Biomedical subjects
Publications and source records attributed to P J Gruber.
We present a patient with a pulmonary artery (PA) aneurysm who has none of the documented causes of PA aneurysm but who is pregnant. We believe that this patient represents a case of primary pregnancy-associated PA aneurysm.
BACKGROUND: The development of improved strategies for efficient and reproducible in vivo gene transfer into the murine heart will ultimately allow the intersection of somatic and germline gene transfer strategies to study complex features of cardiac biology and diseases. METHODS AND RESULTS: For embryonic gene transfer, an adenovirus vector expressing beta-galactosidase was injected in utero into the ventricular cavity of living embryos via microsurgical approaches. The injected embryos were developed to term, and efficient expression of the transgene was detected in all cell types in the heart. For postnatal cardiac gene transfer, adenovirus was injected into the cardiac ventricle of neonatal mice, resulting in efficient expression of the transgene in the outer layer of the myocardium as well as cardiomyocytes in the middle and inner layers of the cardiac wall. Mice examined after 3 weeks displayed a pattern of expression that completely mimicked the pattern seen after 3 days, and gene expression was also found after 6 months. The infected myocytes can be identified by coinfection of an adenovirus expressing green fluorescent protein without affecting their normal physiological function. CONCLUSIONS: We have developed a new strategy to achieve efficient and long-term foreign gene expression in both embryonic and postnatal mouse myocardium via direct intracardiac injection of recombinant adenovirus. The strategy should allow the functional assessment of the expression of dominantly acting exogenous genes, overexpression of wild-type genes, and Cre recombinase-mediated gene ablations at the single-cell level in the context of the intact adult mouse myocardium.
We describe a technique that uses the internal mammary vessels to enhance long-segment jejunal graft blood supply in addition to an intact distal mesenteric vascular arcade. We believe that this technique, called vascular augmentation, improves jejunal graft perfusion and decreases ischemic complications.
Vertebrate cardiogenesis is a complex process involving multiple, distinct tissue types which interact to form a four-chambered heart. Molecules have been identified whose expression patterns co-segregate with the maturation of the atrial and ventricular muscle cell lineages. It is not currently known what role intrinsic events versus external influences play in cardiac chamber morphogenesis. We developed novel, fluorescent-based, myocardial, cellular transplantation systems in order to study these questions in murine embryos and report the irreversible nature of chamber specification with respect to the downregulation of atrial myosin light chain 2 (MLC-2a) and alpha myosin heavy chain (alpha-MHC). Grafting ventricular cells into the atrial chamber does not result in upregulation of MLC-2a expression in ventricular cells. Additionally, wild-type ventricular muscle cells grafted into the wild-type background appropriately downregulate MLC-2a and alpha-MHC. Finally, grafting of RXRalpha gene-deficient ventricular muscle cells into the ventricular chambers of wild-type embryos does not rescue the persistent expression of MLC-2a, providing further evidence that ventricular chamber maturation is an early event. These studies provide a new approach for the mechanistic dissection of critical signaling events during cardiac chamber growth, maturation and morphogenesis in the mouse, and should find utility with other approaches of cellular transplantation in murine embryos. These experiments document the irreversible nature of the downregulation of atrial markers after the onset of cardiogenesis during ventricular chamber morphogenesis and temporally define the response of cardiac muscle cells to signals regulating chamber specification.
We have isolated the mouse homologue of human spliceosome-associated protein SAP49, mSAP49. mSAP49 contains two RNA recognition motifs (RRM) in the N terminus of the predicted amino acid sequence, and a highly basic C terminus rich in glycine/proline. mSAP49 displayed a plastic of expression in cardiac development. In the adult mouse, mSAP49 is widely distributed, although it was found at relatively lower levels in the heart. In situ hybridization analysis of mSAP49 mRNA distribution in staged mouse embryos showed that mSAP49 onset occurs later in the heart than in other embryonic tissues. While mSAP49 expression was found at day 10.0 postconception (pc) in the optic eminence, optic vesicle, hindbrain, and somites, it was not in cardiac structures. mSAP49 was detected in the ventricles at day 11.5, and at day 13.5 it was also detected in the atria. Northern analysis showed that mSAP49 mRNA displayed a peak of expression in the heart at days 14.0-15.0 pc, and its abundance decayed in the adult. This dynamic pattern of cardiac expression suggests that mSAP49 may be contributing to a change in the ratio of spliceosome components during cardiac growth and development, which may have consequences for tissue-specific splicing, RNA stabilization, or translation.
Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.
