Design and development of poly-L/D-lactide copolymer and barium titanate nanoparticle 3D composite scaffolds using breath figure method for tissue engineering applications.

In tissue engineering, the scaffold topography influences the adhesion, proliferation, and function of cells. Specifically, the interconnected porosity is crucial for cell migration and nutrient delivery in 3D scaffolds. The objective of this study was to develop a 3D porous composite scaffold for musculoskeletal tissue engineering applications by incorporating barium titanate nanoparticles (BTNPs) into a poly-L/D-lactide copolymer (PLDLA) scaffold using the breath figure method. The porous scaffold fabrication utilised 96/04 PLDLA, dioleoyl phosphatidylethanolamine (DOPE), and different types of BTNPs, including uncoated BTNPs, Al2O3-coated BTNPs, and SiO2-coated BTNPs. The BTNPs were incorporated into the polymer scaffold, which was subsequently analysed using field emission scanning electron microscopy (FE-SEM). The biocompatibility of each scaffold was tested using ovine bone marrow stromal stem cells. The cell morphology, viability, and proliferation were evaluated using FE-SEM, LIVE/DEAD staining, and Prestoblue assay. Porous 3D composite scaffolds were successfully produced, and it was observed that the incorporation of uncoated BTNPs increased the average pore size from 1.6 µm (PLDLA) to 16.2 µm (PLDLA/BTNP). The increased pore size in the PLDLA/BTNP scaffolds provided a suitable porosity for the cells to migrate inside the scaffold, while in the pure PLDLA scaffolds with their much smaller pore size, cells elongated on the surface. To conclude, the breath figure method was successfully used to develop a PLDLA/BTNP scaffold. The use of uncoated BTNPs resulted in a composite scaffold with an optimal pore size while maintaining the honeycomb-like structure. The composite scaffolds were biocompatible and yielded promising structures for future tissue engineering applications.

A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis.

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.

Structure and NTPase activity of the RNA-translocating protein (P4) of bacteriophage phi 6.

The RNA polymerase complex of bacteriophage phi 6 comprises four proteins, P1, P2, P4 and P7, and forms the core of the virion. Protein P4 is a non-specific NTPase that provides the energy required for RNA translocation (packaging). Characterization of purified recombinant P4 shows that the protein assembles into stable hexamers in the presence of ADP and divalent cations. Image averaging of electron micrographs reveals this hexamer as a slightly skewed ring with outer and inner diameters of 12 and 2 nm, respectively. NTPase activity of P4 is associated only with the hexameric form. Ca2+ and Zn2+ and non-specific single-stranded RNA stimulate the NTPase activity, while Mg2+ acts as a non-competitive inhibitor, presumably via a separate Mg2+ binding site. Binding affinities of different nucleotide mono-, di- and triphosphates and non-hydrolyzable analogs indicate that the beta-phosphate moiety is required for substrate binding. A slight preference for binding of purine nucleotides is also observed. Analysis of P4 by CD and Raman spectroscopy indicates an alpha/beta subunit fold that is altered only slightly by hexamer assembly. Raman markers of P4 secondary and tertiary structures are also largely invariant to nucleotide exchange and hydrolysis, suggesting that the mechanisms of RNA translocation involves movement of subunits relative to one another rather than large scale changes in the alpha/beta subunit fold. The stoichiometry of P4 in the mature phi 6 virion is estimated as 120 copies. Because the recombinant P4 hexamers exhibit hydrodynamic and enzymatic properties that are identical to those of P4 oligomers released from native phi 6, we propose that P4 occurs as hexamers in the native viral core particle.

Protein P7 of phage phi6 RNA polymerase complex, acquiring of RNA packaging activity by in vitro assembly of the purified protein onto deficient particles.

The RNA polymerase complex of double-stranded RNA bacteriophage phi6 is composed of four proteins, P1, P2, P4 and P7. These four proteins are capable of performing all the functions required for the replication of the double-stranded RNAs of the phi6 genome. The polymerase complex containing the three genomic dsRNA segments is the core particle of the phi6 virion. In this study purified protein P7 was found to form highly asymmetric dimers. Using polyclonal anti-P7 antibody, P7 was shown to be accessible on the surface of the nucleocapsid. Treatment of nucleocapsids with polyclonal anti-P7 antibody released coat protein P8 with ensuing activation of the plus strand RNA synthesis from the resulting core particles. Purified P7 could be assembled onto particles lacking P7 and particles lacking both P2 (RNA polymerase) and P7. In both cases RNA packaging activity was acquired. Assembly of P7 onto deficient particles took place also in the absence of host proteins. Protein P7 is known to be necessary for stable packaging of the three genomic phi6 plus strand RNAs into preformed polymerase complex particles. Additionally, protein P7 seems to be involved in the regulation of plus strand synthesis (i.e. transcription) as a fidelity factor. Particles lacking protein P7 produce anomalous size transcripts. Analysis of the polymerase complex stability revealed that proteins P2, P4 and P7 are independently associated with the major structural protein P1. The number of P7 molecules in one virion was estimated to be 60 and a location at the 5-fold symmetry position is proposed.

