Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin

Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule – we named Kartogenin (KGN) – that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFβ superfamily members, particularly TFGβ1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFβ1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFβ1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFβ and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFβ signaling, working in addition to and/or in concert with the filamin A/CBFβ/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.     

Biosynthesis, localization, and macromolecular arrangement of the Plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ～600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.     

Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs

Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-beta-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5-96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-beta-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20-30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.     

Overexpression of LaMDR2, a novel multidrug resistance ATP-binding cassette transporter, causes 5-fluorouracil resistance in Leishmania amazonensis

The ATP-binding cassette (ABC) proteins play an important role in drug resistance and detoxification in various organisms. Here we isolated LaMDR2, a new member of the multidrug resistance (MDR) subfamily of ABC proteins in Leishmania amazonensis. LaMDR2 exhibited 47% amino acid identity to its most closely related protein, LaMDR1, which was previously isolated from the same species. Promastigotes that overexpressed LaMDR2 showed significant resistance to 5-fluorouracil (5-FU), but not to LaMDR1 substrates. Expression of LaMDR2 in the transfectants was relatively higher in the log phase than the stationary phase, and a lower accumulation of [(3)H]5-FU was observed in the log-phase cells. These results suggest that LaMDR2 is involved in extrusion of xenobiotics, but functionally different from LaMDR1.     

Characterization of the <Emphasis Type="Italic">Selaginella remotifolia</Emphasis> MADS-box gene

Recent progress in plant molecular genetics has revealed that floral organ development is regulated by several homeotic selector genes, most of which belong to the MADS-box gene family. Here we report on SrMADS1, a MIKC^c-type MADS-box gene from Selaginella, a spikemoss belonging to the lycophytes. SrMADS1 phylogenetically forms a monophyletic clade with genes of the LAMB2 group, which are MIKC^c genes of the clubmoss Lycopodium, and is expressed in whole sporophytic tissues except roots and rhizophores. Our results and the previous report on Lycopodium MIKC^c genes suggest that the ancestral MIKC^c gene of primitive dichotomous plants in the early Devonian was involved in the development of basic sporophytic tissues such as shoot, stem, and sporangium. Electronic Publication     

Runx3 is required for full activation of regulatory T cells to prevent colitis-associated tumor formation

Inflammation is increasingly recognized as an essential component of tumorigenesis, which is promoted and suppressed by various T cell subsets acting in different ways. It was shown previously in Runx3-deficient mice that differentiation of CD8 T and NK cells is perturbed. In this study, we show that Runx3 is also required for proper differentiation and function of regulatory T cells. In Runx3-deficient mice, T cells were unable to inhibit inflammation and to suppress tumor development. As expected, recombination activating gene 2-deficient mice bearing Runx3-deficient lymphocytes spontaneously developed colon tumors. However, tumor formation was completely blocked by transfer of either regulatory T cells or CD8 T cells derived from wild-type mice to mutant mice or by housing mutant mice in a specific pathogen-free condition. These results indicate that Runx3-deficient lymphocytes and microorganisms act together to induce inflammation and consequently induce the development of colon tumors.     

Rumba and Haus3 are essential factors for the maintenance of hematopoietic stem/progenitor cells during zebrafish hematopoiesis

The hallmark of vertebrate definitive hematopoiesis is the establishment of the hematopoietic stem/progenitor cell (HSPC) pool during embryogenesis. This process involves a defined ontogenic switching of HSPCs in successive hematopoietic compartments and is evolutionarily conserved from teleost fish to human. In zebrafish, HSPCs originate from the ventral wall of the dorsal aorta (VDA), from which they subsequently mobilize to an intermediate hematopoietic site known as the caudal hematopoietic tissue (CHT) and finally colonize the kidney for adult hematopoiesis. Despite substantial understanding of the ontogeny of HSPCs, the molecular basis governing migration, colonization and maintenance of HSPCs remains to be explored fully. Here, we report the isolation and characterization of two zebrafish mutants, rumba(hkz1) and samba(hkz2), that are defective in generating definitive hematopoiesis. We find that HSPC initiation in the VDA and subsequent homing to the CHT are not affected in these two mutants. However, the further development of HSPCs in the CHT is compromised in both mutants. Positional cloning reveals that Rumba is a novel nuclear C2H2 zinc-finger factor with unknown function and samba encodes an evolutionarily conserved protein that is homologous to human augmin complex subunit 3 (HAUS3). Furthermore, we show that these two factors independently regulate cell cycle progression of HSPCs and are cell autonomously required for HPSC development in the CHT. Our study identifies Rumba and Haus3 as two essential regulators of HSPC maintenance during zebrafish fetal hematopoiesis.     

Contribution of pollinators to seed production as revealed by differential pollinator exclusion in Clerodendrum trichotomum (Lamiaceae)

A diverse assemblage of pollinators, such as bees, beetles, flies, and butterflies, will often visit a single plant species. However, evaluating the effect of several insects on fruit and seed production is difficult in plants visited by a variety of insects. Here, we analyzed the effect of three types of pollinators, Papilio spp., Macroglossum pyrrhosticta, and Xylocopa appendiculata on fruit and seed production in Clerodendrum trichotomum by using a flower visitor barrier experiment with nets of specific mesh sizes. As a result, fruit/flower and seed/ovule ratios were significantly lower under Papilio exclusion than under natural conditions. On the other hand, ratios were not significantly different between Papilio excluded and both Papilio and M. pyrrhosticta excluded treatments. Therefore, Papilio and X. appendiculata are effective pollinators, whereas M. pyrrhosticta, which was the most frequent visitor, of C. trichotomum, is not. From our observations of visiting behaviors, we believe that because M. pyrrhosticta probably promotes self- pollination, this species is a non-effective pollinator. This is the first study to separate and compare the contribution of various visitors to the reproductive success of a plant.     

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.