Biomedical subjects

S A Fisher

Publications and source records attributed to S A Fisher.

At least 19 recordsLinked to original sources

PubMed2001

Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation.

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.

Amino Acid Sequence↗

PubMed2001

Courtship behavior of captive American kestrels (Falco sparverius) exposed to polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) adversely affect reproduction in birds. Captive adult male and female American kestrels (Falco sparverius) were studied to investigate the potential behavioral and hormonal alterations during the courtship period resulting from clinical exposure to PCBs. American kestrels ingested 7 mg/kg/body weight/bird/day of a 1:1:1 mixture of Aroclors 1248, 1254, and 1260 through their diet of day-old cockerels. The dietary dosage of Aroclors resulted in environmentally relevant total PCB residues in the eggs, averaging 34.1 microg/g wet weight (geometric mean). There was no difference between treatment and control birds in the circulating levels of total androgens (p = 0.44) or in 17 beta-estradiol (p = 0.29), one week following pairing. Male kestrels exposed to dietary PCBs exhibited significantly more sexual behaviors (p = 0.034) and flight behaviors (p = 0.026) than the control males. Sexual behaviors of male kestrels included; nest-box inspections, solicitation of copulation, the offer of food to the female, and giving the female food. The flight behaviors of the male included; flying from one perch to another and aerial display. In addition, the frequency of male sexual behaviors were correlated (r = 0.605, p = 0.001) with total PCB residues in the eggs of their mates. A concurrent study found that these same PCB-exposed kestrels experienced a delay in clutch initiation as well as a greater number of completely infertile clutches.

Animals↗

PubMed2000

Apoptosis during cardiovascular development.

Morphogenesis and developmental remodeling of cardiovascular tissues involve coordinated regulation of cell proliferation and apoptosis. In the heart, clear evidence points toward focal apoptosis as a contributor to development of the embryonic outflow tract, cardiac valves, conducting system, and the developing coronary vasculature. Apoptosis in the heart is likely regulated by survival and death signals that are also present in many other tissues. Cell type-specific regulation may be superimposed on general cell death/survival machinery through tissue-specific transcriptional pathways. In the vasculature, apoptosis almost certainly contributes to developmental vessel regression, and it is of proven importance in remodeling of arterial structure in response to local changes in hemodynamics. Physical forces, growth factors, and extracellular matrix drive vascular cell survival pathways, and considerable evidence points to local nitric oxide production as an important but complex regulator of vascular cell death. In both the heart and vasculature, progress has been impeded by inadequate information concerning the incidence of apoptosis, its relative importance compared with the diverse cell behaviors that remodel developing tissues, and by our primitive knowledge concerning regulation of cell death in these tissues. However, tools are now available to better understand apoptosis in normal and abnormal development of cardiovascular structures, and a framework has been established that should lead to considerable progress in the coming years.

Animals↗

PubMed2000

Multipoint linkage analysis of a candidate gene locus in rheumatoid arthritis demonstrates significant evidence of linkage and association with the corticotropin-releasing hormone genomic region.

