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PubMed2004

Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm.

Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals.

6-Phytase↗
PubMed1996

Cutaneous cell and extracellular matrix responses to ultraviolet-B irradiation.

The present study examined fibroblasts and keratinocytes in monolayers and cultured within dermal and skin substitutes and their use in assessing the effect of UVB irradiation on cutaneous cells and extracellular matrix organization. Dermal substitutes (DS) were produced by incorporating normal fibroblasts into a collagen lattice and skin substitutes (SS) were obtained by seeding normal keratinocytes onto the DS. Keratinocyte monolayers, fibroblast monolayers, DS, and SS were exposed once a day to a UVB source (10 mJ/cm2). The irradiation protocol was stopped when the keratinocytes of the non-irradiated cultures (control groups) had reached confluence. Microscopic observations revealed that UVB radiation decreased both fibroblast and keratinocyte growth and enhanced their differentiation resulting in (1) less fibroblasts in the DS and SS, and (2) incomplete coverage of the DS by keratinocytes. Microscopic observations and histological analyses revealed major morphological changes. Both cell types became bigger and presented wide nuclei and vacuoles in the cytoplasm. No organized deep epidermal layer was observed in irradiated compared to non-irradiated SS. Irradiated DS and SS extracellular matrices showed an irregular aggregating collagen fiber organization with serious discrepancies suggesting large defects in the structural properties of the extracellular matrix. The present study demonstrated that exposure to a UVB source led to profound morphological and functional disturbances in both cutaneous cells and in the extracellular matrices of the DS and SS. The present technology would be of great interest for step-by-step studies of UVR effects on cutaneous cell morphology and functional properties, and could be an alternative to using animals for pharmacological and toxicological evaluations.

Cell Division↗
PubMed2004

Evaluation of a new generation of plastic culture bottles with an automated microbial detection system for nine common contaminating organisms found in PLT components.

BACKGROUND: A microbial detection system (BacT/ALERT 3D, bioMérieux [formerly Organon Teknika]) has previously been validated with a variety of bacterial contaminants in PLTs. The recovery of nine organisms seeded into PLTs with new plastic culture bottles was studied in comparison to the current glass bottles. The use of plastic instead of glass would be expected to reduce the risk of injury. STUDY DESIGN AND METHODS: Isolates of Bacillus cereus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus viridans, and Propionibacterium acnes were inoculated into Day 2 (>24 hr <48 hr) apheresis PLT units to 10 and 100 CFUs per mL. Replicate samples (4 mL) were inoculated into both current- and new-generation standard aerobic and anaerobic bottles. RESULTS: All organisms (with the exception of P. acnes) were detected in a mean time of 9.3 to 18.9 hours (10 CFUs/mL) or 8.7 to 18.2 hours (100 CFUs/mL). In aggregate (with the exception of P. acnes), the plastic and glass aerobic bottles had a mean difference in detection of 1.2 hours (p < 0.0001), and the plastic and glass anaerobic bottles had a mean difference of 3.3 hours (p < 0.0001). In all cases, the mean detection time was superior or clinically comparable (within 0.1 hr) with the new plastic bottles. P. acnes (an anaerobic organism) was detected with the new and current anaerobic bottles in a mean of 72.8 and 90.4 hours (10 CFUs/mL) or 64.0 and 80.8 hours (100 CFUs/mL), respectively. The narrower bottle neck and smaller inoculation septum present with the new-generation plastic bottles were inoculated with comparable ease to that of the glass bottles. CONCLUSIONS: These data demonstrate that the new plastic bottles are clinically comparable or superior to the current glass standard aerobic and anaerobic culture bottles.

Bacillus cereus↗
PubMed1986

Cultured heart cell reaggregates: a model for studying relationships between aging and lipid composition.

