PERIODONTAL LIGAMENT CELL KINETICS FOLLOWING ORTHODONTIC TOOTH MOVEMENT

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (³H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with ³H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. ³H-TdR-Iabeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bone-forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G₂ block by the orthodontic stimulation.     

Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning

Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains.     

Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.     

Stochastic models of telomere shortening

Shortening of chromosome ends, known as telomeres, is one of the supposed mechanisms of cellular aging and death. We provide a probabilistic analysis of the process of loss of telomere ends. The first work concerned with that issue is the paper by Levy et al. [J. Molec. Biol. 225 (1992) 951-960]. Their deterministic model reproduced the observed frequencies of viable cells in the in vitro experiments. Arino et al. [J. Theor. Biol. 177 (1995) 45-57] reformulated the model of Levy et al. (1992) in the terms of branching processes with denumerable type space. In the present paper, the mathematical results of Arino et al. (1995) are extended to the case in which cell death is present, in cells with telomeres above and below the critical threshold of length, generally with differing probabilities. Both exact and asymptotic results are provided, as well as a discussion of biological relevance of the results.     

Glycosylphosphatidyl-inositols in murine malaria: Plasmodium yoelii yoelii

Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.     

An accurate,sensitive, and scalable method to identify functional sites in protein structures

Functional sites determine the activity and interactions of proteins and as such constitute the targets of most drugs. However, the exponential growth of sequence and structure data far exceeds the ability of experimental techniques to identify their locations and key amino acids. To fill this gap we developed a computational Evolutionary Trace method that ranks the evolutionary importance of amino acids in protein sequences. Studies show that the best-ranked residues form fewer and larger structural clusters than expected by chance and overlap with functional sites, but until now the significance of this overlap has remained qualitative. Here, we use 86 diverse protein structures, including 20 determined by the structural genomics initiative, to show that this overlap is a recurrent and statistically significant feature. An automated ET correctly identifies seven of ten functional sites by the least favorable statistical measure, and nine of ten by the most favorable one. These results quantitatively demonstrate that a large fraction of functional sites in the proteome may be accurately identified from sequence and structure. This should help focus structure-function studies, rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.     

Coincident iterated gene expression in the amphioxus neural tube

SUMMARY The segmental patterning of the vertebrate hindbrain has been intensely investigated, yet the evolutionary origin of hindbrain segmentation remains unclear. In the vertebrate sister group, amphioxus (Cephalochordata), the embryonic neural tube lacks obvious morphological segmentation, but comparative Hox gene expression analysis has suggested the presence of a region homologous to the vertebrate hindbrain. Does this region contain ancient segmental features shared with the vertebrate hindbrain? To help address this question we cloned the paired‐like amphioxus homeodomain gene shox and found that its expression is segmental in the amphioxus neural tube. We also uncovered a previously uncharacterized iterated neural tube expression pattern of the zinc‐finger gene AmphiKrox. We propose that these genes, along with amphioxus islet and AmphiMnx, share a one‐somite width periodicity of expression in the neural tube, the coincidence of which may reflect an underlying segmental organization. We hypothesize that the segmental patterning of neurons in the neural tube was present in the amphioxus/vertebrate ancestor, but the establishment of a bona fide segmented hindbrain may indeed have arisen in the vertebrate lineage.