct lesion (BDL) between high responder BALB/c and low responder DBA/2 mice, the mice of both strains injected with SS twice a week for up to 4 weeks were killed and examined immunopathologically after the 2nd, 4th, 6th and 8th SS-injection, respectively. In BALB/c mice, BDL developed rapidly following the SS-injections, and a slight enlargement of common bile ducts accompanied with infiltration of T helper cells and eosinophils was detected after the 2nd SS-injection. From the 4th injection on, BDL was characterized by proliferation of mucous glands, hyperplasia and hypertrophy of biliary and glandular epithelial cells, periductal fibrosis, infiltration of eosinophils, plasma cells and T helper cells, and increase of mast cells, resulting in more apparent enlargement of common bile ducts. Several hypertrophied biliary and glandular epithelial cells were positive for mouse immunoglobulins and SS. BDL subsided after cessation of the SS-treatment. On the other hand, in DBA/2 mice, immune response and inflammatory reaction were very weak, and only slight BDL were detected.

Process of carboxylation of glutamic acid residues in the gla domain of human des-gamma-carboxyprothrombin.

In the absence of vitamin K (VK) or in the presence of VK antagonists, hepatic VK-dependent carboxylase activity is inhibited and des-gamma-carboxyprothrombin (DCP) is released into the blood. We analyzed the number of glutamic acid (Glu) residues and their positions in the Gla domain (GD) of DCP to investigate the gamma-carboxylation mechanism of VK-dependent carboxylase. Several DCPs were found in each subject studied. The 10 Gla residues of human prothrombin were carboxylated in order from the N-terminal (residues 26, 25, 16, 29, 20, 19, 14, 32, 7 and 6). The process of Glu carboxylation seemed to proceed three-dimensionally from inside to outside the molecule.

Viral-induced cirrhosis: grading of severity using MR imaging.

OBJECTIVE: The purpose of this study was to determine whether MR imaging can be used to grade the severity of cirrhosis. MATERIALS AND METHODS: The MR examinations of 46 patients with cirrhosis were retrospectively reviewed independently by two radiologists and correlated with clinical severity assessed by Child-Pugh classification. MR imaging analysis by reviewers who were unaware of clinical status included comparison of volume indexes (computed as the product of three axis measurements) of the spleen and each segment of the liver, and changes in hepatic contour, iron or fat deposition, and presence of varices and collaterals. RESULTS: Volume index of the spleen and the presence of ascites and varices were significantly and positively correlated (p = .008, .002, .0001, respectively) with the clinical severity of cirrhosis (Child-Pugh classifications), and volume indexes of the posterior, medial, and lateral segments of the liver were significantly and inversely correlated (p = .001, .049, .041, respectively). On an MR scoring system based on four items (volume index of the spleen; volume index of posterior + medial + lateral segments; presence of ascites; and presence of varices and collaterals), averaged total MR scores were 2.5 +/- 0.3, 4.9 +/- 0.6, and 7.9 +/- 0.8 for Child-Pugh grades A, B, and C, respectively (p < .0001). The accuracy of MR scoring in distinguishing between clinical Child-Pugh grade A cirrhosis and further grades was 89%, the sensitivity was 93%, and the specificity was 82%. CONCLUSION: An MR scoring system can be used to grade the severity of cirrhosis.

Hepatocellular carcinoma: association with increased iron deposition in the cirrhotic liver at MR imaging.

PURPOSE: To determine whether the frequency of hepatocellular carcinoma (HCC) in patients with cirrhosis is affected by hepatic iron deposition as detected with magnetic resonance (MR) imaging. MATERIALS AND METHODS: In a retrospective search of MR imaging and histopathology records, 196 patients with histopathologically proved cirrhosis and with (n = 80) or without (n = 116) HCC who underwent T2-weighted conventional or fast spin-echo and gradient-echo (GRE) (echo time > or = 6.0 msec) imaging were identified. MR images were qualitatively and quantitatively evaluated for diffuse hepatic iron deposition and siderotic regenerative nodules to assess their correlation with the presence of HCC. RESULTS: Hepatic parenchymal iron deposition was seen in 79 (40%) patients, and iron deposition in regenerative nodules was seen in 71 (36%) at MR imaging. The mean signal intensity ratio of GRE images in patients with hepatic iron deposition was significantly lower than that in patients without it (P < .001). The frequency of HCC in patients with iron deposition in regenerative nodules (52% [37 of 71 patients]) was significantly higher (P = .015) than that in patients without iron in regenerative nodules (34% [43 of 125 patients]). CONCLUSION: The occurrence of HCC may be associated causally with iron deposition in regenerative nodules in patients with cirrhosis. MR imaging can enable detection of iron deposition in regenerative nodules as a possible risk factor for the development of HCC.