High transfection efficiency of poly(4-vinylimidazole) as a new gene carrier

Poly(4-vinylimidazole) (P4V) was obtained by free radical polymerization of 4-vinylimidazole (4V) prepared by decarboxylation of urocanic acid. P4V formed a complex with DNA that exhibited higher transfection effiency on Hela cells than polyethylenimine (PEI), through the proton sponge mechanism of the imidazole groups in the side chain of the P4V, and low cell toxicity.     

Measurement of protease activity of live Uronema marinun (Ciliata: Scuticociliatida) by fluorescence polarization

The proteolytic activity of live Uronema marinum was analyzed by a fluorescence polarization (FP) technique. Protease activity was measured as a decrease in the FP value using fluorescein isothiocynate (FITC)-casein as a protein substrate. A time-dependent decrease in FP occurred in plate wells containing live U. marinum. Supplementation with the cysteine protease inhibitor E-64 had no significant inhibitory effect on the decrease in FP at any of the concentrations used. In contrast, supplementation with 1,10-phenanthroline resulted in complete inhibition of proteolysis for 30 min at 1 mM and for 1 h at 2 and 5 mM. Effective inhibition of the proteolytic activity of live U. marinum by 1,10-phenanthroline indicated that metalloproteases are the main proteases excreted by U. marinum. As U. marinum has a high potential for systemically invading and destroying fish tissues, the metalloproteases excreted by live U. marinum are likely to be involved in the invasion of host tissues and the pathogenicity of the parasite.     

Transmodulation between phospholipase D and c-Src enhances cell proliferation

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Effect of temperature and salinity on in vitro zoosporulation of Perkinsus sp. in Manila clams Ruditapes philippinarum

The effects of temperature and salinity on in vitro development of Perkinsus sp. prezoosporangia isolated from cultured Manila clams in Korea were investigated, and the difference in resistance to low temperature between prezoosporangia collected in winter and those collected in summer was compared. Temperature and salinity had significant effects on the development of prezoosporangia, and the developmental rates increased with increasing temperature and salinity. Prezoosporangia isolated in winter sporulated and released motile zoospores at 10 degrees C, although the rates were significantly lower than those at 20 and 30 degrees C. However, no prezoosporangia collected in summer sporulated at 10 degrees C. Low salinities (< or = 10/1000) had a significant negative effect on the development of prezoosporangia. A small number of prezoosporangia sampled in summer did sporulate at 5/1000, but further developments including formation and release of zoospores were not observed. However, prezoosporangia sampled in winter and incubated at 5/1000 released motile zoospores, although the rates were significantly lower than those at higher salinities.     

A role for p53 in base excision repair

Wild-type p53 protein can markedly stimulate base excision repair (BER) in vitro, either reconstituted with purified components or in extracts of cells. In contrast, p53 with missense mutations either at hot-spots in the core domain or within the N-terminal transactivation domain is defective in this function. Stimulation of BER by p53 is correlated with its ability to interact directly both with the AP endonuclease (APE) and with DNA polymerase beta (pol beta). Furthermore, p53 stabilizes the interaction between DNA pol beta and abasic DNA. Evidence that this function of p53 is physiologically relevant is supported by the facts that BER activity in human and murine cell extracts closely parallels their levels of endogenous p53, and that BER activity is much reduced in cell extracts immunodepleted of p53. These data suggest a novel role for p53 in DNA repair, which could contribute to its function as a key tumor suppressor.     

Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells

An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.     

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.