Biomedical subjects

A Radu

Publications and source records attributed to A Radu.

At least 19 recordsLinked to original sources

PubMed2001

BAPS prize-1999: Lung growth induced by prenatal tracheal occlusion and its modifying factors: a study in the rat model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Prenatal tracheal occlusion (TO) has been shown to accelerate lung growth in animal models and models of pulmonary hypoplasia. However, these models may not mimic early events in human congenital diaphragmatic hernia (CDH). The authors previously have developed a model of TO in the rat. The purpose of this study was to apply this technique to characterize TO-induced lung growth in the early onset nitrofen-induced model of CDH, and to address the clinically important questions of the effect of timing of TO and maternal infusion of terbutaline on TO-induced lung growth. METHODS: Left-sided CDH was induced in the fetuses of time-dated pregnant Sprague-Dawley rats by feeding 100 mg of nitrofen on day 9 of gestation. TO was performed via maternal laparotomy and hysterotomy at 19 days' gestation. At harvest (21.5 days' gestation), lungs from nitrofen-exposed fetuses without CDH (non-CDH), with CDH (CDH), and with CDH and TO (CDH-TO) were compared by analysis of wet and dry weight, DNA and protein content, and stereologic morphometry. A second study was performed to assess relative lung growth achieved by equal intervals of TO after "early" (19 days) versus "late" (20 days) gestational TO. Finally, the effect of maternal infusion of terbutaline, a commonly used tocolytic for fetal surgery, on TO-induced lung growth was analyzed. RESULTS: Analysis of lung growth showed consistent and significant lung growth in CDH-TO lungs. Lung growth after TO was proliferative and characterized by an increase in parenchymal volume as manifest by increased total saccular number and surface area and radial saccular count. Although visceral reduction was partially achieved, herniated liver was reduced incompletely. The majority of lung growth occurred during the latter half of the TO period. Early gestational age at TO and maternal terbutaline administration adversely influenced lung growth in CDH-TO fetuses. CONCLUSIONS: Prenatal TO induces dramatic lung growth in the early onset, nitrofen-induced rat model of CDH. TO is more effective later in gestation presumably because of the advanced stage of lung development and lung fluid production. This effect could be counterbalanced by the use of beta-mimetic tocolytic, which inhibits fetal lung fluid production late in gestation. Multiple factors including fetal lung fluid production and absorption, pharmacologic agents, space-occupying herniated viscera, and timing and duration of TO may be important clinical variables. The development of the rat model should facilitate further studies into the cellular and molecular mechanisms responsible for TO-induced lung growth.

Animals↗

PubMed2001

CompAc information system for traditional Chinese medicine.

The paper presents a system developed for the assistance of diagnosis and treatment in alternative medicine, based on traditional Chinese methods. The system named CompAc, is a result of an interdisciplinary cooperation and is designed for the physician, specialist in acupuncture. The Compac system allows the determination of the type of energetic imbalance starting from the clinical picture of the patient and establishing whether an organ or any of the viscera are affected. It allows also the indication of different variants of treatment. The diagnosis proposed by the system has to be confirmed by the physician and can be modified by him. The system is also useful for medical training.

Diagnosis, Computer-Assisted↗

PubMed2000

Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.

Adipocytes↗

PubMed2000

Fluorescence pharmacokinetics of Lutetium Texaphyrin (PCI-0123, Lu-Tex) in the skin and in healthy and tumoral hamster cheek-pouch mucosa.

We have investigated the pharmacokinetics (PK) of Lutetium Texaphyrin (Lu-Tex), a second-generation photosensitizer, in the Syrian hamster cheek pouch early cancer model. Ten male hamsters, five with chemically induced early squamous cell cancer of the left cheek pouch, received an intracardiac injection of a 10 mg/ml Lu-Tex solution, resulting in a dose of 12 mg Lu-Tex per kg of bodyweight. The PK of the dye have been measured during the 24 h following the injection with an optical-fiber-based spectrofluorometer on the ventral skin, the healthy and the tumoral cheek-pouch mucosa. The Lu-Tex fluorescence is excited at 460 nm and detected around 740 nm. All the measurements yield very similar pharmacokinetic curves. The fluorescence intensity reaches a maximum between two and three hours after the injection and, at its maximum, it is consistently higher (up to 1.5 times) on the tumor than on the healthy mucosa. It remains smaller on the skin than on cheek-pouch mucosa. After 24 h, the Lu-Tex fluorescence is no longer detectable either on the skin, on the lesion or on the healthy mucosa. Moreover, Lu-Tex clearly displays a significant fluorescence selectivity between early carcinoma and healthy mucosa in this model. Furthermore, the inter-animal fluctuations of the fluorescence signal are small (+/-16% on the tumor-bearing mucosa). Eight-minute-long skin-irradiation tests have been performed 24 h after the injection of the Lu-Tex on the ventral skin of 16 additional animals with a solar simulator. No reaction is observed, either macroscopically or microscopically, which further demonstrates, as suggested by the fluorescence measurements, that this photosensitizer is significantly cleared from the skin after 24 h.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2000

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Adipocytes↗

PubMed2000

Photodynamic therapy of early squamous cell cancers of the esophagus.

