Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides.

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection.

Molecular and functional characterization of an evolutionarily conserved CREB-binding protein in the Lymnaea CNS.

In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long-term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In-situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP-induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.

Real-time visualization of intratumoral necrosis using split-luciferase reconstitution by protein trans-splicing.

Necrosis, a form of cell death, occurs not only with the development of various diseases but also with a tumor tissue response to cancer treatment. Therefore, pursuing progress for cancer therapy through induction of necrosis may be one of the most effective approaches for cancer eradication. We herein describe the development of a real-time imaging system to visualize intratumoral necrosis. The system is composed of two types of cells expressing either one of two necrosis imaging reporters that consist of a DnaE intein sequence linking to one of two split-luciferase fragments. When necrosis occurs in a tumor composed of both of the cells, the two types of leaked reporters can reconstitute the enzymatic activity as a result of protein trans-splicing and thereby emit bioluminescence in the presence of the substrate. This system, which was constructed with shrimp-derived luciferase, allowed in vitro imaging of necrosis. We further confirmed real-time imaging of intratumoral necrosis caused by physical or chemical tissue disruption, validating its application in in vivo necrosis imaging. Thus, the constructed imaging system could be a powerful tool for the optimization of the therapeutic condition for cancer therapy and for the evaluation of novel anticancer drugs targeting necrosis.

Benzenedithiolate-bridged MoFe complexes: structures, oxidation states, and reactivities.

Two benzendithiolate-bridged MoFe complexes, [(Me3P)2(CO)2Mo(µ-S2C6H4)Fe(CO)3] (1) and [(Me3P)(CO)3Mo(µ-S2C6H4)Fe(CO)3] (2), were synthesized by reacting [Mo(S2C6H4)(CO)2(PMe3)2] (3) with Fe(CO)5. Each complex has a direct Mo-Fe bond that is supported by a bridging benzenedithiolate ligand and a semi-bridging carbonyl ligand as elucidated by single-crystal X-ray diffractometry. The structural data and differences in reactivity of these complexes suggest that monophosphine complex 2 is formed via diphosphine complex 1. The reaction of 2 with PMe3 gives the diiron bis(dithiolate) complex, [Fe(S2C6H4)(CO)2(PMe3)]2 (4), rather than 1. 57Fe Mössbauer and X-ray photoelectron spectroscopy studies reveal the oxidation states of the metal centers in 2 to be Fe0 and MoII.

Chemoselective hydrogenation of heteroarenes and arenes by Pd-Ru-PVP under mild conditions.

Monometallic (Pd, Ru or Rh) and bimetallic (Pd0.5-Ru0.5) alloy NPs catalysts were examined for the hydrogenation of quinoline. Pd-Ru alloy catalyst showed superior catalytic activity to the traditional Rh catalyst. The characterization of Pd0.5-Ru0.5 catalysts, HAADF-EDX mapping and XPS analysis suggested that the alloy state of PdRu catalysts remained unchanged in the recovered catalyst. Furthermore, the catalyst was highly selective for the hydrogenation of different arenes.

Distribution of cardiac troponin I in the Japanese general population and factors influencing its concentrations.

BACKGROUND: The 99th percentile of cardiac troponin I level in the general population is accepted as the cut-off for the diagnosis of acute myocardial infarction (AMI). However, it is not clear whether the cut-offs derived in racially and geographically different populations are applicable in Japan. METHODS: Troponin I was determined using the Abbott ARCHITECT STAT high-sensitive troponin I immunoassay in 698 apparently healthy individuals who visited the Japanese Red Cross Medical Center for a health checkup. RESULTS: The 99th percentile of the hsTnI in the overall population was 22.5 (95% confidence interval (CI), 16.8-36.6) pg/mL, 17.7 (95% CI 12.0-22.8) pg/mL for females and 30.6 (95% CI 17.1-53.4) pg/mL for males. The median of the hsTnI in the overall population was 3.2 (95% CI, 3.0-3.3) pg/mL, 2.6 (95% CI 2.4-2.8) pg/mL for females and 4.0 (95% CI 3.8-4.3) pg/mL for males. The age and gender had a significant influence on these values. The troponin I level also showed significant associations with the body mass index (BMI), the gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), estimated glomerular filtration rate (eGFR), and cardiac abnormalities by electrocardiography (ECG) but not with the high-sensitive C-reactive protein (hsCRP) level. CONCLUSIONS: The 99th percentiles of the troponin I measured in the general population in Japan were comparable as the ones derived in the US, Germany, and Singapore. The troponin I level was dependent on the gender, age, BMI, and cardiac abnormalities found by ECG but not by the hsCRP level.

