Bioactive recombinant neuregulin-1, -2, and -3 expressed in Escherichia coli

The neuregulins (NRGs) are a family of signaling proteins that are ligands for receptor tyrosine kinase of the ErbB family (namely ErbB3 and ErbB4). To date, four different neuregulin genes have been identified (neuregulin1-4). While NRG1 isoforms have been extensively studied, little is yet known about the other genes of the family. We report the expression of recombinant NRG1beta1, NRG2alpha, NRG2beta, and NRG3 as recombinant fusion proteins in Escherichia coli. The cDNA encoding for the EGF-like domain of each protein was cloned from the mouse olfactory bulb and inserted into the pET-19b vector allowing for bacterial expression of the protein fused to an N-terminal His tag. The recombinant NRGs expressed in the inclusion bodies were solubilized under denaturing conditions, purified by affinity chromatography, and refolded via dialysis in the presence of reducing agents. Purified recombinant NRGs were active as they bound to their receptors and induced their phosphorylation. In particular, and in agreement with data on the native proteins, all the molecules were able to bind and activate ErbB4 while only the rNRG1 and the two rNRG2 (but not rNRG3) bound ErbB3.     

Seeing fearful body expressions activates the fusiform cortex and amygdala

Darwin's evolutionary approach to organisms' emotional states attributes a prominent role to expressions of emotion in whole-body actions. Researchers in social psychology [1,2] and human development [3] have long emphasized the fact that emotional states are expressed through body movement, but cognitive neuroscientists have almost exclusively considered isolated facial expressions (for review, see [4]). Here we used high-field fMRI to determine the underlying neural mechanisms of perception of body expression of emotion. Subjects were presented with short blocks of body expressions of fear alternating with short blocks of emotionally neutral meaningful body gestures. All images had internal facial features blurred out to avoid confounds due to a face or facial expression. We show that exposure to body expressions of fear, as opposed to neutral body postures, activates the fusiform gyrus and the amygdala. The fact that these two areas have previously been associated with the processing of faces and facial expressions [5-8] suggests synergies between facial and body-action expressions of emotion. Our findings open a new area of investigation of the role of body expressions of emotion in adaptive behavior as well as the relation between processes of emotion recognition in the face and in the body.     

Fate of polydnavirus DNA of the egg-larval parasitoid Chelonus inanitus in the host Spodoptera littoralis

In situ hybridizations show that 5 min after parasitization, polydnavirus DNA is in close vicinity of the parasitoid egg, but 5 h later also in the yolk and partially in the host embryo. Fifteen hours after parasitization, the viral DNA is seen all over the host embryo and hardly in the yolk. The tissue distribution of the viral DNA was analysed and quantified by dot blots in the fifth instar parasitized larvae. On a per host basis, haemocytes and fat body contained the highest amount of viral DNA, while nervous tissue, intestinal tract and carcass contained less. Of the three viral segments tested, all were found in all tissues. Relative to the quantity of host DNA, viral DNA was most abundant in haemocytes, about five times less abundant in fat body and nervous tissue and about 25 times less abundant in intestinal tract. The total quantity of viral DNA per host was 444+/-145 pg which is similar to the quantity injected by the wasp; thus, the viral DNA persists throughout parasitization. The parasitoid larva contains 820+/-80 pg viral DNA integrated in the genome. This illustrates that the dose of viral DNA injected in virions represents approximately one third of the total viral genomic information present in a host at a late stage of parasitism.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.     

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Sulfolobus solfataricus via chemical rescue of an inactive mutant

We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.     

Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene. Production of a functional enzyme by mutations mimicking programmed -1 frameshifting

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.