Incubation In PBS Research Articles

Bone Regeneration in a Rabbit Critical-Sized Calvarial Model Using Tyrosine-Derived Polycarbonate Scaffolds

Porous three-dimensional tyrosine-derived polycarbonate (TyrPC) scaffolds with a bimodal pore distribution were fabricated to mimic bone architecture using a combination of salt-leaching and phase separation techniques. TyrPC scaffolds degraded in register with bone regeneration during the 6-week study period and compressive moduli of the scaffolds were maintained >0.5 MPa at 6 weeks of incubation in PBS at 37 °C. The TyrPC scaffolds either unsupplemented or supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) were implanted in a rabbit calvarial critical-sized defect (CSD) model and the TyrPC scaffolds treated with rhBMP-2 or TyrPC coated with calcium phosphate scaffold alone promoted bone regeneration in a rabbit calvarial CSD at 6 weeks postimplantation. A synthetic TyrPC polymeric scaffold either without a biological supplement or with a minimal dose of rhBMP-2 induced bone regeneration comparable to a commercially available bone graft substitute in a nonrodent CSD animal model.

Submicromolar Aβ42 reduces hippocampal glutamate receptors and presynaptic markers in an aggregation-dependent manner

Synaptic pathology in Alzheimer's disease brains is thought to involve soluble Aβ42 peptide. Here, sterile incubation in PBS caused small Aβ42 oligomer formation as well as heterogeneous, 6E10-immunopositive aggregates of 80–100 kDa. The high molecular weight aggregates (H-agg) formed in a time-dependent manner over an extended 30-day period. Interestingly, an inverse relationship between dimeric and H-agg formation was more evident when incubations were performed at 37 °C as compared to 23 °C, thus providing an experimental strategy with which to address synaptic compromise produced by the different Aβ aggregates. H-agg species formed faster and to higher levels at 37 °C compared to 23 °C, and the two aggregate preparations were evaluated in hippocampal slice cultures, a sensitive system for monitoring synaptic integrity. Applied daily at 80–600 nM for 7 days, the Aβ42 preparations caused dose-dependent and aggregation-dependent declines in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl- d-aspartate (NMDA) receptor subunits as well as in presynaptic components. Unlike the synaptic effects, Aβ42 induced only trace cellular degeneration that was CA1 specific. The 37 °C preparation was less effective at decreasing synaptic markers, corresponding with its reduced levels of Aβ42 monomers and dimers. Aβ42 dimers decayed significantly faster at 37 °C than 23 °C, and more rapidly than monomers at either temperature. These findings indicate that Aβ42 can self-aggregate into potent synaptotoxic oligomers as well as into larger aggregates that may serve to neutralize the toxic formations. These results will add to the growing debate concerning whether high molecular weight Aβ complexes that form amyloid plaques are protective through the sequestration of oligomeric species.

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na +–K +–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.

Proliferation of Chondrocytes on a 3-D Modelled Macroporous Poly(Hydroxyethyl Methacrylate)–Gelatin Cryogel

Tissue-engineering constructs should be designed to mimic the native tissue environment for cells, the scaffold matching to stiffness and strength of the tissues while maintaining an interconnected porous network and a reasonable porosity. This study presents a new single-step protocol for synthesis of a poly(hydroxyethyl methacrylate)–poly(ethylene glycol) diacrylate–gelatin (HPG) macroporous polymeric scaffold with well-controlled porous structure and good mechanical strength. The pore size of these matrices lies in the range of 30 to 100 μm with an average pore diameter of 80 μm and with an interconnected pore structure as analyzed by scanning electron microscopy. Further, interconnectivity was also confirmed by high solvent uptake capacity, as the cryogel reached its equilibrium within 2 min. The gels also showed substantial mechanical integrity, i.e., the average compressive modulus was 32.73 ± 2.36 kPa at 15% compression of their original length. The degree of weight loss of these cryogels was found to be approx. 88% within 8 weeks of incubation in PBS (pH 7.4) at 37°C. Physio-chemically optimized cryogel was further evaluated for in vitro growth and proliferation of isolated primary goat chondrocytes up to 3 weeks. The cell adherence on cryogel was examined by SEM analysis, while cell–matrix interaction was examined by 4-6-diamidino-2-phenylindole and propidium iodide staining. Furthermore, the cell compatibility and proliferation was evaluated using the MTT assay. Increase in total cellular metabolic activity was observed as shown by continuous increase in glycosaminoglycan and collagen contents with time. Collagen type-I and type-II gene expression analysed for over 3 weeks by RT-PCR showed the prominent expression of collagen type-II. These results suggest the use of synthesised cryogel scaffold as a matrix for chondrocyte attachment and proliferation in 3-D environment and as a delivery system in cartilage-tissue engineering.

