A cross-coupling reaction at the carbon vertex of a monocarba-closo-dodecaborate has been developed to allow introduction of various aryl groups and other sp2/sp-hybridized carbon centers. A copper(I) complex facilitated the coupling process with a wide range of electrophiles at room temperature. The reaction was successfully used to prepare a series of C-arylated caborane anion derivatives, some of which showed potent androgen-receptor binding activity and excellent mesogenic properties.
Organometallic compounds and complexes possibly show novel bioactivities unprecedented with organic molecules that consist of C, H, O, and N atoms. We have already reported that an organoantimony compound selectively induces the expression of perlecan, a large heparin sulfate proteoglycan, in cultured vascular endothelial cells. Perlecan consists of a core protein with Mr of approximately 400 kDa and three anticoagulant heparan sulfate glycosaminoglycan chains. However, little is known about the molecular mechanisms of perlecan expression in endothelial cells. Since an organoantimony compound induces the expression of perlecan, we prepared a variety of its derivatives and tried to identify the binding proteins for the organoantimony compound by the drug affinity responsive target stability method, a negative selection method to identify the target proteins. As the results, we found glucocoriticoid receptor as binding protein of the organoantimony compound, which mediates the expression of perlecan in endothelial cells.
Metals are effectively used in biological systems under the strict regulation for exploiting their specific and broad reactivities. For example, manganese (Mn) can induce catecholamines-mediated oxidative biological damage in cooperation with iron (Fe) and/or copper (Cu). In children, the damage could induce developmental disorders such as attention deficit hyperactivity disorder (ADHD). We hypothesize that infant neurons are more labile to metals than adult ones due to the prematured protection systems and sensitive differentiating cells. An experimental system reconstituting neural differentiation is expected to assess the influences of endogenous/exogenous factors including metals. In this study, we investigated an impact of Mn together with Fe and dopamine (DA) on neural differentiation of mouse embryonic stem cells (mESCs). The differentiation of mESCs was initiated by embryoid bodies (EBs) formation in the presence of all-trans retinoic acid, and then EBs were treated with Mn, Fe and/or DA. Then, the mRNA levels of neural differentiation marker genes (Nestin, Emx2, Mtap2, Th, Olig2 and Gfap) were examined using realtime RT-PCR analysis. Mn or DA alone reduced Mtap2, Th and Olig2 expression levels and increased Nestin. Moreover, combined treatment of Mn and DA also increased Nestin expression level. On the other hand, Fe alone reduced Mtap2, Th and Olig2 expression levels, and increased Emx2. Combined treatments of Fe with Mn or DA also tended to increase Emx2 expression level. These effects emerged at about 100 times less concentration than that inducing cytotoxicity in human neuroblastoma. The present study showed that Mn inhibits neural development, and that our mESCs system can be a useful tool to elucidate the toxicity mechanism as well as to evaluate the effects of metals and chemicals on differentiating cells.
Radiolabeled monoclonal antibodies (mAbs) are very useful molecular probes for nuclear imaging technique due to their high-target specificity and high-stability in the bloodstream. MAbs are generally labeled by indirect method using the combination of relatively long-lived metallic radionuclides (including 64Cu, 89Zr, and 111In) and bifunctional chelating agents which are capable of binding both antibodies and radio metals. The indirect radiolabeling method has some advantages such as high labeling efficiency and long-term retention ability within their target cells. However, this conventional labeling method can potentially lead to low-target affinity of the mAb probes, because of the non-site-specific introduction of the bifunctional chelators into the active site of the mAbs. To overcome the shortcoming, we proposed a new direct labeling method utilizing fusion proteins comprising mAbs linked to metal binding peptides at the N- or C-terminus. In this study, we synthesized new peptide derivatives possessing an N-terminal tripeptide sequence (Xaa-Yaa-His) called the amino terminal Cu2+- and Ni2+-binding (ATCUN) motif as 64Cu binding peptides for the proposed labeling method. Moreover, we studied the stability constants of Cu2+-ATCUN peptide complexes by pH titration. From these studies, we found that a low basicity of the N-terminal amine in the peptide resulted in a high stability constant of the complex. This finding may provide valuable guidelines in designing the ATCUN peptide with high-binding affinity toward 64Cu.
