Freezing activities of flavonoids in solutions containing different ice nucleators

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 μL droplets at 0.2 °C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT(50) between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4-4.2 °C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 °C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 °C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 °C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 °C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 °C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.     

An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.     

Dual modulation of airway smooth muscle contraction by Th2 cytokines via matrix metalloproteinase-1 production

Altered contractility of airway smooth muscle (SM) is one of the main causes of allergic asthma, in which the predominance of Th2 over Th1 cytokines plays a central role. In the present study, we examine the effects of Th2 cytokines on airway SM contraction. Treatment with a low concentration of IL-4 (0.2 ng/ml) for 6 h augmented, whereas higher concentrations (2-20 ng/ml) inhibited, agonist-induced contractions of collagen gels containing bovine tracheal SM cells. Another Th2 cytokine (IL-13) showed an augmentation of gel contraction in the concentration range of 20-200 ng/ml. IL-4 and IL-13 increased mRNA expression and protein secretion of matrix metalloproteinase (MMP)-1, but these cytokines did not affect Ca(2+)-mobilizing properties and phosphorylation levels of myosin L chain in bovine tracheal SM cells. These changes were sensitive to wortmannin, an inhibitor of PI3K, but not to leflunomide, an inhibitor of STAT6. Scanning electron microscope observation revealed that collagen fibers twining around SM cells were completely dissolved in 20 ng/ml IL-4-treated gels and reorganized into basket-like structure in 20 ng/ml IL-13-treated gels. Exogenous application of high and low concentrations of MMP-1 also induced the inhibition and augmentation of gel contraction, respectively. Furthermore, nonselective MMP inhibitor galardin suppressed the effects of IL-4 and IL-13 on gel contraction, and MMP-1-targeted small-interfering RNA reversed the inhibitory effects of IL-4 on gel contraction to the augmentation. This indicates that Th2 cytokines modulate airway contraction without affecting cellular contractility but by secreting MMP-1 from the SM cells via PI3K activation and changing cell-to-matrix interactions.     

Evolutionary conservation and changes in insect TRP channels

Background  

TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts.     

Enhanced anti-influenza A virus activity of (-)-epigallocatechin-3-O-gallate fatty acid monoester derivatives: effect of alkyl chain length

A series of fatty acid monoester derivatives of (−)-epigallocatechin-3-O-gallate (EGCG) were prepared by one-pot lipase-catalyzed transesterification. The introduction of long alkyl chains enhanced anti-influenza A/PR8/34 (H1N1) virus activity 24-fold relative to native EGCG.     

Retrospective Proteomic Analysis of a Novel,Cancer Metastasis-Promoting RGD-Containing Peptide

Patients who undergo surgical extirpation of a primary liver carcinoma followed by radiotherapy and chemotherapy leading to complete remission are nevertheless known to develop cancerous metastases 3–10 years later. We retrospectively examined the blood sera collected over 8 years from 30 patients who developed bone metastases after the complete remission of liver cancer to identify serum proteins showing differential expression compared to patients without remission. We detected a novel RGD (Arg-Gly-Asp)-containing peptide derived from the C-terminal portion of fibrinogen in the sera of metastatic patients that appeared to control the EMT (epithelial-mesenchymal transition) of cancer cells, in a process associated with miR-199a-3p. The RGD peptide enhanced new blood vessel growth and increased vascular endothelial growth factor levels when introduced into fertilized chicken eggs. The purpose of this study was to enable early detection of metastatic cancer cells using the novel RGD peptide as a biomarker, and thereby develop new drugs for the treatment of metastatic cancer.     

Monocarboxylates and glucose utilization as energy substrates in rat brain slices under selective glial poisoning – a 31P NMR study

Molecular and Cellular Biochemistry -     

Hypoperfusion of the Adventitial Vasa Vasorum Develops an Abdominal Aortic Aneurysm

The aortic wall is perfused by the adventitial vasa vasorum (VV). Tissue hypoxia has previously been observed as a manifestation of enlarged abdominal aortic aneurysms (AAAs). We sought to determine whether hypoperfusion of the adventitial VV could develop AAAs. We created a novel animal model of adventitial VV hypoperfusion with a combination of a polyurethane catheter insertion and a suture ligation of the infrarenal abdominal aorta in rats. VV hypoperfusion caused tissue hypoxia and developed infrarenal AAA, which had similar morphological and pathological characteristics to human AAA. In human AAA tissue, the adventitial VV were stenotic in both small AAAs (30–49 mm in diameter) and in large AAAs (> 50 mm in diameter), with the sac tissue in these AAAs being ischemic and hypoxic. These results indicate that hypoperfusion of adventitial VV has critical effects on the development of infrarenal AAA.     

Molecular mechanism for activation and regulation of matrix metalloproteinases during bacterial infections and respiratory inflammation

Matrix metalloproteinases (MMPs) are critical mediators of tissue remodeling. Inappropriate regulation of MMPs causes many pathological events, including microbial invasion and inflammatory tissue damage. Some of the bacterial exoproteinases can effectively activate pro-MMPs (inactive zymogens) via limited proteolysis around their autoinhibitory domains. In addition, overproduction of nitric oxide (NO) may contribute to respiratory inflammation via the formation of reactive nitrogen species (RNS). Several studies have identified regulatory properties of NO/RNS on biomolecules due to functional modification of their cysteine residues. In fact, NO/RNS can mediate activation and expression of MMPs, because RNS can interact with a cysteine switch in the autoinhibitory domain, thus converting proMMPs into their active forms without proteolysis. Many studies have indicated that NO/RNS can participate in expression of various genes that affect immune-inflammatory responses, including MMPs. Although NO in some cases upregulates MMPs, S -nitrosothiols downregulate MMP-9 expression by suppressing the NF-kappaB pathway. While microbial proteinases cause excessive activation of MMPs and contribute to microbial pathogenesis, NO/RNS may modulate expression and activation of MMPs as well as various inflammatory mediators, depending on the redox status at sites of inflammation. Therefore, appropriate regulation of MMPs may be of potential therapeutic value for various infections and inflammatory lung diseases.