Preclinical Development of Seriniquinones as Selective Dermcidin Modulators for the Treatment of Melanoma

The bioactive natural product seriniquinone was discovered as a potential melanoma drug, which was produced by the as-yet-undescribed marine bacterium of the rare genus Serinicoccus. As part of a long-term research program aimed at the discovery of new agents for the treatment of cancer, seriniquinone revealed remarkable in vitro activity against a diversity of cancer cell lines in the US National Cancer Institute 60-cell line screening. Target deconvolution studies defined the seriniquinones as a new class of melanoma-selective agents that act in part by targeting dermcidin (DCD). The targeted DCD peptide has been recently examined and defined as a “pro-survival peptide” in cancer cells. While DCD was first isolated from human skin and thought to be only an antimicrobial peptide, currently DCD has been also identified as a peptide associated with the survival of cancer cells, through what is believed to be a disulfide-based conjugation with proteins that would normally induce apoptosis. However, the significantly enhanced potency of seriniquinone was of particular interest against the melanoma cell lines assessed in the NCI 60-cell line panel. This observed selectivity provided a driving force that resulted in a multidimensional program for the discovery of a usable drug with a new anticancer target and, therefore, a novel mode of action. Here, we provided an overview of the discovery and development efforts to date.     

Engineering of Bacillus thuringiensis insecticidal proteins

Bacillus thuringiensis (Bt) has been used as sprayable pesticides for many decades. Bt strains utilized in these products produce multiple insecticidal proteins to complement a narrow insect specificity of each protein. In the late 1990s, genes encoding Bt insecticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry1Ac to control Heliothis virescens (tobacco budworm). Cry1Ab was applied to corn to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry1Ac and Cry1Ab, new Bt proteins are required to overcome the resistance. In order to protect corn furthermore, it is desired to control Diabrotica virgifera (Western corn rootworm), Helicoverpa zea (corn earworm) and Spodoptera frugiperda (fall armyworm). Recently, many new Bt insecticidal proteins have been discovered, but most of them require protein engineering to meet the high activity standard for commercialization. The engineering process for higher activity necessary for Bt crops is called optimization. The seed industry has been optimizing Bt insecticidal proteins to improve their insecticidal activity. In this review, several optimization projects, which have led to substantial activity increases of Bt insecticidal proteins, are described.     

The potential of organoids in toxicologic pathology: Histopathological and immunohistochemical evaluation of a mouse normal tissue-derived organoid-based carcinogenesis model

Recently, we introduced an organoid-based chemical carcinogenesis model using mouse normal tissue-derived organoids. In the present review article, the histopathological and immunohistochemical characteristics of mouse normal tissue-derived organoids and tumors derived from these organoids after their in vitro treatment with genotoxic carcinogens and injection into nude mouse are reviewed. In organoids treated in vitro with genotoxic carcinogens, we confirmed macroscopic tumorigenicity and histopathological findings, including neoplastic characteristics, such as multilayered epithelia and/or invasion of epithelia into the surrounding interstitium. In contrast glandular/cystic structures with monolayered epithelia were clearly demarcated from the surrounding Matrigel/interstitium in the untreated control groups. In addition to macroscopic tumorigenicity, these microscopic epithelial changes, which are characteristic of the early stages of carcinogenesis, are included in the requirements for carcinogenicity-positive judgement of the organoid-based carcinogenesis model. Immunohistochemistry of cytokeratins (CKs), used to determine the origin of epithelia and distribution of extraductal invasive lesions, or oncogenic kinases, which reflect molecular activation in epithelia following chemical treatment, is helpful for accurate diagnosis and molecular evaluation in the early stages of carcinogenesis. This information improves our biological understanding of organoid-based chemical carcinogenesis models.     

