Preparation of polysaccharide supramolecular films by vine-twining polymerization approach

In this study, we investigated the preparation of polysaccharide supramolecular films through the formation of inclusion complexes by amylose in vine-twining polymerization using carboxymethyl cellulose-graft-poly(?-caprolactone) (CMC-g-PCL) as a new guest polymer. First, hydrogels were prepared by phosphorylase-catalyzed enzymatic polymerization in the presence of CMC-g-PCL according to the vine-twining polymerization manner. The XRD result of a powdered sample obtained by lyophilization of the resulting hydrogel indicated the presence of inclusion complexes of amylose with the PCL graft-chains between intermolecular (CMC-g-PCL)s, which acted as supramolecular cross-linking points for the hydrogelation. Then, the supramolecular films were obtained by adding water to the powdered samples, followed by drying. The mechanical properties of the selected films examined by tensile testing were superior to those of a CMC film. The effect of the supramolecular cross-linking structures on the mechanical properties of the films was evaluated further by several investigations.     

Differential population responses of native and alien rodents to an invasive predator,habitat alteration and plant masting

Invasive species and anthropogenic habitat alteration are major drivers of biodiversity loss. When multiple invasive species occupy different trophic levels, removing an invasive predator might cause unexpected outcomes owing to complex interactions among native and non-native prey. Moreover, external factors such as habitat alteration and resource availability can affect such dynamics. We hypothesized that native and non-native prey respond differently to an invasive predator, habitat alteration and bottom-up effects. To test the hypothesis, we used Bayesian state-space modelling to analyse 8-year data on the spatio-temporal patterns of two endemic rat species and the non-native black rat in response to the continual removal of the invasive small Indian mongoose on Amami Island, Japan. Despite low reproductive potentials, the endemic rats recovered better after mongoose removal than did the black rat. The endemic species appeared to be vulnerable to predation by mongooses, whose eradication increased the abundances of the endemic rats, but not of the black rat. Habitat alteration increased the black rat''s carrying capacity, but decreased those of the endemic species. We propose that spatio-temporal monitoring data from eradication programmes will clarify the underlying ecological impacts of land-use change and invasive species, and will be useful for future habitat management.     

Flow threshold for enhanced phorbol ester binding in the ischemic gerbil brain

The correlation between regional phorbol ester binding and cerebral blood flow (CBF) was evaluated in the gerbil brain after 2-hour unilateral common carotid artery occlusion [³H]phorbol 12, 13-dibutyrate (PDBu) was used as a specific ligand for estimating the translocation of protein kinase C (PKC), and CBF was determined by the [¹⁴C]iodoantipyrine method. A quantitative autoradiographic method permitted concurrent measurement of these two parameters in the same brain. In the ischemia group of the animals, statistically significant, inverse correlations were noted between the CBF and PDBu binding in the hippocampus (CA₁ and CA₃ regions and dentate gyrus), the caudate-putamen and lateral nuclei of the thalamus. In these regions, the PDBu binding increased progressively as CBF fell below 35–40 ml/100 g/min. On the other hand, the PDBu binding in the cerebral cortices did not show any significant changes even when CBF was decreased to below 35 ml/100 g/min. The above data suggest that (1) the translocation of PKC to the cell membrane may be regionally specific in response to ischmia and may remain in the regions particularly vulnerable to ischemia such as the hippocampus, caudate-putamen and lateral nuclei of the thalamus in the early ischemic phase; (2) the threshold of CBF below which PKC begins to translocate to the cell membrane in the above regions, may be 35–40 ml/100 g/min in 2-hour ischemia.     

