Cell-seeded polyurethane-fibrin structures--a possible system for intervertebral disc regeneration.

Nowadays, intervertebral disc (IVD) degeneration is one of the principal causes of low back pain involving high expense within the health care system. The long-term goal is the development of a medical treatment modality focused on a more biological regeneration of the inner nucleus pulposus (NP). Hence, interest in the endoscopic implantation of an injectable material took center stage in the recent past. We report on the development of a novel polyurethane (PU) scaffold as a mechanically stable carrier system for the reimplantation of expanded autologous IVD-derived cells (disc cells) to stimulate regenerative processes and restore the chondrocyte-like tissue within the NP. Primary human disc cells were seeded into newly developed PU spheroids which were subsequently encapsulated in fibrin hydrogel. The study aims to analyze adhesion properties, proliferation capacity and phenotypic characterization of these cells. Polymerase chain reaction was carried out to detect the expression of genes specifically expressed by native IVD cells. Biochemical analyses showed an increased DNA content, and a progressive enhancement of total collagen and glycosaminoglycans (GAG) was observed during cell culture. The results suggest the synthesis of an appropriate extracellular matrix as well as a stable mRNA expression of chondrogenic and/or NP specific markers. In conclusion, the data presented indicate an alternative medical approach to current treatment options of degenerated IVD tissue.

Analysis of rpoS mRNA in Salmonella dublin: identification of multiple transcripts with growth-phase-dependent variation in transcript stability.

In Salmonella dublin, rpoS encodes an alternative sigma factor of the RNA polymerase that activates a variety of stationary-phase-induced genes, including some virulence-associated genes. In this work, we studied the regulation and transcriptional organization of rpoS during growth. We found two transcripts, 2.3 and 1.6 kb in length, that represent the complete rpoS sequence. The 2.3-kb transcript is a polycistronic message that also includes the upstream nlpD gene. It is driven by a weak promoter with increasing activity when cells enter early stationary growth. The 1.6-kb message includes 566 bp upstream of the rpoS start codon. It is transcribed from a strong sigma70 RNA polymerase-dependent promoter which is independent of growth. The decay of this transcript decreases substantially in early stationary growth, resulting in a significant net increase in rpoS mRNA levels. These levels are approximately 10-fold higher than the levels of the 2.3-kb mRNA, indicating that the 1.6-kb message is mainly responsible for RpoS upregulation. In addition to the 2.3- and 1.6-kb transcripts, two smaller 1.0- and 0.4-kb RNA species are produced from the nlpD-rpoS locus. They do not allow translation of full-length RpoS; hence their significance for rpoS regulation remains unclear. We conclude that of four transcripts arising from the nlpD-rpoS locus, only one plays a significant role in rpoS expression in S. dublin. Its upregulation when cells enter stationary growth is due primarily to an increase in transcript stability.

Expression profile and subcellular location of the plasmid-encoded virulence (Spv) proteins in wild-type Salmonella dublin.

The plasmid-encoded virulence genes (spvABCD) in nontyphoid Salmonella strains mediate lethal infections in a variety of animals. Previous studies have shown that these genes are transcriptionally regulated by stationary-phase growth. We studied the expression profile and the subcellular locations of the SpvABCD proteins in wild-type S. dublin by using polyclonal antibodies against SpvA, SpvB, SpvC, and SpvD. The cellular levels of the individual proteins were determined during growth by quantitative immunoblotting. As expected, SpvA, SpvB, SpvC, and SpvD were not detectable before the late logarithmic growth phase and appeared in the sequence SpvA, SpvB, SpvC, and SpvD. In contrast to the transcriptional regulation, however, SpvA and SpvB reached their maximal expression shortly after induction and declined during further growth whereas SpvC and SpvD expression remained high throughout the stationary phase, indicating that the Spv proteins are individually regulated at a posttranscriptional level. To localize SpvABCD within the bacteria, the cells were fractionated into the periplasmic, cytoplasmic, inner membrane, and outer membrane components. The cell fractions and the culture supernatant were analyzed by immunoblotting. SpvA was present in the outer membrane, SpvB was present in the cytoplasm and the inner membrane, and SpvC was present in the cytoplasm. SpvD was secreted into the supernatant; however, a substantial portion of this protein was also detected in the cytoplasm and membranes. The molecular weights of SpvD in the supernatant and in the cytoplasm appeared to be equal, suggesting that SpvD is not cleaved upon secretion.

