Magnetic Particle Concentrator Research Articles

Effects of typical PKC subtypes on the proliferation of mouse pulmonary artery smooth muscle cells and the expression of ERK1/2 and Akt induced by hypoxia

Objective: To study the effects of specific isoforms of classic protein kinase C (cPKCs) on hypoxia-induced proliferation and the expression of ERK1/2 and Akt using drug intervention or virus transfection in vitro. Methods: Dynal MPC-1 magnetic particle concentrator was used to separate iron-containing pulmonary arterioles fragments, and the pulmonary artery smooth muscle cells (PASMCs) were primary cultured and identified. The cells were intervened by PKC agonist (PMA), PKCα inhibitor (safingol), PKCβⅠ inhibitor (Go6976) and PKCβⅡ inhibitor (LY333531) respectively, and the changes in protein expressions of cPKCs, and the phosphorylation levels of ERK1/2 and Akt were observed by immunoblotting under the condition of normal oxygen or hypoxia. The lentiviral vectors of PKCα and PKCβ were used to specifically knock-down the activity of target genes by virus transfection techniques, and Western blotting was used to observe the protein expressions of cPKCs, and the phosphorylation levels of ERK1/2 and Akt in hypoxia-induced PASMCs in mice. Results: With Brdu method, the proliferation of PASMCs induced by hypoxia was significantly inhibited by safingol, Go6976 and LY333531 by inhibiting cPKCα, βⅠ and βⅡ respectively. Compared with the hypoxic control group, the rates of Brdu positive cells were (7.35±0.26)% vs (11.28±0.43)%, (3.76±0.25)% vs (7.98±0.28)% and (4.12±0.46)% vs (7.78±0.53)%. We also observed that PMA could significantly promote the proliferation of PASMCs under normoxic condition. Compared with the normoxia control group, the Brdu-positive cell rates were (9.65±0.47)% vs (6.34±0.52)%, (9.34±0.38)% vs (5.42±0.21)% and (7.78±0.53)% vs (4.12±0.46)%. In addition, after transfection with PKCα or PKCβ lentiviral vector, the proliferation of PASMCs was significantly lower in hypoxia transfection group than in the control group. The rates of Brdu positive cells were (3.58±0.54)% vs (5.97±0.63)%, respectively. Using Western blotting, we also observed that after being inhibited by safingol, Go6976 and LY333531 respectively, the phosphorylation levels of ERK1/2 and Akt in PASMCs induced by hypoxia was significantly lower than the control group. After using safingol, the phosphorylation levels of ERK1/2 and Akt were (0.56±0.07) vs (1.08±0.13) and (0.49±0.04) vs (0.97±0.08). After using Go6976, the phosphorylation levels of ERK1/2 and Akt were (0.41±0.09) vs (0.79±0.10) and (0.48±0.09) vs (0.82±0.16), after using LY333531, the phosphorylation levels of ERK1/2 and Akt were (0.42±0.03) vs (0.87±0.06) and (0.34±0.07) vs (0.78±0.05). While PMA could promote the phosphorylation levels of ERK1/2 and Akt under normoxic condition, 1.25±0.12 vs 0.41±0.07 and 0.98±0.06 vs 0.37±0.08, respectively. Using transfection technique to specifically knock down the expression of cPKCα and β, we found that under hypoxic conditions, transfection of PASMCs could significantly lower the phosphorylation levels of ERK1/2, its phosphorylation level was 0.29±0.06 vs 0.76±0.05, with no evident change in the phosphorylation levels of Akt. Conclusions: Hypoxia may lead to phosphorylation of ERK1/2 by promoting the protein expression of cPKCα, cPKCβⅠ and cPKCβⅡ respectively, which eventually induces abnormal proliferation of PASMCs from the distal pulmonary arteries, participating in the development of hypoxic pulmonary hypertension (HPH) of the mice. Regulation of the expression of cPKCα, cPKCβⅠ and cPKCβⅡ may help to attenuate the formation of pulmonary vascular remodeling. Target therapy based on cPKCs is expected to be a new direction for HPH therapy in the future.