The conventional treatment for the cure of esophageal cancer is surgical resection. Esophageal cancer, when detected at an early stage, has a very good probability of being eradicated by less aggressive procedures, and photodynamic therapy has proven to be a safe and effective treatment modality in some carefully selected patients. The indications, outcomes, and future considerations regarding the use of photodynamic therapy for the treatment of superficial squamous cell carcinomas of the esophagus are discussed in this article.

Aminolevulinic Acid↗

PubMed1999

Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing.

Chronic wounds represent a major clinical problem with significant morbidity and healthcare expenditures, but no effective therapies. Topical platelet-derived growth factor-BB trials have required large and repeated doses to achieve only a modest effect. We examined the ability of an adenovirus containing the platelet-derived growth factor-B transgene to improve the rate of wound healing through induction of platelet-derived growth factor-B overexpression in cells participating in the wound healing response. We treated excisional wounds in the ischemic rabbit ear, which have a 60% delay in healing, with vehicle, 106, or 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B per wound (n = 19). At 7 d this resulted in a decrease in the epithelial gap from 3.4 +/- 1 mm (mean +/- SD) in vehicle-treated wounds to 1.9 +/- 1.8 mm (mean +/- SD, p < 0.05) when treated with 106 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B, and 0.7 +/- 1.1 mm (mean +/- SD, p < 0.001) when treated with 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B. Ischemic excisional wounds treated with 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B even healed more rapidly than non-ischemic excisional wounds treated with vehicle (p < 0.05). In contrast, 5 microg of platelet-derived growth factor-BB protein (n = 2) resulted in only modest granulation tissue at the margin, but no significant differences in epithelial gap (3 +/- 0.6 mm, mean +/- SD). Plaque-forming units (106 or 108) of an adenovirus containing the beta-galactosidase transgene (n = 4) impaired wound re-epithelialization with an epithelial gap of 5.11 +/- 0.69 mm, mean +/- SD, p < 0.004, and 3.8 +/- 0.57 mm, mean +/- SD, p < 0.07, respectively. Adenoviral-mediated gene transfer of platelet-derived growth factor-B overcame the ischemic defect in wound healing and offers promise in the treatment of chronic nonhealing wounds. The vulnerary effects of platelet-derived growth factor-B overexpression were sufficient to overcome the adverse effects of the adenovirus or transgene on wound healing.

Adenoviridae↗

PubMed1999

Pulse oximeter as a cause of skin burn during photodynamic therapy.

Photodynamic therapy is increasingly being used for the treatment of various cancers. However, this technique has a major adverse effect, namely skin photosensitization. An unusual case of skin burn associated with pulse oximetry during photodynamic therapy in a patient treated for an early esophageal tumor is described. The patient, who was treated with mTHPC, suffered a second-degree burn on the index finger with subsequent loss of the nail. The only previously reported case of such a complication occurred with Photofrin, whereby no tissue other than skin was damaged.

Burns↗

PubMed1997

RanGTP-mediated nuclear export of karyopherin alpha involves its interaction with the nucleoporin Nup153.

Using binding assays, we discovered an interaction between karyopherin alpha2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin beta1, termed beta1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin alpha from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin alpha antibodies, we found that karyopherin alpha export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin alpha was inhibited by peptides representing the interacting domains of Nup153 and karyopherin alpha2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, beta1*, although it inhibited import, did not inhibit export of karyopherin alpha. Hence, karyopherin alpha import into and export from nuclei are asymmetric processes.

Amino Acid Sequence↗

PubMed1997

Karyopherin beta2 mediates nuclear import of a mRNA binding protein.

We have cloned and sequenced cDNA for human karyopherin beta2, also known as transportin. In a solution binding assay, recombinant beta2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin beta1, karyopherin beta2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both beta1 and beta2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, beta2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of beta2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of beta2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin beta2 inhibited karyopherin alpha/beta1-mediated import of a classical NLS containing substrate and, vice versa, beta1 inhibited beta2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of beta1 and beta2 to repeat nucleoporins.

Amino Acid Sequence↗

PubMed1996

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

Amino Acid Sequence↗

PubMed1996

The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence.

By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of karyopherin alpha. We found that the C-terminal region of karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds karyopherin beta. Surprisingly, karyopherin alpha also contains an NLS. Thus, karyopherin alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of karyopherin alpha overlaps with the binding site of karyopherin alpha for karyopherin beta. Hence, binding of karyopherin beta to karyopherin alpha covers the NLS of karyopherin alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of karyopherin alpha to a heterologous NLS protein in the presence of karyopherin beta.

Amino Acid Sequence↗

PubMed1995

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

Animals↗

PubMed1995

The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex.

