The Tumor Suppressor BCL7B Functions in the Wnt Signaling Pathway

Human BCL7 gene family consists of BCL7A, BCL7B, and BCL7C. A number of clinical studies have reported that BCL7 family is involved in cancer incidence, progression, and development. Among them, BCL7B, located on chromosome 7q11.23, is one of the deleted genes in patients with Williams-Beuren syndrome. Although several studies have suggested that malignant diseases occurring in patients with Williams-Beuren syndrome are associated with aberrations in BCL7B, little is known regarding the function of this gene at the cellular level. In this study, we focused on bcl-7, which is the only homolog of BCL7 gene family in Caenorhabditis elegans, and analyzed bcl-7 deletion mutants. As a result, we found that bcl-7 is required for the asymmetric differentiation of epithelial seam cells, which have self-renewal properties as stem cells and divide asymmetrically through the WNT pathway. Distal tip cell development, which is regulated by the WNT pathway in Caenorhabditis elegans, was also affected in bcl-7-knockout mutants. Interestingly, bcl-7 mutants exhibited nuclear enlargement, reminiscent of the anaplastic features of malignant cells. Furthermore, in KATOIII human gastric cancer cells, BCL7B knockdown induced nuclear enlargement, promoted the multinuclei phenotype and suppressed cell death. In addition, this study showed that BCL7B negatively regulates the Wnt-signaling pathway and positively regulates the apoptotic pathway. Taken together, our data indicate that BCL7B/BCL-7 has some roles in maintaining the structure of nuclei and is involved in the modulation of multiple pathways, including Wnt and apoptosis. This study may implicate a risk of malignancies with BCL7B-deficiency, such as Williams-Beuren syndrome.     

Computer-Aided Prediction of Long-Term Prognosis of Patients with Ulcerative Colitis after Cytoapheresis Therapy

Cytoapheresis (CAP) therapy is widely used in ulcerative colitis (UC) patients with moderate to severe activity in Japan. The aim of this study is to predict the need of operation after CAP therapy of UC patients on an individual level using an artificial neural network system (ANN). Ninety UC patients with moderate to severe activity were treated with CAP. Data on the patients’ demographics, medication, clinical activity index (CAI) and efficacy of CAP were collected. Clinical data were divided into training data group and validation data group and analyzed using ANN to predict individual outcomes. The sensitivity and specificity of predictive expression by ANN were 0.96 and 0.97, respectively. Events of admission, operation, and use of immunomodulator, and efficacy of CAP were significantly correlated to the outcome. Requirement of operation after CAP therapy was successfully predicted by using ANN. This newly established ANN strategy would be used as powerful support of physicians in the clinical practice.     

Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling

To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2^−/− cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE₂-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.     

Intracellular Phospholipase A1 and Acyltransferase, Which Are Involved in Caenorhabditis elegans Stem Cell Divisions, Determine the sn-1 Fatty Acyl Chain of Phosphatidylinositol

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, -9, and -10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of Caenorhabditis elegans, the stearic acid content of PI is reduced, and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A₁ and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.     

Chemo-Mechanical Coupling in F1-ATPase Revealed by Catalytic Site Occupancy during Catalysis

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.     

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.     

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.     

Schistosomicidal and antifecundity effects of oral treatment of synthetic endoperoxide N-89

1,2,6,7-Tetraoxaspiro[7.11]nonadecane (N-89) is a chemically synthesized compound with good efficacy against malaria parasites. We observed strong anti-schistosomal activities of N-89 both in vitro and in vivo. In a murine model with experimental infection of Schistosoma mansoni, orally administered N-89 at the dose of 300 mg/kg resulted in a significant reduction in worm burden (63%) when mice were treated at 2-weeks postinfection. Strong larvicidal effects of N-89 were confirmed in vitro; schistosomula of S. mansoni were killed by N-89 at an EC50 of 16 nM. In contrast, no significant reduction in worm burden was observed when N-89 was administered at 5 weeks postinfection in vivo. However, egg production was markedly suppressed by N-89 treatment at that time point. On microscopic observation, the intestine of N-89-treated female worms seemed to be empty compared with the control group, and the mean body length was significantly shorter than that of controls. Nutritional impairment in the parasite due to N-89 treatment was possible, and therefore quantification of hemozoin was compared between parasites with or without N-89 treatment. We found that the hemozoin content was significantly reduced in N-89 treated parasites compared with controls (P < 0.001). The surface of adult worms was observed by scanning and transmission electron microscopy, but there were no apparent changes. Taken together, these observations suggested that N-89 has strong antischistosomal effects, probably through a unique mode of drug efficacy. As N-89 is less toxic to mammalian host animals, it is a possible drug candidate against schistosomiasis.     

