The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region: studies using chimeric human and fly receptors.

Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.     

Galectin-9 in physiological and pathological conditions

We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings. Published in 2004.     

Inhibitory effect of a synthetic polypeptide, poly(Tyr-Ile-Gly-Ser-Arg), on the metastatic formation of malignant tumour cells

A new type of polypeptide containing the repeating sequence of cell attachment site in laminin, poly(Tyr-Ile-Gly-Ser-Arg), was synthesized by the polymerization procedure with diphenylphosphoryl azide (DPPA). We found that this polypeptide inhibited the metastatic formation in the lung of malignant tumour cells far more effectively than pentapeptide.     

Molecular evolution of catalytic antibodies in autoimmune mice.

Catalytic Abs (catAbs) preferentially evolved in autoimmune MRL/MPJ-lpr/lpr (MRL/lpr) mice upon immunization with the phosphonate transition-state analogue (TSA), but this did not happen in normal BALB/c mice. The majority of the catAbs from MRL/lpr mice were from several independent clones of the same family. Most of them had a lysine at position 95 in the heavy chain (H95), which is at the junctional region. This residue, which interacts with the phosphonate moiety of the TSA and presumably is involved in the catalytic activity, was not changed even after expansive evolution following multiple mutations. By contrast, the majority that arose from BALB/c mice were the non-catAbs, which were quite different in the sequence from the catAbs from MRL/lpr mice, but they were clonally related to one another, so most of them were originated from a single clone. In the MRL/lpr mice, the catalytic subsets that existed in the initial repertoire were effectively captured by the phosphonyl oxygens in the TSA by interacting with the lysine at H95. In the BALB/c mice, however, another noncatalytic subset with only the binding capability directed to a moiety other than the phosphonate moiety was alternatively evolved, because of the lowest abundance or elimination of the catalytic subsets.     

Comparison between parental and variant soybeanBradyrhizobium strains with regard to the production of lipo-chitin nodulation signals,early stages of root infection,nodule occupancy,and N2 fixation rates

Soybean is the most important leguminous crop in Brazil and the nitrogen required for plant growth is supplied byBradyrhizobium bacteria through the symbiotic relation established by the inoculation process. Since 1992, two new strains, CPAC 7 and CPAC 15, which have been shown to increase yields in several field experiments, have been recommended in Brazilian commercial inoculants. CPAC 15 is a natural variant of theB. elkanii SEMIA 566 strain, and was isolated after several years of adaptation to a Brazilian Cerrado soil, while CPAC 7 is a variant ofB. japonicum strain CB 1809, selected under laboratory conditions for higher nodulation and yield. The comparison between parental and variant strains, under greenhouse conditions, showed that both CPAC 15 and CPAC 7 increased N₂ fixation rates in relation to the parental strains. The better performance of CPAC 15 was related to an increase in nodule efficiency (mg N₂ fixed mg^-1 nodule) while with CPAC 7 the higher N₂ fixation rates were due to increased nodulation. Both CPAC 15 and CPAC 7 increased nodule occupancy, when co-inoculated at a ratio of 1:1 withB. elkanii 29w, in relation to their parental strains. Variant strains also differed from parental in their ability to increase numbers of root hairs (Hai phenotype) either when inoculated onto plants, or when supernatants of bacteria exposed to seed exudates were used as inoculants. This results lead to the hypothesis that a modification in some of the “common” nodulation genes had occurred. However, the increase in Hai phenotype with CPAC 7 was dependent on the soybean cultivar, indicating a possible alteration in some genotypic specific nodulation gene. Apparently, there were no differences in Nod metabolites produced by strains CPAC 15 and SEMIA 566, but a more detailed chemical analysis would be required to rule out subtle differences. On the contrary, significant differences were found between CPAC 7 and the parental strain CP 1809, in the profile of Nod metabolites. Consequently, it may be possible that diffusable molecules, responsible for Hai phenotype, would be related to nodulation ability, competiviveness, and N₂ fixation, resulting in the higher yields that have been associated with CPAC 7 and CPAC 15. For the CPAC 7 strain, the increase in Hai phenotype could be atributed to the differences found in the Nod molecules. Consequently, a high degree of physiological and genetic variability can result from the adaptation of rhizobial strains to the soil. Also, this variability can be found under laboratory conditions, when searching single colonies with specific properties. ei]Section editor: R O D Dixon