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Protein Kinase G-I Deficiency Induces Pulmonary Hypertension through Rho A/Rho Kinase Activation

Protein kinase G (PKG) plays an important role in the regulation of vascular smooth cell contractility and is a critical mediator of nitric oxide signaling, which regulates cardiovascular homeostasis. PKG-I-knockout (Prkg1(-/-)) mice exhibit impaired nitric oxide/cGMP-dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG-I deficiency induces pulmonary hypertension. In this study, we characterized the hypertensive pulmonary phenotypes in Prkg1(-/-) mice and delineated the underlying molecular basis. We observed a significant increase in right ventricular systolic pressure in Prkg1(-/-) mice in the absence of systemic hypertension and left-sided heart dysfunction. In addition, we observed marked muscularization of distal pulmonary vessels in Prkg1(-/-) mice. Microangiography revealed impaired integrity of the pulmonary vasculature in Prkg1(-/-) mice. Mechanistically, PKG-I-mediated phosphorylation of Rho A Ser188 was markedly decreased, and the resultant Rho A activation was significantly increased in Prkg1(-/-) lung tissues, which resulted in Rho kinase activation. The i.t. administration of fasudil, a Rho kinase inhibitor, reversed the hypertensive pulmonary phenotype in Prkg1(-/-) mice. Taken together, these data show that PKG-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation-mediated vasoconstriction and pulmonary vascular remodeling.

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Protein Complex Binding to Promoter DNA: Immobilized Template Assay

INTRODUCTIONLarge multiprotein complexes involved in transcription generally cannot be resolved by the methods conventionally used to study cooperative DNA binding by smaller proteins. The immobilized template recruitment assay can be used to circumvent this limitation. In this procedure, a biotinylated DNA fragment is immobilized on streptavidin-coated magnetic beads. DNA-binding proteins are incubated with the immobilized template, and proteins not specifically assembled in a complex are removed by separating the immobilized template with a magnetic particle concentrator and subsequent washing of the magnetic bead/template pellet. Bound proteins are detected by immunoblotting. To detect cooperative binding, subsaturating amounts of two or more proteins or protein complexes are added to the immobilized template. Template binding of a protein in concert with its cooperative binding partner is then compared to the binding of each individual protein or protein complex alone. In addition to studying the binding of protein complexes, the immobilized promoter template can also be used in functional assays such as in vitro transcription, allowing a correlation of complex assembly with function.

Flow-through immunomagnetic separation system for waterborne pathogen isolation and detection: Application to Giardia and Cryptosporidium cell isolation

Simultaneous sample washing and concentration of two waterborne pathogen samples were demonstrated using a rotational magnetic system under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to a fluidic channel carrying magnetic particle suspension allows the trapping and release of particles along the fluidic channel in a periodic manner. Each trapping and release event resembles one washing cycle. The performance of the magnetic separation system (MSS) was evaluated in order to test its functionality to isolate magnetic-labelled protozoan cells from filtered, concentrated tap water, secondary effluent water, and purified water. Experimental protocols described in US Environmental Protection Agency method 1623 which rely on the use of a magnetic particle concentrator, were applied to test and compare our continuous flow cell separation system to the standard magnetic bead-based isolation instruments. The recovery efficiencies for Giardia cysts using the magnetic tube holder and our magnetic separation system were 90.5% and 90.1%, respectively, from a tap water matrix and about 31% and 18.5%, respectively, from a spiked secondary effluent matrix. The recovery efficiencies for Cryptosporidium cells using the magnetic tube holder and our magnetic separation system were 90% and 83.3%, respectively, from a tap water matrix and about 38% and 36%, respectively, from a spiked secondary effluent matrix. Recoveries from all matrices with the continuous flow system were typically higher in glass tubing conduits than in molded plastic conduits.

Effects of Increased Renal Tubular Vascular Endothelial Growth Factor (VEGF) on Fibrosis, Cyst Formation, and Glomerular Disease

The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

Selective enrichment of human DNA from non-human DNAs for DNA typing of decomposed skeletal remains

The degraded or decomposed body remains pose serious challenges for successful short tandem repeat (STR)-based DNA typing for human identification because of contamination with non-human DNAs and environment-induced changes. Owing to their tropical environment, which results in robust microbial infestation, obtaining good quality and quantity of human DNA for body identification by DNA profiling has not been very successful in those regions. To address this issue, we attempted to selectively recover the human DNA from the mixture of non-human DNAs by employing the biotinylated oligonucleotides, designed complementary to the regions flanking the STR loci to selectively capture the respective STR loci that are used for DNA profiling. Initially, the fragmented human DNA was hybridized with the biotinylated oligos, followed by conjugation of biotin with streptavidin-coated paramagnetic beads. After extensive washing to remove non-specific binding, repeat-containing DNA fragments were eluted employing a magnetic particle concentrator. Such repeat-enriched DNA fragments were subjected to PCR amplification to analyze STRs employing commercial human DNA typing kits. Our preliminary analysis has shown that there was selective recovery of STR-containing human DNAs when biotinylated oligos were used.

