Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

Gap formation and regeneration of tropical mangrove forests in Ranong,Thailand

Tropical mangrove forests are characterized by clear zonation along a tidal gradient, and it has been supposed that the zonation is primarily controlled by soil factors. However, effects of disturbance on mangrove forests are still not well understood and may play an important role on the vegetation patterns and forest dynamics in some forest formations. In this study, the pattern of disturbance regime and its effects on regeneration of tropical mangrove forests along a tidal gradient were investigated in Ranong, Thailand. We established one or two 0.5 ha plots in four vegetation zones, i.e. Sonneratia alba–Avicennia alba zone, Rhizophora apiculata zone, Ra – Bruguiera gymnorrhiza zone, Ceriops tagal–Xylocarpus spp. zone. Gap size (percentage gap area to total study area and individual gap size) was the largest in Sa–Aa zone which is located on the most seaward fringe, and it declined from seaward to inland. Canopy trees of S. alba and A. alba had stunted trunks and showed low tree density. On the contrary, canopy dominants in the other three inland zones, e.g. R. apiculata, B. gymnorrhiza, and Xylocarpus spp., had slender trunks and showed high tree density. Accordingly, differences in disturbance regime among the four zones were resulted from the forest structural features of each zone. Disturbance regime matched with regeneration strategies of canopy dominants. Seedlings and saplings of S. alba and A. alba, which need sunny condition for their growth, were abundant in gaps than in understorey. By contrast, R. apiculata, B. gymnorrhiza, and Xylocarpus spp., which can tolerate less light than S. alba and A. alba, had greater seedling and sapling density under closed canopy than gaps. Many large gaps may enhance the abundance of S. alba and A. alba in Sa–Aa zone, and a few small gaps may prevent the light demanding species to establish and grow in the other inland zones. Correspondence of disturbance regime and regeneration strategies (e.g. light requirement) of canopy dominants may contribute to the maintenance of the present species composition in each of the vegetation zones.     

Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.     

Putative "stemness" gene jam-B is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells

Many genes have been identified that are specifically expressed in multiple types of stem cells in their undifferentiated state. It is generally assumed that at least some of these putative "stemness" genes are involved in maintaining properties that are common to all stem cells. We compared gene expression profiles between undifferentiated and differentiated embryonic stem cells (ESCs) using DNA microarrays. We identified several genes with much greater signal in undifferentiated ESCs than in their differentiated derivatives, among them the putative stemness gene encoding junctional adhesion molecule B (Jam-B gene). However, in spite of the specific expression in undifferentiated ESCs, Jam-B mutant ESCs had normal morphology and pluripotency. Furthermore, Jam-B homozygous mutant mice are fertile and have no overt developmental defects. Moreover, we found that neural and hematopoietic stem cells recovered from Jam-B mutant mice are not impaired in their ability to self-renew and differentiate. These results demonstrate that Jam-B is dispensable for normal mouse development and stem cell identity in embryonic, neural, and hematopoietic stem cells.     

Gene pathway analysis of the mechanism by which the Rho-associated kinase inhibitor Y-27632 inhibits apoptosis in isolated thawed human embryonic stem cells

Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1β, TGF-β, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1β, TGF-β, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-β receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.     

Construction of a Genetic Linkage Map and Genetic Analysis of Domestication Related Traits in Mungbean (Vigna radiata)

The genetic differences between mungbean and its presumed wild ancestor were analyzed for domestication related traits by QTL mapping. A genetic linkage map of mungbean was constructed using 430 SSR and EST-SSR markers from mungbean and its related species, and all these markers were mapped onto 11 linkage groups spanning a total of 727.6 cM. The present mungbean map is the first map where the number of linkage groups coincided with the haploid chromosome number of mungbean. In total 105 QTLs and genes for 38 domestication related traits were identified. Compared with the situation in other Vigna crops, many linkage groups have played an important role in the domestication of mungbean. In particular the QTLs with high contribution were distributed on seven out of 11 linkage groups. In addition, a large number of QTLs with small contribution were found. The accumulation of many mutations with large and/or small contribution has contributed to the differentiation between wild and cultivated mungbean. The useful QTLs for seed size, pod dehiscence and pod maturity that have not been found in other Asian Vigna species were identified in mungbean, and these QTLs may play the important role as new gene resources for other Asian Vigna species. The results provide the foundation that will be useful for improvement of mungbean and related legumes.     

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.     

Mapping of quantitative trait loci for a new source of resistance to bruchids in the wild species Vigna nepalensis Tateishi & Maxted (Vigna subgenus Ceratotropis)

Azuki bean breeders have long been interested in producing azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] varieties with bruchid resistance. A new bruchid (Callosobruchus spp.) resistance source was found in V. nepalensis Tateishi & Maxted, a species that is cross compatible with azuki bean. Quantitative trait loci (QTLs) analysis for resistance to C. chinensis (L.) and C. maculatus (F.) was conducted using F(2) (V. nepalensis x V. angularis) and BC(1)F(1) [(V. nepalensis x V. angularis) x V. angularis] populations derived from crosses between the bruchid resistant species V. nepalensis and bruchid susceptible species V. angularis. Resistance was measured using two traits, percentage of seeds damaged by bruchids and the time taken for adult bruchids to emerge from seeds. Based on the results from both populations seven QTLs were detected for bruchid resistance; five QTLs for resistance to C. chinensis and two QTLs for resistance to C. maculatus. The different locations found for some resistance QTL to the two bruchid species suggests different resistance mechanisms. QTLs on linkage group (LG) 1 and LG2 for bruchid resistance to C. chinensis co-localized with seed size QTLs suggesting that incremental increase in seed size accompanied susceptibility to C. chinensis. Based on linked markers the QTL on these two linkage groups appear to be the same as previously reported in other Asian Vigna. However, several other QTLs were newly detected including one on LG4 that appears unrelated to seed size. Transfer of these new sources of bruchid resistance from V. nepalensis to azuki bean will be aided by the progress being made in azuki genome mapping.