A semi-empirical model for scatter field reduction in digital mammography.

X-ray mammography is the gold standard technique in breast cancer screening programmes. One of the main challenges that mammography is still facing is scattered radiation, which degrades the quality of the image and complicates the diagnosis process. Anti-scatter grids, the main standard physical scattering reduction technique, have some unresolved challenges as they increase the dose delivered to the patient, do not remove all the scattered radiation and increase the cost of the equipment. Alternative scattering reduction methods based on post-processing algorithms, have lately been under investigation. This study is concerned with the use of image post-processing to reduce the scatter contribution in the image, by convolving the primary plus scatter image with kernels obtained from simplified Monte Carlo (MC) simulations. The proposed semi-empirical approach uses up to five thickness-dependant symmetric kernels to accurately estimate the scatter contribution of different areas of the image. Single breast thickness-dependant kernels can over-estimate the scatter signal up to 60%, while kernels adapting to local variations have to be modified for each specific case adding high computational costs. The proposed method reduces the uncertainty to a 4%-10% range for a 35-70 mm breast thickness range, making it a very efficient, case-independent scatter modelling technique. To test the robustness of the method, the scattered corrected image has been successfully compared against full MC simulations for a range of breast thicknesses. In addition, clinical images of the 010A CIRS phantom were acquired with a mammography system with and without the presence of the anti-scatter grid. The grid-less images were post-processed and their quality was compared against the grid images, by evaluating the contrast-to-noise ratio and variance ratio using several test objects, which simulate calcifications and tumour masses. The results obtained show that the method reduces the scatter to similar levels than the anti-scatter grids.

Advances, nuances, and potential pitfalls when exploiting the therapeutic potential of RNA interference.

The discovery of RNA interference (RNAi) holds the potential to alter the paradigm of medical therapeutics. With the ability to selectively silence the function of a gene, RNAi not only provides an indispensable research tool for determining the function of a gene, but also offers potential for the development of novel therapeutics that will inhibit specific genes involved in disease. New concepts in therapeutics have been uncovered through the study of RNAi. Nuances have emerged. For instance, global RNAi pathways can be affected by somatic mutations in cancer and cellular stress, such as hypoxia. Also, viral gene therapy can have unexpected effects on endogenous short noncoding RNA pathways. Therefore, it is important to understand where RNAi therapeutics enter the processing pathways. We highlight the evolving use of RNAi as a new class of therapeutics, such as for amyloidosis, and address some of the anticipated challenges associated with its clinical application.

Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos.

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

The prothrombinase activity of FGL2 contributes to the pathogenesis of experimental arthritis.

OBJECTIVE: Fibrin deposition is integral to the pathogenesis of collagen-induced arthritis (CIA), an experimental model of rheumatoid arthritis (RA). Membrane-associated fibrinogen-like protein 2 (mFGL2), a novel inducible prothrombinase, generates fibrin by an alternate pathway and has been reported to be involved in the pathogenesis of a number of immune-mediated diseases. We hypothesized that expression of mFGL2 in inflamed synovium contributes to the fibrin deposition and subsequent inflammation in arthritis. METHODS: DBA/1 mice were immunized with 100 µg bovine collagen type II (CII) emulsified in complete Freund's adjuvant (CFA) followed by lipopolysaccharide (LPS) injection. Expression of mFGL2 prothrombinase in association with fibrin deposition was examined in mice with CIA and CD200-treated mice following induction of CIA. To directly assess the contribution of mFGL2, fgl2(-/-) mice were injected with antibody to CII (anti-CII). RESULTS: Levels of fgl2 mRNA transcripts and mFGL2 protein were markedly up-regulated in joints of mice that developed CIA. Fibrin deposition was prominent within the synovial lining and articular joint space associated with expression of mFGL2. Inhibition of CIA by the immunosuppressant CD200 was associated with decreased expression of fgl2 mRNA and mFGL2 protein and absence of fibrin deposition. Following injection of anti-CII, all fgl2(+/+) mice developed severe arthritis with clinical and histological manifestations characteristic of RA, whereas fgl2(-/-) mice failed to develop any clinical manifestation or histological evidence of arthritis. CONCLUSIONS: This study demonstrates that the prothrombinase activity of mFGL2 contributes to the pathogenesis of experimental arthritis. These studies may have therapeutic implications for patients with RA.