In vivo tracking of transplanted mononuclear cells using manganese-enhanced magnetic resonance imaging (MEMRI)

Background

Transplantation of mononuclear cells (MNCs) has previously been tested as a method to induce therapeutic angiogenesis to treat limb ischemia in clinical trials. Non-invasive high resolution imaging is required to track the cells and evaluate clinical relevance after cell transplantation. The hypothesis that MRI can provide in vivo detection and long-term observation of MNCs labeled with manganese contrast-agent was investigated in ischemic rat legs.

Methods and Findings

The Mn-labeled MNCs were evaluated using 7-tesla high-field magnetic resonance imaging (MRI). Intramuscular transplanted Mn-labeled MNCs were visualized with MRI for at least 7 and up to 21 days after transplantation in the ischemic leg. The distribution of Mn-labeled MNCs was similar to that of ¹¹¹In-labeled MNCs measured with single-photon emission computed tomography (SPECT) and DiI-dyed MNCs with fluorescence microscopy. In addition, at 1–2 days after transplantation the volume of the site injected with intact Mn-labeled MNCs was significantly larger than that injected with dead MNCs, although the dead Mn-labeled MNCs were also found for approximately 2 weeks in the ischemic legs. The area covered by CD31-positive cells (as a marker of capillary endothelial cells) in the intact Mn-MNCs implanted site at 43 days was significantly larger than that at a site implanted with dead Mn-MNCs.

Conclusions

The present Mn-enhanced MRI method enabled visualization of the transplanted area with a 150–175 µm in-plane spatial resolution and allowed the migration of labeled-MNCs to be observed for long periods in the same subject. After further optimization, MRI-based Mn-enhanced cell-tracking could be a useful technique for evaluation of cell therapy both in research and clinical applications.     

Distinct characteristics of circulating vascular endothelial growth factor-a and C levels in human subjects

The mechanisms that lead from obesity to atherosclerotic disease are not fully understood. Obesity involves angiogenesis in which vascular endothelial growth factor-A (VEGF-A) plays a key role. On the other hand, vascular endothelial growth factor-C (VEGF-C) plays a pivotal role in lymphangiogenesis. Circulating levels of VEGF-A and VEGF-C are elevated in sera from obese subjects. However, relationships of VEGF-C with atherosclerotic risk factors and atherosclerosis are unknown. We determined circulating levels of VEGF-A and VEGF-C in 423 consecutive subjects not receiving any drugs at the Health Evaluation Center. After adjusting for age and gender, VEGF-A levels were significantly and more strongly correlated with the body mass index (BMI) and waist circumference than VEGF-C. Conversely, VEGF-C levels were significantly and more closely correlated with metabolic (e.g., fasting plasma glucose, hemoglobin A1c, immunoreactive insulin, and the homeostasis model assessment of insulin resistance) and lipid parameters (e.g., triglycerides, total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C), and non-high-density-lipoprotein cholesterol (non-HDL-C)) than VEGF-A. Stepwise regression analyses revealed that independent determinants of VEGF-A were the BMI and age, whereas strong independent determinants of VEGF-C were age, triglycerides, and non-HDL-C. In apolipoprotein E-deficient mice fed a high-fat-diet (HFD) or normal chow (NC) for 16 weeks, levels of VEGF-A were not significantly different between the two groups. However, levels of VEGF-C were significantly higher in HFD mice with advanced atherosclerosis and marked hypercholesterolemia than NC mice. Furthermore, immunohistochemistry revealed that the expression of VEGF-C in atheromatous plaque of the aortic sinus was significantly intensified by feeding HFD compared to NC, while that of VEGF-A was not. In conclusion, these findings demonstrate that VEGF-C, rather than VEGF-A, is closely related to dyslipidemia and atherosclerosis.