Structural characterization of the 60-kDa bermuda grass pollen isoallergens, a covalent flavoprotein

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20 degrees C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (approximately 180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS-PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.     

Role of the nonspecific DNA-binding region and alpha helices within the core domain of retroviral integrase in selecting target DNA sites for integration

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49-238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain alpha helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49-187 and showed that alpha 3 and alpha 4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the alpha 2 helix (residues 118-121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the alpha 2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.     

Expression of the acidic stretch of nardilysin as a functional binding domain

Kinetic evidence suggests an acidic region in nardilysin binds polyamines and acts as a regulatory domain. The binding of approximately 5 mol of spermine/mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibiting an IC(50) of approximately 118 microM and a Hill coefficient of 1.8. Spermine diminished the tryptophan fluorescence of the enzyme and increased its sensitivity to protease V8. The acidic stretch from mouse and human nardilysin were expressed as glutathione transferase fusion proteins. All fusion proteins bound spermine with an IC(50) of 40 to 110 microM. The mouse fusion protein bound approximately 7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound approximately 5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acidic region fused to the C-terminus of the human acidic region bound approximately 10 mol of spermine, while the opposite chimera bound approximately 4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3--4 mol spermine/mol exhibiting a Hill coefficient of 1.4, while the same region from human nardilysin binds 1 mol of spermine/mol. Spermine enhanced the sensitivity of the mouse acidic domain, but not the human acidic domain, to protease V8. Together the data support a model where the acidic stretch of nardilysin functions as an autonomous domain.     

Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 microM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.     

Inhibition of photosystems I and II and enhanced back flow of photosystem I electrons in cucumber leaf discs chilled in the light

Pre-illumination of cucumber leaf discs at a chilling temperature in low-irradiance white light resulted in accelerated re-reduction of P700(+) [the special Chl pair in the photosystem I (PSI) reaction centre] when the far-red measuring light was turned off. Measurements (in +/- methyl viologen or +/- DCMU conditions) of the re-reduction half time suggest that accelerated re-reduction of P700(+) appeared to be predominantly due to charge recombination and only partly due to reductants sustained by previous cyclic electron flow around PSI. Apparently, charge recombination in PSI was greatly enhanced by inhibition of forward, linear electron flow. Inhibition of PSII electron transport was observed to occur to a lesser extent than that of PSI, but only if the measurement of PSII functionality was free from complications due to downstream accumulation of electrons in pools. We suggest that promotion of controlled charge recombination and cyclic electron flow round PSI during chilling of leaves in the light may partly prevent further damage to both photosystems.     

Efficacy of antifungal agents in tissue conditioners in treating candidiasis

Chronic atrophic candidiasis is prevalent in up to 72% of institutionalized geriatric populations and is causally associated with Candida albicans. Topical antifungal treatments are difficult to implement in some geriatric patients due to cognitive impairment, reduced motor dexterity and memory loss. Objective: This in vitro study incorporated antifungal agents into tissue conditioners to investigate the effectiveness of this method of drug delivery. Design: Combinations of nystatin, fluconazole, itraconazole and Coe Soft, Viscogel, Fitt were tested at 1, 3, 5, 7, 9 and 11wt/wt%, with and without sterilized saliva. 6 mm diameter cores were punched in Sabouraud plates pre-grown with standardized C. albicans. Antifungal agents plus tissue conditioner mixtures were injected into each core. Inhibition diameters were measured for 14 days. Results: Cores with only tissue conditioners acted as negative control and showed no significant inhibition activity (ANOVA, p>0.05). Peak activity was between 65 to 89 hours; followed by a plateau. Itraconazole had greater fungicidal activity than fluconazole; while nystatin was found to have the least fungicidal activity (ANOVA. p<0.05). The most effective concentration for nearly all combinations was 5%wt/wt (ANOVA, p<0.05). Specimens with saliva showed greater antifungal activity than those without (t-test. p<0.001). Itraconazole altered the physical properties of Viscogel hence this combination is not recommended for clinical use. Conclusion: The treatment of chronic atrophic candidiasis by incorporation of antifungal drugs into tissue conditioners is efficacious. 5% wt/wt itraconazole mixed with Coe Soft or Fitt is recommended for clinical study where compliance of patient or care giver cannot be relied upon. Peak antifungal activity at 3 days suggests that mixtures prepared for clinical study may be replaced soon after this time for maximum effectiveness.     

Mitogenic effects of 20-hydroxyecdysone on neurogenesis in adult mushroom bodies of the cockroach, Diploptera punctata

The purpose of this study was to examine the mitogenic effects of 20-hydroxyecdysone on neurogenesis in mushroom bodies of the adult cockroach, Diploptera punctata. The occurrence of neurogenesis was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of BrdU-labeled cells in the mushroom bodies was high shortly after adult ecdysis, then gradually decreased, and proliferation ceased on day 8. 20-Hydroxyecdysone injection during the early adult stages significantly delayed the decrease in mitotic activity. Moreover, 20-hydroxyecdysone injection during the late stage stimulated quiescent mushroom body neuroblasts to initiate their mitotic activity in a dose-dependent manner. These results indicated that the mushroom body neuroblasts of this insect become quiescent in the maturing central nervous system, but retain the capacity for proliferation if exposed to appropriate environmental signals. We conclude that 20-hydroxyecdysone has a mitogenic effect on neurogenesis in mushroom bodies of this insect.     

gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787-793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.     

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.