Structure-function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli

Production of type IV bundle-forming pili by enteropathogenic Escherichia coli (EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily. BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of < or = 65 degrees C. Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB. Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament. Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane. BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion.     

An essential GTPase, der, containing double GTP-binding domains from Escherichia coli and Thermotoga maritima

A gene encoding a putative GTPase containing two tandemly repeated GTP-binding domains from a hyperthermophilic bacterium, Thermotoga maritima, was cloned and expressed in Escherichia coli. The gene (TM1446) termed der is highly conserved in Eubacteria including E. coli. The purified der product (Tm-Der) has GTPase activity but no ATPase activity. GTP, GDP, and dGTP but not GMP, ATP, CTP, and UTP compete for GTP binding to Tm-Der. An optimal condition for the GTPase assay was determined to be pH 7.5 in 400 mm KCl and 5 mm MgCl(2) at 70 degrees C, where K(m), V(max), and k(cat) values were determined to be 110 microm, 3.46 microm/min, and 0.87 min(-1), respectively. A der deletion strain of E. coli was constructed by replacing the der gene (originally annotated yfgK) with a kanamycin resistance gene. The deletion strain was found to form colonies only if the cells harbored a plasmid containing der, indicating that der is essential for E. coli growth.     

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K(i) = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situ cytochemical assay (IC(50) > 1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50) > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K(i) = 0.052 nm) and K (K(i) = 1.57 nm) was also active in both assays (IC(50) = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.     

Effect of the bidistilled modified water on bovine serum albumin conformation. Fluorescent spectroscopy data

It was shown that bidistilled modified water induces a marked decrease in the intensity of intrinsic fluorescence of bovine serum albumin and increases the binding of this protein to the fluorescent probe 1.8 ANS. These effects can be interpreted as a denaturing action of bidistilled modified water on the protein and a change in its conformational state, which is probably caused by changes in the microenvironment of the protein molecule. In addition, a substantial increase in the intrinsic fluorescence of bidistilled modified water, as compared with that of distilled water, was found.     

Gene profile of replicative senescence is different from progeria or elderly donor

In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression.     

Expression and functional analysis of three isoforms of human heterochromatin-associated protein HP1 in Drosophila

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein associated with pericentromeric heterochromatin in Drosophila. HP1-like proteins have also been found associated with heterochromatin in human cells. The goal of this study was to determine whether proteins of the structurally conserved human HP1 family exhibit conserved heterochromatin targeting and silencing properties in Drosophila. We established transgenic lines of Drosophila melanogaster expressing each of the three human HP1 proteins, HP1Hsalpha, HP1HSbeta, and HP1Hsgamma, under the Hsp70 heat shock promoter. We show that all three isoforms of human HP1 are stably expressed in Drosophila and are associated with heterochromatin in Drosophila chromosomes. Like Drosophila HP1, all three human HP1 proteins are delocalized by an HP1-POLYCOMB chimeric protein, implying that both human HP1 and Drosophila HP1 interact in a common protein complex, and that at least some aspects of heterochromatin structure are highly conserved throughout the evolution of eukaryotes. Ectopic expression of two of the three human HP1 family proteins significantly enhances heterochromatic silencing in Drosophila.     

Role of the actin cytoskeleton in T cell absorption and internalization of ligands from APC

A feature of T-APC interaction is that, via either TCR or CD28, T cells can absorb molecules from APC on to the cell surface and then internalize these molecules. Here, using both normal and TCR-transgenic T cells, we investigated the mechanism of T cell absorption of molecules from APC and the role of the cytoskeleton. The results show that although activated T cells could absorb APC molecules in the form of cell fragments, uptake of molecules by resting T cells required direct T-APC interaction. Based on studies with latrunculin B, surface absorption of molecules by resting T cells was crucially dependent upon the actin cytoskeleton for both CD28- and TCR-mediated absorption. Significantly, however, TCR-mediated absorption became strongly resistant to latrunculin B when the concentration of MHC-bound peptide on APC was raised to a high level, implying that the actin cytoskeleton is only important for absorption when the density of receptor/ligand interaction is low. By contrast, in all situations tested, the actin cytoskeleton played a decisive role in controlling T cell internalization of ligands from the cell surface.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.