Cultured heart cells serve as a common model for studying the electronphysiology and pharmacology of intact cells of the myocardium from which they are derived (Sperelakis, N. (1982) in Cardiovascular Toxicology (Van Stel, E.W., ed.), pp. 57-108, Raven Press, New York). In this study, heart cell reaggregates were used for investigating the relationship between lipid composition and aging of the heart cells. Spherical reaggregates were prepared from newborn, 3- and 18-month-old rats, respectively. They were grown for 6 days in culture and then analyzed for their lipid composition and creatine phosphokinase levels. There was an age-related increase in total phospholipids and cholesterol level per unit of cell protein. Due to a relatively greater increase in the cholesterol, the mole ratio of cholesterol to phospholipids increased with animal age. The phospholipid composition was also affected. Thus, sphingomyelin levels increased, while those of phosphatidylcholine decreased; these alterations became much more pronounced with increasing animal age. All these changes could be affected by adding small unilamellar vesicles composed of egg phosphatidylcholine to the growth medium on the 5th day after seeding. Such treatment resulted in a lesser ratio of cholesterol to phospholipid as well as sphingomyelin to phosphatidylcholine, without reducing the total phospholipid per unit protein; the level of creatine phosphokinase was also reduced. This study demonstrated that cultured heart reaggregates can serve as a model for studying aging of the whole animal. Its main advantage is the ability to employ cells from rats of any desired age. Currently this is not possible for cultured heart monolayers.

Aging↗
PubMed2003

Formation of a membrane-active form of amyloid beta-protein in raft-like model membranes.

The conversion of soluble, nontoxic amyloid beta-protein (A beta) to aggregated, toxic A beta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. We have proposed that the aggregation proceeds in the lipid raft containing a ganglioside cluster, the formation of which is facilitated by cholesterol and for which A beta shows a specific affinity. In this study, using fluorescence resonance energy transfer, we found that after A beta binds to raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin (1/1/1), the protein can translocate to the phosphatidylcholine membranes to which soluble A beta does not bind. Furthermore, self-quenching experiments using fluorescein-labeled A beta revealed that the translocation process competes with the oligomerization of the protein in the raft-like membranes. These results suggest that the lipid raft containing a ganglioside cluster serves as a conformational catalyst or a chaperon generating a membrane-active form of A beta with seeding ability.

Alzheimer Disease↗
PubMed1965

Iodination in relation to thyroglobulin maturation and subunit aggregation.

Noniodinated subunits of thyroglobulin can aggregate, but iodination of the aggregate is required for its stabilization (maturation). Rat-thyroid slices incorporate amino acids into subunits, but cannot form mature thyroglobulin from the newly synthesized subunits. This defect leads to an accumulation of 16S and 12S proteins, although the preexisting thyroglobulin is 19S. Accumulation of 16S and 12S proteins can be produced in rat thyroids by the administration to the animals of a thiocarbamide derivative, methimazole. Upon withdrawal of methimazole, iodination of the 16S and 12S proteins proceeds, and 19S protein appears.

Animals↗
PubMed1997

Spatial Patterns of Rhizoplane Populations of Pseudomonas fluorescens.

Geostatistical analysis was used to compare rhizoplane colonization patterns of an antibiotic-producing biological control bacterium versus a non-antibiotic-producing mutant strain. Pea seeds were inoculated with Pseudomonas fluorescens 2-79RN(inf10) or P. fluorescens 2-79-B46 (a phenazine-deficient Tn5 mutant of P. fluorescens 2-79RN(inf10)) (10(sup8) CFU/pea), planted in sterile sand, and incubated at 20(deg)C. After 3 days, seedlings were prepared for scanning electron microscopy. Photomicrographs (x1,000) of the root surface were taken at the seed-root junction and at 0.5-cm intervals to the root tip. Bacterial counts on the root surface were made in 5- by 5-(mu)m sample units over an area which was 105 by 80 (mu)m. Coordinates and number of bacteria were recorded for each sample unit. Spatial statistics were calculated by covariance for the following directions: omnidirectional, 0, 45, 90, and 135(deg). The ranges of spatial influence and nugget (estimator of spatially dependent variation) were determined. For both P. fluorescens 2-79RN(inf10) and P. fluorescens 2-79-B46, spatial structure was evident along the entire root, particularly in the 0(deg) direction (along the root length) (e.g., range = 24 (mu)m, nugget = 0.52). The degree of spatial dependence observed indicated aggregation of bacterial cells. No differences were detected in the spatial patterns of colonies of P. fluorescens 2-79RN(inf10) and P. fluorescens 2-79-B46, indicating that the lack of phenazine production did not influence spatial patterns on the rhizoplane.

Journal Article↗
PubMed2002

Heterogeneous amyloid-formed ion channels as a common cytotoxic mechanism: implications for therapeutic strategies against amyloidosis.