OBJECTIVE: Rheumatoid arthritis (RA) is the most common disabling autoimmune disease, affecting approximately 1% of the population. The disease etiology is unknown, but it involves inflammation and immune dysregulation and is influenced by genetic variation at both HLA and other, as-yet-unidentified genetic loci. Corticotropin-releasing hormone (CRH; or corticotropin-releasing factor), a primary regulator of the hypothalamic-pituitary-adrenal axis and a key element in the response to stress and inflammation, is a strong candidate gene for RA. We examined the role of DNA variation across the region containing this gene in multicase families with RA. METHODS: We genotyped fluorescently labeled simple tandem repeat genetic markers from chromosome 8q13 in 295 families with multiple cases of RA. Singlepoint and multipoint nonparametric linkage analysis and association analysis using transmission disequilibrium testing (TDT) were also used. RESULTS: Single-point linkage analysis using a microsatellite within 30 kb of the CRH locus (CRH.PCR at position 8q13) showed a significant excess of allele sharing in 295 United Kingdom RA families with at least 2 affected members (MapMaker/Sibs logarithm of odds [LOD] 1.4; P = 5.5x10(-3); mean identity by descent [ibd] sharing 55.9%). To provide a more detailed linkage map, a multipoint analysis was conducted with an additional 7 dinucleotide microsatellite markers (average heterozygosity 0.75) flanking the CRH locus. Significant linkage was detected over a 22-cM region between D8S285 and D8S530, with the maximum singlepoint LOD score of 1.77 at D8S1723 (MapMaker/Sibs P = 2.2x10(-3); mean ibd sharing 59.3%). Multipoint analysis showed strongest evidence for linkage at the same marker (multipoint LOD 1.78, P = 2.1x10(-3), mean ibd sharing 55.8%). TDT analysis showed significant association at the CRH locus (P = 2.6x10(-3)). CRH has a sibling relative risk of 1.14, and contributes <10% to the sibling relative risk of RA. CONCLUSION: With the exception of HLA, this is the strongest evidence yet of a genetic locus that is both linked to and associated with RA, and provides an avenue for further genetic characterization and potentially novel therapeutic intervention.

Adult↗

PubMed2000

Genetic polymorphism of IL-12 p40 gene in immune-mediated disease.

Understanding of the genetic basis of autoimmune diseases is currently incomplete. Cytokine gene polymorphisms warrant consideration as factors explaining variation in the human immune and inflammatory responses and as candidate susceptibility genes for related pathological states. Interleukin 12 (IL-12) is a key regulator of the polarisation of immune responses to T helper 1 or 2 categories and plays a role in autoimmune and infectious diseases. Using a bioinformatic strategy, we aligned cDNA and expressed sequence tag sequences to identify putative polymorphic regions of the IL-12 p40 gene. Position 1188 in the 3' untranslated region (UTR) was polymorphic with the frequency of the common allele around 80% in healthy UK Caucasoids. PCR genotyping of multiple Caucasoid groups and an African group showed significant population variation. In a case-control design, the polymorphism was not associated with rheumatoid arthritis, Felty's syndrome or large granular lymphocyte syndrome with arthritis or multiple sclerosis. A nonsignificant increase in the B allele frequency was observed in the rare large granular lymphocyte syndrome without arthritis (odds ratio 2.02 95% CI 0.95-4.3). This new genetic marker could be useful in anthropological studies and should be investigated in other autoimmune, allergic, inflammatory and infectious diseases.

Alleles↗

PubMed2000

Neoadjuvant chemotherapy, radical resection with intraoperative radiation therapy (IORT): improved treatment for gastric adenocarcinoma.

BACKGROUND: Adenocarcinoma of the stomach and gastroesophageal junction results in substantial morbidity, locoregional recurrence, and death. Surgical procedures, even with adjuvant therapy, have not significantly improved survival. This study evaluated the toxicity, response rate, locoregional control, and survival of patients with locally advanced gastric cancer that was treated with neoadjuvant multimodality therapy. METHODS: Patients with stage IIIA or early stage IV gastric adenocarcinoma received neoadjuvant 5-fluorouracil, Leucovorin, Adriamycin, and Cisplatin and underwent gastrectomy or esophagogastrectomy with intraoperative radiotherapy (IORT; 1000 cGY) to the gastric bed and postoperative radiation therapy. RESULTS: Nine of 15 patients (60%) with transmural extension and/or nodal metastases received IORT. There were 2 pathologically complete responses at the primary site. Eleven of 15 patients (73%) had tumor in perigastric lymph nodes; however, 9 of 15 patients (60%) had mucin-filled nodes without tumor cells. Neoadjuvant treatment did not increase operative morbidity rates. Ten of 15 patients (67%) remain free of disease (median, 27 months; range, 6-60 months). Five patients died 13 to 41 months (median, 17 months) after diagnosis. CONCLUSIONS: Neoadjuvant multimodality therapy with neoadjuvant 5-fluorouracil, Leucovorin, Adriamycin, and Cisplatin, radical resection with IORT, and postoperative radiation therapy is safe, can downstage tumors, provides improved locoregional control, and appears to cause significant tumor regression that may result in long-term survival or cure.

