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PubMed2003

Distinct organization of the candidate tumor suppressor gene RFP2 in human and mouse: multiple mRNA isoforms in both species- and human-specific antisense transcript RFP2OS.

In the present study, we describe the human and mouse RFP2 gene structure, multiple RFP2 mRNA isoforms in the two species that have different 5' UTRs and a human-specific antisense transcript RFP2OS. Since the human RFP2 5' UTR is not conserved in mouse, these findings might indicate a different regulation of RFP2 in the two species. The predicted human and mouse RFP2 proteins are shown to contain a tripartite RING finger-B-box-coiled-coil domain (RBCC), also known as a TRIM domain, and therefore belong to a subgroup of RING finger proteins that are often involved in developmental and tumorigenic processes. Because homozygous deletions of chromosomal region 13q14.3 are found in a number of malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), we suggest that RFP2 might be involved in tumor development. This study provides necessary information for evaluation of the role of RFP2 in malignant transformation and other biological processes.

Alternative Splicing↗
PubMed2004

Determination of the binding parameter constants of Renagel capsules and tablets at pH 7 by high performance capillary electrophoresis.

Sevelamer hydrochloride is a cross-linked polymeric amine; it is the active ingredient in Renagel capsules and tablets. Sevelamer hydrochloride is indicated for the control of hyperphosphatemia in patients with end-stage renal disease (ESRD). The binding parameter constants of sevelamer hydrochloride were determined using high performance capillary electrophoresis (HPCE) and the Langmuir approximation for three different dosage forms at pH 7.0. The three dosage forms were Renagel 403 mg capsules, Renagel 400 mg tablets and Renagel 800 mg tablets. The results demonstrate the in vitro bioequivalence of all three dosage forms at pH 7.0. These results are in very good agreement with previously published results obtained by ion chromatography.

Binding Sites↗
PubMed2004

Macular translocation in patients with recurrent subfoveal choroidal neovascularization after laser photocoagulation for nonsubfoveal choroidal neovascularization.

PURPOSE: To report visual outcomes and to examine surgical factors affecting outcomes in patients undergoing macular translocation for recurrent subfoveal choroidal neovascularization after laser photocoagulation for nonsubfoveal choroidal neovascularization. DESIGN: Retrospective, noncomparative, interventional case series. PARTICIPANTS: A consecutive series of 31 eyes of 29 patients who underwent macular translocation for recurrent subfoveal choroidal neovascularization after laser photocoagulation for nonsubfoveal choroidal neovascularization. INTERVENTION: Inferior macular translocation with punctate retinotomy performed by a single surgeon. OUTCOME MEASURES: Surgical and visual outcomes at 3 and 6 months after surgery and complications data are reported. Associations between surgical factors and visual outcomes were analyzed statistically. RESULTS: Effective translocation was achieved in 77.4% of eyes. At 6 months, 54% of eyes achieved visual acuity (VA) better than 20/100, and 46% of eyes gained the equivalent of > or =2 Early Treatment Diabetic Retinopathy Study lines of vision. No association between size of recurrent choroidal neovascularization and visual outcome was identified. Eyes with a larger scar size experienced lower VA at 3 and 6 months, but scar size was not associated with change in VA at 3 and 6 months. Subretinal dissection during surgery to detach the macula was required in 8 of 31 eyes and was associated with a significantly increased incidence of peripheral retinal breaks. However, there was no difference in either VA or change in VA in eyes with and without subretinal dissection. Retinal detachment (RD) occurred in 6 of 31 eyes. No significant difference in the RD rate was observed between groups with or without subretinal dissection (P = 0.30). CONCLUSION: Our pilot data suggest that macular translocation can result in favorable surgical outcomes in patients with recurrent subfoveal choroidal neovascularization after laser photocoagulation for nonsubfoveal choroidal neovascularization. Use of subretinal dissection intraoperatively in these patients does not seem to affect visual outcome adversely, but may be associated with increased risk of peripheral retinal breaks.

Adult↗
PubMed2006

Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition.

Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong beta-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.

Adenocarcinoma↗
PubMed2003

Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes.

