Epitope analysis of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis

Abstract In vitro antigenic reactivity of lipid A from Pseudomonas diminuta and Pseudomonas vesicularis with homologous and heterologous lipid A antibodies including monoclonal antibodies was studied by inhibition test of enzyme-linked immunosorbent assay (ELISA). The results suggest that both Pseudomonas lipid As have very similar epitopes, including species-specific and cross-reactive epitopes as compared with enterobacterial lipid A.     

Site-specific mutagenesis of human interleukin-6 and its biological activity

Amino acid substitutions of human interleukin-6 (IL-6) were performed. Single substitution Met162----Ala and double substitutions Leu159.166----Val resulted in a significant decrease of IL-6 activity in the production of immunoglobulin (Ig) from B-cells. Single substitution Leu166----Val or Leu159----Val gave a slight or no significant decrease in the Ig-induction activity, respectively. The receptor-binding activity of each IL-6 mutant was also examined. It was observed that the decrease of the receptor-binding activity was generally in parallel with that of the Ig-induction activity. We therefore suggest that hydrophobic side-chains existing in Met162, Leu159, and Leu166 are significantly involved in the receptor-binding of IL-6.     

A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin

Pulsed field gradient NMR is a convenient alternative to traditional methods for measuring diffusion of biological macromolecules. In the present study, pulsed field gradient NMR was used to study the effects of calcium binding and hydration on carp parvalbumin. Carp parvalbumin is known to undergo large changes in tertiary structure with calcium loading. The diffusion coefficient is a sensitive guide to changes in molecular shape and in the present study the large changes in tertiary structure were clearly reflected in the measured diffusion coefficient upon calcium loading. The (monomeric) calcium-loaded form had a diffusion coefficient of 1.4 x 10(-10) m(2) s(-1) at 298 K, which conforms with the structure being a nearly spherical prolate ellipsoid from X-ray studies. The calcium-free form had a significantly lower diffusion coefficient of 1.1 x 10(-10) m(2) s(-1). The simplest explanation consistent with the change in diffusion coefficient is that the parvalbumin molecules form dimers upon the removal of Ca(2+) at the protein concentration studied (1 mM).     

Self-contained on-chip cell culture and pretreatment system

In this study, we describe a simple on-chip cell culture and pretreatment system that requires no external machines. Conventional cell culture utilizes culture dishes or microtiter plates, where pipetting and centrifugation are indispensable for washing cells and changing media. However, our microdevice requires no external centrifugation or pump. Utilizing this microdevice, we attained dramatically shorter total analytical time with a high-throughput screening system for proteomic analysis (1 min per 12 samples; one eightieth of the conventional time). Protein expression of Jurkat cells during stress-shock induced apoptosis was readily analyzed using this system. We found that a seaweed extraction effectively induced apoptosis of Jurkat cells.     

A 1H-NMR study of the solution conformation of cyclo(GRGDSPA): conformational effects on the physiological activity.

Solution conformations of cyclo(GRGDSPA) have been analyzed by the use of two-dimensional proton nuclear magnetic resonance spectroscopy and the dynamical simulated annealing calculation. It has been shown that the RGDS segment in cyclo(GRGDSPA) takes a beta-turn conformation. We have concluded that this beta-turn conformation is essential for the physiological activity of cyclo(GRGDSPA).     

Forest characteristics and population structure of Glyptostrobus pensilis,a globally endangered relict species of southeastern China

The Chinese water pine Glyptostrobus pensilis is the sole surviving species of the genus Glyptostrobus. It is endemic to southern China, central Vietnam, and eastern Laos, and today it is nearly extinct in the wild. Forest community characteristics and population structure of G. pensilis in China have remained un-known up to now. We investigated six swamp forest stands and analyzed their forest community characteristics (i.e. vertical stratification, species composition, and diversity) and population structure, including the frequency distribution of DBH (diameter at breast height) and age-classes as found in Fujian Province, southeastern China. The vertical stratifications of all the forest stands were rather simple. The remaining wild specimens ranged from roughly 15 to some 357 years for an average of ca. 85 years, with only a few individuals less than 20 years old. Compared with the stands and populations of G. pensilis in Vietnam, the taxonomic compositions of the stands in the two regions were different, except for the dominant species-G. pensilis. The Shannon-Wiener index showed the overstory of each stand had much lower diversity (0.26 on average) in Fujian Province than that (1.97 on average) in Vietnam, whereas the diversity indices were about the same (around 2.41) for the understories in the two regions. Furthermore, we discovered 18 G. pensilis seedlings at the study sites in Fujian Province. This discovery demonstrates that G. pensilis regeneration is extremely poor and its populations are declining, although these populations are rela-tively healthier than those in Vietnam.     

The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation

The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4(ts) mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol phosphosphingolipid phospholipase C, as an additional target of Slm1 and Slm2 negative regulation. Together, our data suggest that Slm1 and Slm2 define a molecular link between phosphoinositide and sphingolipid signaling and thereby regulate actin cytoskeleton organization.