Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction.

The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor.     

Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment

Electron microscopy (EM) in situ hybridization provides the higher resolution necessary to determine the spatial relationship between a specific mRNA and the organelle containing the protein encoded by that message. EM in situ hybridization was used to determine the subcellular myosin heavy chain (MHC) mRNA distribution with respect to the myofibril in normal cardiac tissue. Sections of frozen or acrylic-embedded tissue were compared for ultrastructural integrity and content of endogenous mRNA. Papillary muscles dissected from hearts of normal rabbits were aldehyde-fixed and either frozen or embedded in LR White. EM in situ hybridization with no riboprobe, vector sequence, same-sense, and anti-sense biotinylated riboprobes was detected by indirect immunocytochemistry. Labeling density using an antisense probe was highest over the intermyofibrillar space, with an average signal five times that of background. Background labeling by nonspecific sense probe was consistently low but not random, also having the highest density of gold clusters over the intermyofibrillar space. Ultracryomicrotomy yielded a higher absolute number of gold clusters, but sections were fragmented and disrupted striated muscle morphology. LR White embedment maintained ultrastructural integrity but gave a lower absolute signal. Fortunately, MHC mRNA is an abundant message and can tolerate the decreased sensitivity of LR White.     

Herpes simplex virus infection can occur without involvement of the fibroblast growth factor receptor.

Basic fibroblast growth factor (bFGF) has been reported to block uptake of herpes simplex virus type 1 (HSV-1) and plaque formation on arterial smooth muscle cells, suggesting a role for the bFGF receptor in HSV entry (R. J. Kaner, A. Baird, A. Mansukhani, C. Basilico, B. D. Summers, R. Z. Florkiewicz, and D. P. Hajjar, Science 248:1410-1413, 1990). We confirmed the effect of bFGF on infection of this cell type with HSV-1 and HSV-2 and found the same result with umbilical vein endothelial cells. However, bFGF does not inhibit plaque formation on any other cell type we tested. Furthermore, there is no correlation between the level of expression of the bFGF receptor and the effect of bFGF. HEp-2 and A431 cells express barely detectable levels of receptor, and yet they are fully permissive for HSV infection in a bFGF-insensitive manner. Thus, interaction of virus with the bFGF receptor is not required for infection of many cell types. In addition, infection of smooth muscle cells is not prevented by incubation of virus with an anti-bFGF antibody, arguing against the hypothesis that virion-associated bFGF acts as a bridge between virus and receptor (A. Baird, R. Z. Florkiewicz, P. A. Maher, R. J. Kaner, and D. P. Hajjar, Nature [London] 348:344-346, 1990).     

Stability of a model beta-sheet in water.

We have used molecular dynamics simulations to determine the stability in water of a model beta-sheet formed by two alanine dipeptide molecules with two intermolecular hydrogen bonds in the closely spaced antiparallel arrangement. In this paper we describe our computations of the binding free energy of the model sheet and a portion of the free energy surface as a function of a reaction co-ordinate for sheet formation. We used the free energy surface to identify stable conformations along the reaction co-ordinate. To determine whether or not the model sheet with two hydrogen bonds is more stable than a single amide hydrogen bond in water, we compared the results of the present calculations to results from our earlier study of linear hydrogen bond formation between two formamide molecules (the formamide "dimer"). The free energy surfaces for the sheet and formamide dimer each have two minima corresponding to locally stable hydrogen-bonded and solvent-separated configurations. The binding free energies of the model sheet and the formamide dimer are -5.5 and -0.34 kcal/mol, respectively. Thus, the model sheet with two hydrogen bonds is quite stable while the simple amide hydrogen bond is only marginally stable. To understand the relative stabilities of the model sheet and formamide dimer in terms of solute-solute and solute-water interactions, we decomposed the free energy differences between hydrogen-bonded and solvent-separated conformations into energetic and entropic contributions. The changes in the peptide-peptide energy and the entropy are roughly twice as large for the sheet as they are for the formamide dimer. The magnitude of the peptide-water energy difference for the sheet is less than twice (by about 3.5 kcal/mol) that for the formamide dimer, and this accounts for the stability of the sheet. The presence of the side-chains and/or blocking groups apparently prevents the amide groups in the sheet from being solvated as favorably in the separated arrangement as in the formamide dimer, where the amide groups are completely exposed to the solvent.     

Structural basis of C3b binding by glycoprotein C of herpes simplex virus.

Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.     

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.     

Protein synthesis modulates the biological effectiveness of the combined action of hyperthermia and high-LET radiation.

The combined effects of heavy-ion radiation and hyperthermia on the survival of CHO-SC1 cells and its temperature-sensitive (ts) mutant tsH1 cells were studied using accelerated neon ions followed by mild heating at 41.5 degrees C. The sequence of application of heat and high-LET radiation is significant to cell-killing effects. Heat applied to cells prior to irradiation with neon plateau ions (LET = 32 keV/microns) was less effective than heat applied immediately after irradiation. The ability of cells to synthesize new proteins plays a key role in this sequence-dependent thermal sensitization. When protein synthesis was shut down in tsH1 cells, the thermal enhancement of cell killing by high-LET radiation was the same regardless of the sequence. The thermal enhancement of radiation-induced cell killing was LET-dependent for the SC1 cells, but this was not clearly demonstrated in the tsH1 cells. Furthermore, the RBE of heated SC1 cells varied with LET and reached a maximum of greater than 3 at 80 keV/microns. In the absence of protein synthesis, the maximum RBE value was reduced to 2.6. These results suggest that the accumulation of cellular damage caused by exposure to densely ionizing particles with increasing LETs can be potentiated with active protein synthesis during postirradiation heat treatment.     

Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N2-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9).(G10-T11-A12-C-13-F14-C15 -A16-T17-G-18) X.F 9-mer duplex. Two-dimensional NMR experiments establish that the X.F 9-mer helix is right-handed with Watson-Crick A.T and G.C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4.C15 and G6.C13 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4.C15 and G6.C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N2-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.