Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1S12OG and nm23-H1P96S. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1P96S or nm23-H1S120G, and another was phosphorylated much more slowly by nm23-H1P96S and by nm23-H1S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1S120G and nm23-H1P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

Thematic Issue on the Hydrological Effects of the Vegetation-Soil Complex

Peer reviewed

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement

Recently, the target function for crystallographic refinement has been improved through a maximum likelihood analysis, which makes proper allowance for the effects of data quality, model errors, and incompleteness. The maximum likelihood target reduces the significance of false local minima during the refinement process, but it does not completely eliminate them, necessitating the use of stochastic optimization methods such as simulated annealing for poor initial models. It is shown that the combination of maximum likelihood with cross-validation, which reduces overfitting, and simulated annealing by torsion angle molecular dynamics, which simplifies the conformational search problem, results in a major improvement of the radius of convergence of refinement and the accuracy of the refined structure. Torsion angle molecular dynamics and the maximum likelihood target function interact synergistically, the combination of both methods being significantly more powerful than each method individually. This is demonstrated in realistic test cases at two typical minimum Bragg spacings (dmin = 2.0 and 2.8 â„«, respectively), illustrating the broad applicability of the combined method. In an application to the refinement of a new crystal structure, the combined method automatically corrected a mistraced loop in a poor initial model, moving the backbone by 4 â„«.

Derivation of pluripotent stem cells from cultured human primordial germ cells

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian CellsV⃞

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.