BACKGROUND: Myocardial infarction results in the death of cardiomyocytes, which are replaced by scar tissue. Cardiomyocytes cannot regenerate because they are terminally differentiated. Mesenchymal cells are pluripotent cells, which have the potential to differentiate to specialized tissues under appropriate stimuli. The aim of this study was to direct differentiation of the adult mesenchymal stem cells isolated from fatty tissue into cardiomyocytes using 5-azacytidine. METHODS: Adult mesenchymal stem cells were isolated from the fatty tissue of New Zealand White rabbits and cultured in RPMI medium. Second-passaged mesenchymal cells were treated with various concentrations of 5-azacytidine and incubated for different intervals of time. The cells were plated in six-well dishes at 500, 5,000, and 50,000 cells/well. These cells were treated with 1-, 3-, 6-, 9-, and 12-micromol/L concentrations of 5-azacytidine and incubated for 12, 24, 48, and 72 hours. Later, the medium was replaced with fresh medium and incubated in a CO2 incubator. The medium was changed once at 3 to 4 days. At 2 months, the cells were fixed with 0.4% glutaraldehyde for 2 hours and later washed with phosphate-buffered saline. The transformed cells were subjected to immunostaining for the myosin heavy chain, alpha actinin, and troponin-I. RESULTS: After treatment with 5-azacytidine, the adult mesenchymal stem cells were transformed into cardiomyocytes. At 1 week, some cells showed binucleation and extended cytoplasmic processes with adjacent cells. At 2 weeks, 20% to 30% of the cells increased in size and formed a ball-like appearance. At 3 weeks, these cells began to beat spontaneously in culture when observed under phase contrast microscope. Immunostaining of the transformed cells for myosin heavy chain, alpha actinin, and troponin-I was positive. The differentiated cells maintained the phenotype and did not dedifferentiate up to 2 months after treatment with 5-azacytidine. CONCLUSIONS: These observations confirm that adult mesenchymal stem cells isolated from fatty tissue can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.

Actinin↗
PubMed2002

Direct molecular dynamics observation of protein folding transition state ensemble.

The concept of the protein transition state ensemble (TSE), a collection of the conformations that have 50% probability to convert rapidly to the folded state and 50% chance to rapidly unfold, constitutes the basis of the modern interpretation of protein engineering experiments. It has been conjectured that conformations constituting the TSE in many proteins are the expanded and distorted forms of the native state built around a specific folding nucleus. This view has been supported by a number of on-lattice and off-lattice simulations. Here we report a direct observation and characterization of the TSE by molecular dynamic folding simulations of the C-Src SH3 domain, a small protein that has been extensively studied experimentally. Our analysis reveals a set of key interactions between residues, conserved by evolution, that must be formed to enter the kinetic basin of attraction of the native state.

Computer Simulation↗
PubMed2002

Thermodynamics and folding kinetics analysis of the SH3 domain form discrete molecular dynamics.

We perform a detailed analysis of the thermodynamics and folding kinetics of the SH3 domain fold with discrete molecular dynamic simulations. We propose a protein model that reproduces some of the experimentally observed thermodynamic and folding kinetic properties of proteins. Specifically, we use our model to study the transition state ensemble of the SH3 fold family of proteins, a set of unstable conformations that fold to the protein native state with probability 1/2. We analyze the participation of each secondary structure element formed at the transition state ensemble. We also identify the folding nucleus of the SH3 fold and test extensively its importance for folding kinetics. We predict that a set of amino acid contacts between the RT-loop and the distal hairpin are the critical folding nucleus of the SH3 fold and propose a hypothesis that explains this result.

Amino Acid Sequence↗
PubMed2002

Molecular dynamics simulation of the SH3 domain aggregation suggests a generic amyloidogenesis mechanism.

We use molecular dynamics simulation to study the aggregation of Src SH3 domain proteins. For the case of two proteins, we observe two possible aggregation conformations: the closed form dimer and the open aggregation state. The closed dimer is formed by "domain swapping"-the two proteins exchange their RT-loops. All the hydrophobic residues are buried inside the dimer so proteins cannot further aggregate into elongated amyloid fibrils. We find that the open structure-stabilized by backbone hydrogen bond interactions-packs the RT-loops together by swapping the two strands of the RT-loop. The packed RT-loops form a beta-sheet structure and expose the backbone to promote further aggregation. We also simulate more than two proteins, and find that the aggregate adopts a fibrillar double beta-sheet structure, which is formed by packing the RT-loops from different proteins. Our simulations are consistent with a possible generic amyloidogenesis scenario.

Amino Acid Sequence↗
PubMed2002

A new 25-gauge instrument system for transconjunctival sutureless vitrectomy surgery.

