In situ inhibition of tyrosine protein kinase by erbstatin

Erbstatin inhibited the kinase activity of the receptor for epidermal growth factor in cultured A431 cells, while it did not alter turnover of the receptor protein. It also inhibited autophosphorylation of the src gene product p60src in Rous sarcoma virus-infected normal rat kidney cells. Erbstatin did not inhibit the binding of epidermal growth factor to its receptor but did inhibit internalization of epidermal growth factor-receptor complexes. It did not inhibit the epidermal growth factor-stimulated phosphatidylinositol turnover in A431 cells. Thus, erbstatin inhibited two oncogene product-related tyrosine protein kinases in situ and thus is a useful tool to study the role of tyrosine protein kinase.     

Inhibition of cellular phosphatidylinosito, turnover by psi-tectorigenin

Psi-tectorigenin, an isoflavonoid, was isolated from a culture filtrate of actinomycetes as an inhibitor of epidermal growth factor-induced phosphatidylinositol turnover in cultured A431 cells. It inhibited phosphatidylinositol turnover with an IC₅₀ of about 1 μg/ml; thus, its inhibitory activity was 6-times stronger than that of genistein or orobol. When added to cultured A431 cells psi-tectorigenin inhibited phosphatidylinositol turnover without inhibiting epidermal growth factor receptor tyrosine protein kinase. Thus, psi-tectorigenin is a specific inhibitor of phosphatidylinositol turnover and may be a useful tool for the functional analysis of phosphatidylinositol turnover.     

Self-adaptive modification of red-cell membrane lipids in lecithin: Cholesterol acyltransferase deficiency. Lipid analysis and spin labeling

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin:cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin:cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin:cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.     

Application of Overall Dynamic Body Acceleration as a Proxy for Estimating the Energy Expenditure of Grazing Farm Animals: Relationship with Heart Rate

Estimating the energy expenditure of farm animals at pasture is important for efficient animal management. In recent years, an alternative technique for estimating energy expenditure by measuring body acceleration has been widely performed in wildlife and human studies, but the availability of the technique in farm animals has not yet been examined. In the present study, we tested the potential use of an acceleration index, overall dynamic body acceleration (ODBA), as a new proxy for estimating the energy expenditure of grazing farm animals (cattle, goats and sheep) at pasture with the simultaneous evaluation of a conventional proxy, heart rate. Body accelerations in three axes and heart rate for cows (n = 8, two breeds), goats (n = 6) and sheep (n = 5) were recorded, and the effect of ODBA calculated from the body accelerations on heart rate was analyzed. In addition, the effects of the two other activity indices, the number of steps and vectorial dynamic body acceleration (VeDBA), on heart rate were also investigated. The results of the comparison among three activity indices indicated that ODBA was the best predictor for heart rate. Although the relationship between ODBA and heart rate was different between the groups of species and breeds and between individuals (P<0.01), the difference could be explained by different body weights; a common equation could be established by correcting the body weights (M: kg): heart rate (beats/min) = 147.263∙M ^-0.141 + 889.640∙M ^-0.179∙ODBA (g). Combining this equation with the previously reported energy expenditure per heartbeat, we estimated the energy expenditure of the tested animals, and the results indicated that ODBA is a good proxy for estimating the energy expenditure of grazing farm animals across species and breeds. The utility and simplicity of the procedure with acceleration loggers could make the accelerometry technique a worthwhile option in field research and commercial farm use.     

Growth inhibition of Helicobacter pylori by apolyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone)

The anti-proliferative effect of methylglyoxal bis(cyclopentyl-amidino-hydrazone) (MGBCP), a multi-enzyme inhibitor of polyamine biosynthesis, on the growth of Helicobacter pylori was investigated. MGBCP inhibited the cell growth of H. pylori in a dose-dependent manner. The inhibition was partially reversed by the addition of spermidine. Synthesis of macromolecules, DNA, RNA and protein, was inhibited in the spermidine-depleted H. pylori cells. These findings suggest that MGBCP exhibits an anti-proliferative effect on H. pylori by suppression of macromolecule synthesis.     

Design,Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives

Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8 have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11) with primary alcohols proceeded regioselectively to furnish the desired N⁹-substituted products in moderate yield. Evaluation of 4-8 for anti-HBV activity in HepG2 cells revealed that the phosphonate derivative 8 was found to exhibit moderated activity (EC₅₀ value of 0.29 μM), but cytotoxicity (CC₅₀ value of 39 μM) against the host cells was also observed.     

Draft Genome Sequence of a Psychrotolerant Sulfur-Oxidizing Bacterium, Sulfuricella denitrificans skB26, and Proteomic Insights into Cold Adaptation

Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.     

Comparative mapping of crawling-cell morphodynamics in deep learning-based feature space

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.     

Preferential involvement of Tim-3 in the regulation of hepatic CD8+ T cells in murine acute graft-versus-host disease

Tim-3, a member of the T cell Ig mucin (TIM) family regulates effector Th1 responses. We examined Tim-3 and its ligand expression as well as the effects of anti-Tim-3 mAb treatment in a murine model of acute graft-vs-host disease (aGVHD). In mice with aGVHD, Tim-3 expression was markedly up-regulated on splenic and hepatic CD4+ and CD8+ T cells, dendritic cells (DCs), and macrophages, and this was especially dramatic in hepatic CD8+ T cells. Both donor- and host-derived CD8+ T cells induced similar levels of Tim-3. Tim-3 ligand expression was also up-regulated in splenic T cells, DCs, and macrophages, but not in the hepatic lymphocytes. The administration of anti-Tim-3 mAbs accelerated aGVHD, as demonstrated by body weight loss, reduction in total splenocyte number, and infiltration of lymphocytes in the liver. IFN-gamma expression by splenic and hepatic CD4+ and CD8+ T cells was significantly augmented by anti-Tim-3 mAb treatment. In addition, the cytotoxicity against host alloantigen by donor CD8+ T cells was enhanced. These results demonstrate that the anti-Tim-3 treatment in aGVHD augmented the activation of effector T cells expressing IFN-gamma or exerting cytotoxicity. Our results suggest that Tim-3 may play a crucial role in the regulation of CD8+ T cells responsible for the maintenance of hepatic homeostasis and tolerance.     

Inputting information about amino acid residues in protein bioinformatics: a case study on predicting helical regions with a neural network

The best way to introduce information about amino acid residues into calculations of protein bioinformatics was examined. That was done for predicting helical regions with a neural network. Several fundamental and instructive ways for information processing were developed and are described.