Layer-by-layer construction of the heparin/fibronectin coatings on titanium surface:stability and functionality

Layer-by-layer assembly as a versatile bottom-up nanofabrication technique has been widely used in the development of biomimetic materials with superior mechanical and biological properties. In this study, layer-by-layer assembled heparin/fibronectin biofunctional films were fabricated on titanium (Ti) surface to enhance the blood anticoagulation and accelerate the endothelialization simultaneously. The wettability and chemical changes of the assembled films were investigated by static water contact angle measurement and fourier transform infrared spectroscopy (FTIR). The morphology of modified Ti surfaces were observed using scanning electron microscopy (SEM). The real time assembly process was in-situ monitored by quartz crystal microbalance with dissipation (QCM-D). The stability of the films was evaluated by measuring the changes in wettability and the quantity of heparin and fibronectin on the surfaces. The anticoagulation properties of the films were quantitatively rated using Activated partial thromboplastin time (APTT) analysis. New peaks of hydroxyl and amino group were observed on the assembled Ti srufaces by FTIR. The contact angles varied among the films with different bilayer numbers, indicating the successful graft of the heparin and fibronectin layer-by-layer. QCM-D results showed that the frequency shift increased with the bilayer numbers, and the heparin and fibronectin could form multilayers. The assembly films were stable after incubation in PBS for 24h based on the results of the contact angle measurement and the quantity of heparin and fibronectin analysis. APTT results suggested that the assembled films kept excellent antithrombotic properties. All these results revealed that the assembled heparin/fibronectin films with stabiltiy and anticoagulation property could be firmly formed on titanium surfaces.Our study further demonstrates that layer-by-layer assembly of heparin and fibronectin will provide a potential and effective tool for biomaterials surface modification.

An in vitro study of plasticized poly(lactic‐co‐glycolic acid) films as possible guided tissue regeneration membranes: Material properties and drug release kinetics

Guided tissue regeneration (GTR), in periodontal therapy, involves the placement of a barrier membrane, to ensure the detached root surface becomes repopulated with periodontal ligament cells capable of regenerating this attachment. GTR procedures exhibit large variability in surgical outcome as a consequence of poor membrane performance. The objective of this study was to evaluate the suitability of plasticized poly(lactic-co-glycolic acid) (PLGA) as a material for GTR membranes. The material was also investigated as a localized controlled release system for the antibiotic, anti-inflammatory agent tetracycline. Films made from PLGA (85:15), plasticized with either 10% w/v methoxypoly(ethyleneglycol) (MePEG) or a diblock copolymer [poly(D,L-lactic acid)-block-methoxypoly(ethyleneglycol)] were loaded with tetracycline base (or hydrochloride salt) and cast by solvent evaporation. Drug release was measured using high performance liquid chromatography (HPLC). The time-course of elasticity changes and swelling were determined using a stress-strain apparatus or gravimetric/dimensional determinations, respectively. Cells extracted from periodontal ligament cell explants were used to evaluate the effect of material and drug loading on cell morphology. Tetracycline·HCl released more rapidly than tetracycline from PLGA films. The addition of either MePEG or diblock caused a concentration dependent increase in release rates for both drugs. Release profiles ranged from a small initial burst phase followed by slow sustained release to almost full drug release after 1 day. After incubation in PBS, the films stiffened and swelled within 30 min. Periodontal ligament cell morphology was not affected by the inclusion of tetracycline. Plasticized PLGA films displayed desired features for possible use as GTR membranes.

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Effect of vorinostat (V)-loaded PLGA nanoparticles (NP) surface modified with a PSMA ligand on prostate cancer (PC) cells.

