Biochemical and electron microscopic observations of vaccinia virus morphogenesis in HeLa cells after hydroxyurea reversal

Examination of thin sections of S3 HeLa cells propagated in suspension cultures and infected with vaccinia virus in the presence of hydroxyurea (HU) showed accumulation of immature virus particles. Upon removal of the drug the typical cytoplasmic development of virions was observed, confirming the usefulness of HU for separating early and late events in the replication cycle of poxvirions in spinner cultured cells and for studying viral morphogenesis under quasisynchronous conditions.Six hours after HU reversal, cytoplasm prepared by lysis of cells in Nonidet P-40 was fractionated by sucrose density centrifugation. Particles obtained by the fractionation procedure were negatively stained and their morphology characterized as follows: fraction I contained complete virions and cores with lateral bodies; fraction II contained particles 2300 × 3000 Å in size which resembled viral cores but lacked some of the surface layers found on such subviral particles prepared in vitro; fraction III contained spherical particles 2600–2800 Å in diameter and tubular structures of varying lengths and 1300 Å in diameter; fraction IV yielded particles which morphologically resembled viral cores but had irregular surfaces and appeared damaged; their sedimentation behavior during density gradient centrifugation and subsequent isolation suggested that they may be membrane bound in the cytoplasm and were mechanically released during isolation.When cytoplasmic samples were centrifuged in velocity gradients under conditions where subviral and viral particles were pelleted, progeny DNA was found throughout the gradient. In sucrose gradients prepared in 10^?3M phosphate buffer, pH 7.2, this DNA could be resolved into three classes. A minor fraction (10% or less of the radioactivity) remained at the top of the gradient, major components sedimented at 50–100 S (about 30–40%), the third > 100 S (10–15%). The remainder of the radioactivity was in the pellet. When the 50–100 S fraction was analyzed in alkaline gradients, the denatured DNA sedimented at about 30 S.Early after HU reversal (1–3 hr), before significant assembly of viral DNA into DNase resistant structures had occurred, 85–90% of the newly synthesized viral DNA could be recovered in the form of virosomes.Thin sections of viral DNA protein complexes when examined in the electron microscope were found to be heterogeneous in composition. Irregular ovoid bodies, 0.8–1.0 μm by 0.5 to 0.7 μm in size and filamentous material often in large aggregates composed of fibrils about 20 Å in diameter comprised the major components observed in virosome preparations.     

A genome-wide scan for a simulated data set using two newly developed methods

A genome-wide scan of a simulated data set for fictitious disease genes was conducted using both semiparametric and nonparametric methods. The semiparametric model-based method, which tests for linkage/linkage disequilibrium separately and together, correctly identified all three underlying disease loci along with two false positives through the linkage analysis. However, the nonparametric model-free method which tests combined linkage/linkage disequilibrium, failed to yield any results due to the lack of linkage disequilibrium information in the data.     

Fetal membranes as a source of stem cells

In recent years, a constant growth of knowledge and clinical applications of stem cells have been observed. Mesenchymal stromal cells, also described as mesenchymal stem cells (MSCs) represent a particular cell type for research and therapy because of their ability to differentiate into mesodermal lineage cells. The most investigated source of MSCs is bone marrow (BM). Yet, collection of BM is an invasive procedure associated with significant discomfort to the patient. The procedure results in a relatively low number of these cells, which can decrease with donor's age. Therefore, it seems to be very important to find other sources of mesenchymal stem cells nowadays. A human placenta, which is routinely discarded postpartum, in spite of its natural aging process, is still a rich source of stem cells capable to proliferate and in vitro differentiate in many directions. Besides homing and differentiation in the area of injury, MSCs there elicit strong paracrine effects stimulating the processes of repair. In this review, we focus on the biology, characteristics and potential clinical applications of cells derived from human fetal membranes: amnion and chorion.     

Use of bifunctional hybrid beta-lactamases for epitope mapping and immunoassay development

Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis beta-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random gene fragments. The procedure presented in this paper allows the selection of large infectious phage libraries with high diversity and efficient beta-lactamase activities. A useful aspect of the proposed technique results from the possibility of using the beta-lactamase activity carried by phages to evaluate the proportion of immobilised phages during the successive enrichment steps of the library or competition experiments with the selected phages. Another advantage of the technique derives from the fact that the epitope is selected as a bifunctional hybrid protein, which can be overproduced and purified. The resulting recombinant protein associates an epitope with a specific and efficient enzymatic activity. This constitutes an original tool for immunoassay development. A virus influenza hemagglutinin (HA1)-gene fragment library has been generated with this system and used to identify a linear epitope.     

Ueber Melanosis lenticularis progressiva

Ohne Zusammenfassung     

Endogenous endophthalmitis following disseminated fungemia due to Fusarium solani in a patient with acute myeloid leukemia

We report the case of a young man with a resistant acute myeloid leukemia (AML) who developed a disseminated fungemia due to Fusarium solani involving the skin and lungs, during the neutropenic phase following a chemotherapy course. Despite continuous therapy with liposomal amphotericin B, he developed a bilateral endophthalmitis that rapidly evolved to complete blindness. The patient underwent two procedures of vitrectomy, with detection of F. solani in the vitreous fluid, and continued antifungal therapy, without any recovery of visual acuity. When he eventually died due to recurrence of leukemia and hemorrhagic shock, autopsy revealed a diffuse fusarial involvement of the central nervous system.