Presence of Lunasin in Plasma of Men after Soy Protein Consumption

Lunasin is a 43-amino acid bioactive peptide from soybean and other plant sources which is reported to possess anti-inflammatory and anticancer properties. The objective of this study was to assess the presence and concentration of lunasin in blood of men fed soy protein products. Five healthy male subjects who were 18-25 years old consumed 50 g of soy protein for 5 days, and blood was taken 30 min and 1 h after soy protein ingestion on day 5. Lunasin was isolated from plasma using strong anion exchange beads in a magnetic particle concentrator and eluted with 20 mM triethanolamine at pH 8.0 with 0.20 M NaCl. The concentration of lunasin in plasma as determined by an enzyme-linked immunosorbent assay ranged in the various subjects from 50.2 to 110.6 ng/mL of plasma (average +/- standard deviation, 66.0 +/- 25.4 ng/mL) for blood taken at 30 min and from 33.5 to 122.7 ng/mL of plasma (71.0 +/- 32.8 ng/mL) for blood withdrawn 1 h after ingestion on day 5. We estimated an average of 4.5% absorption (range of 2.2-7.8%) of lunasin from the total lunasin ingested from 50 g of soy protein. Matrix-assisted laser desorption ionization time-of-flight peptide mass mapping showed that a 5 kDa peptide similar to synthetic lunasin was present in plasma samples of people who consumed soy protein while absent at the baseline plasma samples from the same individuals. Liquid chromatography-tandem mass spectrometry analysis showed the presence of amino acid sequences from lunasin in plasma samples after soy intake for 30 min and 1 h. No peptides from lunasin were present in plasma samples without soy intake. The results of this study suggest that lunasin is bioavailable in humans, an important requirement for its anticancer potential.

Selective Incorporation of Polyanionic Molecules into Hamster Prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro. Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.

A rapid gel agglutination test for the determination of fetomaternal haemorrhage

Determination of fetomaternal haemorrhage (FMH) remains an area of difficulty. In most cases, prophylactic Rh immunoglobulin is usually administered to affected women without testing for foetal red blood cells (RBC). Here, we describe a new particle gel immunoassay (PaGIA) for the determination FMH (FMH-PaGIA). Superparamagnetic particles were coated with monoclonal anti-D and mixed with ethylenediaminetetraacetic acid-anticoagulated blood samples from D-negative pregnant women. The particles were isolated using a magnetic particle concentrator and then placed into the reaction chamber of a gel card. Agglutinated particles on top or dispersed through the gel matrix indicated the presence of D-positive cells. After the test was adapted to detect >or=0.3% D-positive RBC, randomly selected postpartum samples from 208 women were analysed in parallel with the Kleihauer-Betke test (KBT). In addition, all discrepancies were further analysed by flow cytometry. A total of 203 of the 208 postpartum samples were negative in both tests. One sample reacted positive with both assays. Two samples were strongly positive in the new FMH-PaGIA, but negative in the KBT. A serological re-examination revealed that both women were D positive. The KBT gave a false-positive result in two cases because of hereditary persistence of haemoglobin F. The new test is specific, easy to perform and can be done at any time in all laboratories.

Role of pigment epithelium-derived factor on proliferation and migration of choroidal capillary endothelium induced by vascular endothelial growth factor in vitro

Pigment epithelium-derived factor (PEDF) is expressed in several normal organs and identified as an inhibitor of neovascularization. In the present study, we investigated the effect of PEDF in an in vitro model of ocular choroidal neovascularization. Microdissection was used to isolate the human choroidal endothelial cells (CECs), followed by the use of superparamagnetic beads (Dynabeads) coated with the CD31 antibody, which selectively binds to the endothelial cell surface. The mitogenic and motogenic effects of vascular endothelial growth factor (VEGF) on cultured choroidal capillary endothelial cells were examined in the presence or absence of PEDF (1, 10, 100, and 1000 ng/ml) using cell counts and migration assays. Cells bound to the beads were isolated using a magnetic particle concentrator and they were successfully cultured and characterized to be endothelial cells that possessed greater than 95% immunoreactivity to von Willebrand factor. PEDF suppressed the proliferation and migration of VEGF-induced choroidal capillary endothelial cells. However, the concentration of PEDF which we used has little effect on normal CECs. PEDF played an important role on the growth and migration of VEGF-stimulated choroidal endothelial cell. These findings suggest that PEDF may be an effective approach to the treatment of choroidal neovascular disorders.

Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein

The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [(35)S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H(2)O(2) and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [(35)S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H(2)O(2) and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to approximately 75 and approximately 50% of control, respectively. In addition, the effect of H(2)O(2) was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.

