Invasive and angiogenic phenotype of MCF-7 human breast tumor cells expressing human cyclooxygenase-2

To evaluate the direct effect of human cyclooxygenase-2 (hCox-2) on human breast tumor cell proliferation, invasion, and angiogenesis, hCox-2 cDNA was transfected into slow growing, non-metastatic MCF-7 human breast tumor cells that express low levels of Cox-2. Two stable transfectant clones, designated MCF-7/hCox-2 clones 8 and 10, had significantly decreased (P < 0.05) doubling time, with two-fold greater number of cells during exponential growth compared to the MCF-7/vector control. Proliferation of both of the MCF-7/hCox-2 clones was significantly inhibited in a time- and dose-dependent manner by celecoxib. The MCF-7/hCox-2 clones 8 and 10 formed larger and greater numbers of colonies in soft agar than the MCF-7/vector control, with a corresponding increased invasion across an artificial Matrigel basement membrane in response to recombinant human epidermal growth factor (hEGF). The MCF-7/hCox-2 clones 8 and 10 had higher mRNA levels of two splice variants of vascular endothelial growth factor (VEGF), V145 and V165. These results demonstrate that hCox-2 directly increases breast tumor cell proliferation, stimulates invasion across a basement membrane, and induces synthesis of specific heparin binding splice variants of VEGF.     

Genotoxicity,recombinogenicity and cellular preneoplasic transformation induced by vitamin A supplementation

In spite of being one of the first vitamins to be discovered, the full range of biological activities of Vitamin A remains incomplete. A growing body of evidence has demonstrated an apparent enhancement of carcinogenesis, induced by dietary retinol. Since DNA damage is a well-recognized inducer of carcinogenesis, the aim of this study was to test the possible genotoxic effect of dietary retinol, using different types of bioassays. Retinol caused an increased recombinogenic activity in Drosophila melanogaster larvae as measured by the SMART test. In mammalian cell cultures, retinol supplementation-induced DNA double-strands breaks (DSB) and single-strands breaks (SSB), cell cycle progression and proliferative focus formation in terminal-differentiated rat Sertoli cells and increased DNA fragmentation in Chinese hamster lung fibroblasts (V79 cells), as measured by the comet assay. Altogether, our results suggest that retinol causes DNA damage and chromosomal rearrangements, which may disturbs key physiological processes and lead to cell cycle progression and preneoplasic transformation of terminal-differentiated mammalian cells.     

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris)

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 μm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was “type 9”, 1–2 μm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7–9 μm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were “type 1”, thin walled (< 0.5 μm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin‐fixed paraffin‐embedded sections. Dogs were either singly infected with S. caninum or multiply co‐infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).     

Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity

P66, a Borrelia burgdorferi surface protein with porin and integrin‐binding activities, is essential for murine infection. The role of P66 integrin‐binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202–208). Neither change affected surface localization or channel‐forming activity of P66, but both significantly reduced binding to α_vβ₃. Integrin‐binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post‐subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202–208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post‐inoculation, but bacterial burdens recovered to wild‐type levels at 4 weeks post‐inoculation. The delay in tissue colonization correlated with reduced migration of the Del202–208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.     

Host cell heparan sulfate glycosaminoglycans are ligands for OspF‐related proteins of the Lyme disease spirochete

Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous tissues. Host glycosaminoglycans, highly modified repeating disaccharides that are present on cell surfaces and in extracellular matrix, are common targets of microbial pathogens during tissue colonization. While several dermatan sulfate‐binding B. burgdorferi adhesins have been identified, B. burgdorferi adhesins documented to promote spirochetal binding to heparan sulfate have not yet been identified. OspEF‐related proteins (Erps), a large family of plasmid‐encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF‐related, OspEF‐leader peptide (Elp) and OspE‐related subfamilies. We show here that a member of the OspF‐related subfamily, ErpG, binds to heparan sulfate and when produced on the surface of an otherwise non‐adherent B. burgdorferi strain, ErpG promotes heparan sulfate‐mediated bacterial attachment to the glial but not the endothelial, synovial or respiratory epithelial cells. Six other OspF‐related proteins were capable of binding heparan sulfate, whereas representative OspE‐related and Elp proteins lacked this activity. These results indicate that OspF‐related proteins are heparan sulfate‐binding adhesins, at least one of which promotes bacterial attachment to glial cells.     

Elimination of channel-forming activity by insertional inactivation of the p66 gene in Borrelia burgdorferi

P66 is a chromosomally encoded 66-kDa integral outer membrane protein of the Lyme disease agent Borrelia burgdorferi exhibiting channel-forming activity. Herein, we inactivated and subsequently complemented the p66 gene in the B31-A (WT) strain. The P66 protein was also inactivated in two other channel-forming protein mutant strains, P13-18 (Deltap13) and Deltabba01, and then compared with the channel-forming activities of wild-type and various p66 mutant strains. We further investigated the ion-selectivity of native, purified P66. In conclusion, we show that the porin activity of P66 is eliminated by insertional inactivation and that this activity can be rescued by gene complementation.