We report the cDNA deduced primary structure of a wheat germ agglutinin-reactive nuclear pore complex (NPC) protein of rat. The protein, termed Nup98 (for nucleoporin of 98 kDa), contains numerous GLFG and FG repeats and some FXFG repeats and is thus a vertebrate member of a family of GLFG nucleoporins that were previously discovered in yeast. Immunoelectron microscopy showed Nup98 to be asymmetrically located at the nucleoplasmic side of the NPC. Nup98 functions as one of several docking site nucleoporins in a cytosolic docking activity-mediated binding of a model transport substrate. The docking site of Nup98 was mapped to its N-terminal half, which contains all of the peptide repeats. A recombinant segment of this region depleted the docking activity of cytosol. We suggest that the peptide repeat domain of Nup98, together with peptide repeat domains of other nucleoporins, forms an array of sites for mediated docking of transport substrate, and that bidirectional transport across the NPC proceeds by repeated docking and undocking reactions.

Amino Acid Sequence↗

PubMed1995

Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa beta subunit. We now report that the karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin alpha or beta subunit, we showed that neither karyopherin alpha nor karyopherin beta alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized karyopherin alpha and beta subunits and import substrate as a ligand, we found that only karyopherin alpha bound ligand. We suggest that karyopherin beta functions as an adaptor that binds both to karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of karyopherin alpha-substrate across the NPC.

Animals↗

PubMed1995

Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins.

We have identified and characterized a 9S protein complex from a Xenopus ovary cytosolic subfraction (fraction A) that constitutes this fraction's activity in recognizing a model nuclear import substrate and docking it at the nuclear pore complex. Because of its function, the complex is termed karyopherin. The 54- and 56-kDa subunits of the complex are termed alpha 1 and alpha 2, respectively, and the 97-kDa subunit is termed beta. In an alternative approach we have identified karyopherin beta from a rat liver cytosolic subfraction A by using immobilized rat nucleoporin Nup98 in a single, affinity-based enrichment step. We have molecularly cloned and sequenced rat karyopherin beta. Comparison with protein sequence data banks showed no significant similarity to other known proteins. Using nitrocellulose-immobilized rat liver nuclear envelope proteins and nuclear import substrate as a ligand, we found Xenopus fraction A-dependent binding to at least three bona fide nucleoporins (Nup214, Nup153, and Nup98) and to a candidate nucleoporin with an estimated molecular mass of 270 kDa. We propose that these nucleoporins function as docking proteins for karyopherin-mediated binding of substrate in a nuclear import/export pathway across the nuclear pore complex.

Amino Acid Sequence↗

PubMed1995

Gene transfer to the thymus. A means of abrogating the immune response to recombinant adenovirus.

OBJECTIVE: The authors investigated whether adenoviral gene transfer to the thymus could be accomplished in vivo and whether immunologic unresponsiveness to recombinant adenovirus could be induced by intrathymic inoculation. SUMMARY BACKGROUND DATA: A major barrier to the clinical application of adenovirus-mediated gene therapy for diseases requiring long-lasting gene expression is the immunogenicity of adenoviral vectors, which limits the duration of its effects. In other experimental models, intrathymic inoculation of foreign proteins or cells has proven to be an effective means to induce immunologic tolerance. METHODS: The efficiency of gene transfer to the mouse thymus after direct inoculation of recombinant adenovirus was compared with that of several other vectors. Animals inoculated with adenovirus-infected pancreatic islets into the thymus were tested for unresponsiveness to the virus with a subsequent challenge of adenovirus administered into the liver by intravenous injection. RESULTS: Adenovirus accomplished highly efficient gene transfer to the thymus, unlike plasmid DNA, DNA-liposome complexes, retrovirus, and adeno-associated virus. Adenoviral transgene expression was transient in the thymus of immunocompetent mice but persistent in CD8+ T-cell-deficient and severe combined immunodeficiency (SCID) mice, implicating the role of cytotoxic T lymphocytes in viral clearance. Intrathymic transplantation of syngeneic pancreatic islet cells infected with adenovirus impaired the normal antiviral cytotoxic T-lymphocyte response and prolonged hepatic transgene expression after an intravenous challenge with adenovirus. CONCLUSIONS: Recombinant adenovirus accomplishes highly efficient gene transfer to the thymus in vivo. Intrathymic inoculation of adenovirus-infected islets can be used to induce immunologic unresponsiveness to the adenoviral vector and, potentially, to other proteins that it might be engineered to encode.

Adenoviridae↗

PubMed1994

Nup107 is a novel nuclear pore complex protein that contains a leucine zipper.

We have previously described procedures for the isolation of potential nuclear pore complex proteins (nucleoporins or Nups) from rat liver nuclear envelopes and their subsequent subfractionation into those binding to wheat germ agglutinin (WGA) and those that do not. One of these non-WGA-reactive proteins, termed Nup155, was previously molecularly cloned and sequenced and by immunoelectron microscopy shown to be a bona fide nucleoporin. Here we have characterized a second protein of the non-WGA-reactive type and show that it is a Nup as well. Molecular cloning and sequencing revealed that the protein has a calculated molecular mass of 107.2 kDa. It is, therefore, termed Nup107 (for nucleoporin of 107 kDa). Like other Nups that have so far been analyzed, Nup107 contains an abundance of kinase consensus sites. However, the primary structure of Nup107 is unique and not similar to other proteins in the data banks. Its most striking feature is a leucine zipper in its carboxyl-terminal region.

Amino Acid Sequence↗