Population structure of <Emphasis Type="Italic">Fusarium asiaticum</Emphasis> from two Japanese regions and eastern China

Genetic subdivision of Fusarium asiaticum was investigated using a collection of 478 isolates originating from the Kyushu area and Aichi Prefecture, Japan and Zhejiang Province in China. Trichothecene-type determination by a multiplex PCR-test indicated that all isolates were either of a nivalenol (NIV) or a 3-acetyl deoxynivalenol (3ADON) type. The 15-acetyl deoxynivalenol (15ADON) type was not detected in this collection. Based on a Bayesian model-based clustering method using allele data obtained with 11 variable number of tandem repeats (VNTR) markers, we detected three genetic clusters. The majority of isolates in the clusters were NIV isolates from both Japan and China, Japanese 3ADON and Chinese 3ADON isolates, respectively. High levels of fixation indices and low levels of effective number of migrants were observed between the genetic clusters. Data was re-analyzed by classifying the isolates into six groups according to trichothecene type and geographic location. Population analyses of these re-classified groups indicated that the genetic subdivisions of F. asiaticum were correlated with both trichothecene type and geographic differences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Liver metabolomic analysis in broiler chicks: Profiling the metabolites after oral administration of l-citrulline

Hypothermia is directly linked to metabolism; however, it is still unknown how the overall metabolism is altered by oral administration of hypothermic agent, l -citrulline (l -Cit). The present study aimed to determine the characteristics of liver metabolites of chicks orally administered l -Cit to provide a greater understanding of its metabolism. Capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography–time-of-flight mass spectrometry (LC-TOFMS) were conducted on liver samples after oral administration of l -Cit. A total of 361 liver metabolites were identified. Although a small number of samples were used for each group, a principal component analysis and heatmap patterns confirmed that the composition of metabolites could be segregated from each other. Of the 361 compounds detected in the liver, 41 compounds, including amino acids related to the Cit-arginine (Arg) cycle, argininosuccinic acid, Arg, ornithine, and Cit, as well as gamma aminobutyric acid, glycine, histidine, and nicotinamide adenine dinucleotide were abundant in l -Cit-treated livers. In contrast, 24 compounds containing fatty acids, amino acids, and cyclic adenosine monophosphate were lower in the l -Cit group. These data imply that the active Cit-Arg cycle, TCA cycle metabolism, and a low activity in fatty acid metabolism occur in l -Cit-treated broiler chicks.     

Genetic polymorphism of bovine beta-casein gene in Japanese dairy farm herds

The aim of this study was to investigate beta-casein polymorphism among 320 Japanese cows sampled from eight dairy farms. We used a newly-developed genotyping method that involved collecting DNA from hairs and a Cycleave polymerase chain reaction (PCR) assay to detect the A1, A2, and B variants. Results revealed the presence of five genotypes (A1A1, A2A2, A1A2, A1B, and A2B). We found that the most common genotype was A2A2 (0.42), followed by A1A2 (0.39) and A1A1 (0.11). The A1B and A2B genotypes were less frequent (<0.05). The frequencies of alleles A1, A2, and B were calculated to be 0.32, 0.64, and 0.04, respectively. Our study is the first to show the current status of beta-casein polymorphisms in Japanese dairy farms. Given the adverse effects of A1 beta-casein on human health, attempts have been made to develop herds consisting solely of A2A2 cows. Our study provides a reference for improving cow populations in Japanese dairy farms. The Cycleave PCR-based assay we developed here can be used for rapid and reliable genotyping of bovine beta-casein.     

Assessment of Deep Groundwater Quality in Kathmandu Valley Using Multivariate Statistical Techniques

This study was carried out to assess the overall water quality and identify major variables affecting the deep groundwater quality in Kathmandu Valley, Nepal. Forty-two deep wells were sampled during premonsoon and monsoon seasons in 2007 and analyzed for the major physicochemical variables. The water quality variables such as NH ₄ ⁺ -N, Fe, Pb, As, and Cd at most of the sampling locations exceeded the World Health Organization guideline levels for drinking water. Multivariate statistical techniques such as factor analysis and cluster analysis were applied to identify the major factors (variables) corresponding to the different source of variation in deep groundwater quality. Factor analysis indentified six major factors explaining 74.77% of the total variance in water quality; and the major variations are related with the degree of groundwater mineralization, decomposition of organic matter, and reduction of groundwater environment. The water quality of deep groundwater is influenced by the natural hydrogeochemical environment. The wells are broadly divided into two major groups based on the similar groundwater characteristics using cluster analysis. Results show that water quality of deep groundwater does not vary significantly as a function of season.     

