Vascular endothelial growth factor stimulates organ-specific host matrix metalloproteinase-9 expression and ovarian cancer invasion

Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) regulate each other, contributing to tumor progression. We have previously reported that MMP9 induces the release of tumor VEGF, promoting ascites formation in human ovarian carcinoma xenografts. The aim of this study was to investigate whether tumor-derived VEGF regulated the expression of gelatinase by the stroma, influencing the invasive properties of ovarian tumors. Tumor variants derived from 1A9 human ovarian carcinoma, stably expressing VEGF(121) in the sense (1A9-VS-1) and antisense orientations (1A9-VAS-3), were used. In vivo, zymographic analysis of tumors from 1A9-VS-1 implanted in the peritoneal cavity of nude mice showed higher levels of gelatinases, particularly murine MMP9, indicating that VEGF stimulates host expression of the matrix-degrading enzyme. Murine MMP9 expression was also high in the ovaries of mice bearing 1A9-VS-1 tumors. The effect on host MMP9 activity was organ-specific. The levels of host pro-MMP9 in ovaries correlated with the plasma levels of tumor VEGF and with the selective invasion of the ovaries. Induction of host MMP9 expression in tumors and ovaries was independent of the site of tumor growth as it was seen in mice carrying both intraperitoneal and subcutaneous tumors. The anti-VEGF antibody bevacizumab (Avastin) inhibited MMP9 expression and tumor invasion in the ovaries of mice bearing 1A9-VS-1 tumors. These findings point to a complex cross-talk between VEGF and MMPs in the progression of ovarian tumor and suggest the possibility of using VEGF inhibitors to affect MMP-dependent tumor invasion.     

Threatened but understudied: supporting conservation by understanding the genetic structure of the flat-headed cat

The flat-headed cat (Prionailurus planiceps) is a wetland specialist, currently facing habitat loss on a serious scale due to massive destruction of lowland forests and wetlands in Southeast Asia. Despite its ‘endangered’ status in the IUCN Red List, there has virtually been no investigation on the population structure nor on the evolutionary history of the flat-headed cat. To fill this gap, we used full mitochondrial genome sequences, obtained from archival samples, covering the historical distribution of the flat-headed cat. Our data revealed a high genetic differentiation (F _st = 0.81, P?<?0.001) between mitochondrial lineages from Borneo and those from Thai-Malay Peninsula/Sumatra, a split that was dated to ~575 Kya. Such a significant differentiation clearly distinguishes the Bornean flat-headed cat population from all other populations and suggests that there should be a reassessment of the flat-headed cat’s intraspecific taxonomy. However, morphological and nuclear data are required to corroborate our mtDNA results. Until such data become available, we recommend that for future conservation efforts and captive breeding programmes the two genetically distinct flat-headed cat populations are managed separately as two lineages to maintain the original genetic diversity of this endangered species.     

Dissecting the Molecular Origins of Specific Protein-Nucleic Acid Recognition: Hydrostatic Pressure and Molecular Dynamics

The fundamental processes by which proteins recognize and bind to nucleic acids are critical to understanding cellular function. To explore the factors involved in protein-DNA recognition, we used hydrostatic pressure to perturb the binding of the BamHI endonuclease to cognate DNA, both in experiment and in molecular dynamic simulations. A new technique of high-pressure gel mobility shift analysis was used to test the effects of elevated hydrostatic pressure on the binding of BamHI to its cognate recognition sequence. Upon application of a pressure of 500 bar, the equilibrium dissociation constant of BamHI binding to the cognate site was found to increase nearly 10-fold. A challenge has been to link this type of pure thermodynamic measurement to functional events occurring at the molecular level. Thus, we used molecular dynamic simulations at both ambient and elevated pressures to reveal details of the direct and water-mediated interactions between BamHI and cognate DNA, which allow explanation of the effects of pressure on site-specific protein-DNA binding and complex stability.     

Bacterial SOS Checkpoint Protein SulA Inhibits Polymerization of Purified FtsZ Cell Division Protein

Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.     

A yeast strain that uses D-galacturonic acid as a substrate for L-ascorbic acid biosynthesis

Summary The bioconversion of D-galacturonic acid to L-ascorbic acid was demonstrated in a new yeast strain isolated from the Japanese Crystal. Both intact cells and a crude mitochondrial extract yielded L-ascorbic acid when D-galacturonic acid was present.     

