Osteoclast inhibitory lectin,a family of new osteoclast inhibitors

We have identified two novel type II membrane-bound C-lectins, designated mOCILrP1 and mOCILrP2, of 218 and 217 amino acids, respectively, that share substantial identity with the murine osteoclast inhibitory lectin (OCIL). The extracellular domains of mOCILrP1 and mOCILrP2 share 83 and 75% identity, respectively, with the extracellular domain of mOCIL. When the extracellular domains were expressed as recombinant proteins, each inhibited osteoclast formation in murine bone marrow cultures treated with M-CSF and RANKL with similar potencies to mOCIL (IC(50) of 0.2 ng/ml). Distinct but highly related genes encoded the three OCIL family members, with mOCIL and mOCILrP2 controlled by an inverted TATA promoter, and mOCILrP1 by a TTAAAA promoter. However only mOCIL was robustly regulated by calciotropic agents, while mOCILrP1 was not expressed, and mOCILrP2 was constitutively expressed in osteoblasts. Immunohistochemistry using antipeptide antibodies to the intracellular domain of mOCILrP1/mOCILrP2 and to mOCIL demonstrated that mOCIL and mOCILrP1/mOCILrP2 were concordantly expressed in osteoblasts, chondrocytes, and in extraskeletal tissues. Further, their cellular distribution was identical to that of RANKL. The identification of three distinct genes that were functionally related implies redundancy for OCIL, and their concordant expression with that of RANKL suggests that the RANKL:OPG axis may be further influenced by OCIL family members.     

Evaluation of the human melanoma targeting properties of radiolabeled alpha-melanocyte stimulating hormone peptide analogues

The purpose of this study was to evaluate the human MC1 receptor-mediated melanoma targeting properties of two metal cyclized alpha-MSH peptide analogues, (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH. Initially, the presence and density of the MC1 receptor were determined on a bank of human melanoma cell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor at a density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH were evaluated in a cultured TXM13 human melanoma-xenografted Scid mouse model. Biodistribution results demonstrated that 3.06 +/- 0.68 %ID/g of (188)Re-(Arg(11))CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearance of (188)Re-(Arg(11))CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidney. (188)Re-CCMSH exhibited similar tumor uptake and retention in TXM13 human melanoma-xenografted Scid mice as (188)Re-(Arg(11))CCMSH. However, the kidney uptake value of (188)Re-CCMSH was two times higher than that of (188)Re-(Arg(11))CCMSH. The results of this study indicate that the MC1 receptor is present on the surface of a large number of human melanoma cells, which makes the MC1 receptor a good imaging or therapeutic target. Moreover, the biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH highlight their potential as therapeutic agents for human melanoma.     

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Specificity determinants for the pyruvate dehydrogenase component reaction mapped with mutated and prosthetic group modified lipoyl domains

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys(173)), with three analogs substituted for the lipoyl group on Lys(173), and with selected L1 mutants. Besides Lys(173) mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 A on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(173) or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient use by mammalian E1 of cognate lipoyl domains derives from unique surface residues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, particularly Asp(172) adjacent to Lys(173).     

An improved tripod amphiphile for membrane protein solubilization

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a "tripod amphiphile," that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.     

The evolution of the atpbeta-rbcL intergenic spacer in the epacrids (Ericales) and its systematic and evolutionary implications

Sequence data from the noncoding region separating the plastid genes atpbeta and rbcL were gathered for 27 epacrid taxa, representing all previously recognized infrafamilial groups, and four outgroup taxa (Ericaceae), to address several persistent phylogenetic questions in the group. Parsimony analyses were conducted on these data, as well as on a complementary rbcL sequence dataset assembled from the literature and the combined dataset. The atpbeta-rbcL spacer was notable for the high frequency of insertion-deletion mutations (indels); their distributions were coded as binary characters and included as a adjunct matrix in some of the analyses. The phylogenetic patterns derived from the spacer and rbcL data and the combined analyses, both including and excluding the indel data, concur in resolving seven major lineages corresponding to the tribes of Crayn et al. (1998, Aust. J. Bot. 46, 187-200), viz. Prionoteae, Archerieae, Oligarrheneae, Cosmelieae, Richeeae, Epacrideae, and Styphelieae. The relationships of the tribes and within Styphelieae, however, are not convincingly resolved. Minor conflicts in the positions of some taxa between the spacer and the rbcL trees are poorly supported. Among epacrids, the spacer region provided more cladistically informative characters than rbcL and resulted in trees with lower homoplasy. Further, the spacer data, when analyzed alone and when combined with rbcL, resolved several clades that could not be retrieved on rbcL data alone and provided increased support for many other relationships. The evolution of a putative three-base inversion associated with a hairpin secondary structure in the spacer region is discussed in the light of the inferred phylogeny.