Allan R. Sinning

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro.

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

Role of Vitamin A in the Formation of Congenital Heart Defects

Retinoic acid, the biologically active form of vitamin A, is a critical player in normal development. The concentration of retinoic acid is highly regulated by the embryo to prevent either a deficit or an excess of this molecule, conditions that have been shown to produce cardiac defects that vary depending on the severity and the timing of the insult. The vast majority of these defects are associated with the valves or the membranous septa of the heart, suggesting a problem with the formation of the cardiac mesenchyme from both within and outside the heart. While the exact role of retinoic acid in cardiac development is not known, it is believed that retinoic acid influences development by up‐ or down‐regulating cardiac specific genes. This review briefly discusses the role of cardiac mesenchyme and cardiac neural crest in septation of the heart. This is followed by a discussion of vitamin A metabolism and the cardiac defects associated with abnormal levels of retinoic acid. Finally, a mechanism is proposed concerning the ways abnormal levels of retinoic acid lead to similar cardiac defects by disrupting the production of the extracellular matrix. Anat. Rec. (New Anat.) 253:147–153, 1998.

Partial purification of HLAMP‐1 provides direct evidence for the multicomponent nature of the particulate matrix associated with cardiac mesenchyme formation

H‐LAMP‐1 is a 283 kDa protein that is involved in the transformation of endothelial cells into mesenchyme within the AV canal and proximal outflow tract of the heart. This protein is part of the particulate matrix that has been suggested to be composed of multicomponent complexes that have been termed cardiac adherons. However, to date no direct evidence has been provided that these proteins are complexed into an adheron‐like particle. This report provides the first such evidence by showing that purification of hLAMP‐1, under gentle conditions, results in the isolation of multiple bands of similar molecular weight within the fractions that contain anti‐hLAMP‐1 activity. J. Cell. Biochem. 66:112–122, 1997.

A Subset of SBA Lectin-Binding Proteins Isolated from Myocardial-Conditioned Media Transforms Cardiac Endothelium into Mesenchyme

It has been shown that the inductively active proteins for cardiac mesenchyme formation are localized to a particulate form of extracellular matrix that resembles adheron-like complexes. These complexes are extractable from the embryonic heart using EDTA and can be visualized with the lectin SBA (Glycine max). In addition, the growth medium obtained from embryonic myocardial cell cultures has also been shown to support mesenchyme formation. However, except for the identification of EDTA extract and conditioned media, all previous experiments analyzing this system have relied on negative type results (i.e. the loss of biological activity) to show a relationship between the particulate matrix and the transformation process. We report here that SBA affinity chromatography can be used to partially purify a subset of proteins from myocardial conditioned medium which elicits the transformation of endothelial cells into mesenchyme. In addition, a polyclonal antibody made against this subset of proteins is specific for the in situ particulate matrix and recognizes several proteins in conditioned medium and EDTA extracts. This antibody is also specific for matrix particulates in other areas of the embryo that undergo an epithelial/mesenchymal interaction. These results provide the most direct evidence to date that conditioned medium is equivalent to the hypothesized inductively active particulate matrix. In addition, the data provides evidence that conditioned medium can be used to identify the functional role of the components of the particulate matrix in mesenchyme formation.

Antisense approaches for elucidating ranavirus gene function in an infected fish cell line.

Viral virulence/immune evasion strategies and host anti-viral responses represent different sides of the continuing struggle between virus and host survival. To identify virus-encoding molecules whose function is to subvert or blunt host immune responses, we have adapted anti-sense approaches to knock down the expression of specific viral gene products. Our intention is to correlate knock down with loss of function and thus infer the role of a given viral gene. As a starting point in this process we have targeted several structural and catalytic genes using antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA). In proof of concept experiments we show the feasibility of this approach and describe recent work targeting five frog virus 3 genes. Our results indicate that both 46K and 32R, two immediate-early viral proteins, are essential for replication in vitro, and confirm earlier findings that the major capsid protein, the largest subunit of the viral homolog of RNA polymerase II, and the viral DNA methyltransferase are also essential for replication in cell culture.

Identification of a 283-kDa Protein Component of the Particulate Matrix Associated with Cardiac Mesenchyme Formation

The elaboration of cardiac mesenchyme is controlled by a particulate form of extracellular matrix that is produced by the myocardium. This matrix consists of at least 7-10 major proteins or protein subunits. However, at present only a few of these proteins have been identified (i.e., ES130, fibronectin, transferrin). We report here the identification of a 283-kDa protein associated with this complex. This protein was identified using a new monoclonal antibody that is highly specific for this protein. The antibody does not react with human plasma fibronectin, chicken fibronectin or chicken cytotactin. In addition, this antibody inhibits the formation of mesenchyme when used in a bioassay of mesenchyme formation.