Adenocarcinoma↗

PubMed2000

A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch.

Smooth muscle myosin phosphatase dephosphorylates the regulatory myosin light chain and thus mediates smooth muscle relaxation. The activity of this myosin phosphatase is dependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1 have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle section of chicken and rat MYPT1 genes revealed that each gene gave rise to isoforms by cassette-type alternative splicing of exons. In chicken, a 123-nucleotide exon was included or excluded from the mature mRNA, whereas in rat two exons immediately downstream were alternative. MYPT1 isoforms lacking the alternative exon were only detected in mature chicken smooth muscle tissues that display phasic contractile properties, but the isoform ratios were variable. The patterns of expression of rat MYPT1 mRNA isoforms were more complex, with three major and two minor isoforms present in all smooth muscle tissues at varying stoichiometries. Isoform switching was identified in the developing chicken gizzard, in which the exon-skipped isoform replaced the exon-included isoform around the time of hatching. This isoform switch occurred after transitions in myosin heavy chain and myosin light chain (MLC(17)) isoforms and correlated with a severalfold increase in the rate of relaxation. The developmental switch of MYPT1 isoforms is a good model for determining the mechanisms and significance of alternative splicing in smooth muscle.

Animals↗

PubMed2000

Prevention of primary cytomegalovirus disease in organ transplant recipients with oral ganciclovir or oral acyclovir prophylaxis.

BACKGROUND: Optimal prophylaxis against cytomegalovirus (CMV) disease for organ transplant patients at risk for primary infection (donor seropositive, recipient seronegative, D+R-) remains to be determined. We hypothesized that prolonged oral ganciclovir therapy following intravenous therapy would provide increased protection. METHODS: A total of 155 evaluable D+R- organ transplant recipients from 13 transplant centers were entered into the study: all received intravenous ganciclovir (5 mg/kg/day) for 5-10 days and then either oral acyclovir (400 mg tid) or oral ganciclovir (1 g tid) for an additional 12 weeks. Patients were assigned to their treatment groups at a central randomization site, with a separate randomization scheme for each of the organs transplanted (kidney, heart, or liver). In the case of kidney transplants, the patients were stratified according to source of the kidney (living related vs. cadaveric donor). The primary endpoint was the incidence of CMV disease in the first six months post-transplant. RESULTS: Treatment with oral ganciclovir was associated with a significant decrease in the incidence of symptomatic disease or viremia when compared with the oral acyclovir group (32% vs. 50%, P<0.05). This difference was most marked in terms of tissue invasive disease: only 3 of 15 symptomatic patients in the ganciclovir group vs. 10 of 21 in the acyclovir group developed tissue-invasive infection (P<0.05). There was a significant difference in the time to CMV disease or viremia in the two groups: mean time 212+/-17 days post-transplant for the acyclovir group vs. 291+/-13 days for the ganciclovir group (P<0.001). The incidence of allograft rejection was 34% in the ganciclovir group and 46% in the acyclovir group (P=NS). Leukopenia was more common in the ganciclovir group (P<0.05), but in no case did it require drug discontinuation. Ganciclovir resistance did not develop in this study. CONCLUSION: Prophylaxis with oral ganciclovir following a brief course of intravenous ganciclovir provides useful protection against primary CMV disease.

Acyclovir↗

PubMed1999

Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production.

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.

Animals↗

PubMed1999

Growth in the presence of salicylate increases fluoroquinolone resistance in Staphylococcus aureus.

Salicylate and acetylsalicylate slightly increased fluoroquinolone resistance in ciprofloxacin-susceptible and -resistant Staphylococcus aureus. Salicylate allowed a greater number of cells from ciprofloxacin-susceptible and -resistant strains to survive on high fluoroquinolone concentrations. Salicylate also increased the frequency with which a susceptible strain mutated to become more resistant to ciprofloxacin.

Anti-Infective Agents↗

PubMed1998

Developmental remodeling and shortening of the cardiac outflow tract involves myocyte programmed cell death.

