Whole community estimates of macroalgal pigment concentration within two southern New Zealand kelp forests1

Light availability is a fundamental factor that controls the productivity and distribution of macroalgae and is highly variable, both spatially and temporally, in subtidal coastal systems. Our comprehension of how macroalgae respond to such variability is a significant knowledge gap that limits our understanding of how light influences the structure and productivity of these environments. Here, we examined the pigment characteristics of individual species, and for the first time the whole community, within one low‐light, and one high‐light kelp‐forest system in southern New Zealand. The aim was to quantify the range of pigmentation seen within the two kelp‐forests which differed in irradiance regime. Light availability was 33% and 64% greater at the high‐light compared to the low‐light site at 2 and 10 m depth, respectively. Results suggested Phaeophyceae species at deeper depths in the low‐light site may be living at the edge of their photosynthetic ability and pigment synthesis appeared significantly restricted. Even with greater investment in the pigment fucoxanthin, biomass of Phaeophyceae species was significantly lower in the low‐light site. Highly pigmented Rhodophyceae species made a greater proportional contribution to community biomass within the low‐light site where they likely possessed a photosynthetic advantage. This work helps explain discrepancies in community structure between the two study sites and explores the complex relationship between irradiance and photoacclimation. The comparison of community pigment concentration holds potential as a tool for assessing the relative degree of photoacclimation occurring between sites and provides a proxy of photosynthetic cost under a specific light regime.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

Desiccation tolerance in red and green gametophytes of Jamesoniella colorata in relation to photoprotection

This study tests the hypothesis that red-leaved gametophytes of the liverwort Jamesoniella colorata (Lehm.) Schiffn., which are found in relatively dry habitats, are more desiccation tolerant than their green counterparts, which are found in moister environments, through superior photoprotective systems. The potential role of red foliar pigments in relation to water deficits is investigated by measuring cell water-relations, oxidative damage and photosynthetic responses. The presence of red pigments, or other cellular constituents, did not affect cell water-relations during dehydration and thus appear not to be involved in cell osmotic regulation. During drying, both colour morphs showed a similar non-photochemical quenching activity and did not experience significant oxidative damage, as measured by the amounts of ascorbate, malondialdehyde and photosynthetic pigments. However, the levels of oxidative damage increased directly upon rewetting the gametophytes, especially in low light conditions (25 μmol m⁻² s⁻¹). The efficiency of photosystem II only recovered partially after severe water deficits in both phenotypes. However, the red gametophytes recovered faster and more completely from mild water deficits than did the greens. Moreover, they experienced significantly less photobleaching after rehydration in low light. It is suggested that red pigments and/or carotenoids in these gametophytes improve desiccation tolerance by alleviating photooxidative damage.     

Paralogous origin of the rhodopsinlike opsin genes in lizards

Rhodopsinlike opsins constitute a distinct phylogenetic group (Yokoyama 1994, Mol. Biol. Evol. 11:32–39). This RH2 group includes the green-sensitive opsins in chicken and goldfish and the blue-sensitive opsin in a nocturnal lizard gecko. In the present study, we isolated and sequenced the genomic DNA clones for the RH2 opsin gene, rh2 _Ac , of the diurnal lizard Anolis carolinensis. This single-copy gene spans 18.3 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Phylogenetic analysis strongly suggests that rh2 _Ac is more closely related to the chicken green opsin gene than to the gecko blue opsin gene. This gene tree differs from the organismal tree, where the two lizard species should be most closely related, implying that rh2 _Ac and the gecko blue-sensitive opsin genes have been derived from duplicate ancestral genes.Correspondence to: S. Yukiyama     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.