Synergistic activity of the ninth and tenth FIII domains of human fibronectin depends upon structural stability

The ninth and tenth FIII domains (FIII9-10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9-10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu(1408) to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9-10. We show that the introduction of mutations into the synergistic sequence of FIII9-10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin alpha(5)beta(1). Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu(1408) to Pro substitution in mutant FIII9-10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to alpha(5)beta(1). In keeping with the enhancement of functional activity, the Leu(1408) to Pro substitution in the FIII9-10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9-10 mutants and suggest that a Leu(1408) to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed.     

The cleavage of the triphosphate bridge of a model for the 5'-cap mRNA promoted by dinuclear bicyclic complexes with metal ions

Dinuclear bicyclic complexes, which have two active centers, can significantly promote the hydrolysis of the triphosphate bridge in ApppA, a 5'-cap model compound.     

Hybridization properties of support-bound oligonucleotides: the effect of the site of immobilization on the stability and selectivity of duplex formation

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 microm) polymer particles through C5-tethered thymine and N(4)-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.     

A minimal receptor-Ig chimera of human FcepsilonRI alpha-chain efficiently binds secretory and membrane IgE

We constructed a soluble minimal receptor-Ig chimera in which the two extracellular domains of human Fcepsilonhain (D1 and D2) were fused to the dimerizing C-terminal domain of human IgG1 heavy chain (gamma1-CH3). The protein was expressed and actively secreted by Chinese hamster ovary (CHO) cells as a fully glycosylated soluble dimeric protein. It showed efficient binding both to human membrane-bound IgE isoforms and to the two secretory IgE isoforms. Moreover, the dimeric receptor binds IgE with the expected 1:2 stoichiometry. The receptor-Ig chimera, in 2-fold molar excess, inhibited engagement of secretory IgE to rat basophilic leukemia cells expressing the human alphabetagamma receptor. Full self-nature and inability to bind Fcgamma receptors make this protein an attractive candidate for clinical applications and a novel biotechnological tool for atopic allergy research.     

Calvarial bone distraction with a contractile bioresorbable polymer

The aim of the present study was to evaluate the possibility of mobilizing calvarial bone with a fully implantable and bioresorbable device. The animal model used was the New Zealand white rabbit (n = 12). An island bone flap attached to the dura mater was created in the parietal region and amalgam markers were placed in this bone flap and in the ipsilateral frontal bone. In one group of six rabbits (group 1), a specially processed contractile 70L/30D,L polylactic acid plate, 15 x 6 x 0.6 mm, was attached to the island flap by one extremity, and to the fixed ipsilateral frontal bone by the other. In group 2 (control), no plate was added. Bone marker movement was followed with serial radiography. In group 1, there was a progressive reduction in mean marker distance over the first 48 hours, and stability thereafter. In group 2 (control), mean marker distance remained stable until the second postoperative week, after which time there was a slight increase until the end of the experimental period. At 4 weeks, the mean marker separation differed significantly between group 1 (mean, -3.62 mm; SD, 0.79 mm) and group 2 (mean, 0.34 mm; SD, 0.14 mm; p <0.001). In conclusion, a totally implantable and bioresorbable device was successfully used to mobilize calvarial bone. Polymer contractility will likely constitute the basis of a new generation of bioresorbable distractors for use in craniofacial surgery.     

Mercury-binding capacity of organic and inorganic selenium in rat blood and liver

The mercury-binding capacity of seleno-DL-methionine and selenium dioxide was assessed in male Wistar rats. Mercury was supplied as fish loaves made of northern pike or rainbow trout. We used a selenium concentration of 3.4 mg/kg fish, about sixfold compared to the equivalent quantity of mercury. Seleno-DL-methionine had a tendency to increase both methyl mercury and total mercury in blood, although it also seemed to reduce the proportion of methyl mercury of total mercury. Selenium dioxide lowered mercury levels by 24–29% both in the blood and in the liver of rats that were fed with northern pike.     

Refuge sites of voles under owl predation risk: priority of dominant individuals?

In an aviary experiment, we studied whether body size or habitatfamiliarity of field voles (Microtus agrestis) affected predationrisk by Tengmalm's owls (Aegolius funereus). In the field, wecompared the body size of field voles snap-trapped in good (covered)and poor (open) habitats in 1992 and 1994 to determine whetherthere were habitat-related differences in the body size of voles.In the aviary, large individuals occupied the good habitat significantlymore than small individuals both in the control (owl not present)and experimental treatments (owl present). Furthermore, habitat-familiarvoles inhabited the good habitat more than habitat-unfamiliarvoles did when an owl was present Our field data were consistentwith our aviary data: larger field voles were more frequentlyfound in good habitats than in poor habitats. In the aviary,Tengmalm's owl predation risk was higher for small and habitat-unfamiliarvoles. This suggests that large field voles may have priorityto sheltered habitats. Furthermore, habitat familiarity mayplay a central role in avoiding risky habitats.