The embryonic outflow tract is a simple tubular structure that connects the single primitive ventricle with the aortic sac and aortic arch arteries. This structure undergoes a complex sequence of morphogenetic processes to become the portion of the heart that aligns the right and left ventricles with the pulmonary artery and aorta. Abnormalities of the outflow tract are involved in many clinically significant congenital cardiac defects; however, the cellular and molecular processes governing the development of this important structure are incompletely understood. Histologic and tissue-tagging studies indicate that the outflow tract tissues compact and are incorporated predominantly into a region of the right ventricle. The hypothesis tested in the current study was that cell death or apoptosis in the muscular portion of the outflow tract is an important cellular mechanism for outflow tract shortening. The tubular outflow tract myocardium was specifically marked by infecting myocytes of the chicken embryo heart with a recombinant replication-defective adenovirus expressing beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter. Histochemical detection of the beta -gal-labeled outflow tract myocytes revealed that the tubular structure shortened to become a compact ring at the level of the pulmonic infundibulum over several days of development (stages 25-32, embryonic days 4-8). The appearance of apoptotic cardiomyocytes was correlated with OFT shortening by two histologic assays, TUNEL labeling of DNA fragments and AnnexinV binding. The rise and fall in the number of apoptotic myocytes detected by histologic analyses paralleled the change in activity levels of Caspase-3, a protease in the apoptotic cascade, measured in outflow tract homogenates. These results suggest that the elimination of myocytes by programmed cell death is one mechanism by which the outflow tract myocardium remodels to form the proper connection between the ventricular chambers and the appropriate arterial trunks.

Adenoviridae↗

PubMed1997

Palliation of AIDS-related primary lymphoma of the brain: observations from a multi-institutional database.

PURPOSE: To catalogue the presenting symptoms of patients with AIDS who are presumed to have primary central nervous system lymphoma (PCNSL). To document the palliative efficacy of cranial irradiation (RT) relative to the endpoints of complete and overall response for the respective symptoms. METHODS: An analysis of 163 patients with AIDS-related PCNSL who were evaluated at nine urban hospitals was performed. These patients were treated for PCNSL after the establishment of a tissue diagnosis or on a presumptive basis after failing empiric treatment for toxoplasmosis. All patients were treated between 1983 and 1995 with radiotherapy (median dose-fractionation scheme = 3 Gy x 10) and steroids (>90% dexamethasone). Because multiple fractionation schemes were used, prescriptions were converted to biologically effective doses according to the formula, Gy10 = Total Dose x (1 + fractional dose/alpha-beta); using an alpha-beta value of 10. RESULTS: The overall palliative response rate for the entire group was 53%. In univariate analysis, trends were present associating complete response rates with higher performance status (KPS > or = 70 vs. KPS < or = 60 = 17% vs. 5%), female gender (women vs. men = 29% vs. 8%), and the delivery of higher biologically effective doses (BED) of RT (Gy10 > 39 vs. < or = 39 = 20% vs. 5%). In multivariate analysis of factors predicting complete response, both higher KPS and higher BED retained independent significance. A separate univariate analysis identified high performance status (KPS > or = 70 vs. KPS < or = 60 = 71% vs. 47%), and young age (< or = 35 vs. > 35 = 61% vs. 40%) as factors significantly correlating with the endpoint of the overall response. In multivariate analysis, high performance status and the delivery of higher biologically effective doses of irradiation correlated significantly with higher overall response rates. CONCLUSION: Most AIDS patients who develop symptoms from primary lymphoma of the brain can achieve some palliation from a management program that includes cranial irradiation. Young patients with excellent performance status are most likely to respond to treatment. The delivery of higher biologically effective doses of irradiation also may increase the probability of achieving a palliative response.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Multiple overlapping processes underlying short-term synaptic enhancement.

