Intractable gastrointestinal (GI) diseases often develop during infancy. Our group previously reported that natriuretic peptide receptor B (NPR-B)-deficient Npr2
mice exhibit severe intestinal ...dysfunction, such as stenosis and distention, which resembles the dysfunction observed in Hirschsprung's disease-allied disorders. However, the root cause of intestinal dysfunction and the detailed of pathophysiological condition in the intestine are not yet clear. Here, we report that the intestine of preweaning Npr2
mice showed bloodless blood vessels, and nodes were found in the lymphatic vessel. Additionally, the lacteals, smooth muscle, blood vessel, and nerves were barely observed in the villi of preweaning Npr2
mice. Moreover, intramuscular interstitial cells of Cajal (ICC-IM) were clearly reduced. In contrast, villi and ICC-IM were developed normally in surviving adult Npr2
mice. However, adult Npr2
mice exhibited partially hypoplastic blood vessels and an atrophied enteric nervous. Furthermore, adult Npr2
mice showed markedly reduced white adipose tissue. These findings suggest that the cause of GI dysfunction in preweaning Npr2
mice is attributed to defective intestinal development with microcirculation disorder. Thus, it is suggested that NPR-B signaling is involved in intestinal development and control of microcirculation and fat metabolism. This report provides new insights into intractable GI diseases, obesity, and NPR-B signaling.
The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA ...wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo–electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4’ four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone–histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.
Fatty acid synthase (FASN) expression is elevated in several cancers, and this over-expression is associated with poor prognosis. Inhibitors of FASN, such as orlistat, reportedly show antitumor ...effects against cancers that over-express FASN, making FASN a promising therapeutic target. However, large variations in FASN expression levels in individual tumors have been observed, and methods to predict FASN-targeted therapy outcome before treatment are required to avoid unnecessary treatment. In addition, how FASN inhibition affects tumor progression remains unclear. Here, we showed the method to predict FASN-targeted therapy outcome using radiolabeled acetate uptake and presented mechanisms of FASN inhibition with human prostate cancer cell lines, to provide the treatment strategy of FASN-targeted therapy. We revealed that tumor uptake of radiolabeled acetate reflected the FASN expression levels and sensitivity to FASN-targeted therapy with orlistat in vitro and in vivo. FASN-targeted therapy was noticeably effective against tumors with high FASN expression, which was indicated by high acetate uptake. To examine mechanisms, we established FASN knockdown prostate cancer cells by transduction of short-hairpin RNA against FASN and investigated the characteristics by analyses on morphology and cell behavior and microarray-based gene expression profiling. FASN inhibition not only suppressed cell proliferation but prevented pseudopodia formation and suppressed cell adhesion, migration, and invasion. FASN inhibition also suppressed genes involved in production of intracellular second messenger arachidonic acid and androgen hormones, both of which promote tumor progression. Collectively, our data demonstrated that uptake of radiolabeled acetate is a useful predictor of FASN-targeted therapy outcome. This suggests that 1-(11)Cacetate positron emission tomography (PET) could be a powerful tool to accomplish personalized FASN-targeted therapy by non-invasive visualization of tumor acetate uptake and selection of responsive tumors. FASN-targeted therapy could be an effective treatment to suppress multiple steps related to tumor progression in prostate cancers selected by 1-(11)Cacetate PET.
CuCu-diacetyl-bis(
-methylthiosemicarbazone) (
CuCu-ATSM) is a radioactive hypoxia-targeting therapeutic agent being investigated in clinical trials for malignant brain tumors. For the quality ...management of
CuCu-ATSM, understanding trace metal impurities' effects on the chelate formation of
Cu and ATSM is important. In this study, we conducted coordination chemistry studies on metal-ATSM complexes. First, the effects of nonradioactive metal ions (Cu
, Ni
, Zn
, and Fe
) on the formation of
CuCu-ATSM were evaluated. When the amount of Cu
or Ni
added was 1.2 mol or 288 mol, equivalent to ATSM, the labeling yield of
CuCu-ATSM fell below 90%. Little effect was observed even when excess amounts of Zn
or Fe
were added to the ATSM. Second, these metals were reacted with ATSM, and chelate formation was measured using ultraviolet-visible (UV-Vis) absorption spectra. UV-Vis spectra showed a rapid formation of Cu
and the ATSM complex upon mixing. The rate of chelate formation by Ni
and ATSM was lower than that by Cu-ATSM. Zn
and Fe
showed much slower reactions with the ATSM than Ni
. Trace amounts of Ni
, Zn
, and Fe
showed little effect on
CuCu-ATSM' quality, while the concentration of impurity Cu
must be controlled. These results can provide process management tools for radiopharmaceuticals.
Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for ...mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes—
COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and
RPL18A—would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line.
COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore,
COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.
The biological importance for the signaling of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) has been recognized. However, the details remain unclear and are debatable. ...The Npr2 is a gene of NPR-B, and we previously reported a unique phenotype of a spontaneous mutant mouse lacking Npr2 (Npr2.sup.slw/slw), such as severe ileus-like disorder with bloodless blood vessels. In this study, we analyzed the bloodless mesenteric vascular morphology of Npr2.sup.slw/slw by histological observation to clarify the effects of the CNP/NPR-B signal deficiency. Blood vessels in the mesentery were clearly dilated in the preweaning Npr2.sup.slw/slw mice. Additionally, in the Npr2.sup.slw/slw mice, the lacteals were partially dilation or randomly direction mucosal epithelial cells in villi, and mesenteric adipocytes were undeveloped. These findings provide important information for understanding the role of CNP/NPR-B signals on intestine with mesentery.
