Hypersensitivity of an Arabidopsis Sugar Signaling Mutant toward Exogenous Proline Application

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Δ1-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutantrsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS andP5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Δ1-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

Physical activity, body composition, exogenous estrogen use and their association with insulin-like growth factors

Obesity and physical inactivity are modifiable risk factors that are associated with several health issues; they are major factors in up to 30% of major cancers. Elevated levels of circulating insulin-like growth factor-I (IGF-I) have been associated with high body composition measurements and high cancer risk; exogenous estrogen use is associated with low circulating IGF-I levels and high cancer risk. The relationship between physical activity and circulating IGF levels is complex and findings of previous studies of their relationship remain inconsistent; however, these studies included vague definitions of physical activity. In this study, we used cross-sectional data from the Women's Health Initiative to determine the relationship between specific measures of physical activity (e.g., intensity, duration, and frequency) and circulating IGF-I levels, accounting for exogenous estrogen use and body composition. These data were collected from women enrolled at Women's Health Initiative clinical centers at Baylor College of Medicine and Wake Forest University School of Medicine. Multivariate linear regression analysis showed that circulating IGF-I and IGF-binding protein (BP) 3 levels were positively associated with frequency, duration, and intensity of physical activity. Circulating IGF-I levels and the molar IGF-I:IGF-BP3 ratio were significantly associated with frequency of walking, whereas circulating IGF-BP3 levels were significantly associated with strenuous physical activity, suggesting that different aspects of physical activity and their effects on fitness affect members of the IGF family differently. The results from our study support the recommendation of a regular exercise routine, particularly that of strenuous intensity, for postmenopausal women as a means to prevention of cancer.^

Association of exogenous hormonal intake with the risk of development of hepatocellular carcinoma in women

Background. Primary liver cancer, the majority of which is hepatocellular carcinoma, is the third most common cause of mortality from cancer. It has one of the worst prognosis outcomes and an overall 5-year survival of only 5-6%. Hepatocellular carcinoma has been shown to have wide variations in geographic distribution and there is a marked difference in the incidence between different races and gender. Previously low-rate countries, including the US, have shown to have doubled the incidence of HCC during the past two decades. Even though the incidence of HCC is higher in males as compared to females, female hormones, especially estrogens have been postulated to have a role in the development of hepatocellular carcinoma on a molecular level. Despite the frequent usage of oral contraceptive pills (OCP) and previously, hormone replacement therapy (HRT), their role on HCC development has not been studied thoroughly. We aim to examine the association between exogenous hormone intake (oral contraceptives and post-menopausal hormone replacement therapy) and the development of HCC. Methods. This study is part of an ongoing hospital-based case-control study which is conducted at the Department of Gastrointestinal Oncology at The University of Texas M. D. Anderson Cancer Center. From January 2005 up to January 2008, a total of 77 women with pathologically confirmed hepatocellular carcinoma (cases) and 277 healthy women (controls) were included in the investigation. Information about the use of hormonal contraceptives, hormone replacement therapy and risk factors of hepatocellular cancer was collected by personal interview. Univariate and multivariate logistic regression analyses were done to estimate the crude odds ratios (OR) and adjusted odds ratios (AOR). Results. We found statistically significant protective effect for the use of HRT on the development of HCC, AOR=0.42 (95% CI, 0.21, 0.81). The significance was observed for estrogen replacement, AOR=0.43 (95% CI, 0.22, 0.83) and not for progesterone replacement, AOR=0.49 (95% CI, 0.10, 2.35). On the other hand, any hormonal contraceptive use, which encompasses oral contraceptive pills, implants and injections, did not show a statistical significance either in the crude OR=0.58 (95% CI, 0.33, 1.01) or AOR=0.56 (95% CI 0.26, 1.18). Conclusions. As corroborated by previous studies, HRT confers 58% HCC risk reduction among American women. The more important question of the association between hormonal contraceptives and HCC remains controversial. Further studies are warranted to explore the mechanism of the protective effect of HRT and the relationship between hormonal contraception and HCC.^

Surfactants during upwelling events in the sea-surface microlayer and 1 m depth during METEOR cruise M117, Baltic Sea

Results from a study of surfactants (SAS) in the sea surface microlayer (SML) and underlying water (ULW) at different locations in the Baltic Sea. The total SAS concentrations were measured using phase-sensitive alternative current voltammetry with hanging mercury drop electrode (HMDE) in unfiltered samples. The concentrations of SAS were expressed as the equivalent concentration of nonanionic surfactants Triton-X-100. The enrichment factors (EF) of SAS were calculated as the ratio of concentration in SML to the corresponding ULW samples.

Removing exogenous information using pedigree data

Management of certain populations requires the preservation of its pure genetic background. When, for different reasons, undesired alleles are introduced, the original genetic conformation must be recovered. The present study tested, through computer simulations, the power of recovery (the ability for removing the foreign information) from genealogical data. Simulated scenarios comprised different numbers of exogenous individuals taking partofthe founder population anddifferent numbers of unmanaged generations before the removal program started. Strategies were based on variables arising from classical pedigree analyses such as founders? contribution and partial coancestry. The ef?ciency of the different strategies was measured as the proportion of native genetic information remaining in the population. Consequences on the inbreeding and coancestry levels of the population were also evaluated. Minimisation of the exogenous founders? contributions was the most powerful method, removing the largest amount of genetic information in just one generation.However, as a side effect, it led to the highest values of inbreeding. Scenarios with a large amount of initial exogenous alleles (i.e. high percentage of non native founders), or many generations of mixing became very dif?cult to recover, pointing out the importance of being careful about introgression events in population

Treatment of tropical forages with exogenous fibrolytiic enzymes: effects on chemical composition and in vitro rumen fermentation

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24 h before, on the in vitro gas production. In general, the 24 h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24 h reduced (p < 0.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (p < 0.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.

Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor α. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2− production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid–cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4°C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid–cholesterol microdomains in living cells.

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46–61)–Ak or HEL-(116–129)–Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.

Inducible expression of exogenous genes in Dictyostelium discoideum using the ribonucleotide reductase promoter

We report here the development of a regulated gene expression system for Dictyostelium discoideum based on the DNA-damage inducibility of the rnrB gene. rnrB, which codes for the small subunit of the enzyme ribonucleotide reductase, responds to DNA-damaging agents at all stages of the D.discoideum life cycle. Doses that have little effect on development have previously been shown to increase the level of the rnrB transcript by up to 15-fold. Here we show that all elements necessary for DNA-damage induction are contained in a 450 bp promoter fragment. We used a fusion of the rnrB promoter with the gene encoding GFP to demonstrate an up to 10-fold induction at the RNA level, which appears in all aspects similar to induction of the endogenous rnrB transcript. Using a fusion with the lacZ gene we observed an up to 7-fold induction at the protein level. These results indicate that the rnrB promoter can be used to regulate the expression of specific genes in D.discoideum. This controllable gene expression system provides the following new characteristics: the induction is rapid, taking place in the order of minutes, and the promoter is responsive at all stages of the D.discoideum life cycle.