Coinoculation with hepatitis B surface and core antigen promotes a Th1 immune response to a multiepitopic protein of HIV-1

It has been defined that strong and multispecific cellular immune responses correlate with a better prognosis during the course of chronic diseases. A cross-enhancing effect on the resulting immune response obtained by the coadministration of recombinant hepatitis B virus (HBV) surface and core Ag was recently observed. With the objective of studying the effect of such Ag on the immune response to coinoculated heterologous Ag and vice versa, several formulations containing the recombinant HBV Ag and a multiepitopic protein (CR3) composed by CTL and Th epitopes from HIV-1 were evaluated by s.c. and mucosal administration. Combinations of two and three Ag were evaluated for cellular and humoral immune responses. The results showed that the best Ag combination for nasal immunization was the mixture comprising the CR3 recombinant HIV protein and both HBV Ag. Similarly, it was also the best formulation for s.c. immunization in aluminium phosphate adjuvant. In conclusion, it is possible to induce a Th1 stimulation of the cellular immune response specific for a HIV-based recombinant protein by formulating this Ag with the recombinant HBV Ag.     

Lovastatin production by Pleurotus ostreatus: effects of the C:N ratio

The types of carbon source and nitrogen source used as well as the C:N ratio in the medium influenced lovastatin production by Pleurotus ostreatus. The maximum value of the lovastatin yield was obtained in a medium that contained organic nitrogen.     

Tissue expression of glandular kallikrein and its response to 17 beta-estradiol in the acclimatized carp

Cyprinus carpio skeletal muscle kallikrein was isolated to apparent homogeneity, and a polyclonal antiserum against the purified protein was generated. Glandular kallikrein expression and tissue distribution were assessed using both Western blots and immunohistochemistry. A 39-kDa protein was detected in skeletal muscle, the gill, kidney, and pituitary gland, where an additional 72-kDa immunoreactive band was observed. Immunohistochemistry revealed immunoreactive kallikrein in the intermuscle tissue, epithelial gill cells, apical portion of distal and proximal tubular cells in the kidney, mucus and epithelial cells of the skin, intestinal tube, and prolactin-producing cells of the pituitary gland. In addition, the effect of 17beta-estradiol on kallikrein expression was analyzed in three different tissues of winter- and summer-acclimatized male carps. A 2.5-fold (p<0.05) increase in kallikrein immunoreactivity due to estrogen treatment was observed in winter-acclimatized carp muscle, but not in summer-acclimatized fish. In contrast, the gill responded differently, since a 2-fold (p<0.05) increase was found only in summer-acclimatized carps. Kallikrein immunoreactivity in the kidney increased both in summer- (2.5 fold) and in winter-acclimatized carps (1.5 fold). The signals obtained demonstrate the existence of tissue-specific variable responses to estrogen treatment in vivo, between winter and summer-acclimatized carp.     

A hybrid approach for efficient and robust parameter estimation in biochemical pathways

Developing suitable dynamic models of biochemical pathways is a key issue in Systems Biology. Predictive models for cells or whole organisms could ultimately lead to model-based predictive and/or preventive medicine. Parameter estimation (i.e. model calibration) in these dynamic models is therefore a critical problem. In a recent contribution [Moles, C.G., Mendes, P., Banga, J.R., 2003b. Parameter estimation in biochemical pathways: a comparison of global optimisation methods. Genome Res. 13, 2467-2474], the challenging nature of such inverse problems was highlighted considering a benchmark problem, and concluding that only a certain type of stochastic global optimisation method, Evolution Strategies (ES), was able to solve it successfully, although at a rather large computational cost. In this new contribution, we present a new integrated optimisation methodology with a number of very significant improvements: (i) computation time is reduced by one order of magnitude by means of a hybrid method which increases efficiency while guaranteeing robustness, (ii) measurement noise (errors) and partial observations are handled adequately, (iii) automatic testing of identifiability of the model (both local and practical) is included and (iv) the information content of the experiments is evaluated via the Fisher information matrix, with subsequent application to design of new optimal experiments through dynamic optimisation.     