Recently there have been exciting advances in understanding the mechanisms and functional roles of a form of short-term synaptic enhancement (STE) that results from an activity-dependent accumulation of Ca2+ in the presynaptic terminal. This form of STE is composed of at least four processes: fast-decaying facilitation (FI), slow-decaying facilitation (F2), augmentation (AUG) and post-tetanic potentiation (PTP). Recent results suggest that these processes can now be distinguished mechanistically by the site of their induction within the presynaptic terminal: FI and F2 appear to be induced by a rapid, high concentration of Ca2+ at or near the site of exocytosis, whereas AUG and PTP seem to be induced by lower levels of Ca2+ with slower kinetics, possibly within the core of the terminal. STE is highly conserved across diverse species, and appears to serve as a flexible mechanism for temporal information processing in systems ranging from peripheral motor control to higher cortical integration.

Animals↗

PubMed1997

Endothelin-1 alters the contractile phenotype of cultured embryonic smooth muscle cells.

Smooth muscle tissues may be classified into phasic (fast) or tonic (slow) contractile phenotypes. This study was initiated to examine the specification of these phenotypes during development and the role of growth factors in this process. We used myosin light chain 17 (MLC17) and myosin heavy chain transcript splice variants as markers of the tonic (aortic) and phasic (intestinal) smooth muscle phenotypes in chick embryos. By reverse transcription-polymerase chain reaction, we determined embryonic days 6 to 16 to be a critical period for the establishment of these phenotypes. During this period, endothelin-1 is present at 40-fold-higher levels in aortic compared with intestinal tissues. To test the hypothesis that endothelin-1 may be involved in establishing the aortic (tonic) phenotype, we developed a system in which embryonic smooth muscle cells exhibit phasic and tonic contractile properties in vitro. Single-cell force measurements showed that cultured embryonic gizzard (phasic) cells developed force more rapidly (8 +/- 2 seconds) and achieved greater force (3.0 +/- 0.7 microN) than did cultured embryonic aortic (tonic) cells (20 +/- 0.7 seconds, 0.76 +/- 0.01 microN; P < .05) in response to depolarization. Chronic exposure of the phasic (gizzard) cells to endothelin-1 prolonged the time to peak force (24 +/- 3 seconds) and reduced the peak force (1.0 +/- 0.1 microN), so that the contraction resembled the tonic type. This effect, mediated by the endothelin-A receptor, was associated with a shift in MLC17 splicing to the tonic pattern. These results demonstrate that endothelin-1 is highly enriched in developing aortic compared with intestinal tissues and can convert phasic smooth muscle cells to the tonic type in vitro, suggesting a role for this growth factor during development in determining the contractile phenotype of smooth muscle cells.

Angiotensin II↗

PubMed1997

Forced expression of the homeodomain protein Gax inhibits cardiomyocyte proliferation and perturbs heart morphogenesis.

The development of the tubular heart into a complex four-chambered organ requires precise temporal and region-specific regulation of cell proliferation, migration, death and differentiation. While the regulatory mechanisms in heart morphogenesis are not well understood, increasing attention has focused on the homeodomain proteins, which are generally linked to morphogenetic processes. The homeodomain containing gene Gax has been shown to be expressed in heart and smooth muscle tissues. In this study, the Gax protein was detected in the nuclei of myocardial cells relatively late in chicken heart development, at a time when myocyte proliferation is declining. To test the hypothesis that the Gax protein functions as a negative regulator of cardiomyocyte proliferation, a replication-defective adenovirus was used to force its precocious nuclear expression during chicken heart morphogenesis. In experiments in which Gax- and beta-galactosidase-expressing adenoviruses were co-injected, clonal expansion of myocytes was reduced, consistent with inhibition of myocyte proliferation. This effect on proliferation was corroborated by the finding that the percentage of exogenous Gax-expressing myocytes that were positive for the cell cycle marker PCNA decreased over time and was lower than in control myocytes. The precocious nuclear expression of Gax in tubular hearts resulted in abnormal heart morphology, including small ventricles with rounded apices, a thinned compact zone and coarse trabeculae. These results suggest a role for the Gax protein in heart morphogenesis causing proliferating cardiomyocytes to withdraw from the cell cycle, thus influencing the size and shape that the heart ultimately attains.

Adenoviridae↗

PubMed1996

Characterization of cardiac gene cis-regulatory elements in the early stages of chicken heart morphogenesis.