Disease-related Prion Protein Forms Aggresomes in Neuronal Cells Leading to Caspase Activation and Apoptosis

The molecular basis for neuronal death in prion disease is not established, but putative pathogenic roles for both disease-related prion protein (PrP(Sc)) and accumulated cytosolic PrP(C) have been proposed. Here we report that only prion-infected neuronal cells become apoptotic after mild inhibition of the proteasome, and this is strictly dependent upon sustained propagation of PrP(Sc). Whereas cells overexpressing PrP(C) developed cytosolic PrP(C) aggregates, this did not cause cell death. In contrast, only in prion-infected cells, mild proteasome impairment resulted in the formation of large cytosolic perinuclear aggresomes that contained PrP(Sc), heat shock chaperone 70, ubiquitin, proteasome subunits, and vimentin. Similar structures were found in the brains of prion-infected mice. PrP(Sc) aggresome formation was directly associated with activation of caspase 3 and 8, resulting in apoptosis. These data suggest that neuronal propagation of prions invokes a neurotoxic mechanism involving intracellular formation of PrP(Sc) aggresomes. This, in turn, triggers caspase-dependent apoptosis and further implicates proteasome dysfunction in the pathogenesis of prion diseases.

High factor VIII (FVIII) levels in venous thromboembolism: role of unbound FVIII.

Theoretically, von Willebrand factor (VWF) should be capable of binding all factor VIII (FVIII), but an unbound FVIII (uFVIII) plasma fraction remains. In patients' status post deep-vein thrombosis (DVT), an altered uFVIII fraction and high FVIII levels might be indicative of dysfunctional FVIII regulation. Out of 928 consecutive DVT patients, 321 were found to have high FVIII levels. After excluding 183 patients with known causes for high FVIII levels, plasma samples with unexplainably high FVIII levels were available from 84 patients. To capture the FVIII-VWF-complex, superparamagnetic polystyrene beads with covalently attached streptavidin were coated with biotinylated anti-rabbit Ig and incubated with rabbit anti-human VWF-Ig. Slowly thawed plasma samples were added to cooled beads, which were then separated by a magnetic particle concentrator. The uFVIII fraction was calculated by dividing the FVIII activity in the supernatant of the FVIII-VWF-complex-free sample by the FVIII activity in the supernatant of the control sample. Additionally, the VWF residuum in the supernatant was determined. Compared to age- and sex-matched blood donors, thrombosis patients showed a significantly higher plasma FVIII/VWF ratio (median: 1.3 vs. 1.0, p<0.001). uFVIII fraction data were adjusted for VWF residuum. After forward stepwise logistic regression, uFVIII had an odds ratio of 0.48 (95% CI 0.34-0.65), i.e. the uFVIII fraction was reduced in thrombosis patients. Analysis of covariance confirmed these results: In thrombosis patients, the estimated mean of the uFVIII fraction was significantly lower (6.34% vs. 7.58%, p<0.001). In conclusion, thrombosis patients with high FVIII levels showed a higher FVIII/VWF ratio, similar to mice with defective FVIII clearance. The clearly reduced uFVIII fraction lends further support to the hypothesis of a modified FVIII clearance.

THE USE OF TIME‐RESOLVED FLUOROIMMUNOASSAY TO SIMULTANEOUSLY DETECT ESCHERICHIA COLI O157:H7, SALMONELLA ENTERICA SEROVAR TYPHIMURIUM AND SALMONELLA ENTERICA SEROVAR ENTERIDITIS IN FOODS1

Immunomagnetic beads (IMB) were used to capture and concentrate Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteriditis. The captured bacteria were allowed to form sandwiched complexes with europium (Eu) labeled anti‐E. coli O157 antibodies and/or samarium (Sm) labeled anti‐Salmonella antibodies. After washing the complexes utilizing a magnetic particle concentrator (MPC), the lanthanide labels were extracted from the cell/antibody complexes utilizing PerkinElmer Wallac Enhancement Solution (PerkinElmer Wallac, Turku, Finland) which causes the release of the Eu or Sm from the antibodies and the formation of highly fluorescent Eu‐(2‐NTA)3(TOPO)2–3 or Sm‐(2‐NTA)3(TOPO)2,3, micellar complexes. The specific time‐resolved fluorescence (TRF) associated with Eu and Sm were measured to estimate the quantity of E. coli and Salmonella, respectively. The sensitivity of the method was in the range of 103 to 104 CFU/mL for pure cultures of E. coli O157:H7 and Salmonella enterica serovar Typhimurium or Salmonella enterica serovar Enteriditis. The approach of applying sandwich complexes yielded a minimum cross‐reactivity with non‐targeted bacteria. After an enrichment of spiked hamburger, turkey or pork for 4.5 h at 37C in EC medium containing novobiocin, one CFU/g of either E. coli or Salmonella or both was detected. The results demonstrated that low levels of E. coli O157:H7, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteriditis in foods could be detected alone or in‐pair by the TRF approach within 8h.