Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins

Galectin-9 and galectin-8, members of beta-galactoside-binding animal lectin family, are promising agents for the treatment of immune-related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin-9 and galectin-8.     

Quantitative structure-activity studies of octopaminergic 2-(Arylimino)thiazolidines and Oxazolidines against the nervous system of Periplaneta americana L.

The quantitative structure-activity relationship (QSAR) of octopaminergic 2-(arylimino)thiazolidines (AITs) and 2-(arylimino)oxazolidines (AIOs) against the thoracic nerve cord of the American cockroach, Periplaneta americana L., was analysed using reported physicochemical parameters and regression analysis. The more electron-donating, the less bulky at m-position, and the more hydrophobic the substituent, the greater the activity. The plots of observed log V_max values against calculated log V_max values having substituents on the m-position deviated downwards from those of compounds having substituents at the o- and/or p-positions. The more hydrophobic and the more electron-withdrawing the substituent, the greater the activity. AIO with a 2,3,4-trichlorophenyl group (58) was more active than its thiazolidine derivative, 2-(2,3,4-trichlorophenylimino)thiazolidine (38) in terms of V_max:V_max of 58 was 30% relative to octopamine (OA), whereas that of 38 has been 9% relative to OA, respectively. Superimposition of energy-minimized OA and 58 revealed structural and conformational similarities that might account for the high activity of 58.     

Fine root biomass in two black spruce stands in interior Alaska: effects of different permafrost conditions

Key message

In black spruce stands on permafrost, trees and understory plants showed higher biomass allocation especially to ‘thin’ fine roots (diam. < 0.5 mm) when growing on shallower permafrost table.

Abstract

Black spruce (Picea mariana) forests in interior Alaska are located on permafrost and show greater below-ground biomass allocation than non-permafrost forests. However, information on fine roots (roots <2 mm in diameter), which have a key role in nutrient uptake and below-ground carbon flux, is still limited especially for effects of different permafrost conditions. In this study, we examined fine root biomass in two black spruce stands with different depths to the permafrost table. In the shallow permafrost (SP) plot, fine root biomass of black spruce trees was 70 % of that in the deep permafrost (DP) plot. In contrast, ratio of the fine root biomass to above-ground biomass was greater in the SP plot than in the DP plot. Understory plants, on the other hand, showed larger fine root biomass in the SP plot than in the DP plot, whereas their above-ground biomass was similar between the two plots. In addition, biomass proportion of ‘thin’ fine roots (diam. <0.5 mm) in total fine roots was greater in the SP plot than in the DP plot. These results suggest that black spruce trees and understory plants could increase biomass allocation to fine roots for efficient below-ground resource acquisition from colder environments with shallower permafrost table. In the SP plot, fine roots of understory plants accounted for 30 % of the stand fine root biomass, suggesting that understory plants such as Ledum and Vaccinium spp. would have significant contribution to below-ground carbon dynamics in permafrost forests.

     

Giant proteoliposomes prepared by freezing-thawing without use of detergent: reconstitution of biomembranes usually inaccessible to patch-clamp pipette microelectrode

When sarcoplasmic reticulum membrane vesicles or synaptosomes were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing, giant vesicles of up to 50 microns in diameter were formed. When the biomembrane vesicles were labeled with a covalently binding fluorescent dye, the resultant giant vesicles were fluorescent, thereby suggesting that the freezing-thawing process induces fusion of phospholipid and biomembrane vesicles. When membranes of the giant proteoliposomes thus prepared were studied using the patch-clamp technique, potassium channels of the biomembranes were detectable. The present method of the giant proteoliposome preparation is simple and rapid, and provides a system suitable for the study of ion channels of various biomembranes usually inaccessible to a patch-pipette microelectrode.     

Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposomes could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential. Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.     

Stereochemistry and Reactions of Aziridinyl- phosphinothionates Derived from Amino Acids

Aziridinylphosphinothionates were prepared from optically active ethyl hydrogen phenylphos- phonothionates and l-bromo-2-alkanamines derived from leucine or valine. The aziridine ring was opened by the action of some nucleophiles. Refluxing the aziridinylphosphinothionates in acetone with sodium iodide caused hydrolysis accompanied by the rearrangement of the sulfur atom to give β -mercaptoethylphosphonamidates. The reaction mechanism was discussed with stereochemical considerations. The insecticidal activity of the products was also examined.     

Enhancing effect of magnesium ion on cell-free synthesis of read-through protein of bacteriophage Qbeta.

This report describes the enhancing effect of magnesium ion on the synthesis of read-through protein of bacteriophage Qβ in a cell-free protein synthesizing system from $\text{E. coli}$. At 6 mM of magnesium acetate, the major product was coat protein. At 12 mM of magnesium, it was replaced by read-through protein. This enhanced synthesis was substituted by the addition of 0.25 mM of spermine or 1 mM of spermidine to 6 mM of magnesium. These results suggest that magnesium or combination of magnesium and polyamines causes leaky termination at the end of the coat protein cistron of Qβ-RNA.     

Dissection of the ATP-Dependent Conformational Change Cycle of a Group II Chaperonin

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1–2 s after mixing. Only in the presence of K⁺ that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K⁺. Without K⁺, a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K⁺, a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.