ARHGAP30 is a Wrch-1-interacting protein involved in actin dynamics and cell adhesion

It has previously been reported that shedding of the PTPκ ectodomain drives enhanced motility of colon cancer cells. Herein, we provide mechanism underlying the regulation of PTPκ shedding by galectin-3 binding protein. PTPκ was inarguably scissored by the processed form of proprotein convertase 5 (subtilisin/kexin type 5), and galectin-3 binding protein which is over-produced in colon cancer cells and tissues contributed to increased cancer cell motility by acting as a negative regulator of galectin-3 at the cell surface. The high expression ratio of galectin-3 binding protein to galectin-3 was clinically correlated to lymphatic invasion. These results suggest that galectin-3 binding protein may be a potential therapeutic target for treatment of, at least, colon cancer patients with high expression of galectin-3 binding protein.     

Localization of decorin gene expression in normal human breast tissue and in benign and malignant tumors of the human breast

The small extracellular matrix proteoglycan decorin which possesses a potent antitumor activity has been shown to be present in various amounts in the stroma of several tumors including those of the breast. Regarding decorin in breast malignancies the published data are conflicting, i.e., whether breast cancer cells express it or not. Here, we first compared decorin gene expression levels between healthy human breast tissue and selected types of human breast cancer using GeneSapiens databank. Next, we localized decorin mRNA in tissue specimen of normal human breast, intraductal breast papillomas and various histologic types of human breast cancer using in situ hybridization (ISH) with digoxigenin-labeled RNA probes for decorin. We also examined the effect of decorin transduction on the behavior of cultured human breast cancer MCF7 cells. Analysis of GeneSapiens databank revealed that in various human breast cancers decorin expression is significant. However, ISH results clearly demonstrated that human breast cancer cells independently of the type of the cancer do not express decorin mRNA. This was also true for papilloma-forming cells of the human breast. Indeed, decorin gene expression in healthy human breast tissue as well as in benign and malignant tumors of human breast was shown to take place solely in cells of the original stroma. Decorin transduction using decorin adenoviral vector in decorin-negative MCF7 cells resulted in a significant decrease in the proliferation of these cells and changed cell cohesion. Decorin-transduced MCF7 cells also exhibited increased apoptosis. In conclusion, our study shows that in human breast tissue only cells of the original stroma are capable of decorin gene expression. Our study also shows that transduction of decorin in decorin-negative human breast cancer cells markedly modulates the growth pattern of these cells.     

In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.     

Angiotensin II response in afferent arterioles of mice lacking either the endothelial or neuronal isoform of nitric oxide synthase

The aim of the study is to evaluate the impact of nitric oxide (NO) produced by endothelial NO synthase (eNOS) and neuronal NOS (nNOS) on the angiotensin II response in afferent arterioles (Af). Dose responses were assessed for angiotensin II in microperfused Af of mice homozygous for disruption of the eNOS gene [eNOS(-/-)], or nNOS gene [nNOS(-/-)], and their wild-type controls, eNOS(+/+) and nNOS(+/+). Angiotensin II at 10(-8) and 10(-6) mol/l reduced the lumen to 69% and 68% in eNOS(+/+), and to 59% and 50% in nNOS(+/+). N(G)-nitro-L-arginine methyl ester (L-NAME) did not change basal arteriolar diameters, but augmented angiotensin II contraction, reducing diameters to 23% and 13% in eNOS(+/+), and 7% and 10% in nNOS(+/+) at 10(-8) and 10(-6) mol/l. The response to angiotensin II was enhanced in nNOS(-/-) mice (41% and 25% at 10(-8) and 10(-6) mol/l) and even more enhanced in eNOS(-/-) mice (12% and 9%) compared with nNOS(+/+) and eNOS(+/+). L-NAME led to complete constriction of Af in these groups. Media-to-lumen ratios of Af did not differ between controls and gene-deficient mice. mRNA expression of angiotensin II receptor types 1A and 1B and type 2 also did not differ. The results reveal that angiotensin II-induced release of NO from both eNOS and nNOS significantly contributes to the control of Af. Results also suggest that eNOS-derived NO is of greater importance than nNOS-derived NO in this isolated arteriolar preparation.     

Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.     

Can isotopic fractionation during respiration explain the 13C-enriched sporocarps of ectomycorrhizal and saprotrophic fungi?

The mechanism behind the (13)C enrichment of fungi relative to plant materials is unclear and constrains the use of stable isotopes in studies of the carbon cycle in soils. Here, we examined whether isotopic fractionation during respiration contributes to this pattern by comparing delta(13)C signatures of respired CO(2), sporocarps and their associated plant materials, from 16 species of ectomycorrhizal or saprotrophic fungi collected in a Norway spruce forest. The isotopic composition of respired CO(2) and sporocarps was positively correlated. The differences in delta(13)C between CO(2) and sporocarps were generally small, < +/-1 per thousand in nine out of 16 species, and the average shift for all investigated species was 0.04 per thousand. However, when fungal groups were analysed separately, three out of six species of ectomycorrhizal basidiomycetes respired (13)C-enriched CO(2) (up to 1.6 per thousand), whereas three out of five species of polypores respired (13)C-depleted CO(2) (up to 1.7 per thousand; P < 0.05). The CO(2) and sporocarps were always (13)C-enriched compared with wood, litter or roots. Loss of (13)C-depleted CO(2) may have enriched some species in (13)C. However, that the CO(2) was consistently (13)C-enriched compared with plant materials implies that other processes must be found to explain the consistent (13)C-enrichment of fungal biomass compared with plant materials.     

The fundamental role of ecological feedback mechanisms for the adaptive management of seagrass ecosystems – a review

Seagrass meadows are vital ecosystems in coastal zones worldwide, but are also under global threat. One of the major hurdles restricting the success of seagrass conservation and restoration is our limited understanding of ecological feedback mechanisms. In these ecosystems, multiple, self‐reinforcing feedbacks can undermine conservation efforts by masking environmental impacts until the decline is precipitous, or alternatively they can inhibit seagrass recovery in spite of restoration efforts. However, no clear framework yet exists for identifying or dealing with feedbacks to improve the management of seagrass ecosystems. Here we review the causes and consequences of multiple feedbacks between seagrass and biotic and/or abiotic processes. We demonstrate how feedbacks have the potential to impose or reinforce regimes of either seagrass dominance or unvegetated substrate, and how the strength and importance of these feedbacks vary across environmental gradients. Although a myriad of feedbacks have now been identified, the co‐occurrence and likely interaction among feedbacks has largely been overlooked to date due to difficulties in analysis and detection. Here we take a fundamental step forward by modelling the interactions among two distinct above‐ and belowground feedbacks to demonstrate that interacting feedbacks are likely to be important for ecosystem resilience. On this basis, we propose a five‐step adaptive management plan to address feedback dynamics for effective conservation and restoration strategies. The management plan provides guidance to aid in the identification and prioritisation of likely feedbacks in different seagrass ecosystems.     

BMP‐2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin‐3 (NT‐3) and also glial cell line‐ derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum‐free conditions, none of the factors tested, including NT‐3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein‐2 (BMP‐2), promoted the survival of tyrosine hydroxylase (TH)‐immunoreactive neurons at 6 days in vitro. However, when BMP‐2 was coadministered with any of these factors the number of LC TH‐positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT‐3, bFGF, or BMP‐2. The strongest effect (a fourfold increase in the number of TH‐positive cells) was induced by cotreatment with forskolin, BMP‐2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 291–304, 2002; DOI 10.1002/neu.10034