Activation of ETB receptors regulates the abundance of ET-1 mRNA in vascular endothelial cells.

BACKGROUND AND PURPOSE: The factors that influence the cellular levels of endothelin-1 (ET-1) include transcription, mRNA localization, stability and translation, post-translational maturation of preproET-1 and degradation of ET-1. We investigated the regulation of ET-1 mRNA abundance by extracellular ET-1 in porcine aortic endothelial cells (PAECs). EXPERIMENTAL APPROACH: Passsage one cultures of PAECs were incubated in starving medium in the presence or absence of ET-1 and antagonists or pharmacological inhibitors. PreproET-1 mRNA, endothelin-1 promoter activity, Erk and p38 MAPK activation were determined. KEY RESULTS: Exogenous ET-1 reduced cellular ET-1 mRNA content: a reduction of 10 000-fold was observed after 4 h. ET-1 simultaneously reduced the stability of ET-1 mRNA and increased the loading of RNA Polymerase II at the endothelin-1 promoter. In the absence of exogenous ET-1, the ETB-selective antagonist, BQ788, increased ET-1 mRNA. An ETA-selective antagonist had no effect. ET-1 mRNA returned to control levels within 24 h. Whereas activation of p38 MAPK induced by ET-1 peaked at 30 min and returned to control levels within 90 min, Erk1/2 remained active after 4 h of stimulation. Inhibition of p38 MAPK prevented the ET-1-induced decrease in ET-1 mRNA. In contrast, Erk1/2 inhibition increased ET-1 mRNA. Similarly, inhibition of receptor internalization increased ET-1 mRNA in the presence or absence of exogenous ET-1. CONCLUSIONS AND IMPLICATIONS: These results suggest that extracellular ET-1 regulates the abundance of ET-1 mRNA in PAECs, in an ETB receptor-dependent manner, by modulating both mRNA stability and transcription via mechanisms involving receptor endocytosis and both ERK and p38 MAPK pathways.

Endothelial nitric oxide synthase: insight into cell-specific gene regulation in the vascular endothelium.

The vascular endothelium plays a crucial role in regulating normal blood vessel physiology. The gene products responsible are commonly expressed exclusively, or preferentially, in this cell type. However, despite the importance of regulated gene expression in the vascular endothelium, relatively little is known about the mechanisms that restrict endothelial-specific gene expression to this cell type. While significant progress has been made towards understanding the regulation of endothelial genes through cis/trans paradigms, it has become apparent that additional mechanisms must also be operative. For example, chromatin-based mechanisms, including cell-specific DNA methylation patterns and post-translational histone modifications, have recently been demonstrated to play important roles in the cell-specific expression of endothelial nitric oxide synthase (eNOS). This review investigates the involvement of epigenetic regulatory mechanisms in vascular endothelial cell-specific gene expression using eNOS as a prototypical model, and will address the possible contributions of these pathways to diseases of the vasculature.

Precession and motional slowing of spin evolution in a high mobility two-dimensional electron gas.

Optical spin-dynamic measurements in a high-mobility n-doped GaAs/AlGaAs quantum well show oscillatory evolution at 1.8 K consistent with a quasi-collision-free D'yakonov-Perel'-Kachorovskii regime. Above 5 K evolution becomes exponential as expected for collision-dominated spin dynamics. Momentum scattering times extracted from Hall mobility and Monte Carlo simulation of spin polarization agree at 1.8 K but diverge at higher temperatures, indicating the importance of electron-electron scattering and an intrinsic upper limit for the spin-relaxation rate.

Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.

Genomic characterization, localization, and functional expression of FGL2, the human gene encoding fibroleukin: a novel human procoagulant.