OBJECTIVES: To introduce and evaluate the infusion and aspiration rates and operative times of the 25-gauge transconjunctival sutureless vitrectomy system (TSV) DESIGN: In vitro experimental and comparative interventional study. PARTICIPANTS AND CONTROLS: Twenty eyes of 20 patients underwent a variety of vitreoretinal procedures using the 25-gauge TSV, including idiopathic epiretinal membrane (n = 10), macular hole (n = 4), rhegmatogenous retinal detachment (n = 3), branch retinal vein occlusion (n = 2), diabetic vitreous hemorrhage (n = 1), and 20 cases similar in diagnosis and severity were matched to provide comparison between duration of individual portions of the surgical procedures with the existing 20-gauge vitrectomy system. METHODS: Description of the 25-gauge TSV is provided; infusion and aspiration rates of the 25-gauge and standard 20-gauge vitrectomy system were measured in vitro using balanced saline solution and porcine vitreous for several levels of aspirating power and bottle height, and operating times of individual portions of surgical procedures were measured for the 25-gauge and 20-gauge vitrectomy system. MAIN OUTCOME MEASURES: Infusion, aspiration rates, and operative times of the 20-gauge and 25-gauge vitrectomy system. RESULTS: Infusion and aspiration rates of the 25-gauge TSV system were reduced by an average of 6.9 and 6.6 times, respectively, compared with the 20-gauge system when balanced saline solution was used. The average flow rate of the Storz 25-gauge cutter (at 500 mmHg, 1500 cuts per minute [cpm]) was 40% greater than that of the 20-gauge pneumatic cutter (at 250 mmHg, 750 cpm) but about 2.3 times less than the 20-gauge high-speed cutter (at 250 mmHg, 1500 cpm). Mean total operative time was significantly greater for the 20-gauge high-speed cutter (26 minutes, 7 seconds) than for the 25-gauge vitrectomy system (17 minutes, 17 seconds) (P = 0.011). CONCLUSIONS: Although the infusion and aspiration rates of the 25-gauge instruments are lower than those for the 20-gauge high-speed vitrectomy system, the use of 25-gauge TVS may effectively reduce operative times of select cases that do not require the full capability of conventional vitrectomy.

Diabetes Complications↗
PubMed2002

Changes of major carotenoids in gonads of sea urchins (Strongylocentrotus intermedius and S. nudus) at maturation.

Changes in the carotenoid content in gonads of two sea urchins species were investigated during maturation. The content of echinenones and carotenes, the two major carotenoid fractions in gonads, is highest for Strongylocentrotus intermedius at the spawning gametogenic stage of gonad maturation for both sexes. For S. nudus, the content of these pigments is highest at stages of active gametogenesis and spawning for males and at the growth stage for females. A comparison of the carotenoid content dynamics during maturation of gonads for males, females and animals at the resting (sexual inactivity) stage was also carried out.

Animals↗
PubMed2004

Outcome of craniopharyngioma in children: long-term complications and quality of life.

Childhood craniopharyngiomas are histologically benign tumours arising from remnants of Rathke's pouch in the hypothalamic-pituitary region. The two common treatment approaches are primary total resection or limited resection followed by radiotherapy. To study the outcome after a primary surgical approach, we followed 25 consecutive patients (10 females, 15 males) under 16 years of age who were treated in a single institution with a management policy of radical tumour excision (mean age at diagnosis 9 years 2 months, SD 4 years 3 months; range 2 years 9 months to 15 years 11 months). Mean follow-up after primary surgery was 11 years 3 months (SD 7 years 7 months). Tumour control, and neurological, endocrine, and hypothalamic complications and their impact on health-related quality of life were assessed (medical follow-up, semi-structured interview, and questionnaires). Results of tumour control were generally good, however, local failure was observed in 6 of 25 patients, and severe late-treatment complications decreased quality of life for many long-time survivors. Endocrine deficiency occurred in 24/25, visual complications in 16/24, neurological complications in 8/24, obesity in 14/23, increased daytime sleepiness in 6/21, and significant school problems in 10/20. Patients with craniopharyngioma rated their health-related quality of life as considerably lower than healthy controls; the domains of social and emotional functioning were particularly affected. Parents' ratings were considerably lower than those of the patients. Poor functional outcome was associated with large tumours infiltrating or displacing the hypothalamus, the occurrence of hydrocephalus, and young age at diagnosis, but also with multiple operations due to tumour recurrence. Alternative treatment strategies should be considered, especially in very young patients with large tumours.

Activities of Daily Living↗