We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.Centromeres are eukaryotic cellular structures that are essential for faithful chromosomal segregation during mitotic and meiotic cell division. The kinetochore complex is a defined multiprotein structure on the mitotic chromosome that adheres to the centromere (18, 61). The kinetochore serves as the site of attachment for spindle microtubules, which facilitate the alignment and separation of chromosomes during mitosis (12,13). Although the centromere's function is highly conserved among eukaryotes, centromeric morphology varies significantly, ranging from small, simple kinetochores in the budding yeast Saccharomyces cerevisiae to complex centromeres in multicellular eukaryotes (14). In mammalian cells, the centromere forms a visible primary constriction during metaphase and the kinetochore is a distinct structure that can be resolved into subregions (45,47,67). Finally, in holokinetic organisms such as the nematode Caenorhabditis elegans, multiple centromeres are dispersed throughout the chromosomes (1, 2). The primary nucleotide sequences of centromeric DNAs are also not conserved across phylogenies. The absolute size of the centromeric DNA can vary from ca. 125 bp in S. cerevisiae to tens of megabases in higher eukaryotes (11). Beyond the lack of size and sequence conservation between organisms, the centromere's function may be established not only at predefined sequences, but also at noncentromeric DNA elements, as illustrated by neocentromeres in human (11) and plant (93) cells. Finally, while in budding yeast the centromere DNA alone can nucleate centromere formation de novo, centromeres of metazoan cells strongly depend on epigenetic factors rather than DNA sequences for their activity (90). Thus, there is no primary sequence determinant in centromeric DNA that is conserved among eukaryotic species.At the protein level, a series of kinetochore components show homology to proteins...

The objective of this study was to develop novel absorbable films suitable for use as a tissue-engineering scaffold for keratinocytes as a therapy for replacement of damaged skin. Poly(4-hydroxybutyrate) (P(4HB)) and poly (3-hydroxybutyrate) (P(3HB)) were blended with small amounts of the polysaccharides hyaluronic acid (HA), chitosan (CH), pectin and alginic acid, and were solution cast to produce porous films. The resulting composites had favorable mechanical properties, and these films were compared with two commercially available implantable films made of poly(L-lactide-co-D,L-lactide) (PLA copolymer) and HA benzyl ester. Tensile testing demonstrated that a high level of flexibility of P(4HB) was retained in the P(4HB)-polysaccharide composite films, whereas the P(3HB) film and its polysaccharide composites were stiffer and more brittle. The proliferation kinetics of adherent HaCaT keratinocytes on the films was examined in vitro. The porous surface of the P(4HB) and P(3HB) films blended with HA or CH promoted the growth of keratinocytes significantly. The order of maximum cell numbers on these films was P(4HB)/HA > P(4HB)/CH > P(3HB)/HA > P(3HB)/CH > P(3HB)/pectin > P(3HB)/alginic acid. Scanning electron microscopy and confocal laser scanning microscopy revealed differences in cell growth. Cells formed clusters on P(3HB) and its composites, while the cells grew as a confluent layer on P(4HB) and its composites. HaCaT cells formed large numbers of filaments only on P(4HB) films, indicating the excellent biocompatibility of this material. For the nonporous PHB films, the proliferation rate of cells was found to increase with decreasing hydrophobicity in the order: P(4HB) > P(3HB)/P(4HB) blend > P(3HB).

Photodynamic antimicrobial chemotherapy (PACT) and antimicrobial peptides (AMPs) are two promising strategies to combat the increasing prevalence of antibiotic-resistant bacteria. To take advantage of these two strategies, we integrated a novel antimicrobial peptide (WLBU2) and a potent generation II photosensitizer (temoporfin) into liposomes by preparing WLBU2-modified liposomes, aiming at bacteria targeted delivery of temoporfin for PACT. WLBU2 was successfully coupled to temoporfin-loaded liposomes using a functional phospholipid. The delivery of temoporfin to bacteria was confirmed by fluorescence microscopy and flow cytometry, thus demonstrating that more temoporfin was delivered to bacteria by WLBU2-modified liposomes than by unmodified liposomes. Consequently, the WLBU2-modified liposomes eradicated all methicillin-resistant Staphylococcus aureus (MRSA) and induced a 3.3 log(10) reduction of Pseudomonas aeruginosa in the in vitro photodynamic inactivation test. These findings demonstrate that the use of AMP-modified liposomes is promising for bacteria-targeted delivery of photosensitizers and for improving the PACT efficiency against both gram-positive and gram-negative bacteria in the local infections.