The amyloidoses consist of human and animal chronic, progressive, and sometimes fatal diseases that are characterized by the deposition of insoluble proteinaceous amyloid fibrils in various tissues. Despite the biochemical diversity of amyloids, they share certain properties. The amphipathic and the charged nature of many amyloid-forming peptides point to their intrinsic ability to form diverse beta-sheet-based aggregates and channel types in negatively charged membranes. We hypothesize that the formation of heterogeneous channels represents a common cytotoxic mechanism that accentuates the changes in the signal transduction that underlie amyloid-induced cell malfunction. One group of amyloid-forming peptides that could mediate their action via the formation of heterogeneous channels includes the extensively examined prions and amyloid beta protein that are associated with conformational neurodegenerative diseases. The aim of this study is to examine heterogeneous channels formed in bilayers with amyloid-forming peptides as a common mechanism of malfunction of signal transduction. The observed amyloid-formed channel types include the following. (1) Natriuretic peptides: (i) 68-pS H2O2- and Ba2+-sensitive channel with fast kinetics. The fast channel had three modes (spike mode, burst mode, and open mode), which differ in their kinetics but not in their conductance properties; (ii) a 273-pS inactivating large conductance channel; and (iii) a 160-pS transiently activated channel. (2) Prions: (i) a 140-pS GSSG- and TEA-sensitive channel with fast kinetics; (ii) a 41-pS dithiothreitol (DTT)-sensitive channel with slow kinetics; (iii) a 900 to 1444-pS large channel. (3) Amyloid beta protein: (i) a 17 to 63-pS AbetaP[1-40]-formed "bursting" fast cation channel, (ii) the AbetaP[1-40]-formed "spiky" fast cation channel with a similar kinetics to the "bursting" fast channel except for the absence of the long intraburst closures, (iii) 275-pS AbetaP[1-40]-formed medium conductance channel, and (iv) 589- to 704-pS AbetaP[1-40]-formed inactivating large conductance channel. This heterogeneity is one of the most common features of these charged cytotoxic amyloid-formed channels, reflecting these channels' ability to modify multiple cellular functions. Although the diversity of these aggregated-peptide-formed channels may indicate that a stochastic mechanism governs their formation, the fact that certain channel types are often observed point to preferential channel protein conformations. In addition, the fact that other amyloids have similar structural properties (e.g. hydrophobicity, charged residues, and beta-structural linkages, suggests that, despite the intrinsic ability to form diverse conformations, certain conformations and, hence, certain channel types could be a common pathologic conformation among these amyloid-forming peptides. It is concluded that conformation-based channel diversity is an important mechanism for enhancing the toxicity of amyloid-forming peptides. The cytotoxic nature of these self-associated beta-based protein channels suggests that under normal physiological conditions cells employ well-evolved protective mechanisms against seeding and/or propagation of channel-forming peptides; for example, (a) compartmentalization of these peptides as membrane bound in internal vesicles and/or (b) degradation of these peptides by enzymes. The pharmacological diversity of the amyloid-forming channels implies that multiple therapeutic interventions may be necessary for blocking and reversing heterogeneous channel formations and preventing their associated diseases.

Amyloid↗
PubMedDate not supplied

Rheologic characterization of vegetal lectins by dissociation of induced erythrocyte agglutinates.

Energy evolved from hemagglutination reaction or spent in dissociating erythrocyte agglutinates has been proved to be an excellent parameter for analyzing cell-cell interactions mediated by bridging molecules such as antibodies or lectins. We developed a new rheo-optical method to estimate the energy of dissociation of red blood cell agglutinates. In a Couette shear field agglutinates can be dissociated until a suspension of monodispersed cells is obtained. Intensity of light backscattered by suspended agglutinates increases during their mechanical dissociation. Variation of backscattered light intensity correlates with the energy spent in the process. The adhesive energy of erythrocyte agglutination induced by lectins has been estimated by applying this method. Two specific lectins (Dolichus Biflorus agglutinin and Ulex Europaeus agglutinin) and a new lectin obtained from Amarantus Cruentus seeds which specificity is unknown were studied. Results obtained in this work for Dolichus Biflorus lectin are comparable with values published by other authors. An asymptotic decrease of adhesive energy was observed when the mechanical dissociation was applied several times on the same sample. Our results suggest that the cell detachment is accompanied by the extraction of membrane receptors. This finding is consistent with results obtained by other authors.