To study transcriptional regulation during early stages of cardiogenesis, stage 10-17 chicken embryo hearts were transfected efficiently within the intact embryo by application of plasmid DNA complexed to liposomes. Viral regulatory sequences and the skeletal alpha-actin, SERCA2 and ANF promoters activated expression of reporter genes in the primitive heart tube. Deletion and mutation analysis of the skeletal alpha-actin promoter revealed the importance of CArG cis-regulatory elements in enhancing transcription of the gene during early heart development. These results demonstrate the utility of this method for the identification of gene regulatory elements that specify the cardiac phenotype during early stages of heart morphogenesis.

Actins↗

PubMed1996

Immediate-early gene responses to different cardiac loads in the ejecting rabbit left ventricle.

Clinical and experimental observations in humans and animals have shown that different cardiac adaptations occur in response to different types of hemodynamic overload. However, very little is known about how different hemodynamic loads lead to these different cardiac adaptations. Accordingly, we studied the acute response of ejecting isolated rabbit hearts to independently varied systolic and diastolic mechanical loads at constant coronary perfusion pressure. We studied the combined effects of low end-diastolic volume (EDV) and low systolic ejection pressure (Pej), compared to low EDV and high Pej, high EDU and low Pej, and high EDV and high Pej, on the expression of c-fos, c-jun, and egr-1. Further, although we did not seek to clarify the role of these immediate-early genes in cardiac hypertrophy, we hypothesized that they should not all respond in the same manner to these different mechanical loads. In these ejecting hearts we found that the expression of these immediate-early genes did not all respond alike to the different mechanical loads: both c-fos and egr-1 were strongly induced at both 30 and 60 min. However, at 30 min only c-fos depended on the level of EDV (P = 0.01). Neither c-fos nor egr-1 was influenced by EDV at 60 min. The expression of c-jun was largely insensitive to all loading conditions. We conclude that EDV, independent of Pej, influences the pattern and time course of expression of some immediately-early genes and that these different immediate-early genes do not respond in parallel to changes in cardiac loading.

Animals↗

PubMed1996

Expression of exogenous protein and analysis of morphogenesis in the developing chicken heart using an adenoviral vector.

OBJECTIVES: Recombinant retroviral vectors have been shown to be useful tools for marking cells so as to follow their fates during development. The aim of this study was to determine the utility and advantages of an adenoviral vector as a tool to study the heart as it develops from a simple tube into a complex four-chambered organ. METHODS: Replication-defective adenovirus (10(7) pfu) expressing beta-galactosidase (beta-gal) under the control of the RSV-LTR was applied to the external surface of embryonic stage 13-21 chick hearts in ovo. Embryos were incubated for up to an additional 96 h. Hearts were harvested at 12-24 h intervals and (1) whole-mount-stained for beta-gal and sectioned, (2) examined by electron microscopy and (3) homogenized and beta-gal activity measured with a luminescent assay. RESULTS: beta-gal expression peaked at 48 h, when a significant percentage of the myocytes in the atrial and ventricular walls expressed the protein, and it comprised 0.5% of total heart protein. Significant levels were still expressed at 96 h. When applied to early-stage (13-16) embryos, expression occurred predominantly in cardiomyocytes. beta-gal marking of cells enabled us to identify the following morphogenic patterns: (1) cells of the conus region compact into the bulbis cordis; (2) by applying the virus at later stages, e.g. 21-22, it was evident that the epicardium invests the heart, after stage 17, in a dorsal to ventral and caudal to rostral direction; (3) at lower titers (10(5) pfu), the virus serves as a clonal marker through several cell divisions, with an estimated cell doubling time of 24 h. CONCLUSIONS: Application of an adenoviral vector to early-stage embryonic chick heart results in substantial expression of exogenous protein in a significant percentage of cardiomyocytes without grossly affecting heart development. Adenoviral vectors are useful for following the fate of cells as the heart develops from a simple tube into a complex four-chambered organ and hold promise for enabling the expression of exogenous proteins which might alter cell behavior.

Adenoviridae↗