Export of the pneumococcal phage SV1 lysin requires choline‐containing teichoic acids and is holin‐independent

Streptococcus pneumoniae bacteriophages (phages) rely on a holin–lysin system to accomplish host lysis. Due to the lack of lysin export signals, it is assumed that holin disruption of the cytoplasmic membrane allows endolysin access to the peptidoglycan. We investigated the lysis mechanism of pneumococcal phage SV1, by using lysogens without holin activity. Upon phage induction in a holin deficient background, phage lysin was gradually targeted to the cell wall, in spite of lacking any obvious signal sequence. Our data indicate that export of the phage lysin requires the presence of choline in the teichoic acids, an unusual characteristic of pneumococci. At the bacterial surface, the exolysin remains bound to choline residues without inducing lysis, but is readily activated by the collapse of the membrane potential. Additionally, the activation of the major autolysin LytA, which also participates in phage‐mediated lysis, is equally related to perturbations of the membrane proton motive force. These results indicate that collapse of the membrane potential by holins is sufficient to trigger bacterial lysis. We found that the lysin of phage SV1 reaches the peptidoglycan through a novel holin‐independent pathway and propose that the same mechanism could be used by other pneumococcal phages.     

Anti-Inflammatory Effect of Targeted Delivery of SOD to Endothelium: Mechanism,Synergism with NO Donors and Protective Effects In Vitro and In Vivo

Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation.     

Genetic distance of two fibrillar collagen loci, COL3A1 and COL5A2, located on the long arm of human chromosome 2

Two of the human fibrillar collagen genes, proa1(III) (COL3A1) and proa2(V) (COL5A2), map to the same region of the long arm of chromosome 2. To establish the genetic distance between the two loci, we analyzed the segregation of COL3A1 and COL5A2 RFLPs in five families informative for the two loci specific markers. We found that the maximum lod score was 9.33 at a recombination frequency of theta = 0.00. The data therefore provide strong evidence for tight linkage between two evolutionarily related fibrillar collagen genes on the 2q14----2q32 segment of chromosome 2.     

Strong magnesium binding sites in yeast phenylalanine transfer RNA.

To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op....Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m2G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it links the dihydrouridine arm with the extra (V) loop.     

Technique for Restoration of Mite (Acari) Preparations in Deteriorated Hoyer’s Medium

The Acari Collection of Instituto Butantan (IBSP), São Paulo, Brazil, includes many types and other identified mite specimens that were mounted in Hoyer’s medium, mainly in the first part of last century. An effort to restore degraded preparations was initiated in 1996. In this process, an improved technique was developed, allowing the adequate cleaning of specimens mounted up to 50–70 years before. Types and other identified specimens of Trombidiformes (Harpirhynchidae and Trombiculidae), Sarcoptiformes (Acaridae, Atopomelidae, Listrophoridae, and Psoroptidae) and Mesostigmata (Dermanyssidae, Ixodorhynchidae, Laelapidae, Macronyssidae, and Spinturnicidae) deposited at IBSP Collection have been satisfactorily restored.     

Evaluation of Hyperpolarized [1-13C]-Pyruvate by Magnetic Resonance to Detect Ionizing Radiation Effects in Real Time

Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.     

Specific expression in adult mice and post-implantation embryos of a transgene carrying the histone H1° regulatory region

Abstract. Histone H1°, a variant of the H1 group, has been found associated with the repressed state of chromatin and its content is increased in terminally differentiated cells. We have cloned a mouse H1° histone gene and introduced the promoter region, ligated to the β-galactosidase reporter gene, into transgenic mice. By histochemistry we demonstrated a strong expression of the transgene in adult kidney, testis and brain. Intestine, uterus and ovarium were also positive. This expression followed the same pattern as that of the endogenous H1° gene, as demonstrated by in situ hybridization with a non-coding fragment of the mRNA, by Northern analysis, and by immunofluorescence with specific antibodies. In post-implantation embryos, the expression was very low up to day ten p.c. At this time, most of the X-Gal staining was found in the brain, retina and some of the large blood vessels. Hence, expression of the transgene as well as of the endogenous H1° gene is not exclusively linked to a differentiated phenotype or to a reduced cell proliferation capacity.