RNA binding, packaging and polymerase activities of the different incomplete polymerase complex particles of dsRNA bacteriophage phi 6.

phi 6 is an enveloped dsRNA bacterial virus. Its segmented genome resides inside the virion associated polymerase complex which is formed by four proteins (P1, P2, P4 and P7) encoded by the viral L segment. Complete and incomplete polymerase complex particles can be produced using cDNA copies of this largest genome segment. We have analysed the capacity of the different purified particles to (1) package phi 6 (+) sense genomic precursors and unspecific RNA, (2) synthesize (-) and (+) strands and (3) bind phi 6 specific and unspecific RNAs. Both (-) and (+) strand synthesis polymerase activities were found to be associated with protein P2. In addition to complete particles, particles lacking protein P2 were found to package and protect genomic precursor ssRNAs. Protein P7 was needed for efficient packaging. Regulation and specificity of the packaging were found to be independent of P2. Particles composed of proteins P1 and P4 did not package or protect RNA but did bind phi 6 genomic (+) strand RNAs. The three phi 6 (+) strands bound in equal amounts to the particles when tested alone in a filter binding assay. In competition experiments they competed each other for binding, indicating that individual binding sites for the three genomic (+) strands do not exist. Differences in RNA binding competition among the four particles were observed, suggesting that packaging specificity is achieved by complex interactions of proteins and genomic (+) strand RNAs during the advancement of the packaging process after the initial binding events.

Topology of the major capsid protein P3 of bacteriophage PRD1: analysis using monoclonal antibodies and C-terminally truncated proteins.

Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid-protein vesicle containing the viral dsDNA genome. In this study we characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes in the region of amino acids 159-168, which is part of the first predicted beta-barrel structure of P3. The third antigenic region is in the second predicted beta-barrel, between amino acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.

Protein P4 of double-stranded RNA bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein.

Protein P4, an early protein of double-stranded RNA bacteriophage phi 6, is a component of the virion-associated RNA polymerase complex and possesses a nucleoside triphosphate (NTP) phosphohydrolase activity. We have produced and characterized a panel of 20 P4-specific monoclonal antibodies. Epitope mapping using truncated molecules of recombinant P4 revealed seven linear epitopes. The accessibility of the epitopes on the phi 6 nucleocapsid (NC) surface showed that at least the C terminus and an internal domain, containing the consensus sequence for NTP binding, protrude the NC shell. Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8. This finding suggests a close contact between these two proteins. The dissociation of the NC led to the activation of the virion-associated RNA polymerase. The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization.

Cloning, sequence and expression of a beta-tubulin-encoding gene in the homobasidiomycete Schizophyllum commune.

The beta-tubulin (beta Tub)-encoding gene (tub-2) of Schizophyllum commune is the first tubulin gene isolated, cloned and sequenced from higher filamentous fungi (homobasidiomycetes). The S. commune tub-2 gene is organized into nine exons and eight introns. The introns vary from 48 to 107 nt in length, and are distributed throughout the gene. The tub-2 exons code for a protein of 445 amino acids (aa), which shows great homology with beta Tubs of filamentous ascomycetes, plants, and animals, but less homology with yeasts. The codon usage of tub-2 from S. commune is biased, as it is in most beta Tub-encoding genes of filamentous fungi. The S. commune beta Tub shows a conserved aa sequence in the C-terminal domain, which is suggested to interact with microtubule-associated proteins in animals. In contrast, the S. commune beta Tub deviates from most known beta Tubs by having a Cys165 residue, which might be significant for the insensitivity of S. commune haploid strains to the antimicrotubule drug, benomyl. In tub-2 of different haploid strains, sequence polymorphisms occur in the 5' and 3' flanking regions. The expression of tub-2 is high in young mycelium, which has a high number of extending apical cells, but decreases with the aging of the mycelium. No significant difference in the hybridization signal intensity for the tub-2 transcripts was recorded either during intercellular nuclear migration at early mating, or in mycelia with a mutation in the B mating-type gene.(ABSTRACT TRUNCATED AT 250 WORDS)