Synthesis and characterization of injectable, thermally and chemically gelable, amphiphilic poly(N-isopropylacrylamide)-based macromers

In this study, we synthesized and characterized a series of macromers based on poly( N-isopropylacrylamide) that undergo thermally induced physical gelation and, following chemical modification, can be chemically cross-linked. Macromers with number average molecular weights typically ranging from 2000-3500 Da were synthesized via free radical polymerization from, in addition to N-isopropylacrylamide, pentaerythritol diacrylate monostearate, a bifunctional monomer containing a long hydrophobic chain, acrylamide, a hydrophilic monomer, and hydroxyethyl acrylate, a hydrophilic monomer used to provide hydroxyl groups for further chemical modification. Results indicated that the hydrophobic-hydrophilic balance achieved by varying the relative concentrations of comonomers used during synthesis was an important parameter in controlling the transition temperature of the macromers in solution and stability of the resultant gels. Storage moduli of the macromers increased over 4 orders of magnitude once gelation occurred above the transition temperature. Furthermore, chemical cross-linking of these macromers resulted in gels with increased stability compared to uncross-linked controls. These results demonstrate the feasibility of synthesizing poly( N-isopropylacrylamide)-based macromers that undergo tandem gelation and establish key criteria relating to the transition temperature and stability of these materials. The data suggest that these materials may be attractive substrates for tissue engineering and cellular delivery applications as the combination of mechanistically independent gelation techniques used in tandem may offer superior materials with regard to gelation kinetics and stability.     

Backbone dynamics and hydrogen exchange of Pseudomonas aeruginosa ferricytochrome c 551

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c(551) dynamics based on (15)N R(1), (15)N R(2), and [(1)H]-(15)N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (< S(2)>=0.924+/-0.005) and displays little variation in picosecond-nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (< S(2)>=0.80+/-0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties.     

High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.     

Stabilization of RNA through Absorption by Functionalized Mesoporous Silicate Nanospheres

The potential for encapsulating RNA within tunable, semi-permeable structures for storage and transportation purposes offers an interesting approach to the reduction of stringent storage requirements that often hamper the field application of genetic analysis methods. In this study, we assessed the potential for application of functionalized, porous silicate sorbents in maintaining nucleic acid integrity. Mesoporous silica nanoparticles (MSNs) with and without incorporated stabilizing reagents were used to encapsulate triosephosphate isomerase mRNA of Arabidopsis thaliana. The absorption, elution, and the long-term stability of the RNA were monitored by using quantitative real-time RT-PCR. The results indicate that adsorbed RNA can be eluted from the sorbents using simple buffers and employed directly for downstream molecular diagnostic assays without any further processing. RNA integrity can be maintained for extended time periods under refrigeration temperatures in the presence of covalently immobilized stabilizing compounds. This study provides initial evidence of the potential for application of MSNs in transportation and storage. They may also have utility in sample collection and processing in restrictive environments.     

The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system.

Phosphorylation of the myelin-associated glycoprotein (MAG) in the peripheral nervous system is demonstrated by immunoprecipitation from myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Phosphoamino acid analysis of immunoprecipitated MAG revealed the presence of radioactivity in phosphoserine, but not in phosphothreonine or phosphotyrosine. Only the shorter isoform of MAG (S-MAG) was detected by immunostaining of nitrocellulose sheets with anti-MAG anti-serum after enzymatic deglycosylation of immunoprecipitated MAG labeled in nerve slices. Autoradiography of the same Western blots revealed that most of the radioactive phosphate was in S-MAG, demonstrating that the polypeptide backbone of S-MAG is phosphorylated in the PNS.     

Myelin-Associated Calpain II

Anti-chicken muscle calpain (calcium-activated neutral protease) antibody (ACAb) was found to be absorbed by purified human brain myelin when titrated by enzyme-linked immunosorbent assay, suggesting the close association of the protease with myelin. To confirm this, calcium-dependent protease was extracted from myelin membrane and purified on a phenyl Sepharose CL 4B column. It was activated by calcium ion in the millimolar range, and therefore was determined to be calpain II. This enzyme fraction was electrophoresed and immunostained with ACAb, resulting in staining as a single band with apparent molecular weight of 80K. This protease degraded exogenous myelin-associated glycoprotein. From the present results, it is suggested that calpain is bound to myelin membrane and involved in the turnover of myelin proteins.