Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.     

Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes

Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2‐Cre or VE‐cadherin‐Cre constructs to facilitate fate‐mapping of LSECs in liver regeneration. Some YFP‐positive LSECs were observed to convert into hepatocytes following a two‐thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non‐hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte‐like (iHep) cells may provide a new approach to tissue engineering.     

Reduced genetic diversity and increased reproductive isolation follow population‐level loss of larval dispersal in a marine gastropod

Population‐level consequences of dispersal ability remain poorly understood, especially for marine animals in which dispersal is typically considered a species‐level trait governed by oceanographic transport of microscopic larvae. Transitions from dispersive (planktotrophic) to nondispersive, aplanktonic larvae are predicted to reduce connectivity, genetic diversity within populations, and the spatial scale at which reproductive isolation evolves. However, larval dimorphism within a species is rare, precluding population‐level tests. We show the sea slug Costasiella ocellifera expresses both larval morphs in Florida and the Caribbean, regions with divergent mitochondrial lineages. Planktotrophy predominated at 11 sites, 10 of which formed a highly connected and genetically diverse Caribbean metapopulation. Four populations expressed mainly aplanktonic development and had markedly reduced connectivity, and lower genetic diversity at one mitochondrial and six nuclear loci. Aplanktonic dams showed partial postzygotic isolation in most interpopulation crosses, regardless of genetic or geographic distance to the sire's source, suggesting that outbreeding depression affects fragmented populations. Dams from genetically isolated and neighboring populations also exhibited premating isolation, consistent with reinforcement contingent on historical interaction. By increasing self‐recruitment and genetic drift, the loss of dispersal may thus initiate a feedback loop resulting in the evolution of reproductive isolation over small spatial scales in the sea.     

In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats

Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver.     

植物乳杆菌素L-1对单核细胞增生李斯特氏菌作用机理的研究

以凝胶层析纯化的植物乳杆菌素作用单核细胞增生李斯特氏菌,结果表明该细菌素可以导致能量化的敏感细胞胞内K 、无机磷离子、乳酸脱氢酶、紫外吸收物质和ATP发生不同程度的泄漏,相应地破坏了膜Δψ和部分ΔpH,引起PMF的耗散,结果导致细胞的死亡。综合所测指标,可以推测植物乳杆菌素L-1对单增李斯特氏菌的作用目标主要是细胞膜,通过形成非选择性孔洞使得选择性离子和小分子生命物质外泄,从而打破原有平衡,最终引起细胞的衰亡。     

Protein Profiles of Somatic Embryos and Regenerated Plants from NaCl Selected and Control Cultures of Orchardgrass

The protein profile of cells of control somatic embryos was compared to that of embryos that have become selected and maintained on 200 mM NaCl in order to detect salt inducible proteins. Two proteins (60 and 51.5 kDa) were more abundant in the selected embryos and one protein with molecular mass 18 kDa was unique to the selected embryos. Enhanced content of 27 kDa protein was observed in all somatic embryos indicating its involvement in the embryonal state. Similar pattern of salt inducible proteins in selected somatic embryos and the plantlets regenerated from such embryos was found. This revised version was published online in July 2006 with corrections to the Cover Date.     

Early Plant Succession in Abandoned Pastures in Ecuador1

We compared early plant succession in four abandoned pastures of differing age since abandonment and a nearby secondary forest site in northwestern Ecuador. Two “Open” pastures had no tree canopy covering, and two “Guava” pastures had a well-developed canopy cover of Psidium guajava. No site had been seeded with pasture grasses. All pastures were compared in a chronological sequence; two were monitored for 18 months. Species richness was consistently higher in Guava sites than in Open sites and it continued to increase over time, whereas it remained static in Open sites. Species richness was highest in secondary forest. Recruitment of tree saplings in Guava sites was lower than in secondary forest; however, it was nearly absent in Open sites. The seed bank contained predominantly herbaceous species at all sites, and was highly dissimilar to aboveground vegetation. Dominance-diversity curves for Guava sites showed a more equitable distribution of species that increased over time. In contrast, dominance-diversity curves for Open sites were static and indicated dominance by a few aggressive species. Soil characteristics among sites were variable; however, a principal components analysis on soils isolated the older Open site from all others. The older Open site had the lowest species richness and was dominated by Baccharis trinervis, an aggressive shrub species. The site appears to be in a state of arrested succession and some form of restorative intervention may be necessary to initiate succession toward a forested condition. Succession in Guava sites appears headed toward secondary forest, whereas it does not in Open sites.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Structural diversity of Papaver somniferum L. cell surfaces in vitro depending on particular steps of plant regeneration and morphogenetic program

Culture of Papaver somniferum in vitro was used for a characterisation of cell surface structures and mode of cell adhesion and cell separation during cell differentiation and plant regeneration in somatic embryogenesis and shoot organogenesis. In early stages of somatic embryogenesis, cell type-specific and developmentally regulated change of cell morphogenesis was demonstrated. Cell wall of separated embryonic cells were self-covered with external tubular network, whereas morphogenetic co-ordination of adhered cells of somatic proembryos was supported by fine and fibrillar external cell wall continuum of peripheral cells, interconnecting also local sites of cell separation. Such type of cell contacts disappeared during histogenesis, when the protodermis formation took place. Tight cell adhesion of activated cells with polar cell wall thickening, and production of extent mucilage on the periphery were the crucial aspects of meristemoids. Fine amorphous layer covered developing shoot primordia, but we have not observed such comparable external fibrillar network. On the contrary intercellular separation of differentiated cells in regenerated organs, and accepting distinct developmental system of somatic embryogenesis and shoot organogenesis, cell adhesion in early stages and ultrastructural changes associated with tissue disorganisation, and the subsequent reorganisation into either embryos or shoots appear to be regulatory morphogenetical events of plant regeneration in vitro.     

Plant regeneration of mangosteen via nodular callus formation

Nodular callus was induced at a high frequency on young purple red, 5–15 mm long laminae taken from in vitro grown plants of mangosteen. The optimal medium was composed of Murashige and Skoog (MS) nutrients supplemented with 2.22 μM benzyladenine (BA), 2.25 μM thidiazuron (TDZ), 500 mg l^-1 polyvinylpyrrolidone (PVP 360 000) and 3% sucrose. A multiplication rate of two–three was obtained by subculture of the nodular callus at 3–4-week intervals. Plantlet regeneration from the nodules was achieved by transfer to woody plant medium (WPM) with 500 mg l^-1 PVP, 0.4 μM BA and 3% sucrose and overlaying with half strength liquid MS containing 0.32 μM naphthaleneacetic acid (NAA), 0.13 μM BA and 3% sucrose. Elongated shoots were rooted to 100% when wounded at the base of shoot, dipped in 4.4 mM indolebutyric acid (IBA) solution in the dark for 15 min and cultured on WPM supplemented with 1.11 μM BA, 0.25% activated charcoal, 34.5 μM phloroglucinol (PG) and 3% sucrose. This revised version was published online in June 2006 with corrections to the Cover Date.