Hydration and packing effects on prion folding and beta-sheet conversion. High pressure spectroscopy and pressure perturbation calorimetry studies

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the alpha-helical and beta-sheet conformations of recombinant murine prion protein, designated as alpha-rPrP and beta-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel beta-sheet (beta-rPrP), a conformation that shares structural similarity with PrP(Sc). alpha-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar = 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of alpha-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of beta-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from alpha-rPrP. The higher susceptibility to pressure of beta-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of alpha-rPrP is much higher than that of beta-rPrP, which explains the lower degree of hydration of beta-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.     

Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products

Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.     

Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment

Bone morphogenetic proteins (BMPs) promote astrocytic differentiation of cultured subventricular zone stem cells. To determine whether BMPs regulate the astrocytic lineage in vivo, transgenic mice were constructed that overexpress BMP4 under control of the neuron-specific enolase (NSE) promoter. Overexpression of BMP4 was first detectable by Western analysis on embryonic day 16 and persisted into the adult. The overexpression of BMP4 resulted in a remarkable 40% increase in the density of astrocytes in multiple brain regions accompanied by a decrease in the density of oligodendrocytes ranging between 11 and 26%, depending on the brain region and the developmental stage. No changes in neuron numbers or the pattern of myelination were detected, and there were no gross structural abnormalities. Similar phenotypes were observed in three independently derived transgenic lines. Coculture of transgenic neurons with neural progenitor cells significantly enhanced astrocytic lineage commitment by the progenitors; this effect was blocked by the BMP inhibitor Noggin, indicating that the stimulation of astrogliogenesis was due to BMP4 release by the transgenic neurons. These observations suggest that BMP4 directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage. Further, differentiation of radial glial cells into astrocytes was accelerated, suggesting that radial glia were a source of at least some of the supernumerary astrocytes. Therefore, BMPs are likely important mediators of astrocyte development in vivo.     

Effects of midazolam on the contraction and relaxation of segments of thoracic aorta stripped of endothelium and stimulated by adrenaline – experimental study in rabbits

To evaluate the effects of midazolam on the angiokinesis of segments of rabbits' thoracic aorta stripped of endothelium and stimulated by adrenaline.Two groups of aortic rings removed from albinic rabbits anesthetized with thiopental were used (Group I – 6 animals; Group II – 12 animals), stripped of endothelium, studied in an organ chamber, perfused by Krebs-Henseleit solution. The groups were stimulated by adrenaline, recording the maximum contraction and dT/dt at 12, 36, 60 and 120. When the plateau phase was reached, the vessel was washed with perfusion solution, recording relaxation at 2, 4 and 6. When the base values were reached, Group I underwent a new adrenergic stimulus; and Group II was stimulated with midazolam and then with adrenaline, and the same values were recorded. T test was applied as a statistical analysis when two variables were studied. When studying more than two variables the Anova test was used, supplemented by the Tuckey test.Group I did not show any significant difference between the two stimuli. Group II – the midazolam significantly reduced the maximum contraction induced by adrenaline (83.01 ± 4.11%) (p < 0.01). The dT/dt was reduced at 12 (57.06 ± 8.47%), and also at 36 (70.59 ± 5.26%). There was no significance at 60 and 120 (p < 0.01).The relaxation increased significantly at all measurements – at 2-adrenaline 39.31 ± 9.60%; adrenaline/midazolam: 44.06 ± 9.62% (p < 0.05). At 4-adrenaline: 53.08 ± 8.3%; adrenaline/midazolam: 61.68 ± 8.50% (p < 0.01). At 6-adrenaline: 76.26 ± 5.45%; adrenaline/midazolam: 84.20 ± 7.96% (p < 0.01).Midazolam significantly reduced the maximum contraction obtained by the adrenergic stimulus as well as the dT/dt in the initial phases of contraction. The relaxation speed also increased.     

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.Publication of the Millennium Institute for Tissue Bioengineering. The study was supported by PRONEX, CNPq and FINEP grants from the Brazilian Ministry of Science and Technology and a FAPERJ grant from the Rio de Janeiro State Government.     

Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves

In this study cashew (Anacardium occidentale) plants were exposed to a short- and long-term exposure to NaCl in order to establish the importance of the salt-induced proteolysis and the glutamine synthetase activity on the proline accumulation. The cashew leaf showed a prominent proline accumulation in response to salt stress. In contrast, the root tissue had no significant changes in proline content even after the drastic injury caused by salinity on the whole plant. The leaf proline accumulation was correlated to protease activity, accumulation of free amino acid and ammonia, and decrease of both total protein and chlorophyll contents. The leaf GS activity was increased by the salt stress whereas in the roots it was slightly lowered. Although the several amino acids in the soluble pool of leaf tissue have showed an intense increment in its concentrations in the salt-treated plants, proline was the unique to show a proportional increment from 50 to 100 mol m-3 NaCl exposure (16.37 to 34.35 mmol kg-1 DM, respectively). Although the leaf glutamate concentration increased in the leaves of the salt-stressed cashew plants, as compared to control, its relative contribution to the total amino acid decreased significantly in stressed leaves when compared to other amino acids. In addition, when the leaf discs were incubated with NaCl in the presence of exogenous precursors (Glu, Gln, Orn or Arg) involved in the proline synthesis pathways, the glutamate was unique in inducing a significant enhancement of the proline accumulation compared to those discs with precursor in the absence of NaCl. These results, together with the salt-induced increase in the GS activity, suggest an increase in the de novo synthesis of proline probably associated with the increase of the concentration of glutamate. Moreover, the prominent salt-induced proline accumulation in the leaves was associated with the higher salt-sensitivity in terms of proteolysis and salt-induced senescence as compared to the roots. In conclusion, the leaf-proline accumulation was due, at least in part, to the increase in the salt-induced proteolysis associated with the increments in the GS activity and hence the increase in the concentration of glutamate precursor in the soluble amino acid pool.     

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.     

Bradykinin B1 receptor stimulates the proximal tubule Na(+)-ATPase activity through protein kinase C pathway

Recently, our group described a B1-mediated stimulatory effect of des-Arg(9)-bradykinin (DABK) on the Na(+)-ATPase activity of proximal tubule basolateral membranes (BLM) [Biochim. Biophys. Acta 1431 (1999) 483.]. Data in the present report suggest the participation of a phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway as the molecular mechanism of DABK-mediated stimulation of the Na(+)-ATPase activity since (i) 10(-8) M DABK activates PI-PLC activity; (ii) 10(-9) M U73122, a PI-PLC inhibitor, abolishes the effect of 10(-8) M DABK on the Na(+)-ATPase activity; (iii) 10(-8) M DABK increases phosphoprotein formation by 34%. This effect is completely reversed by 10(-7) M calphostin C, an inhibitor of PKC; (iv) 20 ng/ml TPA, an activator of PKC, and 10(-8) M DABK stimulate the Na(+)-ATPase activity in a similar and nonadditive manner. Furthermore, the effect of 10(-8) M DABK is completely reversed by calphostin C; (v) 10(-8) M DABK increases phosphoserine residue levels by 54%. This effect is completely reversed by 10(-7) M calphostin C.     

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.     

The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor

The adapter protein Grb10 belongs to a superfamily of related proteins, including Grb7, -10, and -14 and Caenorhabditis elegans Mig10. Grb10 is an interacting partner of the insulin-like growth factor I receptor (IGF-IR) and the insulin receptor (IR). Previous work showed an inhibitory effect of mouse Grb10 (mGrb10alpha) on IGF-I-mediated mitogenesis (A. Morrione et al., J. Biol. Chem. 272:26382-26387, 1997). With mGrb10alpha as bait in a yeast two-hybrid screen, mouse Nedd4 (mNedd4-1), a ubiquitin protein ligase, was previously isolated as an interacting protein of Grb10 (A. Morrione et al., J. Biol. Chem. 274:24094-24099, 1999). However, Grb10 is not ubiquitinated by Nedd4 in cells. Here we show that in mouse embryo fibroblasts overexpressing Grb10 and the IGF-IR (p6/Grb10), there is a strong ligand-dependent increase in ubiquitination of the IGF-IR compared with that in parental cells (p6). This increased ubiquitination is associated with a shorter half-life and increased internalization of the IGF-IR. The IGF-IR is stabilized following treatment with both MG132 and chloroquine, indicating that both the proteasome and lysosomal pathways mediate degradation of the receptor. Ubiquitination of the IGF-IR likely occurs at the plasma membrane, prior to the formation of endocytic vesicles, as it is insensitive to dansylcadaverine, an inhibitor of early endosome formation in IGF-IR endocytosis. Grb10 coimmunoprecipitates with the IGF-IR and endogenous Nedd4 in p6/Grb10 cells, suggesting the presence of a Grb10/Nedd4/IGF-IR complex. Ubiquitination of the IGF-IR in p6/Grb10 cells is severely impaired by overexpression of a catalytically inactive Nedd4 mutant (Nedd4-CS), which also stabilizes the receptor. Likewise, overexpression of a Grb10 mutant lacking the Src homology 2 (SH2) domain impaired ubiquitination of the IGF-IR in parental p6 and p6/Grb10 cells, indicating that Grb10 binding to Nedd4 is critical for ubiquitination of the receptor. These results suggest a role for the Grb10/Nedd4 complex in regulating ubiquitination and stability of the IGF-IR, and they suggest that Grb10 serves as an adapter to form a bridge between Nedd4 and the IGF-IR. This is the first demonstration of regulation of stability of a tyrosine kinase receptor by the Nedd4 (HECT) family of E3 ligases.