Tailoring cardiac resynchronization therapy using interventricular asynchrony. Validation of a simple model

This study explores the use of interventricular asynchrony (interVA) for optimizing cardiac resynchronization therapy (CRT), an idea emerging from a simple pathway model of conduction in the ventricles. Measurements were performed in six dogs with chronic left bundle branch block (LBBB) and in 29 patients of the Pacing Therapies for Congestive Heart Failure (PATH-CHF)-I study. In the dogs, intraventricular asynchrony (intraVA) was determined using left ventricular (LV) endocardial activation maps. In dogs and patients, the maximum rate of rise of LV pressure (LV dP/dt(max)) and the pulse pressure (PP) and interVA [time delay between upslope of LV and right ventricular (RV) pressure curves] were measured during LV, RV, and biventricular (BiV) pacing with various atrioventricular (AV) delays. Measurements in the canine hearts supported the pathway model in that optimal resynchronization occurred at approximately 50% reduction of intraVA and at an interVA value halfway that during LBBB and LV pacing. In patients with significant hemodynamic response during pacing (n = 22), intrinsic interVA and interVA at peak improvement (interVA(p)) varied widely between patients (from -83 to -15 ms and from -42 to +31 ms, respectively). However, the model predicted individual interVA(p) accurately (SD of +/-6 ms and +/-12 ms for LV dP/dt(max) and PP, respectively). At equal interVA, LV and BiV pacing produced equal hemodynamic response, but in 11 of 22 responders, BiV pacing reduced interVA insufficiently to reach the maximum hemodynamic response. LV pacing at short AV delay proved to result in better hemodynamics than predicted by the model, indicating that additional factors determine hemodynamics during LV preexcitation. Guided by a simple pathway model, interVA measurements accurately predict optimal hemodynamic performance in individual CRT patients.     

Ca2+-binding allergens from olive pollen exhibit biochemical and immunological activity when expressed in stable transgenic Arabidopsis

Employing transgenic plants as alternative systems to the conventional Escherichia coli, Pichia pastoris or baculovirus hosts to produce recombinant allergens may offer the possibility of having available edible vaccines in the near future. In this study, two EF-hand-type Ca2+-binding allergens from olive pollen, Ole e 3 and Ole e 8, were produced in transgenic Arabidopsis thaliana plants. The corresponding cDNAs, under the control of the constitutive CaMV 35S promoter, were stably incorporated into the Arabidopsis genome and encoded recombinant proteins, AtOle e 3 and AtOle e 8, which exhibited the molecular properties (i.e. MS analyses and CD spectra) of their olive and/or E. coli counterparts. Calcium-binding assays, which were carried out to assess the biochemical activity of AtOle e 3 and AtOle e 8, gave positive results. In addition, their mobilities on SDS/PAGE were according to the conformational changes derived from their Ca2+-binding capability. The immunological behaviour of Arabidopsis-expressed proteins was equivalent to that of the natural- and/or E. coli-derived allergens, as shown by their ability to bind allergen-specific rabbit IgG antiserum and IgE from sensitized patients. These results indicate that transgenic plants constitute a valid alternative to obtain allergens with structural and immunological integrity not only for scaling up production, but also to develop new kind of vaccines for human utilization.     

Truncated HP1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting

Packaging of the eukaryotic genome into higher order chromatin structures is tightly related to gene expression. Pericentromeric heterochromatin is typified by accumulations of heterochromatin protein 1 (HP1), methylation of histone H3 at lysine 9 (MeH3K9) and global histone deacetylation. HP1 interacts with chromatin by binding to MeH3K9 through the chromodomain (CD). HP1 dimerizes with itself and binds a variety of proteins through its chromoshadow domain. We have analyzed at the single cell level whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. We used a lac-operator array-based system in mammalian cells to target EGFP-lac repressor tagged truncated HP1α and HP1β to a lac operator containing gene-amplified chromosome region in living cells. After targeting truncated HP1α or HP1β we observe enhanced tri-MeH3K9 and recruitment of endogenous HP1α and HP1β to the chromosome region. We show that CD-less HP1α can induce chromatin condensation, whereas the effect of truncated HP1β is less pronounced. Our results demonstrate that after lac repressor-mediated targeting, HP1α and HP1β without a functional CD are able to induce heterochromatinization.