The generation of bio‐targetable photosensitizers is of utmost importance to the emerging field of photodynamic therapy and antimicrobial (photo‐)therapy. A synthetic strategy is presented in which chelating dipyrrin moieties are used to enhance the known photoactivity of iridium(III) metal complexes. Formed complexes can thus be functionalized in a facile manner with a range of targeting groups at their chemically active reaction sites. Dipyrrins with N‐ and O‐substituents afforded (dipy)iridium(III) complexes via complexation with the respective Cp*‐iridium(III) and ppy‐iridium(III) precursors (dipy=dipyrrinato, Cp*=pentamethyl‐η5‐cyclopentadienyl, ppy=2‐phenylpyridyl). Similarly, electron‐deficient [IrIII(dipy)(ppy)2] complexes could be used for post‐functionalization, forming alkenyl, alkynyl and glyco‐appended iridium(III) complexes. The phototoxic activity of these complexes has been assessed in cellular and bacterial assays with and without light; the [IrIII(Cl)(Cp*)(dipy)] complexes and the glyco‐substituted iridium(III) complexes showing particular promise as photomedicine candidates. Representative crystal structures of the complexes are also presented.

IntroductionAntigenic stimulation of T cells is induced by T-cell-receptor ligation and results in the release of immune mediators such as cytokines and chemokines. This activation initiates a nuclear program that results in activation of early growth response factors (EGRs), NF-κB, NF-AT, AP-1 and STAT proteins, and de novo transcription of the corresponding genes. In general terms, these immediate-early genes encode DNA-binding transcription factors. Following transcription, protein expression and nuclear translocation, these effector proteins bind to their cognate promoters and initiate transcription of cytokine, chemokine and inflammatory genes. The precise mechanism of how, in lymphocytes, individual transcription factors interact with coactivators and basal transcription factors, and how these DNA-binding proteins control transcription of inflammatory genes is currently unclear.The four EGR zinc-finger transcription factors (EGR-1 to EGR-4) are transiently and coordinately induced in T cells upon antigenic stimulation and the individual proteins regulate expression of genes encoding immune effectors like interleukin 2 (IL-2) (Skerka et al., 1995), tumor necrosis factor α (TNF-α) (Krämer et al., 1994), the β chain of the IL-2 receptor (Lin and Leonard, 1997), Fas (Dinkel et al., 1997) and the Fas ligand (Li-Weber et al., 1999). In addition, EGR proteins are expressed in distinct cell types and regulate transcription of a wide range of genes, including genes involved in the control of cell growth and apoptosis (Gashler and Sukhatme, 1995).There is a large panel of EGR-regulated genes and these target genes show tissue-specific expression (Gashler and Sukhatme, 1995). The restricted expression pattern of the target genes contrasts with the ubiquitous expression of EGR proteins and it is hypothesized that the tissue-specific transcription is mediated by additional nuclear proteins that cooperate with EGR proteins. We have previously shown that, in T cells, EGR-1 and EGR-4 form stable complexes with the nuclear factor of activated T cells (NFAT) (Decker et al., 2003).NF-κB is a transcription factor that plays a key role in inflammation and the immune response. The NF-κB family has five members, including RelA (p65), RelB, c-Rel, NF-κB1 (p105-p50) and NF-κB2 (p100-p52) (Hayden and Gosh, 2004). 3203Here, we characterize the basis for the T-cell-specific activity of the human zinc-finger protein early growth response factor 4 (EGR-4). A yeast two-hybrid screen showed interaction of EGR-4 with NF-κB p50. Using recombinant proteins, stable physical complex formation was confirmed for EGR-4 and EGR-3 with p50 and with p65 using glutathione-S-transferase pull-down assays and surface-plasmon-resonance and peptide-spot analyses. In vivo interaction of EGR-4 and EGR-3 with NF-κB p65 was demonstrated by immunoprecipitation experiments and fluorescence-resonance-energy transfer (FRET) analysis showing interaction in the nucleus of transfected Jurkat T cells. In transfection assays, EGR-p50 complexes were transcriptionally...