Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.     

Gastrointestinal stromal tumor. A study of 158 cases: clinicopathological features and prognostic factors

OBJECTIVE: To increase knowledge on the behavior of gastrointestinal stromal tumors (GISTs) and factors influencing therapy. STUDY DESIGN: The clinicomorphological features of 158 GISTs were analyzed. Survival analysis was performed on the whole series, as well as on a selected group of patients with high risk GIST who did not receive imatinib mesylate. The impact of imatinib mesylate on the prognosis was investigated. RESULTS: Most of the GISTs had a benign behavior. The risk class was a powerful prognostic factor but was unable to predict the outcome in a single case; even patients in the high risk class not receiving imatinib mesylate had a low mortality rate. In this group, it was the mitotic activity that better correlated with prognosis, and a cut point of 10 mitoses per 50 high-power field can be fixed to discriminate cases with favorable or unfavorable outcome. Patients with GISTs presenting as aggressive disease received great benefit from imatinib mesylate therapy. CONCLUSION: Mitotic activity is important in predicting the outcome of patients with high risk GIST who present at diagnosis without dissemination. This finding can have therapeutic implications.     

Preferred conformation of peptides rich in Ac8c,a medium-ring alicyclic Cα,α-disubstituted glycine

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C^α,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac₈c), and two Ala/Ac₈c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and ¹H-NMR. The molecular structures of the amino acid derivative Z-Ac₈c-OH, the dipeptide pBrBz- (Ac₈c)₂-OH and the tripeptide pBrBz-(Ac₈c)₃-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac₈c-NHMe. Taken together, the results obtained strongly support the view that the Ac₈c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of C^{α, α}-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–7) investigated so far. The implications for the use of the Ac₈c residue in peptide conformational design are considered.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.     

Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes

Background

Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo.

Methods

The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71.

Results

The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71.

Conclusion

The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine.     

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.     

Multiple restrictions of human immunodeficiency virus type 1 in feline cells

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.     

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

Volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H₂O₂ plays an essential role in the activation of these channels and that H₂O₂ per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl⁻ current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H₂O₂ VSOR Cl⁻ current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H₂O₂, exogenous addition of ATP in the presence of extracellular Ca²⁺ resulted in a decrease in the half-time for VSOR Cl⁻ current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl⁻ current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl⁻ current onset in a extracellular Ca²⁺-dependent manner.     

"Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence

Background

Bovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process.

Results

We demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads.

Conclusion

Together, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.