Increased susceptibility to tumor necrosis factor-α in butyric acid-induced apoptosis is caused by downregulation of cFLIP expression in Jurkat T cells

Butyric acid is one of the major extracellular metabolites of periodontopathic Gram-negative bacteria. We previously demonstrated that butyric acid induced apoptosis in human T cells. In the present study, we examined the interaction between butyric acid and TNF-α in Jurkat T-cell apoptosis. Simultaneous treatment with TNF-α enhanced butyric acid-induced apoptosis by promoting caspase activity more than was achieved by either reagent alone. We examined which genes were associated with the increased susceptibility to TNF-α caused by butyric acid, and revealed that expression of cFLIP decreased with increased concentrations of butyric acid. Furthermore, exogenous expression of cFLIP protein suppressed the enhancing effect by TNF-α in the apoptosis. These results suggest that butyric acid downregulates cFLIP expression and increases the susceptibility to TNF-α by activating caspases via the death receptor signal.     

A procedure to introduce protein molecules into living mammalian cells

Although several methods are now available by which to introduce macromolecules into cultured living mammalian cells, each has limitations on its adoption as a general means, for a variety of purposes. We describe here a simple procedure to introduce protein molecules into various living mammalian cells. This procedure is based upon the finding that mammalian cells, after exposure to a low concentration of a phospholipid (L-alpha-lysophosphatidylcholine) in the presence of high (hypertonic) concentrations of glycerol became permeable to protein molecules and that a significant portion of the exposed cells regain their viability following incubation in the appropriate growth medium. We have demonstrated that diphtheria toxin (A fragment), horseradish peroxidase and antibodies against SV40 T-antigens are incorporated into living mouse erythroleukemia (Friend) cells, baby hamster kidney (BHK) cells and mouse fibroblasts (C3H), respectively. The volume introduced into a single cell (mouse Friend cells) is approx. 3 X 10(-15) liter, which is comparable to those with other systems. Parameters affecting permeability to protein molecules and viability of the treated cells were also investigated with these and other cell lines.     

Unwinding of DNA induced by fluorescein mercuric acetate

Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double-strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10^?5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single-strand breaks and a smaller number of double-strand breaks, also increased the initial rate. However, this increase was due only to the double-strand breaks, that is, single-strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 10⁵ erg/mm², suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.     

Differential recruitment of CD63 and Rab7‐interacting‐lysosomal‐protein to phagosomes containing Mycobacterium tuberculosis in macrophages

M.tb is an intracellular pathogen which survives within the phagosomes of host macrophages by inhibiting their fusion with lysosomes. Here, it has been demonstrated that a lysosomal glycoprotein, CD63, is recruited to the majority of M.tb phagosomes, while RILP shows limited localization. This is consistent with the author's findings that CD63, but not RILP, is recruited to the phagosomes in macrophages expressing the dominant negative form of Rab7. These results suggest that M.tb phagosomes selectively fuse with endosomes and lysosomes to escape killing activity while acquiring nutrients.     

Alleviation of High-Fat Diet-Induced Fatty Liver Damage in Group IVA Phospholipase A2-Knockout Mice

Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A₂ (IVA-PLA₂), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA₂-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA₂-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA₂-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA₂-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA₂-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E₂, which has a fat storage effect, was lower in IVA-PLA₂-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA₂ alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA₂ metabolites, such as prostaglandin E₂. IVA-PLA₂ could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.     

Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.     

Green tea extracts reduce leukocyte cell–Derived chemotaxin 2 and selenoprotein P levels in the livers of C57BL/6J mice fed a high-fat diet

Epidemiological studies suggest that green tea extracts (GTEs), including catechins such as epigallocatechin gallate and epicatechin gallate, have a beneficial effect on obesity, hyperglycemia, insulin resistance, endothelial dysfunction, and inflammation. Although several studies have shown that catechins directly modulate the cellular and molecular alterations in the liver tissue, the contributions of indirect mechanisms underlying these systemic effects of catechins remain unclear. In this study, we report that, in the C57BL/6J mouse liver, GTEs reduce high-fat diet-induced increases in the levels of hepatokines, liver-derived secretary proteins such as leukocyte cell-derived chemotaxin 2 and selenoprotein P production, which have been shown to induce systemic adverse effects, including several metabolic diseases. These findings suggest that the systemic effects of GTEs involve the regulation of hepatokine production as an indirect mechanism.