For diseases in which thrombosis plays a pivotal role, such as virus-induced fulminant hepatitis, fetal loss syndrome, and xenograft rejection, the major procoagulant has remained elusive. Here we describe the isolation and functional expression of a distinct human prothrombinase, termed FGL2. The murine fgl2 gene product has been implicated in the pathophysiology of murine fulminant hepatitis. The predicted ORF corresponds to a 439-amino-acid type II integral membrane protein that contains a carboxy-terminal Fibrinogen-related domain. Functional analysis showed that FGL2-encoded protein is indeed a prothrombinase. This enzyme is a serine protease and directly cleaves prothrombin to thrombin. The FGL2 gene is a single-copy gene in the haploid human genome and has two exons separated by a 2195-bp intron expressing two mRNA transcripts of 1.5 and 5.0 kb. The 5'-flanking region contains putative cis-elements including a TATA box, an AP1 site, CEBP sites, Sp1 site, and Ets binding domains. By both radiation hybrid analyses and fluorescence in situ hybridization, human FGL2 was localized to 7q11.23.

In vivo expression profile of an endothelial nitric oxide synthase promoter-reporter transgene.

Endothelium-derived nitric oxide (NO) is primarily attributable to constitutive expression of the endothelial nitric oxide synthase (eNOS) gene. Although a more comprehensive understanding of transcriptional regulation of eNOS is emerging with respect to in vitro regulatory pathways, their relevance in vivo warrants assessment. In this regard, promoter-reporter insertional transgenic murine lines were created containing 5,200 bp of the native murine eNOS promoter directing transcription of nuclear-localized beta-galactosidase. Examination of beta-galactosidase expression in heart, lung, kidney, liver, spleen, and brain of adult mice demonstrated robust signal in large and medium-sized blood vessels. Small arterioles, capillaries, and venules of the microvasculature were notably negative, with the exception of the vasa recta of the medullary circulation of the kidney, which was strongly positive. Only in the brain was the reporter expressed in non-endothelial cell types, such as the CA1 region of the hippocampus. Epithelial cells of the bronchi, bronchioles, and alveoli were scored as negative, as was renal tubular epithelium. Cardiac myocytes, skeletal muscle, and smooth muscle of both vascular and nonvascular sources failed to demonstrate beta-galactosidase staining. Expression was uniform across multiple founders and was not significantly affected by genomic integration site. These transgenic eNOS promoter-reporter lines will be a valuable resource for ongoing studies addressing the regulated expression of eNOS in vivo in both health and disease.

Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in viral hepatitis.

In the present studies, we report the cloning and structural characterization of the HFGL2 gene and its functional role in human fulminant hepatitis. The HFGL2 gene is approximately 7 kb in length with 2 exons. The putative promoter contains cis element consensus sequences that strongly suggest the inducibility of its expression. From the nucleotide sequence of the human gene, a 439-amino acid long protein is predicted. The overall identity between the murine fgl2 and hfgl2 coded proteins is over 70%. About 225 amino acids at the carboxyl end of these molecules are almost 90% identical, and correspond to a well-conserved fibrinogen-related domain. Both HFGL2 and FGL2 encode a type II transmembrane protein with a predicted catalytic domain toward the amino terminus of the protein. Transient transfection of Chinese hamster ovary (CHO) cells with a full-length cDNA of HFGL2 coding region resulted in high levels of prothrombinase activity. Livers from 8 patients transplanted for fulminant viral hepatitis were examined for extent of necrosis, inflammation, fibrin deposition, and HFGL2 induction. In situ hybridization showed positive staining of macrophages in areas of active hepatocellular necrosis. Fibrin stained positively in these areas and was confirmed by electron microscopy. These studies define a unique prothrombinase gene (HFGL2) and implicate its importance in the pathogenesis of fulminant viral hepatitis.

RNA diversity has profound effects on the translation of neuronal nitric oxide synthase.

A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5'-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5'-untranslated regions, which have profound effects on translation both in vitro and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.

Germline polymorphisms in cardiovascular medicine. Is that the whole story?

A wealth of recent data points to the importance of germline mutations and polymorphisms in the pathophysiology of heart and blood vessel disease. In addition, new data suggest that somatic mutations and perturbation in epigenetic pathways may be involved in diseases of the cardiovascular system. This focused discussion uses examples in hypertension and atherosclerosis to highlight these emerging concepts.

Neuronal NOS: gene structure, mRNA diversity, and functional relevance.

