Solution conformation of wild-type and mutant IgG3 and IgG4 immunoglobulins using crystallohydrodynamics: possible implications for complement activation

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.     

The Use of Kinetic and Dynamic Data in Risk Assessment of Drugs

The risk assessment process for non-carcinogens must incorporate all available scientific information, including toxicokinetic and toxicodynamic data. The framework for exposure limit setting proposed by Renwick and the International Programme on Chemical Safety (IPCS) subdivides traditional 10X uncertainty factors (UFs) into separate partial-log default values based on kinetic and dynamic considerations and allows for incorporation of compound-specific data when available. In this investigation, an extensive literature search was conducted on nine pharmaceuticals in order to incorporate information on kinetics and dynamics to allow extrapolation across species and among susceptible humans. The drugs are diazepam, oxazepam, midazolam, buspirone, fluoxetine, venlafaxine, amlodipine, felodipine, and nifedipine. The composite factors were calculated using the highest ratio or the average ratio for appropriate parameters and default subfactor. For the drugs examined, most of the subfactors for kinetics and dynamics were less than the proposed values by Renwick and IPCS, and the composite factors were far less than 100. From this study, it was concluded that relevant compound-specific kinetic and dynamic data can reduce uncertainties associated with interspecies differences and interindividual variability.     

Light-induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center.

The behaviour of cytochrome (cyt) b559 during acceptor- and donor-side photoinhibition has been investigated in oxygen-evolving and non-evolving photosystem II (PSII) membranes. Strong illumination at 20 degrees C under aerobiosis induced a strong decrease in the absorbance of the cyt b559 alpha-band in the two preparations. This absorbance decline was observed only in non-oxygen-evolving PSII samples when illumination was performed under aerobiosis but at 4 degrees C, or under anaerobiosis at 20 degrees C. These results suggest that acceptor-side photoinhibition induces the degradation of cyt b559 by a mechanism related to an enzymatic reaction mediated by singlet oxygen. Donor-side photoinhibition may induce, however, a non-enzymatic photocleavage of the protein.     

Biomineralization of Carbonates by Marinococcus albus and Marinococcus halophilus Isolated from the Salar de Atacama (Chile)

We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile). They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20%. The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy. M. halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations. M. albus did not precipitate at 20% and showed a maximum at 7.5%. This behavior is similar to that of other Gram-positive bacteria and differs from that found in Gram-negative bacteria. The bioliths precipitated were spherical, generally isolated, with a size of 10–100 μm, varying with salinity. They were of magnesium calcite (CO₃ Ca_1-x Mg_x) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium. These results demonstrate the active role played by M. halophilus and M. albus in the precipitation of carbonates. Received: 3 November 1998 / Accepted: 9 March 1999     

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide stimulate the induction of Th2 responses by up-regulating B7.2 expression.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunologic functions. We have recently demonstrated that VIP and PACAP enhance the macrophage costimulatory activity for naive CD4+ T cells exposed to allogeneic or anti-CD3 stimuli through the differential regulation of the B7 costimulatory molecules. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for Ag-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP up-regulate the costimulatory activity of macrophages for Ag-primed CD4+ T cells. VIP-/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5 and reduce Th1-type cytokines such as IFN-gamma and IL-2. In vivo administration of VIP or PACAP in Ag-immunized mice reduce the numbers of IFN-gamma-secreting cells and enhance the numbers of IL-4-secreting cells. One of the consequences of the VIP-/PACAP-induced shift in cytokine profile is a change in the Ag-specific Ig isotype, increasing IgG1 and decreasing IgG2a levels. Finally, the preferential differentiation into Th2 effector cells after Ag stimulation induced by VIP-/PACAP-treated macrophages is mediated through the up-regulation of B7.2 expression.     

Genome‐wide SNP identification in Fraxinus linking genetic characteristics to tolerance of Agrilus planipennis

Ash (Fraxinus spp.) is one of the most widely distributed tree genera in North America. Populations of ash in the United States and Canada have been decimated by the introduced pest Agrilus planipennis (Coleoptera: Buprestidae; emerald ash borer), having negative impacts on both forest ecosystems and economic interests. The majority of trees succumb to attack by A. planipennis, but some trees have been found to be tolerant to infestation despite years of exposure. Restriction site‐associated DNA (RAD) sequencing was used to sequence ash individuals, both tolerant and susceptible to A. planipennis attack, in order to identify single nucleotide polymorphism (SNP) patterns related to tolerance and health declines. de novo SNPs were called using SAMtools and, after filtering criteria were implemented, a set of 17,807 SNPs were generated. Principal component analysis (PCA) of SNPs aligned individual trees into clusters related to geography; however, five tolerant trees clustered together despite geographic location. A subset of 32 outlier SNPs identified within this group, as well as a subset of 17 SNPs identified based on vigor rating, are potential candidates for the selection of host tolerance. Understanding the mechanisms of host tolerance through genome‐wide association has the potential to restore populations with cultivars that are able to withstand A. planipennis infestation. This study was successful in using RAD‐sequencing in order to identify SNPs that could contribute to tolerance of A. planipennis. This was a first step toward uncovering the genetic basis for host tolerance to A. planipennis. Future studies are needed to identify the functionality of the loci where these SNPs occur and how they may be related to tolerance of A. planipennis attack.     

Validation of a spatial agent-based model for Taenia solium transmission (“CystiAgent”) against a large prospective trial of control strategies in northern Peru

BackgroundThe pork tapeworm (Taenia solium) is a parasitic helminth that imposes a major health and economic burden on poor rural populations around the world. As recognized by the World Health Organization, a key barrier for achieving control of T. solium is the lack of an accurate and validated simulation model with which to study transmission and evaluate available control and elimination strategies. CystiAgent is a spatially-explicit agent based model for T. solium that is unique among T. solium models in its ability to represent key spatial and environmental features of transmission and simulate spatially targeted interventions, such as ring strategy.Methods/Principal findingsWe validated CystiAgent against results from the Ring Strategy Trial (RST)–a large cluster-randomized trial conducted in northern Peru that evaluated six unique interventions for T. solium control in 23 villages. For the validation, each intervention strategy was replicated in CystiAgent, and the simulated prevalences of human taeniasis, porcine cysticercosis, and porcine seroincidence were compared against prevalence estimates from the trial. Results showed that CystiAgent produced declines in transmission in response to each of the six intervention strategies, but overestimated the effect of interventions in the majority of villages; simulated prevalences for human taenasis and porcine cysticercosis at the end of the trial were a median of 0.53 and 5.0 percentages points less than prevalence observed at the end of the trial, respectively.Conclusions/SignificanceThe validation of CystiAgent represented an important step towards developing an accurate and reliable T. solium transmission model that can be deployed to fill critical gaps in our understanding of T. solium transmission and control. To improve model accuracy, future versions would benefit from improved data on pig immunity and resistance, field effectiveness of anti-helminthic treatment, and factors driving spatial clustering of T. solium infections including dispersion and contact with T. solium eggs in the environment.