The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA.

Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5'UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Δ40p53 isoform, respectively. Here, we report that DAP5, a translation initiation factor shown to positively regulate the translation of various IRES containing mRNAs, promotes IRES-driven translation of p53 mRNA. Upon DAP5 depletion, p53 and Δ40p53 protein levels were decreased, with a greater effect on the N-terminal truncated isoform. Functional analysis using bicistronic vectors driving the expression of a reporter gene from each of these two IRESs indicated that DAP5 preferentially promotes translation from the second IRES residing in the coding sequence. Furthermore, p53 mRNA expressed from a plasmid carrying this second IRES was selectively shifted to lighter polysomes upon DAP5 knockdown. Consequently, Δ40p53 protein levels and the subsequent transcriptional activation of the 14-3-3σ gene, a known target of Δ40p53, were strongly reduced. In addition, we show here that DAP5 interacts with p53 IRES elements in in vitro and in vivo binding studies, proving for the first time that DAP5 directly binds a target mRNA. Thus, through its ability to regulate IRES-dependent translation of the p53 mRNA, DAP5 may control the ratio between different p53 isoforms encoded by a single mRNA.

Shelhigh No-React porcine pulmonic valve conduit: a new alternative to the homograft.

BACKGROUND: The Shelhigh No-React pulmonic valve conduit is a new porcine conduit that is glutaraldehyde-treated and detoxified using a proprietary heparin process. In our institution it has been implanted in 25 patients. The aim of this present contribution is to evaluate the short-term follow-up after its implantation. METHODS: From November 1997 to August 1999, 25 patients (mean age, 20.2 years; range, 0.6 to 28.3 years) were operated on using this conduit. Seventeen patients underwent a Ross procedure for aortic valve disease, with the conduits implanted in anatomic position; 6 patients underwent right ventricular outflow tract reconstruction; 2 patients underwent the Rastelli operation. The follow-up was complete. Preoperative and postoperative two-dimensional echocardiography data were collected. RESULTS: There were two non-conduit-related deaths. Two conduits needed to be exchanged because of an increase in the gradient. Overall, all patients were improved in terms of New York Heart Association class. Comparison of preoperative and postoperative two-dimensional echocardiography gradient showed significant improvement. At the 30-month follow-up, no calcification was seen on the explanted conduits or on the two-dimensional echocardiography, although many of the patients are children. CONCLUSIONS: The Shelhigh conduits seem to be an alternative to homograft especially in infants. These experiences are preliminary, and longer follow-up is required.

Chloride conductance across toad skin: effects of ionic acclimations and cyclic AMP and relationship to mitochondria-rich cell density.

The anionic conductance across toad (Bufo viridis) skin was studied using the voltage-clamp technique following long-term (more than 10 days) acclimation to NaCl and KCl solutions. The non-specific baseline conductance was approximately 0.6 mS cm(-)(2) and was similar in skins from all acclimation conditions. The voltage-activated Cl(-) conductance (G(Cl)) was maximal in skins from distilled-water- and KCl-acclimated toads (>3 mS cm(-)(2)) and was greatly reduced following acclimation to NaCl solutions. Cyclic AMP (EC(50)=13 micromol l(-)(1)) and isobutylmethyl xanthine (IBMX) (EC(50)=69 micromol l(-)(1)) exerted different effects on the activated conductance. IBMX only sensitized the activated conductance, whereas cyclic AMP (CPTcAMP) at high concentrations induced an increase in anionic conductance that was insensitive to electrical potential. Furthermore, external Cl(-) was not required for the stimulatory effect of cyclic AMP, and the conductive pathway had low selectivity. The effects of the two agonists were reversible and depended on the acclimation conditions. Following electrical measurements, the skin of the toads was removed and stained with silver to measure mitochondria-rich cell density (D(mrc)). There was no correlation between D(mrc) and Cl(-) conductance in the present study.

Mitochondria-rich cells in anuran amphibia: chloride conductance and regional distribution over the body surface.