C-type natriuretic peptide (CNP) exerts its main biological effects by binding to natriuretic peptide receptor B (NPR-B), a membrane-bound guanylyl cyclase receptor that produces cyclic guanosine ...monophosphate (cGMP). CNP is known to cause gastrointestinal (GI) smooth muscle relaxation. Experimental evidence suggests a connection between CNP signaling and GI function, with reactive regions in the GI tract possibly affecting transit; however, this relation has not yet been conclusively shown. Here, we show that CNP plays important region-specific roles in the GI tract of mice. We found that treatment with CNP (1 or 2 mg/kg) increased transient cGMP production in the pylorus, colon, and rectum, with the higher dose (2 mg/kg) enhancing gastric emptying in mice; this increase in cGMP levels was however absent in NPR-B-deficient short-limbed dwarfism (SLW) mouse. Furthermore, we found that NPR-B is highly expressed in the pylorus, colon, and rectum, being localized to nerve fibers and to the nuclei and cytoplasm of smooth muscle cells of the GI tract and blood vessels. Our in vivo findings showed that NPR-B-mediated cGMP production after CNP administration specifically acted on the pylorus, colon, and rectum and contributed to gastric emptying. CNP may thus be a potential therapeutic agent for GI motility/transit disorders such as ileus and pyloric stenosis.
Natriuretic peptide receptor B (NPR-B), which has high affinity for C-type natriuretic peptide (CNP) and synthesizes intracellular cGMP, may be involved in gastrointestinal tract (GIT) regulation. A ...mutant allele of the NPR-B-encoding gene ( Npr2 ) is responsible for the phenotype of the short-limb dwarfism (SLW) mouse. Homozygosity for this autosomal-recessive gene ( slw / slw ) leads to dwarfism and death before weaning because of milk retention in the stomach and intestinal distention. To elucidate the relationship between CNP/NPR-B signaling and GIT function, we investigated the association between Npr2 mutation and the GIT phenotype in slw / slw mice. The pylorus and large intestine of the mutants did not respond to CNP stimulation; further, they showed pyloric lumen narrowing with randomly aligned circular muscle cells. Comparison of the cGMP and neuronal marker distribution in GIT tissues confirmed cGMP expression in neuronal tissues. An Auerbach’s plexus and submucosal tissues of the mutants didn’t express cGMP and expressed Ca2+ . In contrast, those of normal mice (controls) expressed both cGMP and Ca2+ . Sequencing revealed that the causative Npr2 mutation was a 7-base deletion in exon 8, resulting in a frameshift and premature termination codon appearance. Therefore, the GIT phenotype of slw / slw mice is because of a CNP/NPR-B-signaling defect caused by an Npr2 mutation. These results facilitate better understanding of the role of CNP/NPR-B signaling in GIT motility.
(64)Cu-diacetyl-bis (N (4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising theranostic agent that targets hypoxic regions in tumors related to malignant characteristics. Its diagnostic ...usefulness has been recognized in clinical studies. Internal radiotherapy (IRT) with (64)Cu-ATSM is reportedly effective in preclinical studies; however, for clinical applications, improvements to reduce radiation exposure in non-target organs, particularly the liver, are required. We developed a strategy to reduce radiation doses to critical organs while preserving tumor radiation doses by controlled administration of copper chelator penicillamine during (64)Cu-ATSM IRT.
Biodistribution was evaluated in HT-29 tumor-bearing mice injected with (64)Cu-ATSM (185 kBq) with or without oral penicillamine administration. The appropriate injection interval between (64)Cu-ATSM and penicillamine was determined. Then, the optimal penicillamine administration schedule was selected from single (100, 300, and 500 mg/kg) and fractionated doses (100 mg/kg×3 at 1- or 2-h intervals from 1 h after (64)Cu-ATSM injection). PET imaging was performed to confirm the effect of penicillamine with a therapeutic (64)Cu-ATSM dose (37 MBq). Dosimetry analysis was performed to estimate human absorbed doses.
Penicillamine reduced (64)Cu accumulation in the liver and small intestine. Tumor uptake was not affected by penicillamine administration at 1 h after (64)Cu-ATSM injection, when radioactivity was almost cleared from the blood and tumor uptake had plateaued. Of the single doses, 300 mg/kg was most effective. Fractionated administration at 2-h intervals further decreased liver accumulation at later time points. PET indicated that penicillamine acts similarly with the therapeutic (64)Cu-ATSM dose. Dosimetry demonstrated that appropriately scheduled penicillamine administration reduced radiation doses to critical organs (liver, ovaries, and red marrow) below tolerance levels. Laxatives reduced radiation doses to the large intestine.
We developed a novel strategy to reduce radiation exposure in critical organs during (64)Cu-ATSM IRT, thus promoting its clinical applications. This method could be beneficial for other (64)Cu-labeled compounds.
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