Preferential Release of Newly Synthesized Insulin Assessed by a Multi-Label Reporter System Using Pancreatic β-Cell Line MIN6

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP^−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.     

Humans but Not Chimpanzees Vary Face-Scanning Patterns Depending on Contexts during Action Observation

Human and nonhuman primates comprehend the actions of other individuals by detecting social cues, including others’ goal-directed motor actions and faces. However, little is known about how this information is integrated with action understanding. Here, we present the ontogenetic and evolutionary foundations of this capacity by comparing face-scanning patterns of chimpanzees and humans as they viewed goal-directed human actions within contexts that differ in whether or not the predicted goal is achieved. Human adults and children attend to the actor’s face during action sequences, and this tendency is particularly pronounced in adults when observing that the predicted goal is not achieved. Chimpanzees rarely attend to the actor’s face during the goal-directed action, regardless of whether the predicted action goal is achieved or not. These results suggest that in humans, but not chimpanzees, attention to actor’s faces conveying referential information toward the target object indicates the process of observers making inferences about the intentionality of an action. Furthermore, this remarkable predisposition to observe others’ actions by integrating the prediction of action goals and the actor’s intention is developmentally acquired.     

Carotenoid synthesis: Retrospect and recent progress

Carotenoid synthesis during the past ∼30 years is summarized. Total syntheses of peridinin, fucoxanthin and halocynthiaxanthin, and biomimetic total syntheses of mytiloxanthin, crassostreaxanthin B, capsanthin, capsorubin, cucurbitaxanthin A, cycloviolaxanthin and capsanthin 3,6-epoxide are discussed.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

Synthesis of 6-Sulfur Analogues of Oxanosine and Closely Related Derivatives Thereof

Abstract

Novel β-D-ribofuranosides having a 5-substituted imidazo [4,5-d] [1,3]thiazine ring, including the S⁶-congener 3 of oxanosine 2, were synthesized for screening their anticancer and antiviral activities.     

Distributions of cadmium,zinc, and polyphenols in Gamblea innovans

Gamblea innovans is a Cd- and Zn-accumulating deciduous tree widely distributed in the secondary forests of Japan. We aimed to understand the characteristics of Cd and Zn accumulation in G. innovans in order to effectively utilize the species for phytoremediation. To accomplish that, we studied the relationship between secondary metabolite concentrations and the accumulation and distributions of Cd and Zn in G. innovans leaves and basal stems using micro-X ray fluorescence (µ-XRF). Our results showed a negative correlation between Zn leaf concentrations and polyphenol/2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. This finding might be related to stress or the manifestation of a mechanism for tolerance to Cd and Zn accumulation. In addition, we observed that Cd accumulated primarily in the apoplastic region of surface tissues such as bark and the epidermis of leaves, whereas Zn accumulated in both apoplastic and symplastic regions. Thus, it might be possible that G. innovans can distinguish between Cd and Zn and control their translocation.     

Enhanced epithelial-mesenchymal transition-like phenotype in N-acetylglucosaminyltransferase V transgenic mouse skin promotes wound healing

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the β1,6 branching of N-acetylglucosamine on N-glycans. GnT-V expression is elevated during malignant transformation in various types of cancer. However, the mechanism by which GnT-V promotes cancer progression is unclear. To characterize the biological significance of GnT-V, we established GnT-V transgenic (Tg) mice, in which GnT-V is regulated by a β-actin promoter. No spontaneous cancer was detected in any organs of the GnT-V Tg mice. However, GnT-V expression was up-regulated in GnT-V Tg mouse skin, and cultured keratinocytes derived from these mice showed enhanced migration, which was associated with changes in E-cadherin localization and epithelial-mesenchymal transition (EMT). Further, EMT-associated factors snail, twist, and N-cadherin were up-regulated, and cutaneous wound healing was accelerated in vivo. We further investigated the detailed mechanisms of EMT by assessing EGF signaling and found up-regulated EGF receptor signaling in GnT-V Tg mouse keratinocytes. These findings indicate that GnT-V overexpression promotes EMT and keratinocyte migration in part through enhanced EGF receptor signaling.     

A novel carborane analog, BE360, with a carbon-containing polyhedral boron-cluster is a new selective estrogen receptor modulator for bone

Carboranes are a class of carbon-containing polyhedral boron-cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors. Estrogen deficiency results in marked bone loss due to increased osteoclastic bone resorption in females, but estrogen replacement therapy is not generally used for postmenopausal osteoporosis due to the risk of uterine cancer. We synthesized a novel carborane compound BE360 to clarify its anti-osteoporosis activity. BE360 showed a high binding affinity to estrogen receptors (ER), ERα and ERβ. In ovariectomized (OVX) mice, femoral bone volume was markedly reduced and BE360 dose-dependently restored bone loss in OVX mice. However, BE360 did not exhibit any estrogenic activity in the uterus. BE360 also restored bone loss in orchidectomized mice without androgenic action in the sex organs. Therefore, BE360 is a novel selective estrogen receptor modulator (SERM) that may offer a new therapy option for osteoporosis.