Spheroid formation and invasion capacity are differentially influenced by co-cultures of fibroblast and macrophage cells in breast cancer

Interactions with stromal components influence the growth, survival, spread, and colonization capacities of tumor cells. Fibroblasts and macrophages which are responsible for the stroma production and maintenance are of the basic elements found in tumor microenvironment. Cellular density and ratio of stromal cells to tumor cells can also have modulatory effects in cancer. Here, the contribution of fibroblast and/or macrophage cells on the malignant behavior of breast cancer cells was modeled in co-culture systems. Co-cultures were established at different cell densities and ratios with 4T1 breast cancer, NIH/3T3 or 3T3-L1 fibroblast, and J774A.1 monocyte/macrophage cell lines. Flow cytometry-based proliferation, 3D growth on alginate matrix, and matrigel invasion assays were performed to determine the change in the malignant assets of tumor cells. The data were also supported by immunocytochemical and morphological analyses. Co-culturing with fibroblasts (especially, NIH/3T3 cells) significantly supported the proliferation, scattering, and invasiveness of 4T1 cells whereas inclusion of macrophages disrupted this positive influence. On the other hand, the invasion capacity of 4T1 cells was not enhanced in the co-cultures with fibroblasts whose motility were inhibited with pertussis toxin pretreatment. Particularly at low-density seeding in 3D cultures, 4T1 cells could form substantially more spheroids than that of in the co-cultures with fibroblasts. Only, increasing the amount of fibroblasts could restore the 3D-growth. Intriguingly, co-existence of macrophage, fibroblast, and tumor cells in 3D cultures provided a convenient stroma sustaining the spheroid formation and growth. In conclusion, fibroblasts can form a favorable environment for tumor cells’ spread and motility whereas restricting their 3D-growth capacity. On the other hand, presence of macrophages may disrupt the influence of fibroblasts and enhance the spheroid formation by the tumor cells.     

The role of trematode parasites in larval anuran communities: an aquatic ecologist’s guide to the major players

Conservation strategies depend on our understanding of the ecosystem and community dynamics. To date, such understanding has focused mostly on predator–prey and competitor interactions. It is increasingly clear, however, that parasite–host interactions may represent a large, and important, component of natural communities. The need to consider multiple factors and their synergistic interactions if we are to elucidate the contribution of anthropogenic factors to loss in biodiversity is exemplified by research into present-day amphibian declines. Only recently has the role of factors such as trematode parasite infections been incorporated into studies of the population and community dynamics of aquatic systems. We argue that this is due, at least in part, to difficulties faced by aquatic ecologists in sifting through the complex systematics that pervade the parasite literature. We note that two trematode species are of dominant importance with regard to North American larval anuran communities, and provide in this review a clear explanation of how to distinguish between the infective stages of these two parasites. We describe the general biology and life history of these parasites, as well as what is known about their effect on larval anurans, and the interactive effects of environmental stressors (typically anthropogenic in nature) and parasites on larval anurans. We hope that this review will convince the reader of the potential importance of these parasites to aquatic communities in general, and to amphibian communities specifically, and will also provide the information necessary for aquatic ecologists to more frequently consider the role of these parasites in their studies of aquatic ecology.     

Serotonin-Synthesizing Neurons in the Rostral Medullary Raphé/Parapyramidal Region Transneuronally Labelled After Injection of Pseudorabies Virus into the Rat Tail

Serotonin-synthesizing raphé/parapyramidal neurons (5-HT neurons) may function as sympathetic premotor neurons regulating sympathetic outflow to the cutaneous vascular bed. In the present study a genetically engineered pseudorabies virus (PRV) expressing green fluorescent protein (GFP) was injected into the rat tail. After survival for 3–4 days the medulla oblongata was examined using double-label immunohistochemistry, with an antibody against GFP for the virus and an antibody against phenylalanine hydroxylase 8 (PH8) for 5-HT synthesis. Sections were examined using light microscopy, and conventional and confocal fluorescence microscopy. There were two subpopulations of PRV+ve neurons in the raphé/parapyramidal region: a more dorsally and laterally located subgroup of medium-sized and large neurons, mainly non-serotonergic, and a more ventrally located subgroup of small mainly serotonin-synthesizing neurons, including those just dorsal to the pyramids, those in raphé pallidus, and those in close relationship to the ventral surface in the parapyramidal-subependymal zone.Special issue dedicated to Miklós Palkovits.