STUDY OBJECTIVE: To determine the frequency of fever and leukocytosis in patients presenting to the emergency department with acute cholecystitis (AC). METHODS: We carried out a retrospective review of charts from 1990 to 1993 at a university-affiliated hospital. Our subjects were ED patients with hepato-iminodiacetic acid (HIDA) scans interpreted as showing AC and who had undergone cholecystectomy during hospitalization. Final diagnosis was determined on the basis of the pathology report. Fever was defined as an oral temperature of 100 degrees F (37.7 degrees C) or greater or a rectal temperature of 100.4 degrees F (38.0 degrees C) or greater. Leukocytosis was defined as a WBC count of 11,000/mm3 or greater. RESULTS: Of the 198 cases studied, the pathologic diagnosis of nongangrenous AC was made in 103 (52%), gangrenous AC was diagnosed in 51 (26%), and chronic cholecystitis was diagnosed in 44 (22%). In patients with nongangrenous AC, 71% were afebrile, 32% lacked leukocytosis, and 28% lacked fever and leukocytosis. In patients with gangrenous AC, 59% were afebrile, 27% lacked leukocytosis, and 16% lacked fever and leukocytosis. CONCLUSION: We found that patients with AC diagnosed in the ED frequently lacked fever or leukocytosis. The clinician should not rely on the presence of these signs in making the diagnosis of acute cholecystitis.
The vitamin A metabolite retinoic acid (RA) is utilized as a signalling molecule in wide variety of developmental processes, defined by defects which occur after nutritional vitamin A deficiency or after exposure to excess vitamin A. We have initiated a genetic analysis of RA function through the establishment of lines of mice which carry germline mutations in the genes which encode retinoid receptors. Defects which result from developmental RA deficiency or excess have been recovered in embryos which are deficient in various combinations of retinoid receptors. In this chapter, our current understanding of the role of RA and retinoid receptors in cardiovascular and limb development are described, as for these our level of understanding is most advanced.
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Overproduction of Apo CIII causes elevated plasma triglyceride levels in transgenic animals and is associated with hypertriglyceridemia in humans. The regulation of apo CIII production is likely to play an important role in controlling plasma triglyceride levels. As an initial step in determining the role of transcriptional regulation in the production of apo CIII and in triglyceride metabolism, we have begun to characterize the activity of specific transcriptional regulatory elements in the CIII promoter. In the current study, we have identified and characterized an NF-kappa B regulatory element located 150 nucleotides upstream from the transcriptional start site of the apo CIII gene. Purified NF-kappa B, as well as an NF-kappa B protein in HepG2 cell nuclear extracts, bound specifically to this sequence element. The hepatic protein was induced by phorbol ester (PMA), and reacted with antibodies to the p50 and p65 subunits of NF-kappa B. The NF-kappa B element conferred PMA and IL1-beta inducible transcriptional activity to a heterologous promoter/reporter construct when transfected into HepG2 cells. Analysis of the full length CIII promoter demonstrated that the inducible activity of the NF-kappa B element was suppressed by sequences in the apo CIII enhancer element located approximately 500 nucleotides upstream of the NF-kappa B binding site. A deletion removing the enhancer restored the PMA inducible activity of the NF-kappa B binding site. These results indicate that apo CIII gene expression is regulated by NF-kappa B, and suggest that apo CIII production may be modulated by cellular signals, like inflammatory cytokines, that activate NF-kB.
Although the presence of a matrix in plant nuclei has been reported, major questions remain about its structural and biochemical features. We have used an intermediate filament antibody of broad specificity to explore whether Daucus carota (carrot) nuclei and nuclear matrices contain intermediate filament/lamin antigens and, if so, where specifically they are localized. SDS-PAGE and Western blotting revealed two bands, at 60 and 65 kDa, that were highly immunoreactive with the intermediate filament antibody (IFA) of Pruss et al. (1981, Cell 27, 419-428). This pattern was observed consistently, not only with carrot cell-free nuclei and nuclear matrices, but also with nuclear preparations from Vicia faba (broad bean) and Pisum sativum (pea). Immunofluorescence studies with whole carrot nuclei localized the IFA antigens to the nucleoplasm and disclosed no accentuated peripheral labeling. Agarose-embedded nuclear matrices showed not only fluorescence throughout the nucleoplasm but also heavy labeling surrounding the nucleoli and suggestions of peripheral labeling. At the ultrastructural level, immunogold results from pre- and postembedment treatments supported the conclusion that IFA antigens occur throughout the nucleoplasm, with possibly a slight concentration at the periphery. These combined results provide substantial evidence that plant nuclei and their matrices possess at least two major intermediate filament antigens with molecular weights characteristic of animal lamins. Whether or not these antigens represent plant lamins, their nonperipheral localization hints at significant differences among the eukaryotic kingdoms in nuclear organization.
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The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 microm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 microm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.
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