Energy Metabolism↗
PubMed2000

Bioglass 45S5 stimulates osteoblast turnover and enhances bone formation In vitro: implications and applications for bone tissue engineering.

We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.

Alkaline Phosphatase↗
PubMed2004

The interaction of Vatairea macrocarca and Rhizobium tropici: net H+ efflux stimulus and alteration of extracellular Na+ concentration.

We studied the effect of a lectin isolated from seeds of the legume Vatairea macrocarpa on net H+ efflux in Rhizobium tropici, a bacterium capable of nodulating legume Phaseolus vulgaris. V. macrocarpa lectin (VML) was observed to temporarily stimulate the specific net H(+) efflux in R. tropici. When VML was present at 32 microg ml(-1), with or without 2 microM galactose (Gal), a specific net efflux >2.4 pM H+(min)(-1) mg dry biomass(-1) was induced. There was no detectable net H+ efflux when bovine serum albumin (16 microg ml(-1)) was tested. Addition of 16 microgVMLml(-1) resulted in a 700% increase of the extracellular Na+ concentration. The soluble proteins in the supernatant containing VML extract indicate a maximum immobilization of +/-10 microgVMLml(-1), with a minimum of 36,600 dimers or 8500 larger aggregates of VML binding in each bacterium. Our data suggest that VML activates Rhizobium as a bioenergetic substrate molecule, resulting in potential alterations of the external bacterial membrane.

Dimerization↗
PubMed1996

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Amyloid beta-Peptides↗
PubMed2003

In vitro characterization of lactoferrin aggregation and amyloid formation.

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.

Amino Acid Sequence↗
PubMed1996

Inhibition of cultured cell growth by vascular endothelial cadherin (cadherin-5/VE-cadherin).

Endothelial cell proliferation is inhibited by the establishment of cell to cell contacts. Adhesive molecules at junctions could therefore play a role in transferring negative growth signals. The transmembrane protein VE-cadherin (vascular endothelial cadherin/cadherin-S) is selectively expressed at intercellular clefts in the endothelium. The intracellular domain interacts with cytoplasmic proteins called catenins that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton. Transfection of VE-cadherin in both Chinese hamster ovary (CHO) and L929 cells confers inhibition of cell growth. Truncation of VE-cadherin cytoplasmic region, responsible for linking catenins, does not affect VE-cadherin adhesive properties but abolishes its effect on cell growth. Seeding human umbilical vein endothelial cells or VE-cadherin transfectants on a recombinant VE-cadherin amino-terminal fragment inhibited their proliferation. These data show that VE-cadherin homotypic engagement at junctions participates in density dependent inhibition of cell growth. This effect requires both the extracellular adhesive domain and the intracellular catenin binding region of the molecule.

Animals↗
PubMed2002

Artocarpus hirsuta lectin. Differential modes of chemical and thermal denaturation.

Unfolding, inactivation and dissociation of the lectin from Artocarpus hirsuta seeds were studied by chemical (guanidine hydrochloride, GdnHCl) and thermal denaturation. Conformational transitions were monitored by intrinsic fluorescence and circular dichroism. The gradual red shift in the emission maxima of the native protein from 335 to 356 nm, change in the ellipticity at 218 nm and simultaneous decrease in the sugar binding activity were observed with increasing concentration of GdnHCl in the pH range between 4.0 and 9.0. The unfolding and inactivation by GdnHCl were partially reversible. Gel filtration of the lectin in presence of 1-6 m GdnHCl showed that the protein dissociates reversibly into partially unfolded dimer and then irreversibly into unfolded inactive monomer. Thermal denaturation was irreversible. The lectin loses activity rapidly above 45 degrees C. The exposure of hydrophobic patches, distorted secondary structure and formation of insoluble aggregates of the thermally inactivated protein probably leads to the irreversible denaturation.

Circular Dichroism↗
PubMed1994

Influence of Korean pine (Pinus koraiensis)-seed oil containing cis-5,cis-9,cis-12-octadecatrienoic acid on polyunsaturated fatty acid metabolism, eicosanoid production and blood pressure of rats.