e13629 Background: Polymeric NP can be tuned for controlled drug delivery and SM for cell-specific targeting. To overcome V concentration and time dependent reversible growth inhibition of PC cells, we designed a NP to deliver V to PC by polymeric NP that were SM with a ligand to prostate specific membrane antigen (PSMA). Methods: V was encapsulated in poly(lactic-co-glycolic acid) (PLGA) using oil in water emulsion. Pegylated (PEG) phospholipid was added to the aqueous phase for SM, electrostatic and steric stabilization, and longer circulation in vivo. For PSMA targeting, the lipid amino end was covalently modified to possess a glutamate urea with known pM Ki binding affinity to PSMA. Four NP designs were used in cytotoxicity assays : (1) V loaded PLGA NP (V-NP), (2) V loaded PLGA NP and PSMA functionalized (V-NP-PSMA), (3) no drug and PSMA functionalized NP (Bk-NP-PSMA), (4) V loaded and unmodified PEG phospholipid (V-NP-DSPE). LNCaP and PC-3 cells (104 /well) were cultured with V (0, 0.1, 0.5, 1, 5, 10, and 100 μM), or equivalent μM concentration of V-NP, V-NP-PSMA, Bk-NP-PSMA, V-NP-DSPE for up to 72hrs. Controlled release (CR) profiles were determined from incubation in PBS, cell proliferation was measured by MTT assay, and IC50 was calculated. Results: NP mean sizes were 110-130nm, and controlled release data show at least 50% release in 7 days and are ongoing. V loading of 9.5 μg V/mg PLGA was improved to over 150μg V/mg PLGA using the PEG phospholipid method. Nonlipid formulations yielded LNCaP IC50 of 8μM, whereas unencapsulated V and functionalized PEG phospholipid formulations yielded IC50 <1μM for LNCaP and PC-3. At 24hrs, V-NP-DSPE showed no cell kill in LNCaP (PSMA+) cells at concentrations of 10μM, whereas V-NP-PSMA did show suppression to less than 80% survival. By 48hrs, 10μM V-NP-PSMA induced complete cell kill compared to 40% survival in the V-NP-DSPE treated group. Conclusions: This novel SM NP design loads a high concentration of V and effective PC cell kill with non-inferior IC50 to unencapsulated drug. Ongoing studies involve characterizing the selective PSMA binding, controlled drug release properties, and optimizing these NP qualities by fraction of PEG phospholipid. No significant financial relationships to disclose.

Surface Modification of PHBV Scaffolds via UV Polymerization to Improve Hydrophilicity

Surface modification of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with polyacrylamide was achieved with the aim to improve their hydrophilicity. PHBV scaffolds were prepared using the particle-leaching technique, with a porogen size of 100–200, 200–300, 300–400 and 400–500 μm, respectively. After UV polymerization, the modified PHBV scaffolds had interconnected pores with flaky substances partially filled inside. They had a lower porosity, ranging from 80.3 to 86.6%, and higher density, 0.22–0.25 g/cm3, than the PHBV scaffolds. Mercury intrusion porosimetry results showed that the pore sizes at the peak volume increased with the increase of porogen sizes for the modified scaffolds. No extensive weight loss of the PHBV was found after 360 days incubation in PBS at 37°C. However, the weight loss of the modified PHBV increased with time before 90 days incubation. After that period, it ranged only between 5 and 7.5%, with the maximum value at 240 days. During 360 days incubation, the pH value of degradation fluid fluctuated between 7.1 and 7.2. Sheep chondrocytes were cultured on the PHBV and modified PHBV scaffolds. Results showed that the modified PHBV scaffolds provided a better surface for chondrocyte adhesion and spread.

Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

PBS incubation, a simple technique to release miracidia of Opisthorchis-like eggs for DNA extraction

This study describes a simple technique for releasing miracidia from Opisthorchis-like eggs before DNA extraction by keeping PBS washed specimens at 4 °C for two days. Using PCR amplification, the sensitivity of the PBS incubation technique was as good as that obtained from freeze–thaw technique. Moreover, this low-cost technique is less laborious.

Injectable, Highly Flexible, and Thermosensitive Hydrogels Capable of Delivering Superoxide Dismutase

