Empirical calculation of the relative free energies of peptide binding to the molecular chaperone DnaK

We describe a methodology to calculate the relative free energies of protein-peptide complex formation. The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions. A free energy-surface area relationship served to calculate the nonpolar free energy term. The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains. We applied this methodology to a series of DnaK*peptide complexes. On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes. Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy. The calculated relative binding free energies were compared with the experimentally determined free energies. Linear scaling of the calculated terms was applied to fit them to the experimental values. The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86. The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues. Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand. Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation. The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available.     

Modelling T-cell-mediated suppression dependent on interactions in multicellular conjugates

Tolerance to peripheral body antigens involves multiple mechanisms, namely T-cell-mediated suppression of potentially autoimmune cells. Recent in vivo and in vitro evidence indicates that regulatory T cells suppress the response of effector T cells by a mechanism that requires the simultaneous conjugation of regulatory and effector T cells with the same antigen-presenting cell (APC). Despite this strong requirement, it is not yet clear what happens while both cells are conjugated. Several hypotheses are discussed in the literature. Suppression may result from simple competition of regulatory and effector cells for activation resources on the APC; regulatory T cells may deliver an inhibitory signal to effector T cells in the same conjugate; or effector T cells may acquire the regulatory phenotype during their interaction with regulatory T cells. The present article tries to further our understanding of T-cell-mediated suppression, and to narrow-down the number of candidate mechanisms. We propose the first general formalism describing the formation of multicellular conjugates of T cells and APCs. Using this formalism we derive three particular models, representing alternative mechanisms of T-cell-mediated suppression. For each model, we make phase plane and bifurcation analysis, and identify their pros and cons in terms of the relationship with the large body of experimental observations on T-cell-mediated suppression. We argue that accounting for the quantitative details of adoptive transfers of tolerance requires models with bistable regimes in which either regulatory cells or effectors cells dominate the steady state. From this analysis, we conclude that the most plausible mechanism of T-cell-mediated suppression requires that regulatory T cells actively inhibit the growth of effector T cells, and that the maintenance of the population of regulatory T cells is dependent on the effector T cells. The regulatory T cell population may depend on a growth factor produced by effector T cells and/or on a continuous differentiation of effector cells to the regulatory phenotype.     

Sequential analysis of cell differentials and IFN-gamma production of splenocytes from mice infected with Toxoplasma gondii

To assess the relationship between the changes of cellular components and the production of Th1 cytokine in the immune tissue, inbred C57BL/6 mice were orally infected with 40 cysts of 76K strain of Toxoplasma gondii. The sequential change of cell differentials and IFN-gamma production of splenocytes were analyzed by Diff-Quik stain and RT-PCR. There were no significant proportional changes of cellular components of splenocytes until day 4 postinfection (PI) as compared to those of day 0, and the relative percentage of macrophages and neutrophils/eosinophils increased significantly (p < 0.01) thereafter. The expression of IFN-gamma mRNA of CD3- cells was observed from day 1 PI at a low level. However, IFN-gamma production of CD3+ cells increased significantly from day 4 PI (p < 0.01) which progressively increased thereafter. These findings provide the relative percentages of granulocytes and macrophages were increased in conjunction with increase of total number of splenocytes after oral infection with T. gondii in the susceptible murine hosts, and lymphocytes were the major cellular components and the important source of IFN-gamma.     

Immunohistochemical distribution of connexin 43 in the cartilage of rats and mice

Using fluorescence immunohistochemistry, the distribution of connexin 43 was examined in hyaline cartilage and in the perichondrium of mouse and rat knee joints. In addition, rat chondrocytes were shown to be coupled in dye transfer studies with Lucifer Yellow. Connexin 43 was detected between chondrocytes in the outer layer of knee joint cartilage, between chondrocytes of the growth plate and between fibrocartilage-like cells at tendon and ligament insertions and in the tendons and ligaments proper. However, in the hyaline cartilage of the hind limbs of mature rats, the degree of connexin 43 immunoreactivity was diminished. These data suggest a possible involvement of connexins in cartilage development. © 1998 Chapman & Hall     

Cardiorespiratory Responses and Prediction of Peak Oxygen Uptake during the Shuttle Walking Test in Healthy Sedentary Adult Men

BackgroundThe application of the Shuttle Walking Test (SWT) to assess cardiorespiratory fitness and the intensity of this test in healthy participants has rarely been studied. This study aimed to assess and correlate the cardiorespiratory responses of the SWT with the cardiopulmonary exercise testing (CEPT) and to develop a regression equation for the prediction of peak oxygen uptake (VO2 peak) in healthy sedentary adult men.MethodsIn the first stage of this study, 12 participants underwent the SWT and the CEPT on a treadmill. In the second stage, 53 participants underwent the SWT twice. In both phases, the VO2 peak, respiratory exchange ratio (R), and heart rate (HR) were evaluated.ResultsSimilar results in VO2 peak (P>0.05), R peak (P>0.05) and predicted maximum HR (P>0.05) were obtained between the SWT and CEPT. Both tests showed strong and significant correlations of VO2 peak (r = 0.704, P = 0.01) and R peak (r = 0.737, P<0.01), as well as the agreement of these measurements by Bland-Altman analysis. Body mass index and gait speed were the variables that explained 40.6% (R2 = 0.406, P = 0.001) of the variance in VO2 peak. The results obtained by the equation were compared with the values obtained by the gas analyzer and no significant difference between them (P>0.05) was found.ConclusionsThe SWT produced maximal cardiorespiratory responses comparable to the CEPT, and the developed equation showed viability for the prediction of VO2 peak in healthy sedentary men.     

Bovine Milk Proteome in the First 9 Days: Protein Interactions in Maturation of the Immune and Digestive System of the Newborn

In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.     

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.     

Adsorption of Cationic Peptides to Solid Surfaces of Glass and Plastic

Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects.     

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms

BackgroundTreatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.MethodologyWe used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.ConclusionsFunctional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.