The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.     

Dendritic cells support sequential reprogramming of chemoattractant receptor profiles during naive to effector T cell differentiation

T cells undergo chemokine receptor switches during activation and differentiation in secondary lymphoid tissues. Here we present evidence that dendritic cells can induce changes in T cell expression of chemokine receptors in two continuous steps. In the first switch over a 4-5 day period, dendritic cells up-regulate T cell expression of CXCR3 and CXCR5. Additional stimulation leads to the second switch: down-regulation of lymphoid tissue homing related CCR7 and CXCR5, and up-regulation of Th1/2 effector tissue-targeting chemoattractant receptors such as CCR4, CCR5, CXCR6, and CRTH2. We show that IL-4 and IL-12 can determine the fate of the secondary chemokine receptor switch. IL-4 enhances the generation of CCR4(+) and CRTH2(+) T cells, and suppresses the generation of CXCR3(+) T cells and CCR7(-) T cells, while IL-12 suppresses the level of CCR4 in responding T cells. Furthermore, IL-4 has positive effects on generation of CXCR5(+) and CCR7(+) T cells during the second switch. Our study suggests that the sequential switches in chemokine receptor expression occur during naive T cell interaction with dendritic cells. The first switch of T cell chemokine receptor expression is consistent with the fact that activated T cells migrate within lymphoid tissues for interaction with B and dendritic cells, while the second switch predicts the trafficking behavior of effector T cells away from lymphoid tissues to effector tissue sites.     

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.     

Correlation of tissue distribution, developmental phenotype, and intestinal homing receptor expression of antigen-specific B cells during the murine anti-rotavirus immune response

The intestinal homing receptor, alpha(4)beta(7), helps target lymphocytes to Peyer's patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing alpha(4)beta(7). In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD(-)B220(low)alpha(4)beta(7)(-)and alpha(4)beta(7)(+) cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD(-)B220(high)alpha(4)beta(7)(-)cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD(-)B220(low)alpha(4)beta(7)(+) cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express alpha(4)beta(7). Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that alpha(4)beta(7) is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues.     

Minor proteins,mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid

Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.     

Biological activity of palladium(II) and platinum(II) complexes of the acetone Schiff bases of S-methyl- and S-benzyldithiocarbazate and the X-ray crystal structure of the [Pd(asme)2] (asme=anionic form of the acetone Schiff base of S-methyldithiocarbazate) complex

Palladium(II) and platinum(II) complexes of general empirical formula, [M(NS)(2)] (NS=uninegatively charged acetone Schiff bases of S-methyl- and S-benzyldithiocarbazate; M=Pt(II) and Pd(II)) have been prepared and characterized by a variety of physicochemical techniques. Based on conductance, IR and electronic spectral evidence, a square-planar structure is assigned to these complexes. The crystal and molecular structure of the [Pd(asme)(2)] complex (asme=anionic form of the acetone Schiff base of S-methyldithiocarbazate) has been determined by X-ray diffraction. The complex has a distorted cis-square planar structure with the ligands coordinated to the palladium(II) ions as uninegatively charged bidentate NS chelating agents via the azomethine nitrogen and the mercaptide sulfur atoms. The distortion from a regular square-planar geometry is attributed to the restricted bite angles of the ligands. Antimicrobial tests indicate that the Schiff bases exhibit strong activities against the pathogenic bacteria, Bacillus subtilis (mutant defective DNA repair), methicillin-resistant Staphylococcus aureus, B. subtilis (wild type) and Pseudomonas aeruginosa and the fungi, Candida albicans (CA), Candida lypotica (2075), Saccharomyces cerevisiae (20341) and Aspergillus ochraceous (398)-the activities exhibited by these compounds being greater than that of the standard antibacterial and antifungal drugs, streptomycin and nystatin, respectively. The palladium(II) and platinum(II) complexes are inactive against most of these organisms but, the microbe, Pseudomonas aeruginosa shows strong sensitivity to the platinum(II) complexes. Screening of the compounds for their cytotoxicities against T-lymphoblastic leukemia cancer cells has shown that the acetone Schiff base of S-methyldithiocarbazate (Hasme) exhibits a very weak activity, whereas the S-benzyl derivative (Hasbz) is inactive. However, the palladium(II) complexes exhibit strong cytotoxicities against this cancer; their activities being more than that of the standard anticancer drug, tamoxifen. The [Pt(asme)(2)] complex exhibits a very weak cytotoxicity, whereas [Pt(asbz)(2)] is inactive against leukemic cells.     

Effect of age on susceptibility to post-traumatic infection in the elderly

Previous work has demonstrated an age-related decline in neutrophil function, including a decline in phagocytic capacity, with age in healthy individuals. This decline in function may contribute to increased susceptibility to bacterial infections in the elderly population. The present study has investigated the effects of age on susceptibility to infection and neutrophil function in elderly humans following mild trauma. Specifically, we have measured neutrophil function in 44 patients, all of whom had no significant co-morbidity, were over 65 years old (mean age 82.5 years) and had sustained a fractured neck of femur. We obtained neutrophils and examined the process of microbial engulfment by phagocytosis and the bactericidal mechanism of superoxide production. In the 5-week period after trauma, almost half of the elderly trauma patients succumbed to bacterial or fungal infection, with a predominance of chest and urinary tract infections. When examining neutrophil function, a decline in superoxide production was observed in neutrophils from the elderly trauma group at the time of hip fracture when compared with those from healthy elderly controls, and this was maintained 5 weeks after trauma. This was accompanied by an age-related reduction in phagocytic function during this period. We propose that trauma and an age-related decline in neutrophil function combine to decrease the immune response to bacteria in the elderly.