Synthesis and characterization of water soluble biomimetic chitosans for bone and cartilage tissue regeneration.

Chitosan, a polysaccharide derived from the exoskeleton of crustaceans, insects, the cell walls of fungi, the radulas of mollusks and the internal shells of cephalopods, has been shown to promote osteogenesis. Arginine functionalized chitosan, a water soluble derivative of chitosan, was successfully sulfated with a degree of sulfur incorporation of up to 9% with substitution at the 2-N position. This degree of sulfation replicated those of naturally occurring growth factor binding glycosaminoglycans. Sulfated chitosan-arginine was found to bind and signal fibroblast growth factor 2. Chitosan-arginine promoted an osteogenic phenotype in primary human fetal chondroblasts over a period of 7 days in the absence of osteogenic medium while sulfated chitosan-arginine promoted a chondrogenic phenotype in these same cells. Together these data demonstrate that fine control over progenitor cell phenotype can be achieved in the presence of sulfate modified chitosan-arginine that promotes further investigation and potential development in the future for applications requiring osteo-chondral repair.

Anti-angiogenic activity of heparin functionalised cerium oxide nanoparticles.

Cerium oxide nanoparticles (nanoceria) are widely reported to be non-cytotoxic and modulate intracellular reactive oxygen species (ROS). In this study, nanoceria (dxRD = 12 nm) were functionalised with either 130 or 880 molecules of unfractionated heparin using the organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria with a low level of heparin functionalisation were found to scavenge intracellular ROS to the same extent as unfunctionalised nanoceria and significantly more than cells exposed to medium only. In contrast, nanoceria with the highest level of heparin functionalisation were not as effective at scavenging intracellular ROS. Nanoceria were localised predominantly in the cytoplasm, while heparin-nanoceria were localised in both the cytoplasm and lysosomes. Together these data demonstrated that the level of nanoceria surface functionalisation with heparin determined the intracellular localisation and ROS scavenging ability of these particles. Additionally, heparin-nanoceria were effective in reducing endothelial cell proliferation indicating that they may find application in the control of angiogenesis in cancer in the future.

Essential role of one-carbon metabolism and Gcn4p and Bas1p transcriptional regulators during adaptation to anaerobic growth of Saccharomyces cerevisiae.

The transcriptional activator Gcn4p is considered the master regulator of amino acid metabolism in Saccharomyces cerevisiae and is required for the transcriptional response to amino acid starvation. Here it is shown that Gcn4p plays a previously undescribed role in regulating adaptation to anaerobic growth. A gcn4 mutant exhibited a highly extended lag phase after a shift to anaerobiosis that was the result of l-serine depletion. In addition, the one-carbon metabolism and purine biosynthesis transcriptional regulator Bas1p were strictly required for anaerobic growth on minimal medium, and this was similarly due to l-serine limitation in bas1 mutants. The induction of one-carbon metabolism during anaerobiosis is needed to increase the supply of l-serine from the glycine and threonine pathways. Using a number of experimental approaches, we demonstrate that these transcription regulators play vital roles in regulating l-serine biosynthesis in the face of increased demand during adaptation to anaerobiosis. This increased l-serine requirement is most likely due to anaerobic remodeling of the cell wall, involving de novo synthesis of a large number of very serine-rich mannoproteins and an increase in the total serine content of the cell wall. During anaerobic starvation for l-serine, this essential amino acid is preferentially directed to the cell wall, indicating the existence of a regulatory mechanism to balance competing cellular demands.

IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion.

The family of interferon-induced transmembrane protein (Ifitm/mil/fragilis) genes encodes cell surface proteins that may modulate cell adhesion and influence cell differentiation. Mouse Ifitm1 and -3, which are expressed in primordial germ cells (PGCs), are implicated to have roles in germ cell development, but the specific functions have been unclear. Our results show that Ifitm1 activity is required for PGC transit from the mesoderm into the endoderm, and that it appears to act via a repulsive mechanism, such that PGCs avoid Ifitm1-expressing tissues. In contrast, Ifitm3, which is expressed in migratory PGCs, is sufficient to confer autonomous PGC-like homing properties to somatic cells. These guidance activities are mediated by the N-terminal extracellular domain of the specific IFITM, which cannot be substituted by that of another family member. Complex homo- and/or heterotypic intercellular interactions among various IFITMs in PGCs and neighboring cells may underpin coordinated germ cell guidance in mice.

A conserved noncoding intronic transcript at the mouse Dnm3 locus.