We examined the role of Metallothionein (MT) and metal transporter on Cadmium (Cd) transport from mother to fetus. In this study, female Wistar rats were given Cd (as CdCl2) at a dose of 0, 1, 2 and 5 mg Cd/kg/d. Animals were sacrificed on the pregnant 19-21 d. The concentrations of Cd and MT, and gene expression of MT and metal transporters were determined in the placenta and in the fetuses. In mother rats, the concentration of Cd in the placenta increased dose-dependently, significantly. The Cd of MT-Cd form and the Cd of non-MT-Cd form increased also dose-dependently. In pathological examination of placenta tissue, syncytial trophoblast cells were damaged dose-dependently, particularly in 5 mg Cd/kg group. In fetuses, the Cd concentration in the total body, liver and kidney increased dose-dependently. The Cd concentration in liver of fetus was significantly higher than that in kidney. In gene expression of metal transporter, gene expression of Zip-8 and ATP7B increased dose-dependently in placenta in 0 mg, 1 mg and 2 mg groups, but the increase was not statistically significant. Based on these results, it was speculated that Cd in placenta is trapped to transport into fetus by MT in placenta. However, non-bound Cd by MT is leaked to fetus by damage of placenta tissue, and it was speculated that non-MT-Cd accumulated in liver of fetus. Namely, it was suggested the possibility that the non-bound Cd by MT is transported by metal transporter of zinc and copper, especially Zip-8 and ATP7B or leaked from the placenta as caused by damage of synsytial trophoblast.
Blood vessels are composed of endothelial cells and other cell types and they are ubiquitously present in every organ. It is important to study the toxicity of cadmium to endothelial cells for understanding organic cadmium toxicity. Although vascular toxicity of cadmium has been studied previously, little is known about the underlying toxicity mechanism. Cadmium-sensitive mice have been reported to show high expression of zinc transporter, ZIP8, in endothelial cells of the testis vasculature, which produces genetic difference in the response to the testicular cadmium toxicity. ZIP8 is involved in the transport of zinc, manganese, and cadmium from the extracellular space or intracellular compartments into the cytosol. In this report, we have described how cadmium is one of the inducers of ZIP8 expression in vascular endothelial cells, and that cadmium cytotoxicity partly depends on the ZIP8 expression levels.
Zinc is the essential trace element and important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway are not poorly understood. Here, we indicate that the ZIP9 induces increase in intracellular zinc level and plays an important role in the phosphorylation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation requires intracellular zinc. To clarify this event, we used chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and Erk phosphorylation significantly decreased in cZip9KO cells treated with zinc pyrithione (ZnPy), and overexpressing the human Zip9 gene restored these biochemical events. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. Additionally, intracellular zinc was localized at the Golgi, even if it was treated with ZnPy in cZip9KO cells. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation.
Organic-inorganic hybrid molecules can exhibit biological activities by controlling the activities of their individual components. These molecules can be applied as seed/lead compounds in drug development, and can be used as molecular probes in chemical biology for the elucidation of the molecular mechanisms of biological systems. In this review, we introduce copper diethyldithiocarbamate (Cu10), a copper complex, as a compound that activates nuclear factor erythroid 2-related factor 2 (Nrf2) and induces metallothionein, which are the key cellular defense factors against toxic metals and oxidative stress in vascular endothelial cells. It was found that neither the complexes with the same ligand and other metal ions (including zinc and iron) nor the ligand alone was inactive. Therefore, both copper ion and the ligand of Cu10 are essential components for the biological functioning of the complex. In addition, Cu10 may be useful in clarifying the molecular mechanism underlying metallothionein induction in vascular endothelial cells.