Neuronal nitric oxide synthase (nNOS) has been implicated in a wide variety of physiological and pathological processes. These include neurotransmission, neurotoxicity, skeletal muscle contraction, sexual function, body fluid homeostasis and atherosclerosis, among others. Consistent with the involvement of nNOS in such varied aspects of cellular biology, nNOS mRNA and protein are expressed in numerous tissues. Both its gene structure and expressional regulation are exceedingly complex. Characterization of the genomic organization of the human nNOS has revealed that the transcription unit of 29 exons spans a region greater than 240 kb at 12q24.2. The gene produces multiple mRNA transcripts via a variety of intriguing mechanisms: alternate promoter usage, alternative splicing, cassette insertions/deletions, and varied sites for 3'-UTR cleavage and polyadenylation. Allelic diversity in mRNA structure also exists. Some, but not all, of these various transcripts affect the encoded amino acid sequence and translate into nNOS protein isoforms with altered structural and functional properties. Interestingly, much of this diversity is restricted to the untranslated regions of the mRNA transcript and may affect its translation or stability. Taken together, these properties present nNOS as one of the most complex human genes described to date. Given the importance of nNOS in human health and disease, understanding this intricate genetic regulation has been a major focus in nNOS research. This review addresses the structure of the nNOS gene, its mRNA diversity, and overall genetic regulation with an emphasis on their biological implications.

The nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene.

Using a set of parental and recombinant murine hepatitis virus strains, we demonstrate that the nucleocapsid protein induces transcription of the novel fgl2 prothrombinase gene and elevated procoagulant activity in those strains that produce fulminant hepatitis. Chinese hamster ovary cells cotransfected with a construct expressing nucleocapsid protein from susceptible strains and with a luciferase reporter construct containing the fgl2 promoter showed a 6-fold increase in luciferase activity compared with nontransfected cells or cells cotransfected with a construct expressing nucleocapsid protein from resistant strains. Two deletions found at coding sites 111-123 and 1143-1145 of structural domains I and III, respectively, of the nucleocapsid gene may account for the differences between pathogenic and nonpathogenic strains. Preliminary mapping of the fgl2 promoter has defined a region from -372 to -306 upstream from the ATG translation initiation site to be responsive to nucleocapsid protein. Hence, mapping of genetic determinants in parental and recombinant strains demonstrates that the nucleocapsid protein of strains that induce fulminant hepatitis is responsible for transcription of the fgl2 prothrombinase gene. These studies provide new insights into the role of the nucleocapsid gene in the pathogenesis of viral hepatitis.

Characterization of the human endothelial nitric-oxide synthase promoter.

Understanding transcription initiation of the endothelial nitric-oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present study therefore focused upon a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of constitutive expression of the human eNOS gene in vascular endothelium. Two tightly clustered cis-regulatory regions were identified in the proximal enhancer of the TATA-less eNOS promoter using deletion analysis and linker-scanning mutagenesis: positive regulatory domains I (-104/-95 relative to transcription initiation) and II (-144/-115). Analysis of trans-factor binding and functional expression studies revealed a surprising degree of cooperativity and complexity. The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional domain studies in Drosophila Schneider cells and endothelial cells revealed examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ. Therefore, multiprotein complexes are formed on the activator recognition sites within this 50-base pair region of the human eNOS promoter in vascular endothelium.

hBRAG, a novel B cell lineage cDNA encoding a type II transmembrane glycoprotein potentially involved in the regulation of recombination activating gene 1 (RAG1).

The different display reverse transcription-PCR (DD RT-PCR) technique was used to identify novel cDNA detecting mRNA transcripts co-expressed with human recombination activating gene-1 (RAG1). A 5.0-kb transcript detected by the differential display amplicon 3G1 was found to correlate strongly with RAG1 mRNA expression in various human cell lines. Subsequent screenings of a pre-B cDNA library with 3G1 led to the identification of a complete cDNA we have termed hBRAG (human B-cell RAG-Associated Gene). The hBRAG cDNA encodes a 503-amino acid (aa) protein with no known homology to any nucleotide or protein sequence. The predicted molecular mass of 55 kDa was confirmed by in vitro translation. Based on sequence analysis, the predicted open reading frame encodes for a type II transmembrane spanning glycoprotein with the N-terminal 81 -aa in the cytoplasm, a 17-aa transmembrane domain, and a C-terminal 405-aa extracellular domain with four potential N-glycosylation sites. Northern blot analysis indicated a close association of the 5.0-kb hBRAG mRNA transcript with RAG1 in numerous human pro-B, pre-B and mature B cell lines assessed, but not in human T cell lines. In human tissues, hBRAG is expressed at highest levels in B cell-enriched tissues, but is not expressed in fetal or adult thymus. Southern blotting analysis revealed that this gene is conserved across eukaryotes, is expressed as a single copy in the human genome, and is likely not a multigene family member. The hBRAG gene was localized to the long arm of chromosome 10 (10q26). Transfection of the full-length hBRAG cDNA increased levels of human RAG1 transcripts in the B cell line OCI LY8-C3P, but not in the non-lymphoid line K562, suggesting a B cell-specific role for the hBRAG product in regulating RAG expression.