The distribution and density (D(mrc)) of mitochondria-rich cells (MR cells) in skin epithelium, were determined over the whole body surface in nine species of anuran Amphibia that live in a variety of habitats. It was found that the more terrestrial species (beginning with Hyla arborea) have a higher density of MR cells in their pelvic region. In the skin of aquatic (Xenopus laevis) or fossorial (Pelobates syriacus) species, D(mrc) is evenly distributed over the whole body surface. In dorsal skin pieces of H. arborea that lack detectable MR cells, transepithelial voltage activation did not induce Cl(-) conductance as it did in ventral pieces. Skins from Bufo viridis and X. laevis, both have MR cells in their skin, differ markedly in their biophysical properties: a Cl(-) specific current conductance is predominant in the skin epithelium of B. viridis, and is absent in X. laevis. In the latter, anionic conductance is due to glandular secretion. The biophysical properties cannot therefore be related solely to the presence or density of MR cells. Mitochondria-rich cells are sites of Cl(-) conductance across the skin of those amphibians that show this property, but must have different function(s) in other species. It is suggested that the specific zonal distribution of MR cells in the species that were examined in this study could be due to ion exchange activity and water conservation in more terrestrial environments.

Lectin binding patterns in amphibian skin epithelium.

Seven lectins (PNA, DBA, WGA, UEA-I, RCA, SBA, Con A) were used to localize glycoconjugates in the skin of 10 species of Amphibia, 7 anurans (Bufo marinus, Bufo bufo, Rana ridibunda, Rana pipiens, Hyla arborea, Pelobates syriacus and Xenopus laevis) and 3 urodeles (Salamandra salamandra, Triturus vulgaris and Ambystoma mexicanum). It was found that every lectin has a specific binding pattern in the skin of each species. No common pattern could be established, either among frogs or toads, nor for a particular lectin. Each lectin bound specifically and selectively to a particular epithelial component, which differed from one species to the other. A number of lectins showed selective binding to mitochondria-rich cells, but, again, a pattern in positivity could not be found. It is concluded that lectin histochemistry does correlate with cellular function. Our data can be applied in studies of epithelium and skin development, and of changes that occur during adaptation to the environment by amphibian species.

Lectin binding pattern and band 3 localization in toad skin epithelium and the effect of salt acclimation.

Seven lectins were employed to localize glycoconjugates in the skin of a toad (Bufo viridis). Each of the lectins exhibited a particular, specific and selective binding pattern. Peanut lectin (PNA) and WGA bound to mitochondria-rich (MR) cells, but WGA bound also abundantly, in the dermis. Band 3-like protein, as indicated by the reaction with polyclonal anti band 3 antibody, was localized exclusively in MR cells. Ionic acclimation (200 mmol/L NaCl, or 50 mmol/L KCl) affected profoundly the binding pattern of the lectins. High NaCl acclimation resulted also in diminishing anti band 3 antibody binding, whereas in skins of KCl-acclimated toads the staining remained similar to the control. The binding of WGA but not PNA, corresponded with the same cells that stained with anti band 3 antibody. PNA in concentration of > 10 micrograms/mL reduced reversibly, both the resting and activated Cl- conductance by 25-30%. Based on differential binding of band 3, WGA and PNA, these observations provide conclusive verification of the presence of at least two populations of MR cells in the toad skin epithelium. It is suggested that the PNA positive MR cells may correspond to a beta-type MR cell. The information can be used to study molecular mechanisms that are involved in ionic acclimation.

No-react detoxification process: a superior anticalcification method for bioprostheses.

