Phosphorylcholine on Streptococcus pneumoniae R36a is responsible for in vitro polyclonal antibody secretion by human peripheral blood lymphocytes

We have shown that Pc on the C-polysaccharide of Streptococcus pneumoniae R36a is responsible for polyclonal PFC responses induced in vitro by this bacterium in humans. R36a grown in media containing EA instead of CL, and therefore having phosphorylethanolamine instead of Pc in their C-polysaccharide, were unable to induce substantial PFC responses. When EA-substituted bacteria were chemically conjugated with Pc, their ability to induce polyclonal PFC was restored. Specific removal of Pc from the surface of the bacteria by the use PLC also resulted in abrogation of the polyclonal antibody response. These data are consistent with our hypothesis that polyclonal activation resulting from R36a stimulation may be mediated by a recently described Pc-binding receptor that is distributed on the surface of a subpopulation of B lymphocytes in humans and mice.     

Real-time interactive frequency filtering of molecular dynamics trajectories

Molecular dynamics simulations of atomic motion in protein and nucleic acid molecules must be done on a femtosecond time-scale. Much of this rapid motion is unimportant for the slower changes that are most relevant to biological function (conformational changes, substrate binding, protein folding). The high-frequency motion makes simulations computationally expensive. More importantly, the high frequencies obscure visualization of the relevant dynamics processes. Sessions, Dauber-Osguthorpe and Osguthorpe presented a method for removing high-frequency motions from atomic co-ordinates of trajectories generated by simulation. While that study used fast Fourier methods and emphasized the use of filtering for analysis of trajectories, this communication describes a new method that makes it much easier to use frequency filtering in programs that display trajectories as a sequence of moving images. Tests of the method on systems extending from pure water to proteins and nucleic acid molecules in vacuo and in solution have demonstrated its general utility. Impressed with the power and simplicity of the new method, we wish to present it in sufficient detail to allow others to implement it themselves.     

Theoretical studies of enzymic reactions: Dielectric,electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme

A general method for detailed study of enzymic reactions is presented. The method considers the complete enzyme-substrate complex together with the surrounding solvent and evaluates all the different quantum mechanical and classical energy factors that can affect the reaction pathway. These factors include the quantum mechanical energies associated with bond cleavage and charge redistribution of the substrate and the classical energies of steric and electrostatic interactions between the substrate and the enzyme. The electrostatic polarization of the enzyme atoms and the orientation of the dipoles of the surrounding water molecules is simulated by a microscopic dielectric model. The solvation energy resulting from this polarization is considerable and must be included in any realistic calculation of chemical reactions involving anything more than an isolated molecule in vacuo. Without it, acidic groups can never become ionized and the charge distribution on the substrate will not be reasonable. The same dielectric model can also be used to study the reaction of the substrate in solution. In this way the reaction in solution can be compared with the enzymic reaction.In this paper we study the stability of the carbonium ion intermediate formed in the cleavage of a glycosidic bond by lysozyme. It is found that electrostatic stabilization is an important factor in increasing the rate of the reaction step that leads to the formation of the carbonium ion intermediate. Steric factors, such as the strain of the substrate on binding to lysozyme, do not seem to contribute significantly.     

Modeling nucleic acids

Nucleic acids are an important class of biological macromolecules that carry out a variety of cellular roles. For many functions, naturally occurring DNA and RNA molecules need to fold into precise three-dimensional structures. Due to their self-assembling characteristics, nucleic acids have also been widely studied in the field of nanotechnology, and a diverse range of intricate three-dimensional nanostructures have been designed and synthesized. Different physical terms such as base-pairing and stacking interactions, tertiary contacts, electrostatic interactions and entropy all affect nucleic acid folding and structure. Here we review general computational approaches developed to model nucleic acid systems. We focus on four key areas of nucleic acid modeling: molecular representation, potential energy function, degrees of freedom and sampling algorithm. Appropriate choices in each of these key areas in nucleic acid modeling can effectively combine to aid interpretation of experimental data and facilitate prediction of nucleic acid structure.     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.     

Genome maintenance defects in cultured cells and mice following partial inactivation of the essential cell cycle checkpoint gene Hus1

Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1(neo), with either wild-type or null (Hus1(Delta1)) alleles. Primary Hus1(neo/Delta1) embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1(neo/neo) cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1(neo/Delta1) cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1(neo/neo) and Hus1(neo/Delta1) cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1(neo/neo) and Hus1(neo/Delta1) cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1(neo/Delta1) and Hus1(neo/neo) cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1(neo/Delta1) mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.