Systematic cytological evaluation and immunocytochemistry of minichromosome maintenance protein 2 and p53 significantly improve cytological diagnosis of pancreaticobiliary adenocarcinoma.

Endoscopic retrograde cholangiopancreatography (ERCP) brushing cytology often cannot distinguish adenocarcinoma from reactive epithelial changes. We attempted to improve the diagnostic sensitivity of ERCP using the following methods: systematic cytological evaluation, immunocytochemical examination of minichromosome maintenance proteins (MCM) 2 and p53, and a combination of these methods. ERCP specimens from 53 patients (13 benign and 40 malignant cases) were studied. First, we reclassified the cases into three categories according to the systematic cytological evaluation: negative, suspicious, and positive. Secondly, immunocytochemistry was performed for MCM 2 and p53. The cut-off values were set at 25% labeling index (LI) for MCM 2 and 10% LI for p53, respectively. We evaluated the sensitivity, specificity, and diagnostic accuracy. The sensitivity of the systematic cytological evaluation alone did not improve significantly, compared with the original screening examination (77% vs. 68%). The sensitivity of immunocytochemistry for MCM 2 and p53 was 90% (P < 0.05) and 68%, respectively. Applying only the suspicious or positive categories, the sensitivity improved significantly to 93% for the combination of systematic cytological evaluation and immunocytochemistry for MCM 2 and p53 (P < 0.01). In conclusion, the combination of morphology and immunocytochemistry for MCM 2 and p53 may help to overcome the diagnostic cytological difficulties of pancreaticobiliary adenocarcinoma.

Significance of nuclear p-Akt in endometrial carcinogenesis: rapid translocation of p-Akt into the nucleus by estrogen, possibly resulting in inhibition of apoptosis.

INTRODUCTION: Endometrial endometrioid adenocarcinoma (endometrial cancer) develops through endometrial hyperplasia caused by estrogenic hyperstimulation. Estrogen is known to activate growth factor signaling pathways, resulting in cellular proliferation, but precisely how has not been clarified. The aim of this study was to investigate the significance of estrogen and downstream factors such as the MAPK (MEK, ERK) and Akt pathways in endometrial carcinogenesis. METHODS: The expression of p-MEK, p-ERK, and p-Akt was analyzed immunohistochemically in normal, hyperplastic, and neoplastic endometria. The estrogenic effect on p-Akt was examined using an endometrial cancer cell line (Ishikawa cells). The estrogenic effect on the apoptosis of Ishikawa cells was assessed by the TUNEL method. RESULTS: Phospho-MEK (p-MEK) and p-ERK expression levels were similar among histological types but correlated with each other. The nuclear p-Akt labeling index (LI) was higher in cancer than in normal endometrium and hyperplasia. The nuclear p-Akt LI of well-differentiated cancer (G1) was higher than that of moderately (G2) or poorly (G3) differentiated cancers. The nuclear expression of p-Akt was correlated with that of estrogen receptor α (ER-α). The nuclear p-Akt level was significantly correlated with prognosis in cases of G1. In Ishikawa cells transfected with ERα, p-Akt was translocated into the nucleus from the cytoplasm in 1 to 3 hours after estrogenic stimulation. Further, apoptosis induced by H2O2 was inhibited by estrogen in the ER-α-positive cells. CONCLUSIONS: The translocation of p-Akt into the nucleus by estrogen may be related to the suppression of apoptosis by estrogen and consequently to endometrial carcinogenesis and prognosis in G1 endometrial cancer.