BACKGROUND: Glutaraldehyde pretreatment of bioprosthetic heart valves is the major pathogenic factor in their calcific degeneration. This comparative study investigates the merit of the No-React aldehyde detoxification process as an alternative modifier of xenograft tissues. METHODS: Glutaraldehyde- and No-React-pretreated porcine aortic valve cusps were implanted subcutaneously in 6-week-old rats (n = 20). At 3, 6, and 14 weeks, randomly selected animals were sacrificed and the explants underwent mineral and morphologic analyses. Glutaraldehyde- and No-React-treated bovine pericardium and porcine aortic valve cusp were incubated in fibroblast cell culture plates. Cell viability was observed under reversed microscope at 6, 24, 48, and 96 hours. Erythrosin B dye exclusion test was used to validate percent cell death. RESULTS: Pretreatment with No-React significantly inhibited calcification of aortic cusp subcutaneous implants throughout the 14-week period (mean tissue Ca2+ content = 1.3 +/- 0.7 micrograms/mg at 14 weeks.) Glutaraldehyde-treated cusps underwent protracted calcification (Ca2+ content = 190.6 +/- 89.5 micrograms/mg; p < 0.01). Morphologic findings correlated with mineral analyses. One-hundred percent of fibroblast cells survived in the presence of No-React-treated tissue, with a growth pattern indistinguishable from control cell culture (ie, in the presence of no tissue). The cells incubated with glutaraldehyde-treated tissue showed signs of nonviability by 6 hours, with 100% cell death by 48 hours. Dye exclusion tests validated these findings. CONCLUSIONS: The No-React detoxification process completely abolishes the cytotoxicity of the xenograft tissue and inhibits calcific degeneration.

Calcification of bovine pericardium: glutaraldehyde versus No-React biomodification.

BACKGROUND: Calcific degeneration is the most frequent cause of clinical dysfunction of glutaraldehyde (GA)-pretreated bioprosthetic heart valves. The No-React (NR) process has been shown to be a promising anticalcification treatment. In this comparative study, our objective was to delineate the advantages of the NR treatment over GA. METHODS: Bovine pericardial strips pretreated with GA and NR were individually incubated in calcium phosphate solution for 21 days at 37 degrees C. The pretreated bovine pericardium then was implanted subcutaneously in rats and retrieved at 14, 21, and 35 days after-implantation. Mineral and morphologic analyses were performed on each specimen. RESULTS: The NR-treated pericardium revealed significantly reduced in vitro calcification compared with the GA-treated tissue (mean tissue calcium content 1.3 +/- 0.2 versus 5.9 +/- 0.7 micrograms/mg; p < 0.001). Mineral analysis showed progressive calcification of the GA-pretreated pericardium over the period of implantation (calcium content increasing from 49.6 +/- 9.6 micrograms/mg after 2 weeks to 134.3 +/- 9.1 micrograms/mg at 5 weeks after-implantation). The NR-treated implants had calcified significantly less (p < 0.05) at each corresponding interval. Moreover, morphologic examinations demonstrated a protracted inflammatory response in the form of giant cell and mononuclear cell infiltration associated with intrinsic collagen disruption in the GA-treated tissue; the NR-treated pericardium maintained morphologic integrity with a mild inflammatory response. CONCLUSIONS: The NR biochemical process appears not only to attenuate pericardial calcification, but also to abort the host's destructive inflammatory response to the xenograft.

Comparative in-vitro fluid dynamics characterization of heart valve bioprostheses under accelerated fatigue conditions and under physiologic conditions.

An accelerated fatigue testing system and a pulse duplicator (heart simulator) were used to evaluate the fluid dynamics characteristics of 12 cardiac bioprostheses. Pressure differences and pulsatile flow rates across the valves, as well as machine rates, were measured using a real-time on-line data-acquisition system. All other valve hydrodynamic parameters were internally calculated. For the same pressure difference, the pulsatile flow rate was higher at higher pulse rates. Regurgitation fraction values were higher for valves tested at higher speeds. Closing volumes, however, remained fairly constant. Mean transvalvular pressure difference (delta p) and root mean square pulsatile flow rate (QRMS) under accelerated testing conditions were related according to the particular case of a parabolic regression through the origin of the form (type 1), delta p = Co Q2RMS. Prosthetic valves tested in the fatigue tester presented in general a more effective area for flow than did valves tested in the pulse duplicator. Calculated discharge coefficients and performance indexes were accordingly higher. In both testing devices pericardial valves had higher effective orifice areas, discharge coefficients, and performance indexes than did porcine xenografts, and large valves performed more efficiently than small valves. Differences among regression coefficients for the same valves tested in both machines appeared to be significant in 100% of the cases. There were differences in the degrees of stenosis among valves tested in the pulse duplicator and in the fatigue tester. Understanding of fluid dynamics data obtained for undamaged valves at accelerated speeds and their relation to data obtained at physiologic speeds permitted the detection and quantification of rupture and malfunctioning of these valves without removing them from the fatigue tester.