Plasmid virulence gene expression induced by short-chain fatty acids in Salmonella dublin: identification of rpoS-dependent and rpo-S-independent mechanisms.

The Salmonella plasmid virulence spvABCD genes are growth phase regulated and require RpoS for maximal expression in stationary phase. We identified a growth phase-independent expression of spv which is mediated by short-chain fatty acids. During this fatty acid-mediated expression of spv, RpoS is required for induction only during exponential phase. In stationary phase, an rpoS-independent mechanism is responsible for expression of spv.

Evidence for H(+)-induced insertion of influenza hemagglutinin HA2 N-terminal segment into viral membrane.

Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral fusion protein hemagglutinin. At 0 degree C, in a first kinetically resolvable step, the hemagglutinin polypeptide 2 (HA2) N-terminal segment (fusion peptide) is exposed and inserts into the target membrane (Tsurudome, M., Glück, R., Graf, R., Falchetto, R., Schaller, U., and Brunner, J. (1992) J. Biol. Chem. 267, 20225-20232). We now report studies of the changes taking place at pH 5.0 and 37 degrees C, conditions that result in fusion or, in the absence of a target membrane, in inactivation of the virus' fusion capacity. To this end, we synthesized the new photosensitive phospholipid, 1-palmitoyl-2-[decanedioyl mono-[2-(125I)iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)-benzyl]e ster]- sn-glycero-3-phosphocholine (specific radioactivity, > 2000 Ci/mmol), and worked out a protocol to incorporate this lipid into the viral membrane. Subsequent photoactivation of the reagent resulted in selective labeling of the C-terminal portion of the HA2 polypeptide chain, in agreement with the membrane topology of hemagglutinin. When, however, prior to reagent activation, the viruses were exposed at pH 5.0, 37 degrees C, both the HA2 C-terminal and the N-terminal regions were labeled, suggesting that the HA2 N-terminal segment (fusion peptide) inserted into the viral membrane. Possible implications for fusion and virus inactivation are discussed.

Refractive indices of TiO(2) films produced by reactive evaporation of various titanium-oxygen phases.

In this paper experiments with reactive evaporation of the starting materials Ti, TiO, Ti(2)O(3), Ti(3)O(5), and TiO(2) to obtain nonabsorbing TiO(2) films are under discussion. For the starting materials TiO and Ti(3)O(5) the dependence of the TiO(2) film refractive index on the substrate temperature, oxygen pressure, and deposition rate was measured. For TiO dispersion curves of the resulting TiO(2) films as a function of the substrate temperature during film formation were determined. The successive evaporation of the different starting materials resulted in the formation of lambda/4 TiO(2) films with different refractive indices. This phenomenon was most obvious during the first evaporation. It disappeared after several evaporations in two groups of TiO2 films with different refractive indices. From the beginning only the starting materials Ti and Ti(3)O(5) resulted in TiO(2) films with constant refractive indices. The first material produced a high, the latter a lower film index. Depending on the number of evaporations performed, both types of TiO(2) films can be obtained with TiO. The films and residues in the crucibles were also subjected to chemical analyses. An attempt was made to explain the optical properties of the resulting TiO(2) films with regard to crystal structure, chemical composition, packing density influenced by the molecular composition of the vapor beam, chemical reaction with the crucible, substrate temperature, O(2) pressure, and deposition rate.