The effects of dietary Korean pine (Pinus koraiensis)-seed oil containing a peculiar trienoic acid (cis-5,cis-9,cis-12-18:3, pinolenic acid, approximately 18%) on various lipid variables were compared in rats with those of flaxseed (Linum usitatissimum L.) oil, safflower (Carthamus tinctorius L.) oil and evening primrose (Oenothera biennis L.) oil under experimental conditions where the effects of different polyunsaturated fatty acids could be estimated. In Sprague-Dawley rats fed on diets containing 100 g fat and 5 g cholesterol/kg, the hypocholesterolaemic activity of pinolenic acid was intermediate between alpha-linolenic and linoleic acids. Analysis of the fatty acid composition of liver phosphatidylcholine indicated that, in contrast to alpha-linolenic acid, pinolenic acid does not interfere with the desaturation of linoleic acid to arachidonic acid. However, the effects on ADP-induced platelet aggregation and aortic prostacyclin production were comparable. When spontaneously hypertensive rats were fed on diets containing 100 g fat/kg but free of cholesterol, gamma-linolenic and pinolenic acids, as compared with linoleic acid, increased prostacyclin production and tended to reduce platelet aggregation. In addition, pinolenic acid attenuated the elevation of blood pressure after 5 weeks of feeding. Thus, the results of the present studies indicate the beneficial effects of pinolenic acid on various lipid variables.

Animals↗
PubMed2006

Experimental metastasis and primary tumor growth in mice with hemophilia A.

During experimental lung metastasis, tumor cells adhere to the pulmonary microvasculature and activate coagulation via surface-expressed tissue factor (TF), leading to local fibrin deposition and platelet aggregation. While interventional studies have demonstrated great efficacy of anticoagulants and antiplatelet agents in inhibiting metastasis, no information is available on how tumor biology may be affected by congenital bleeding disorders such as hemophilia A. We therefore used a syngeneic model to study experimental metastasis and primary tumor growth in factor VIII (FVIII)-deficient mice. By conventional reverse transcription-polymerase chain reaction, flow cytometry, and one-stage clotting assays, we demonstrated constitutive expression of TF mRNA, antigen, and procoagulant activity in the murine B16F10 melanoma cell line. In hemophilic mice, B16F10 lung metastasis was significantly (P < 0.001) enhanced by a single dose of human FVIII (100 U kg(-1)), suggesting that FVIII played a critical role during the early blood-borne phase of the metastatic cascade. In contrast, lung seeding was significantly (P < 0.05) reduced by lepirudin, a direct thrombin inhibitor, suggesting that thrombin generation contributed to pulmonary metastasis even in the absence of FVIII. Consistent with this finding, intravenous injection of B16F10 cell-evoked laboratory changes of a hemolytic thrombotic microangiopathy and consumptive coagulopathy in both hemophilic and non-hemophilic mice. Subcutaneous implantation of B16F10 cells into mice with hemophilia A gave rise to primary tumors in an exponential growth pattern similar to that observed in non-hemophilic mice. Although TF expression by B16F10 cells may promote thrombin-dependent metastasis in mice with hemophilia A, amplification of coagulation by host FVIII appears to be necessary for maximum lung seeding.

Animals↗
PubMed2006

Radioactive sputter cathodes for 32P plasma-based ion implantation.

The development of clinical treatments involving the use of beta-emitting millimetric and sub-millimetric devices has been a continuing trend in nuclear medicine. Implanted a few nanometers below the surface of endovascular implants, seeds or beads, beta-emitting radioisotopes can be used in a variety of biomedical applications. Recently, new technologies have emerged to enable the rapid and efficient activation of such devices. A pulsed, coaxial electron cyclotron resonance plasma reactor was designed and tested to demonstrate the feasibility of plasma-based radioactive ion implantation (PBRII). It has been shown that such plasma reactors allow for the implantation of radioisotopes (32P) into biomedical devices with higher efficiencies than those obtained with conventional ion beams. Fragments containing radioactive atoms are produced in the implanter by means of a negatively biased solid sputter cathode that is inserted into an argon plasma. Dilute orthophosphoric acid solutions (H3(32)PO4) are used for the fabrication of flat sputter targets, since they offer a high radioisotope content. However, the aggregation of the radioactive solute into highly hygroscopic ring-like deposits rather than flat, thin radioactive films is observed on certain substrates. This article describes the effect of this nonuniform distribution of the radioisotopes on the efficiency of PBRII, and presents a technique which enables a better distribution of 32P by coating the substrates with iron. The iron coating is shown to enable optimal radioisotope sputtering rates, which are essential in 32P-PBRII for the efficient activation of millimetric biomedical devices such as stents or coils.

Gold↗