Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.     

Recombinant goldfish growth hormones (gfGH-I and -II) expressed in Escherichia coli have similar biological activities

Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA. The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea. The recombinant hormones were further refolded by gradually removing the urea. Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study. The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay. Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents.     

Selective release of calpain produced alphalI-spectrin (alpha-fodrin) breakdown products by acute neuronal cell death

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.     

Symptomatic relief precedes improvement of myocardial blood flow in patients under spinal cord stimulation

Background

Spinal cord electrical stimulation (SCS) has shown to be a treatment option for patients suffering from angina pectoris CCS III-IV although being on optimal medication and not suitable for conventional treatment strategies, e.g. CABG or PTCA. Although many studies demonstrated a clear symptomatic relief under SCS therapy, there are only a few short-term studies that investigated alterations in cardiac ischemia. Therefore doubts remain whether SCS has a direct effect on myocardial perfusion.

Methods

A prospective study to investigate the short- and long-term effect of spinal cord stimulation (SCS) on myocardial ischemia in patients with refractory angina pectoris and coronary multivessel disease was designed. Myocardial ischemia was measured by MIBI-SPECT scintigraphy 3 months and 12 months after the beginning of neurostimulation. To further examine the relation between cardiac perfusion and functional status of the patients we measured exercise capacity (bicycle ergometry and 6-minute walk test), symptoms and quality of life (Seattle Angina Questionnaire [SAQ]), as well.

Results

31 patients (65 ± 11 SEM years; 25 male, 6 female) were included into the study. The average consumption of short acting nitrates (SAN) decreased rapidly from 12 ± 1.6 times to 3 ± 1 times per week. The walking distance and the maximum workload increased from 143 ± 22 to 225 ± 24 meters and 68 ± 7 to 96 ± 12 watt after 3 months. Quality of life increased (SAQ) significantly after 3 month compared to baseline, as well. No further improvement was observed after one year of treament. Despite the symptomatic relief and the improvement in maximal workload computer based analysis (Emory Cardiac Toolbox) of the MIBI-SPECT studies after 3 months of treatment did not show significant alterations of myocardial ischemia compared to baseline (16 patients idem, 7 with increase and 6 with decrease of ischemia, 2 patients dropped out during initial test phase). Interestingly, in the long-term follow up after one year 16 patients (of 27 who completed the one year follow up) showed a clear decrease of myocardial ischemia and only one patient still had an increase of ischemia compared to baseline.

Conclusion

Thus, spinal cord stimulation not only relieves symptoms, but reduces myocardial ischemia as well. However, since improvement in symptoms and exercise capacity starts much earlier, decreased myocardial ischemia might not be a direct effect of neurostimulation but rather be due to a better coronary collateralisation because of an enhanced physical activity of the patients.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

Sensitivity of ex situ and in situ spectral surface plasmon resonance sensors in the analysis of protein arrays

We have investigated the sensitivity of ex situ (analysis under air condition) and in situ (analysis under liquid condition) spectral SPR sensors, which were self-constructed with fiber optic spectrometers. The sensitivity of SPR sensors was analyzed in the wavelength range of 550-780 nm by the interactions of streptavidin and biotinylated IgG, and the sensitivity was dependent on the wavelength of measurements. The sensitivity of an ex situ SPR sensor operated at the long wavelength range from 712 nm was approximately 2.6 times higher than that at the short wavelength range from 571 nm. In addition, the sensitivity of an ex situ spectral SPR sensor was about twice as high as that of an in situ spectral SPR sensor for the same resonance wavelength range. This was interpreted in that the difference in sensitivity between two SPR sensors was significantly caused by the evanescent field intensity at the metal/dielectric interface. Thus, it was suggested that ex situ spectral SPR sensors operated at the long wavelength range are sensitive biosensors for the high-throughput analysis of protein interactions on protein arrays.     

Suppression of NF-kappaB-mediated beta-defensin gene expression in the mammalian airway by the Bordetella type III secretion system

Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.     

Formation of core/shell nanoparticles with a lipid core and their application as a drug delivery system

A novel preparation method for core/shell nanoparticles with a drug-loaded lipid core was designed and characterized. The lipid core is composed of lecithin and a drug, and the polymeric shell is composed of Pluronics (poly(ethylene oxide)-poly (propylene oxide)-poly(ethylene oxide) triblock copolymer, F-127). For the formation of stabilized core/shell nanoparticles, freeze-drying was performed in the presence of trehalose used as a cryoprotectant. Cryogenic transmittance electron microscopy (cryo-TEM), differential scanning calorimetry (DSC), and a particle size analyzer were used to observe the formation of the stabilized core/shell nanoparticles. For the application of the core/shell nanoparticles as a drug carrier, paclitaxel, a potent anticancer drug, was loaded into the core/shell nanoparticles, and the drug loading amount and the drug release pattern were observed.     

Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: thermal stability of protein domains and unfolding pathway

Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations. The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure. The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding. This region, which is made up of part of the N terminus domain and part of the large domain of the E. coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains. The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway. This observation is compared with the recently proposed hierarchical folding model. Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E. coli dihydrofolate reductase involves sequential formation of the substrate binding sites.