A 6-kb antisense transcript (Dnm3os) contained within an intron of the mouse Dnm3 gene has been identified in a screen for genes that may be regulated by the basic helix-loop-helix transcription factor Twist during mouse development. The antisense transcript is highly conserved between vertebrate species, but does not appear to encode a protein. We show that expression of the Dnm3 and Dnm3os transcripts overlaps during embryogenesis and in adult tissues, except that Dnm3 is most highly expressed in adult brain and testis and expressed at lower levels in embryos, whereas the antisense transcript is most strongly expressed in embryos and gravid uterus. Both Dnm3 and Dnm3os are downregulated in branchial arch tissue of Twist-null embryos. The conservation and restricted expression pattern of this noncoding transcript suggest that it may perform an important function during embryonic development and further suggest a connection between the regulation of Dnm3 and this novel intronic, antisense transcript.

A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling.

PDGF-C is a member of the platelet-derived growth factor (PDGF) family, which signals through PDGF receptor (PDGFR) alphaalpha and alphabeta dimers. Here we show that Pdgfc(-/-) mice die in the perinatal period owing to feeding and respiratory difficulties associated with a complete cleft of the secondary palate. This phenotype was less severe than that of Pdgfra(-/-) embryos. Pdgfc(-/-) Pdgfa(-/-) embryos developed a cleft face, subepidermal blistering, deficiency of renal cortex mesenchyme, spina bifida and skeletal and vascular defects. Complete loss of function of both ligands, therefore, phenocopied the loss of PDGFR-alpha function, suggesting that both PDGF-A and PDGF-C signal through PDGFR-alpha to regulate the development of craniofacial structures, the neural tube and mesodermal organs. Our results also show that PDGF-C signaling is a new pathway in palatogenesis, different from, and independent of, those previously implicated.

Restricted expression of ETn-related sequences during post-implantation mouse development.

In a screen for potential targets of regulation by TWIST in mouse embryos we isolated a fragment with homology to type II early transposon (ETn) and type D endogenous provirus (MusD) elements. Whole-mount in situ hybridization to E7.5-E13.5 mouse embryos reveals a tissue- and stage-specific expression pattern that contrasts with the previously reported lack of expression of ETn elements in mouse embryos beyond late gastrulation. Transcripts were detected in the epiblast at E7.5 and in the neural tube from E8.5 to E10.5. Later expression is predominantly confined to the mesodermal tissues of craniofacial structures, limb buds and somites. The tissue specificity of expression suggests tight regulation of the activity of this early transposon element during embryogenesis.

Twist is required for patterning the cranial nerves and maintaining the viability of mesodermal cells.

Twist encodes a basic helix-loop-helix transcription factor that is required for normal craniofacial morphogenesis in the mouse. Loss of Twist activity in the cranial mesenchyme leads to aberrant migratory behaviour of the neural crest cells, whereas Twist-deficient neural crest cells are located in an inappropriate location in the first branchial arch and display defective osteogenic and odontogenic differentiation (Soo et al. [2002] Dev. Biol. 247:251-270). Results of the present study further show that loss of Twist impacts on the patterning of the cranial ganglia and nerves but not that of the peripheral ganglia and nerves in the trunk region of the body axis. Analyses of the expression of molecular markers of early differentiation of the paraxial mesoderm and the histogenetic potency of somites of Twist(-/-) embryos reveal that Twist-deficient somites can differentiate into muscles, cartilage, and bones, albeit less prolifically. Twist function, therefore, is not essential for mesoderm differentiation. The poor growth of the Twist-deficient somites after transplantation to the ectopic site may be attributed to reduced proliferative capacity and extensive apoptosis of the paraxial mesoderm, suggesting that Twist is required for maintaining cell proliferation and viability in the mesodermal progenitors.

Effect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures.

Factors affecting antibody accumulation and stability were investigated in transgenic plant cell cultures. Whereas IgG(1) antibody was stably maintained in media used for animal cell culture, there was a rapid loss over a period of 1-2 h of antibody added to sterile plant culture media. Antibody stability in Gamborg's B5 medium was improved in the absence of Mn. Tobacco suspensions producing IgG(1) antibody were used to test various medium-based strategies for improving antibody accumulation in plant culture. Even though growth was suppressed, antibody levels in the biomass and medium were increased in media containing mannitol at osmolalities up to approximately 450 mOsm x (kg of water)(-1). Adding gibberellic acid and haemin to the cultures was also beneficial, but the effects were not as great as those obtained under hyperosmolar conditions. Moderate increases in antibody accumulation were found by culturing plant cells in B5 medium without Mn.