Retinoic acid inhibition of cardiac mesenchyme formation in vitro correlates with changes in the secretion of particulate matrix from the myocardium

Retinoic acid has been associated with a variety of cardiac defects. A percentage of these defects are related to changes in the endocardial cushions. Studies in mice and older chick embryos have shown a decrease in mesenchymal cell formation attributable to retinoic acid and have suggested that retinoic acid was affecting the extracellular matrix. In this study we have tested the effect of retinoic acid on cardiac mesenchyme formation in vitro and then tested retinoic acid treated myocyte cultures for changes in the expression of hLAMP‐1, fibronectin and transferrin members of the particulate matrix that is required for mesenchyme formation. Initial experiments tested the effect of retinoic acid on mesenchymal cell formation first in atrioventricular canal and outflow tract explant cultures and then in AV endothelial monolayer cultures using myocyte conditioned media or the particulate matrix fraction from retinoic acid treated myocyte cultures. In all cases, mesenchymal cell formation was suppressed while no suppression was observed when MyoCM was included with retinoic acid. Protein analysis showed that retinoic acid had a stimulatory effect on protein synthesis. ELISA assays revealed that retinoic acid treated myocyte cultures contained significantly more hLAMP‐1 and fibronectin than either normal or DMSO controls. However, transferrin was not affected by retinoic acid treatment in these experiments. Our results suggest that retinoic acid affects the expression of the particulate matrix and that these changes may be responsible for the observed decrease in mesenchymal cell formation. Anat Rec 258:186–197, 2000.

Retinoic acid administration is associated with changes in the extracellular matrix and cardiac mesenchyme within the endocardial cushion

Retinoic acid has been associated with a number of cardiac defects, some of which seem to be related to changes in the endocardial cushions. Studies in mice and older chick embryos have suggested that these defects may be associated with a decrease in mesenchymal cell formation within the cushion. In a previous report we showed that retinoic acid lowered the number of mesenchymal cells in a culture bioassay of mesenchyme formation and that this response was due to retinoic acid modifying the production of particulate matrix from the myocardium. In this study, we have extended these observations to the embryo by implanting a retinoic acid coated bead into the embryo and examined the effect on cardiac mesenchyme formation and in the production of the particulate matrix. In all cases the addition of retinoic acid resulted in a decrease in the number of mesenchymal cells invading the endocardial cushions. In addition retinoic acid increased the production of hLAMP‐1 and fibronectin but not transferrin, confirming our earlier report. Finally, we measured the volume of the cushion and calculated the cell density of both the inferior and superior cushions. The results suggest that the superior cushion is more sensitive to retinoic acid treatment than the inferior cushion. Collectively, these results support our earlier work that suggests that the mechanism of retinoic acid cardiac abnormalities involves a disruption in the production of particulate matrix from the myocardium and a subsequent decrease in cardiac mesenchyme cells that results in a malformed cardiac cushions. Anat Rec 263:53–61, 2001.

Visualization of stereoscopic anatomic models of the paranasal sinuses and cervical vertebrae from the surgical and procedural perspective

Recent improvements in three‐dimensional (3D) virtual modeling software allows anatomists to generate high‐resolution, visually appealing, colored, anatomical 3D models from computed tomography (CT) images. In this study, high‐resolution CT images of a cadaver were used to develop clinically relevant anatomic models including facial skull, nasal cavity, septum, turbinates, paranasal sinuses, optic nerve, pituitary gland, carotid artery, cervical vertebrae, atlanto‐axial joint, cervical spinal cord, cervical nerve root, and vertebral artery that can be used to teach clinical trainees (students, residents, and fellows) approaches for trans‐sphenoidal pituitary surgery and cervical spine injection procedure. Volume, surface rendering and a new rendering technique, semi‐auto‐combined, were applied in the study. These models enable visualization, manipulation, and interaction on a computer and can be presented in a stereoscopic 3D virtual environment, which makes users feel as if they are inside the model. Anat Sci Educ 10: 598–606.

The hLAMP-1-positive particulate matrix involved in cardiac mesenchyme formation in the chick does not include BMP-2.

Early heart development involves the transformation of endocardial cells in the atrioventricular canal and outflow tract regions into mesenchymal cells, a process called endocardial mesenchymal transformation (EMT). This process is initiated by factors, termed the particulate matrix, that are secreted by the myocardium. The particulate matrix causes a subset of endocardial cells to hypertrophy, lose their cell-cell contacts, form migratory processes, transform into mesenchymal cells, and migrate into the underlying endocardial cushions. The particulate matrix can be extracted using EDTA and the EDTA extract can initiate the EMT process. Earlier reports from our laboratory have shown that the particulate matrix can be detected with the hLAMP-1 antibody in immunostaining and Western blot analysis. In addition, similar proteins have been isolated from the growth media of stage 15-16 chick embryo myocardial cultures (MyoCM). Since other investigators have identified a possible role for bone morphogenetic protein (BMP)-2 during the EMT process in the heart, we asked whether BMP-2 is a part of the chick hLAMP-1-positive particulate matrix. To answer this question, we double stained stage 15-16 chick embryo sections with hLAMP-1 and BMP-2 antibodies. We found that BMP-2 signals do not colocalize with hLAMP-1-stained particles. In addition, using immunoprecipitation-Western blot analysis, we demonstrated no association of BMP-2 with the hLAMP-1-bound fraction of the EDTA extract or MyoCM. Our results indicate that BMP-2 is not a component of the hLAMP-1-positive particulate matrix in the chick.