A system for in-vitro characterization of heart valve bioprostheses under accelerated fatigue conditions and under physiologic conditions.

An accelerated fatigue testing system and a pulse duplicator (heart simulator) were used in a set-up in which pressure differences and pulsatile flow rates across prosthetic heart valves, as well as machine rates, could be measured using a real-time on-line data-acquisition system. With this information available, an immediate in-vitro characterization of the fluid dynamics of prosthetic heart valves could be obtained under both physiologic and accelerated conditions. In addition to the fatigue tester and the pulse duplicator, a signal conditioner, a DC amplifier, an analog-to-digital converter, and a digital microcomputer comprised the essential hardware. For the purpose of this study, special acquisition software was developed. By means of the same computational algorithms, all quantitative fluid dynamics data could be calculated using information from both the pulse duplicator and the fatigue tester, so that direct comparisons and correlations could be approached. Pressure difference-flow rate relationships for the two machines were comparable.

Developmental changes in the heterocellular epidermis of Pelobates syriacus integument.

Changes in characteristic components of the skin epidermis of the large tadpole of Pelobates syriacus were studied throughout its development. The fate of two specific cells in the skin epidermis was followed, from the young tadpole to the adult was studied. It was found that flask-shaped type cells in the tadpole epidermis which are PAS-positive, stain with peanut lectin (PNA). There is no detectable band 3 in the premetamorphosed stages, and mitochondria-rich cells are very rare. This pattern of staining changes completely upon metamorphosis: the PAS-positive cells, specific to the tadpole epidermis disappear, and the mitochondria-rich (MR) cells in the adult skin epithelium react with polyclonal anti-band 3 antibody. Western blot analysis showed the presence of a band 3-like protein of about 95 kDa, only in the adult epithelial extract, corroborating the immunocytochemical observations. The finding of the presence of band 3-like protein in the MR cells of Pelobates, is similar to the observations made in the skin of other amphibian species. On the other hand, the binding of peanut lectin to MR cells is species-specific, since it does not react with the MR cells in the skin epithelium of Pelobates syriacus.

New outlook on pericardial substitution after open heart operations.

The difficulties of reoperation owing to adhesions are well known. Clinical attempts to solve this problem using synthetic materials or glutaraldehyde-fixed pericardial xenograft have been less than satisfactory. Although experimental animal results have been good, they have not considered the influence of cardiopulmonary bypass (CPB) on adhesion formation. This study addressed the influence of CPB on the formation of adhesions and evaluated biodegradable polyglycolic acid as a material to reduce adhesions and as a pericardial substitute. Forty-five weanling sheep received implants of pericardial xenograft and polyglycolic acid with and without CPB. The pericardial xenograft showed no adhesions when implanted without CPB, but severe adhesions formed and a thick fibrinous layer covered the heart when CPB was used, making identification of coronary arteries at reoperation very difficult. The polyglycolic acid mesh implanted without CPB was absorbed and replaced with newly formed host collagen. With CPB, the polyglycolic acid was more rapidly absorbed, and a thinner layer of host collagen formed. Therefore, future animal studies must include CPB. In agreement with reported clinical results, glutaraldehyde-fixed pericardial tissue implanted with CPB resulted in severe epicardial reaction and therefore is not an ideal pericardial substitute.

Controversies in management of sternal tumors.