Injectable hydrogels are attractive for cell and drug delivery. In this work, we synthesized a family of injectable, biodegradable, fast gelling and thermosensitive hydrogels based on N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), dimethyl-gamma-butyrolactone acrylate (DBA), and 2-hydroxyethyl methacrylate-poly(trimethylene carbontate) (HEMAPTMC) macromer. Type I collagen was composited with the hydrogels to improve their biocompatibility. The hydrogel copolymer solutions were readily injectable at 4 degrees C. The solutions exhibited thermal transition temperatures ranging from 23.6 to 24.5 degrees C and were capable of gelation within 7 s at 37 degrees C to form highly flexible and soft hydrogels with moduli from 39 to 119 KPa and breaking strains >1000%, depending on the copolymer composition and collagen addition. After 2 weeks incubation in PBS, the hydrogels demonstrated weight losses ranging from 10-20%. The completely degraded hydrogels had thermal transition temperatures >40 degrees C and were soluble at body temperature. Superoxide dismutase (SOD) was encapsulated in the hydrogels for the purpose of capturing superoxide within the inflammatory tissue after being delivered in vivo. The hydrogels demonstrated a sustained release profile during a 21-day release period. The release kinetics was dependent on the SOD loading, collagen addition, hydrogel degradation and water content. The released SOD remained bioactive during the entire release period. To test in vitro if the loaded SOD could protect cells encapsulated within the hydrogel from attack by superoxide, human mesenchymal stem cells (MSC) were encapsulated in SOD-loaded hydrogels and cultured in medium containing superoxide generated by activated macrophages. It was found that SOD loading largely suppressed superoxide penetration into the hydrogel and cell membrane. Under normal culture conditions, SOD loading stimulated MSC growth. The SOD-loaded hydrogel exhibited significantly higher cell numbers than the non-SOD loaded hydrogel during a 7-day culture period. These results demonstrated that the developed hydrogels could be used as delivery vehicles for stem cell therapy and drug delivery.

Nitroxyl Radical Incorporated Electrospun Biodegradable Poly(ester Amide) Nanofiber Membranes

Biodegradable amino-acid-based poly(ester amide) (PEA) ultra-fine fibers pre-loaded with a nitroxyl radical model compound, 4-amino-2.2.6.6-tetramethylpiperidine-1-oxy (4-amino-TEMPO), were prepared by electrospinning. The fiber size and morphology were shown to be greatly affected by the composition ratio of the solvent mixture (chloroform to DMF) prepared for electrospinning. Nano-size PEA fibers (approx. 640 nm) were obtained when PEA dope was electrospun from the chloroform/DMF solvent mixture at a volume ratio of 2 to 1 vs. 3.5 μm size PEA fibers obtained from chloroform-based electrospun dope. Due to the low glass transition temperature and completely amorphous structures, the PEA electrospun fibrous membranes gradually lost their fiber characteristic during 1 month incubation in PBS buffer at 37°C. The glass transition temperature and heat of fusion of PEA electrospun fibers increased with an increasing incubation time and the most significant change occurred in the first day of incubation in PBS. A sustained release of 4-amino-TEMPO from the electrospun PEA nanofiber membranes was observed over the 1-month incubation period in PBS buffer at 37°C and 38% of the incorporated 4-amino-TEMPO (initial loading level 10 mg/g PEA fibers) was released in one month. During this 1 month incubation in PBS buffer, there were only 1.2% weight loss and 11.7% molecular weight reduction for the electrospun PEA fibrous membranes. In an α-chymotrypsin medium (0.1 mg/ml PBS), however, the same electrospun PEA fibrous membranes showed more than 80% weight loss within 6 days and a complete release of encapsulated 4-amino-TEMPO within 5 days.

Fluorocarbon chain end‐capped poly(carbonate urethane)s as biomaterials: Blood compatibility and chemical stability assessments

Previous work has shown the synthesis of fluorocarbon chain (CF(3)(CF(2))(6)CH(2)O-) end-capped poly(carbonate urethane)s (FPCUs) and confirmed the presence of a novel bilayered surface structure in FPCUs, that is, the top fluorocarbon and subsurface hard segment layers (Xie et al., J Biomed Mater Res Part A 2008; 84:30-43). In this work, the effects of such surface structure on blood compatibility were investigated using hemolytic test and platelet adhesion analysis. The chemical stability of the polymers was also determined by Zhao's glass wool-H(2)O(2)/CoCl(2) test and phosphate-buffered saline (PBS, pH = 3.1-3.3) treatment. One of the FPCUs, FPCU-A, and two control materials, a poly(ether urethane) (PEU) and a poly(carbonate urethane) (PCU), were investigated. No significant difference in hemolytic indices was observed among the three materials, whereas the adherent density and deformation of platelets were much lower on FPCU-A compared with on PCU and PEU. Severe surface cracking and surface buckling developed in prestressed PEU and PCU films after H(2)O(2)/CoCl(2) treatment, respectively, whereas smooth surface was observed for the FPCU-A. PBS incubation resulted in parallel ridge-like morphology in PCU whereas PEU and FPCU-A retained their smooth surfaces. Under relatively high stress conditions, all the materials developed well-oriented strip-like surface patterns. Results from ATR-FTIR spectra revealed a surface oxidation mechanism as described in literature. However, observations of universal decrease of molecular weights under stress conditions further suggested the presence of another bulk stress oxidation mechanism. Regardless the degradation mechanisms involved, the unique bilayered surface structure really improved the blood compatibility and chemical stability of FPCU-A, indicating that further in vivo investigations are worthwhile.

