Newt lungs: a versatile system for the study of mucociliary transport

Lungs of the newt, Taricha granulosa, provide a versatile system for studying mucociliary transport. The mucociliary epithelium is restricted to a ventral strip of epithelium which extends the entire length of the lung. The transport process can be studied in the isolated lung or in a variety of functional components derived from it. These components include: (1) isolated sheets of the mucociliary epithelium, (2) flattened epithelial sheets grown in long term primary culture, (3) dissociated ciliated and mucous cells, (4) functional, demembranated ciliary tufts, and (5) isolated populations of discrete, demembranated axonemes. The latter two models can be reactivated with MgATP and made to beat at frequencies comparable to those measured in intact cells. Featuring large cells (25-30 microns in diameter) and cilia (12-13 microns in length) in comparison to other mucus-transporting systems, coupled with the ability to perform 'physiological' experiments at ambient temperatures, newt lungs afford a convenient system to study mechanisms involved in the control of ciliary beat frequency, waveform and coordination at several levels of organization.     

Gastric pouches and the mucociliary sole: setting the stage for nervous system evolution

Prerequisite for tracing nervous system evolution is understanding of the body plan, feeding behaviour and locomotion of the first animals in which neurons evolved. Here, a comprehensive scenario is presented for the diversification of cell types in early metazoans, which enhanced feeding efficiency and led to the emergence of larger animals that were able to move. Starting from cup-shaped, gastraea-like animals with outer and inner choanoflagellate-like cells, two major innovations are discussed that set the stage for nervous system evolution. First, the invention of a mucociliary sole entailed a switch from intra- to extracellular digestion and increased the concentration of nutrients flowing into the gastric cavity. In these animals, an initial nerve net may have evolved via division of labour from mechanosensory-contractile cells in the lateral body wall, enabling coordinated movement of the growing body that involved both mucociliary creeping and changes of body shape. Second, the inner surface of the animals folded into metameric series of gastric pouches, which optimized nutrient resorption and allowed larger body sizes. The concomitant acquisition of bilateral symmetry may have allowed more directed locomotion and, with more demanding coordinative tasks, triggered the evolution of specialized nervous subsystems. Animals of this organizational state would have resembled Ediacarian fossils such as Dickinsonia and may have been close to the cnidarian–bilaterian ancestor. In the bilaterian lineage, the mucociliary sole was used mostly for creeping, or frequently lost. One possible remnant is the enigmatic Reissner''s fibre in the ventral neural tube of cephalochordates and vertebrates.     

Ophiopogon root (Radix Ophiopogonis) prevents ultra-structural damage by SO2 in an epithelial injury model for studies of mucociliary transport

We studied the action of the herb, Ophiopogon root (OR) in a epithelial injury model, hypothesizing that it may have beneficial effects on mucociliary transport following injury to the palate induced by sodium metabisulphite (MB) which releases SO(2) on contact with water. OR (extract from 1g of root/ml)-incubated palates and non-incubated palates were compared to assess the effect of MB on mucociliary clearance on the bull frog palate. MB 10(-1) M, acutely increased mucociliary clearance time (MCT) by 254.5 +/- 57.3% in untreated and 243.3 +/- 98.5% in OR-incubated palates, (over all significance assessed by one-way ANOVA, F = 12.82, p < 0.001, df = 8,54 for MB and F = 10.56, p < 0.001, df = 8,54 for OR). MCT returned to normal during recovery in OR-treated palates following MB. In untreated palates, MCT did not return to control values during a similar recovery period. ANOVA comparing MCTs in the recovery period in untreated vs OR-treated palates was significantly different (F = 2.92, p < 0.03, df = 5,36). SEM images of epithelial tissue, analyzed by morphometry, showed a 25 +/- 12% loss of ciliated cells in untreated palates and little or no damage to cilia in OR-treated palates. Intact groups of ciliated cells were found in SEM micrographs of mucus from MB-treated palates. We conclude that the loss of cilia or ciliated cells prevented full recovery of MCT after MB in untreated palates. In OR-incubated palates, mucociliary transport was completely restored within 20 min after topical application of MB, possibly through a protective action on the extra-cellular matrix.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli K-12: isolation and properties of mutants defective in leucine-repressible transport activities.