Studies of tissue-specific gene expression have suggested that tissue-specific transcription factors or tissue-specific combinations of non-cell-type-specific transcription factors regulate tissue-specific gene expression. Although the studies of endothelial cell (EC)-specific gene expression has identified several transcriptional activators such as SP1, ETS family proteins, and GATA proteins, their expression and combinations are not likely to be EC-specific. To investigate the mechanism of EC-specific gene expression, we analyzed the regulation mechanism of an EC-specific gene, Roundabout4 (Robo4). We identified the 3-kb Robo4 promoter and several transcription factors including SP1 and GABP that bind to the Robo4 promoter and promote Robo4 gene expression. However, we could not explain the mechanism for EC-specific Robo4 gene expression with only those factors because their expression and combination are not EC-specific. Therefore, we hypothesized the contribution of other mechanisms, especially epigenetic control, to Robo4 gene regulation and demonstrated the importance of DNA methylation for EC-specific Robo4 expression. In this review, we summarize our recent studies and discuss the novel regulation model of Robo4 gene expression by transcription factors and DNA methylation.
This study aimed to determine how much time can be saved with the use of unit-of-use packaging for prescription drugs as compared with bulk packaging in community pharmacies as well as to determine the number of errors. In a simulation, mock prescriptions were dispensed either in unit-of-use packages or by transferring medication from a bulk container, and a time study was conducted to measure the time spent on dispensing and prescription auditing by pharmacists. Pharmacists' and patients' degree of satisfaction was also surveyed. The time saved with unit-of-use packaging was 66.25 s per prescription. The sole dispensing error that was found in the study occurred with bulk dispensing. Among both pharmacists and patients, many were of the opinion that dispensing with unit-of-use packaging was preferable to bulk dispensing. Unit-of-use packaging shortens the time that pharmacists spend on dispensing activities and increases the efficiency of their work. Unit-of-use packaging is also thought to reduce the number of counting errors.
Type 2 diabetes caused by chronic obesity is a major lifestyle-related disease. The present study aimed to determine the pathological changes in hepatocytes in chronic obesity. To develop our type 2 diabetes mouse model, we induced chronic obesity to mice by monosodium glutamate. By overeating, the mice significantly increased their body weight compared with age-matched healthy animals. To analyze the pathological changes in hepatocytes of chronic obesity before preclinical stage of type 2 diabetes, the mice were analyzed by hematoxylin-eosin staining of tissue sections at 15 w of age. In these mice, we observed eosin-negative accumulations of hepatocytes around central veins in the hepatic lobule. By Oil-Red O staining, the eosin-negative granules were identified in the lipid droplets. We then ascertained whether these lipid droplets of hepatocytes in the obese mice could be modified by diet. After 24 h of diet restriction, the lipid droplets of hepatocytes in the obese mice were swollen. Furthermore, after 48 h of the diet restriction, the lipid droplets continued swelling and the autophagy-like structures that were found in the healthy mice under the same condition in the obese mice were not observed. These results suggest that the obese mice might have delayed energy metabolism, which might have influenced the mechanisms of hepatocytes. These findings provide new insight into the functional changes in chronic obesity-induced type 2 diabetes and it is possible that the pathological feature make a contribution to promise the target of pharmacological therapy.
In order to provide an opportunity for community pharmacists to actively learn about infection control, this study created learning materials through a board game format and verified characteristics of learning by determining and comparing evaluation according to viewpoint and motivational effects between a lecture and the game. To create the board game, we collected cases of infection from 30 community pharmacists. The game was created using collected and created case studies, and we held a workshop on infection control. Participants were assigned to a lecture (n=32) or game group (n=27) and completed a questionnaire before and after the workshop. The questionnaire included the evaluation according to viewpoint based on the ministry's curriculum guidelines and the motivational effect of Keller's ARCS motivation model. In the evaluation according to viewpoint, the lecture group scores were significantly higher on “knowledge and understanding” than the game group scores. In the comparison of the motivational effects, the game group was significantly higher in three out of the four items of the ARCS motivation model, “Attention”, “Relevance”, and “Satisfaction”. These results indicate that learning through the game aroused the curiosity of the learners, increased the learning outcome, and maintained certain levels of motivation. In addition, the evaluation according to viewpoint showed that the lecture group understood the key concepts and knowledge regarding infection control, whereas there was a possibility that the game group required additional motivational factors for learning and maintaining motivation level.