A Generic Approach for the Purification of Signaling Complexes That Specifically Interact with the Carboxyl-terminal Domain of G Protein-coupled Receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.

Heterodimerization with Jun Family Members Regulates c-Fos Nucleocytoplasmic Traffic

c-Fos proto-oncoprotein forms AP-1 transcription complexes with heterodimerization partners such as c-Jun, JunB, and JunD. Thereby, it controls essential cell functions and exerts tumorigenic actions. The dynamics of c-Fos intracellular distribution is poorly understood. Hence, we have combined genetic, cell biology, and microscopic approaches to investigate this issue. In addition to a previously characterized basic nuclear localization signal (NLS) located within the central DNA-binding domain, we identified a second NLS within the c-Fos N-terminal region. This NLS is non-classic and its activity depends on transportin 1 in vivo. Under conditions of prominent nuclear localization, c-Fos can undergo nucleocytoplasmic shuttling through an active Crm-1 exportin-independent mechanism. Dimerization with the Jun proteins inhibits c-Fos nuclear exit. The strongest effect is observed with c-Jun probably in accordance with the relative stabilities of the different c-Fos:Jun dimers. Retrotransport inhibition is not caused by binding of dimers to DNA and, therefore, is not induced by indirect effects linked to activation of c-Fos target genes. Monomeric, but not dimeric, Jun proteins also shuttle actively. Thus, our work unveils a novel regulation operating on AP-1 by demonstrating that dimerization is crucial, not only for active transcription complex formation, but also for keeping them in the compartment where they exert their transcriptional function.

Membrane-type 1-Matrix Metalloproteinase Regulates Intracellular Adhesion Molecule-1 (ICAM-1)-mediated Monocyte Transmigration

We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.

Synthesis and characterization of MeO–PEG–PLGA–PEG–OMe copolymers as drug carriers and their degradation behavior in vitro

The objective of this study was to characterize the methylpoly (ethylene glycol)-poly (lacticacid-co-glycolicacid)-poly (ethylene-glycol) (MeO-PEG-PLGA-PEG-OMe, abbreviation as PELGE) copolymers as intravenous injection drug delivery carriers and their degradation behavior in vitro. A series of MeO-PEG-PLGA-PEG-OMe copolymers with various molar ratios of lactic to glycolic acid and various molecular weights and different MeO-PEG contents were synthesized by ring-opening polymerization in the presence of MeO-PEG with molar masses of 2000 and 5000, using stannous octoate as the catalyst. The hydrophilicity of PELGE copolymers, evaluated by contact angle measurements, was found to increase with an increase in their MeO-PEG contents. Methylpoly (ethylene glycol)-poly (lacticacid-co-glycolicacid) (MeO-PEG-PLGA, abbreviation as PELGA) nanoparticles and PELGE nanoparticles were prepared using the emulsion-solvent evaporation technique (o/w) with Pluronic F68 (Poloxamer 188 NF) as emulsifier in the external aqueous phase. The degradation behavior of the nanoparticles was evaluated by the lactate generation with time upon their in vitro incubation in PBS (pH 7.4). The rate of in vitro degradation of the PELGE or PELGA nanoparticles depended on their composition, increasing with an increase in the proportion of MeO-PEG or LA in the copolymer chains. The degradation rate was slower at higher lactide: glycolide ratio. The lower the molecular weight of PELGE; the higher the degradation rate of the nanoparticles.

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

In vitro and in vivo degradation behaviors of synthetic absorbable bicomponent monofilament suture prepared with poly( p-dioxanone) and its copolymer

A synthetic absorbable bicomponent monofilament suture (MonoFlex), composed of poly( p-dioxanone) and its copolymer, was prepared by a conjugate spinning method, and its degradation behavior was investigated in vitro and in vivo. MonoFlex degraded by hydrolysis, and retained approximately 55% of its original strength after four weeks of incubation in PBS at 37 °C. About 70% of the original strength was maintained after four weeks of implantation in rats, and the suture material was completely absorbed after 180–210 days post-implantation in rats. No remarkable tissue reactions were observed during degradation, and foreign body reactions were similar to those of commercially available suture materials composed of poly( p-dioxanone). This study to monitor the degradation behavior of monofilament sutures in vitro as well as in vivo may be useful in the development of novel suture materials for extended wound support.