Monoclonal antibody 45-2D9 recognizes a cell surface glycoprotein on a human c-Ha-ras transformed cell line (45-342) and a shared epitope on human tumors

A monoclonal antibody (45-2D9) produced after immunization of BALB/c mice with the c-Ha-ras NIH 3T3 tertiary transfectant (45-342) recognized a determinant expressed by the primary, three of three secondary, and one of three tertiary transfectants, but not by NIH 3T3 cells. The determinant was present on the cell surface and was distinct from murine leukemia virus gp70 by absorption studies. Biosynthetic labeling and immunoprecipitation studies with [35S]methionine and [3H]glucosamine demonstrated that 45-2D9 recognizes a 74,000 Mr glycoprotein with minor bands of 90,000 and 180,000 Mr on SDS-PAGE. Pulse chase studies demonstrated a 68,000 Mr precursor molecule that incorporated only [35S]methionine. The distribution of the epitope recognized by 45-2D9 was assessed by immunoperoxidase staining. The antigen was not detected on 10 primary and metastatic murine tumors or 11 transformed murine cell lines. However, a variety of surgically excised human tumors demonstrated intense staining, whereas staining of normal tissues was minimal or not detectable. Thus a human oncogene-transfected cell can express a new cell surface determinant apparently unrelated to the oncogene product, which is also selectively expressed by human tumors.     

Phlorizin binding to isolated enterocytes: Membrane potential and sodium dependence

Summary Phlorizin binding is studied in isolated intestinal epithelial cells of the chick. Cells are ATP depleted to allow extensive manipulation of ionic gradients and membrane potential (). Phlorizin binding is assayed at steady state. Carrier specific phlorizin binding is defined asd-glucose (90 mM) inhibitable binding. Specific binding displays simple Michaelian kinetics as a function of phlorizin. indicating the presence of a single homogeneous binding site. Sodium concentrations and modify the apparent binding affinity but not the maximum number of binding sites. In contrast, the activation curve as a function of sodium concentrations is sigmoid and the apparent maximum number of binding sites at saturating sodium is phlorizin dependent. The rate of phlorizin association is both and sodium-concentration dependent. Dissociation is sodium-concentration dependent but not dependent. Theoretical analysis indicates binding order of substrates is random. In addition, data suggests that the phlorizin/sodium stoichiometry is 2:1. The dependence can be explained by two models: either translocation is the -dependent step and the free carrier is anionic, or sodium binding is the -dependent step.     

Water as a substrate for the development of Paracoccidiodes brasiliensis mycelial form

Two isolates of P. brasiliensis in the mycelial form were studied for their capacity to survive and grow in sterile distilled water (SDW). Inoculum for the experiments consisted of a spectrophotometrically-standardized suspension of washed and homogenized mycelial fragments; these had been obtained from 2-week old cultures grown in a synthetic medium (SM). Series of tubes with SDW and SM were incubated with the above suspension and kept stationary for 6 months at either 4 °C or room temperature (RT). Growth was measured by dry weight (DW) and turbidity (OD) determinations; additionally, CFU and ultrastructural appearance by transmission electron microscope (TEM) were assesed for one of the isolates. In general, cultures in SM at RT, grew exponentially after 2 weeks, becoming stationary for 7 weeks and then, declining abruptly. In SDW, fungal development was slow for 5 months when an increase in mass was recorded. When incubated at 4° C, both SDW and SM cultures required longer time to develop but mass also increased. Morphologically, mycelial elements in SDW at RT exhibited increased lipid vacuoles and glycogen deposits but were otherwise normal up to 6 weeks when they presented the inter-hyphae-hyphae phenomenum. In SDW P. brasiliensis appears to utilize debris from its degenerated fungal partners to continue growing.     

Virulence of Paracoccidioides brasiliensis: The influence of in vitro passage and storage

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidioidomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated from patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.Abbreviations ATCC American Type Culture Collection - CFU colony-forming units - LD Lethal dose - MMv-M modified McVeigh Morton - PMN polymorphonuclear neutrophil     

Short interfering RNA delivered by a hybrid nanoparticle targeting VEGF: Biodistribution and anti-tumor effect

BackgroundThe use of RNA interference (iRNA) therapy has proved to be an interesting target therapy for the cancer treatment; however, siRNAs are unstable and quickly eliminated from the bloodstream. To face these barriers, the use of biocompatible and efficient nanocarriers emerges as an alternative to improve the success application of iRNA to the cancer, including breast cancer.ResultsA hybrid nanocarrier composed of calcium phosphate as the inorganic phase and a block copolymer containing polyanions as organic phase, named HNPs, was developed to deliver VEGF siRNA into metastatic breast cancer in mice. The particles presented a rounded shape by TEM images with average size measured by DLS suitable and biocompatible for biomedical applications. The XPS and EDS spectra confirmed the hybrid composition of the nanoparticles. Moreover, after intravenous administration, the particles accumulated mainly in the tumor site and kidneys, which demonstrates the tumor targeting accumulation through the Enhanced Permeability and Retention Effect (EPR). A significant decrease in size of the tumors treated with the nanoparticles containing siVEGF (HNPs-siVEGF) was observed and the reduction was related to enhanced tumor accumulation of siRNA as well as in vivo VEGF silencing at gene and protein levels.ConclusionThe hybrid system prepared was successful in promoting the RNAi effect in vivo with very low toxicity.General significanceThis study shows the valuable development of a hybrid nanoparticle carrying VEGF siRNA, as well as their tumor targeting, accumulation and reduction in mice triple-negative breast cancer.     