Resistance to murine hepatitis virus strain 3 is dependent on production of nitric oxide.

The strain-specific spectrum of liver disease following murine hepatitis virus type 3 (MHV-3) infection is dependent on inflammatory mediators released by macrophages. Production of nitric oxide (NO) by macrophages has been implicated in resistance to a number of viruses, including ectromelia virus, vaccinia virus, and herpes simplex virus type 1. This study was undertaken to define the role of NO in MHV-3 infection. Gamma interferon-induced production of NO inhibited growth of MHV-3 in a murine macrophage cell line (RAW 264.7). Viral inhibitory activity was reproduced by the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP), whereas N-acetyl-DL-pencillamine (NAP), an inactive analog of SNAP, had no effect. Electron microscopy studies confirmed the inhibitory effects of NO on viral replication. Peritoneal macrophages isolated from A/J mice known to be resistant to MHV-3 produced a fivefold-higher level of NO and higher levels of mRNA transcripts of inducible NO synthase in response to gamma interferon than macrophages from susceptible BALB/cJ mice. SNAP inhibited growth of MHV-3 in macrophages from both strains of mice to similar degrees. In vivo inhibition of NO by N-monomethyl-L-arginine resulted in loss of resistance to MHV-3 in A/J mice. These results collectively demonstrate a defect in the production of NO in macrophages from susceptible BALB/cJ mice and define the importance of endogenous NO in resistance to MHV-3 infection in resistant A/J mice.

Verotoxin and ricin have novel effects on preproendothelin-1 expression but fail to modify nitric oxide synthase (ecNOS) expression and NO production in vascular endothelium.

Interaction of bipartite Escherichia coli O157-derived verotoxins (VTs) 1 and 2 (Shiga toxin 1 and 2) with vascular endothelium is believed to play a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome and of E. coli O157-associated hemorrhagic colitis. We defined the effects of VTs on the expression of potent endothelial cell-derived regulators of vascular wall function, namely endothelin-1 (ET-1) and nitric oxide (NO). In quiescent bovine aortic endothelial cells, both VT1 and VT2, but not receptor-binding VT B-subunit which lacks N-glycosidase activity, induced concentration-dependent (0.1-10 nM) increases in steady state preproET-1 mRNA transcript levels, an effect that was maximal at 12-24 h. Metabolic-labeling experiments indicated that VTs increased preproET-1 mRNA transcript levels at concentrations that had trivial effects on nascent DNA, RNA, and protein synthesis. In contrast to preproET-1, endothelin converting enzyme-1 and endothelial constitutive NO synthase mRNA transcript levels remained unchanged. Consistent with these findings, VTs failed to modulate immunoreactive endothelial constitutive NO synthase expression and basal and calcium-dependent L-[14C]arginine to L-[14C]citrulline conversion or the NO chemiluminescence signal. The plant-derived toxin ricin, which shows a similar molecular mechanism of enzymatic ribosomal modification to VTs, caused comparable effects on these endothelial vasomediators and metabolite incorporation, at 3 log orders lower concentrations. Nuclear transcription and actinomycin D chase experiments indicated that VTs stabilize labile preproET-1 mRNA transcripts in endothelial cells. Therefore, VTs potently increase select mRNA transcript levels in endothelial cells at concentrations of toxins that have minimal effects on protein synthesis. Perturbed expression of endothelial-derived vasomediators may play a pathophysiologic role in the microvascular dysfunction that is the hallmark of hemolytic uremic syndrome and hemorrhagic colitis.