The rarity of sternal tumors makes a good comparative study a difficult task. Controversies exist on questions of method of surgical diagnosis, the extent of the surgical procedure, and the different ways and means of reconstruction. We describe a case that was believed to be a clear case of chondrosarcoma but was actually a plasmacytoma. From the evidence of this case and a review of the literature we conclude that almost any plasmacytoma should be considered generalized multiple myeloma. Biopsy should be performed in all cases of sternal tumor before any surgical action is taken.

Reducing the variability in durability of heart valve bioprostheses. Key factor for future improvement.

Two observations concerning the Meadox unicusp bovine pericardial valve were presented. First, calcification has not been a major problem because of the relatively short implant durations (less than 5 years). The second observation was that thromboembolism was found to be practically nonexistent. The third observation was that the leaflets of certain valves that had been removed showed leaflet stretching, which resulted in excess stress in certain loci of the leaflet. The studies reported here showed that the bovine pericardial sac was distensible to various degrees. Strips from various sections of pericardium had a large spectrum of strain (5-45%), at very small stress levels (less than or equal to 4 g/mm2). Further, within any section the material was anisotropic, with strain differences of 25%. Finally, histologic studies of clinical grade Ionescu-Shiley bovine pericardial valves showed marked discrepancies in structure and staining characteristics, further documenting that this material is not homogeneous. New methods of tissue selection are mandatory and have been designed to improve uniformity and ultimately reduce the viability in performance in the clinical setting. This method can be applied to all pericardial valves.

Do heart valve bioprostheses degenerate for metabolic or mechanical reasons?

Although heart valve bioprostheses provide a normal quality of life, their durability is still of great concern. Their durability failure is defined as "degeneration," which is considered to be a consequence of metabolic factors. In this study, we demonstrate that mechanical and design factors can also be responsible for bioprosthesis failure. Large numbers of porcine and pericardial bioprostheses were tested in a fatigue-testing system in which the test conditions were proved to be reproducible and accurate by a laser Doppler anemometer. The results have allowed us to define causes of failure, previously insufficiently stressed, in each type of valve tested. There is a clear difference in factors influencing tissue disruption between porcine and pericardial valves. We have compared these in vitro results with in vivo clinical findings. The main inferences are as follows: (1) Bioprostheses rupture and fail in the same fashion in both in vitro and in vivo studies. (2) Mechanical and design factors are involved in tissue failure. (3) The in vitro/in vivo durability ratio is not 1:1. This ratio depends on the test conditions. (4) Pericardial valves fail because of damage during closure, whereas porcine valves are damaged during both opening and closing (mostly opening) because of design features. (5) Once one cusp fails and prolapses, the other cusps will fail in an accelerated fashion. (6) In vitro durability of 100 X 10(6) cycles can be considered excellent and is an achievable goal. (7) Variability is the key impediment to predicting the durability of bioprostheses. Valves can fail within 2 to 3 million cycles or can last more than 100 million cycles. Similarly, bioprostheses may require explantation within a few months or can last 10 to 13 years in patients. (8) Fatigue testing is an excellent and valuable tool to elucidate the mechanical factors responsible for this variability.

Mitochondria-rich cells and carbonic anhydrase content of toad skin epithelium.

The density and carbonic anhydrase (CA) content of the mitochondria-rich cells (MRCs) in the skin epithelium of the toad, Bufo viridis, were studied under conditions of acclimation to various chlorinities. Long-term (days to weeks) acclimation to chloride-free solutions induced a great increase in the MRC density and the area occupied by the apical portion of these cells on the surface of the epithelium. The CA content of the epithelium, and individual MR cells, showed a 5- to 10-fold reduction after acclimation to solutions containing high chloride levels. The MRC density and their relative apical surface area correlated with the chloride permeability of the skin in acclimated (long-term) toads. It is concluded that the MRCs are the principal site of chloride permeability across the amphibian skin, and they respond in an adaptive manner to long-term changes in environmental chloride levels.