Chromosome 10 in the tomato plant carries clusters of genes responsible for field resistance/defence to Phytophthora infestans

The main objective of the present study was to reanalyse tomato expression data that was previously submitted to the Tomato Expression Database to dissect the resistance/defence genomic and metabolic responses of tomato to Phytophthora infestans under field conditions. Overrepresented gene sets belonging to chromosome 10 were identified using the Gene Set Enrichment Analysis, and we found that these genes tend to be located towards the end of the chromosome 10. An analysis of syntenic regions between Arabidopsis thaliana chromosomes and the tomato chromosome 10 allowed us to identify conserved regions in the two genomes. In addition to allowing for the identification of tomato candidate genes participating in resistance/defence in the field, this approach allowed us to investigate the relationships of the candidate genes with chromosomal position and participation in metabolic functions, thus offering more insight into the phenomena occurring during the infection process.     

Using Multidetector Computed Tomography in a Swine Model to Assess the Effects of Sublingual Nitroglycerin and Intravenous Adenosine on Epicardial Coronary Arteries

This study examines the effects of intravenous infusion of adenosine and sublingual nitroglycerin on coronary angiograms obtained by current-generation multidetector computed tomography. We assessed coronary vasodilation at baseline and after intravenous adenosine (140 µg/kg/min) or sublingual nitroglycerin spray (800 µg) in 7 female swine (weight, 40.9 ± 1.4 kg) by using electrocardiogram-gated coronary angiography with a 64-detector scanner (rotation time, 400 ms; 120kV; 400 mA) and intravenous contrast (300 mg/mL iohexol, 4.5 mL/s, 2 mL/kg). Cross-sectional areas of segments in the left anterior descending, circumflex, and right coronary arteries were evaluated in oblique orthogonal views. Images were acquired at an average heart rate of 73 ± 11 beats per minute. Changes in aortic pressure were not significant with nitroglycerin but decreased (approximately 10%) with adenosine. Of the 76 segments analyzed (baseline range, 2 to 39 mm²), 1 distal segment could not be assessed after adenosine. Segment cross-sectional area increased by 11.3% with nitroglycerin but decreased by 9.6% during adenosine infusion. The results of the present study are consistent with the practice of using sublingual nitroglycerin to enhance visualization of epicardial vessels and suggest that intravenous adenosine may hinder coronary artery visualization. This study is the first repeated-measures electrocardiogram-gated CT evaluation to use the same imaging technology to assess changes in coronary cross-sectional area before and after treatment with a vasodilator. The nitroglycerin-associated changes in our swine model were modest in comparison with previously reported human studies.Abbreviations: LAD, left anterior descending artery; MDCT, multidetector computed tomographyRecent advances in multidetector computed tomography (MDCT) for coronary angiography, their increased availability, and the pending release of a new generation of CT scanners contribute to this methodology''s potential for revolutionizing the early diagnosis and functional assessment of coronary artery disease.^15,16,25 However, the benefits of methodologic advances do not diminish the need to validate and assess the safety, efficacy, and costs of technology.¹⁵ In this regard, the present study uses an animal model with coronary anatomy analogous to that of humans^11,43 to assess the effects of sublingual nitroglycerin and intravenous adenosine on coronary epicardial vessel visualization by using baseline imaging as a control. The radiation exposure associated with CT procedures precludes human studies that involve repeated measures on the same subjects.Sublingual nitroglycerin is hypothesized to enhance the visualization of epicardial coronary vessels due to its vasodilatory properties.^8-10 Nitroglycerin acts as a nitric oxide generator to induce relaxation of vascular smooth muscle independent of endothelial function.^1,18,20 Current evidence supporting improved coronary visualization with sublingual nitroglycerin is derived from clinical cross-sectional studies that compare results in different groups of patients with heterogeneous coronary lesions.^5,6,16,27,44 A recent survey reported that more than 80% of facilities in the United States routinely use nitroglycerin in cardiac MDCT angiography.²³In contrast to the action of nitroglycerin on epicardial vascular smooth muscle, intravenous adenosine frequently is used as a pharmacologic stress to affect vasodilatation within the coronary microcirculation.²⁰ Although current-generation CT scanners have limited capability to assess myocardial tissue perfusion, research efforts from advanced imaging centers suggest that future-generation CT scanners soon will permit myocardial perfusion imaging.^13,14,29 No studies to date have assessed epicardial vessel changes by MDCT with intravenous doses of adenosine appropriate for myocardial perfusion imaging.The objective of this study was to provide an animal model for MDCT studies that permits repeated measures of epicardial vessels without the limitations of cross-sectional designs or the complications of heterogeneity of vascular lesions within clinical populations. This study used routes of administration of nitroglycerin and adenosine that are consistent with clinical practice.     

Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

Background

Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta.

Methodology/Principal Findings

We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family.

Conclusions/Significance

We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.