Effects of pH, ionic strength, and solutes on DNA adsorption by andosols

The adsorption of DNA by two andosols was investigated as function of solution pH, ionic strength in solution and some solutes, so as to understand the behavior of extracellular DNA molecules in two andosols. DNA adsorptions greatly decreased by increasing pH of suspensions in a pH range of 3.0–9.0. The adsorption of DNA molecules by both andosols was not affected by ionic strength from 0.1 to 0.5 mol L⁻¹ NaCl. However, the DNA adsorption increased proportionally by increasing Mg²⁺ concentration in the suspension. Addition of phosphate decreased DNA adsorption, indicating competition between DNA molecules and phosphate ion in the adsorption by andosols. These results suggest that there are several DNA adsorption mechanisms in soil.     

Enhanced absorption of anthocyanins after oral administration of phytic acid in rats and humans

Many studies on the bioavailability of polyphenols have been reported. However, the relative urinary excretions of AC are also low, ranging from 0.004% to 0.1%. By contrast, other polyphenols show higher urinary excretion levels. Here, we studied the enhancing effects of phytic acid (IP6) on absorption of blackcurrant anthocyanins (BCAs) in rats and humans. In rats after oral administration of BCAs (as 241 mg of AC/kg body weight) in IP6 (0%, 0.25%, 0.5%, 1%, 2.5%) solution, the ACs recovery in urine was increased dependent on IP6 dose. These results suggest that the IP6 enhances gastrointestinal absorption of ACs. At the further analysis of IP6 enhancement effect in rat, whereas BCAs were normally passed through the stomach and duodenum within 2 h, in IP6 group, after 2-6 h post-administration, stomach and jejunum content's weights were specifically heavy, and large amounts of ACs were also detected in stomach, duodenum, and jejunum. These results suggested that the mixture of BCAs and IP6 reduced the gastrointestinal motility. Prolongation of ACs residue in gastrointestinal tract then caused the enhancing effects of IP6 on absorption of AC. In the human study, each subject was orally administrated a BCA beverage containing BCA concentrate (AC 4 mg/kg body weight), 1% of IP6, and 1% of sodium citrate as a pH stabilizer. Both the plasma level and the urinary excretion of AC were increased as compared to BCA administration without IP6. AC intake with IP6 may increase the bioavailability of AC to the comparative level as other polyphenols. Yet, phytic acid, being a strong chelator of important minerals, contributes to mineral deficiencies. An interference with iron uptake has been reported. Safety tests are therefore necessary before high dose IP6 can be used in foods.     

Amla (Emblica officinalis Gaertn.) attenuates age-related renal dysfunction by oxidative stress

To investigate the effects of amla on renal dysfunction involved in oxidative stress during the aging process, we employed young (2 months old) and aged (13 months old) male rats and administered SunAmla (Taiyo Kagaku Co., Ltd., Japan) or an ethyl acetate (EtOAc) extract of amla, a polyphenol-rich fraction, at a dose of 40 or 10 mg/kg body weight/day for 100 days. The administration of SunAmla or EtOAc extract of amla reduced the elevated levels of serum creatinine and urea nitrogen in the aged rats. In addition, the tail arterial blood pressure was markedly elevated in aged control rats as compared with young rats, while the systolic blood pressure was significantly decreased by the administration of SunAmla or EtOAc extract of amla. Furthermore, the oral administration of SunAmla or EtOAc extract of amla significantly reduced thiobarbituric acid-reactive substance levels of serum, renal homogenate, and mitochondria in aged rats, suggesting that amla would ameliorate oxidative stress under aging. The increases of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression in the aorta of aging rats were also significantly suppressed by SunAmla extract or EtOAc extract of amla, respectively. Moreover, the elevated expression level of bax, a proapoptotic protein, was significantly decreased after oral administration of SunAmla or EtOAc extract of amla. However, the level of bcl-2, an antiapoptotic protein, did not show any difference among the groups. The expressions of renal nuclear factor-kappaB (NF-kappaB), inhibitory kappaB in cytoplasm, iNOS, and COX-2 protein levels were also increased with aging. However, SunAmla or EtOAc extract of amla reduced the iNOS and COX-2 expression levels by inhibiting NF-kappaB activation in the aged rats. These results indicate that amla would be a very useful antioxidant for the prevention of age-related renal disease.