A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Molecular cloning and characterization of all RND-type efflux transporters in Vibrio cholerae non-O1

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram-negative bacteria, including Vibrio cholerae non-O1. There are six operons for putative RND-type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non-O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (DeltaacrAB, DeltaydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC-deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolC(Vc) of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolC(Vc) showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF-TolC(Vc), and the activity required Na(+). Thus, VexEF-TolC (Vc) is either a Na(+)-activated or a Na(+)-coupled transporter. To our knowledge, this is the first report on the requirement of Na(+) for an RND-type efflux transporter.     

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

RAG1 and RAG2 proteins are key components in V(D)J recombination. The core region of RAG1 is capable of catalyzing the recombination reaction; however, the biological function of non-core RAG1 remains largely unknown. Here, we show that in a murine-model carrying the RAG1 ring-finger conserved cysteine residue mutation (C325Y), V(D)J recombination was abrogated at the cleavage step, and this effect was accompanied by decreased mono-ubiquitylation of histone H3. Further analyses suggest that un-ubiquitylated histone H3 restrains RAG1 to the chromatin by interacting with the N-terminal 218 amino acids of RAG1. Our data provide evidence for a model in which ubiquitylation of histone H3 mediated by the ring-finger domain of RAG1 triggers the release of RAG1, thus allowing its transition into the cleavage phase. Collectively, our findings reveal that the non-core region of RAG1 facilitates chromosomal V(D)J recombination in a ubiquitylation-dependent pathway.     

Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes

Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg²⁺, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.     

Control of lysyl oxidase activity through site-specific deuteration of lysine

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d₂-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V_max/K_m as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D₂Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.     

Pyrazolopyridine inhibitors of B-RafV600E. Part 2: structure-activity relationships

Structure-activity relationships around a novel series of B-Raf(V600E) inhibitors are reported. The enzymatic and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and3-methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-Raf(V600E) inhibitor 1 was extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies the small lipophilic pocket formed by an outward shift of the αC-helix, was expanded to a series of arylsulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accommodate the bulkier aryl group.