Weak base dispiro-1,2,4-trioxolanes: potent antimalarial ozonides

Thirty weak base 1,2,4-dispiro trioxolanes (secondary ozonides) were synthesized. Amino amide trioxolanes had the best combination of antimalarial and biopharmaceutical properties. Guanidine, aminoxy, and amino acid trioxolanes had poor antimalarial activity. Lipophilic trioxolanes were less stable metabolically than their more polar counterparts.     

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.     

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.     

Clonal multipotency and effect of long-term in vitro expansion on differentiation potential of human hair follicle derived mesenchymal stem cells

Hair follicle harbors a rich stem cell pool with mesenchymal lineage differentiation potential. Although previous studies with rodent cells demonstrated that hair follicle sheath and papilla cells possess multi-lineage differentiation potential, human hair follicle derived mesenchymal stem cells (hHF-MSCs) have not been characterized in detail in terms of their multipotency. In addition, it is not clear whether these cells are true stem cells that can differentiate along multiple lineages or whether they represent a collection of progenitor cells with restricted differentiation potential. Here we report that hHF-MSCs are highly proliferative cells that can be maintained in culture for ~ 45 population doublings before they start to show signs of cellular senescence. Under appropriate culture conditions, hHF-MSCs differentiated along the myogenic, osteogenic, adipogenic and chondrogenic lineages, as demonstrated by kinetic gene expression profiling and functional assays. Interestingly, the differentiation potential decreased with time in culture in a lineage-specific manner. Specifically, myogenesis and chondrogenesis showed a moderate decrease over time; osteogenesis was maximum at intermediate passages and adipogenesis was highly sensitive to long-term culture and was diminished at late passages. Finally, hHF-MSCs were clonally multipotent as the majority of hHF-MSCs clones (73%) demonstrated bi- or tri-lineage differentiation potential. These results suggest that hHF-MSCs may present as an alternative source of easily accessible, autologous stem cells for tissue engineering and regenerative medicine.     

Gamma radiation-induced proteome of Deinococcus radiodurans primarily targets DNA repair and oxidative stress alleviation

The extraordinary radioresistance of Deinococcus radiodurans primarily originates from its efficient DNA repair ability. The kinetics of proteomic changes induced by a 6-kGy dose of gamma irradiation was mapped during the post-irradiation growth arrest phase by two-dimensional protein electrophoresis coupled with mass spectrometry. The results revealed that at least 37 proteins displayed either enhanced or de novo expression in the first 1 h of post-irradiation recovery. All of the radiation-responsive proteins were identified, and they belonged to the major functional categories of DNA repair, oxidative stress alleviation, and protein translation/folding. The dynamics of radiation-responsive protein levels throughout the growth arrest phase demonstrated (i) sequential up-regulation and processing of DNA repair proteins such as single-stranded DNA-binding protein (Ssb), DNA damage response protein A (DdrA), DNA damage response protein B (DdrB), pleiotropic protein promoting DNA repair (PprA), and recombinase A (RecA) substantiating stepwise genome restitution by different DNA repair pathways and (ii) concurrent early up-regulation of proteins involved in both DNA repair and oxidative stress alleviation. Among DNA repair proteins, Ssb was found to be the first and most abundant radiation-induced protein only to be followed by alternate Ssb, DdrB, indicating aggressive protection of single strand DNA fragments as the first line of defense by D. radiodurans, thereby preserving genetic information following radiation stress. The implications of both qualitative or quantitative and sequential or co-induction of radiation-responsive proteins for envisaged DNA repair mechanism in D. radiodurans are discussed.     

Linkers of Cell Polarity and Cell Cycle Regulation in the Fission Yeast Protein Interaction Network

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify ‘linker’ proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted ‘linkers’ also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes.     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

Uptake and release of bilirubin by skin

1. Skin epithelium of albino rat, mouse and guinea pig was shown to accumulate bilirubin from a medium containing free or bound bilirubin. 2. The K(m) values for bound bilirubin were 2.22x10(-3), 1.33x10(-3) and 9.5x10(-4)m for rat, mouse and guinea pig respectively and the corresponding K(m) values for free bilirubin were 5.2x10(-4), 4.0x10(-4), 1.8x10(-4)m; the V(max.) values of bound and free bilirubin were unchanged. 3. The uptake showed saturation kinetics. Bound bilirubin was released together with serum proteins. Free bilirubin bound to skin was not released into the medium. 4. Freezing and thawing of skin epithelium did not cause any significant lowering of the uptake of bilirubin but heat-denatured skin epidermis took up only 50% of the bound bilirubin or free bilirubin taken up by control unheated skin epithelium. 5. The uptake of free and bound bilirubin was prevented by HgCl(2) but not by sodium arsenate, NaCN, NaF, cycloheximide, 2,4-dinitrophenol or iodoacetate. 6. Most of the free bilirubin was bound to the lipids or lipoprotein fraction of skin epithelium and could be extracted by solvents. 7. Rat skin showed the highest accumulation and efflux of bilirubin.