The characteristics of a mutant (hrbA) of Escherichia coli K-12 that is defective in a leucine-nonrepressible transport system, the LIV-3 system, for branched-chain amino acids were described previously (I. Yamato et al., J. Bacteriol 138:24-32, 1979). New mutants requiring a high concentration of isoleucine for growth were isolated from strain B763 (hrbA ileA) after mutagenesis with ethyl methane sulfonate. These mutants had a defect of the leucine-repressible transport activities for branched-chain amino acids of the parental strain. One of these mutants, strain B7634, had defects of two independent genetic loci (hrbBC and hrbD). The genes hrbBC were mapped at min 76 near malT, and the gene hrbD mapped at min 77 near xyl on the E. coli genetic map. The substrate specificity, kinetic properties, and source of coupling energy of the transport system coded for by each of these genes were studied using cytoplasmic membrane vesicles and intact cells. The results identified three transport systems with characteristic features other than the LIV-3 system. The hrbB and hrbC systems are responsible for the uptake activites of the LIV-2 system, with a high Km value, and the LIV-1 system, with a low Km value, respectively. Both activities are repressed by leucine and inhibited by threonine and the b(--) isomer of 2-aminobicycloheptyl-2-carboxylic acid. They both utilize adenosine 5'-triphosphate as coupling energy and are not detected in cytoplasmic membrane vesicles. The hrbD system is responsible for the LIV-4 system, with a high Km value. Its activity is repressed by leucine and partially inhibited by threonine. It is detected in cytoplasmic membrane vesicles with a proton motive force as the driving energy.     

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: Evidence for a signalling role of docosahexaenoic acid

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport.We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced d-[³H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with α-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[³H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on d-[³H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions.These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.     

L-asparaginase of Thermus thermophilus: Purification,properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.     

2D-MH: A web-server for generating graphic representation of protein sequences based on the physicochemical properties of their constituent amino acids

Introduction of graphic representation for biological sequences can provide intuitive overall pictures as well as useful insights for performing large-scale analysis. Here, a new two-dimensional graph, called “2D-MH”, is proposed to represent protein sequences. It is formed by incorporating the information of the side-chain mass of each of the constituent amino acids and its hydrophobicity. The graphic curve thus generated is featured by (1) an one-to-one correspondence relation without circuit or degeneracy, (2) better reflecting the innate structure of the protein sequence, (3) clear visibility in displaying the similarity of protein sequences, (4) more sensitive for the mutation sites important for drug targeting, and (5) being able to be used as a metric for the “evolutionary distance” of a protein from one species to the other. It is anticipated that the presented graphic method may become a useful vehicle for large-scale analysis of the avalanche of protein sequences generated in the post-genomic age. As a web-server, 2D-MH is freely accessible at http://icpr.jci.jx.cn/bioinfo/pplot/2D-MH, by which one can easily generate the two-dimensional graphs for any number of protein sequences and compare the evolutionary distances between them.     

Colonization of the Hawaiian Archipelago via Johnston Atoll: a characterization of oceanographic transport corridors for pelagic larvae using computer simulation

Larval transport between Johnston Atoll and the Hawaiian Archipelago was examined using computer simulation and high-resolution ocean current data. The effects of pelagic larval duration and spawning seasonality on long-distance transport and local retention were examined using a Lagrangian, individual-based approach. Retention around Johnston Atoll appeared to be low, and there appeared to be seasonal effects on both retention and dispersal. Potential larval transport corridors between Johnston Atoll and the Hawaiian Archipelago were charted. One corridor connects Johnston Atoll with the middle portion of the Hawaiian Archipelago in the vicinity of French Frigate Shoals. Another corridor connects Johnston Atoll with the lower inhabited islands in the vicinity of Kauai. Transport appears to be related to the subtropical countercurrent and the Hawaiian Lee countercurrent, both located to the west of the archipelago and flowing to the east. A new analytical tool, termed CONREC–IRC is presented for the quantification of spatial patterns.     

Understanding polysaccharide production and properties using seed coat mutants: future perspectives for the exploitation of natural variants

Background

The epidermal cells of the seed coat of certain species accumulate polysaccharides during seed development for cell wall reinforcement or release on imbibition to form mucilage. Seed-coat epidermal cells show natural variation in their structure and mucilage production, which could explain the diverse ecophysiological roles proposed for the latter. Arabidopsis mucilage mutants have proved to be an important tool for the identification of genes involved in the production of seed-coat polysaccharides.

Scope

This review documents genes that have been characterized as playing a role in the differentiation of the epidermal cells of the arabidopsis seed coat, the natural variability in polysaccharide features of these cells and the physiological roles attributed to seed mucilage.

Conclusions

Seed-coat epidermal cells are an excellent model for the study of polysaccharide metabolism and properties. Intra- and interspecies natural variation in the differentiation of these epidermal cells is an under-exploited resource for such studies and promises to play an important part in improving our knowledge of polysaccharide production and ecophysiological function.     

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high‐salt protein fraction for quantitative proteome analysis

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high‐molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high‐salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2‐D DIGE, MS and Western blotting.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

Suppression and restoration of primordial germ cell marker gene expression in channel catfish,Ictalurus punctatus,using knockdown constructs regulated by copper transport protein gene promoters: Potential for reversible transgenic sterilization

Complementary DNA overexpression and short hairpin RNA interference approaches were evaluated for decreasing expression of primordial germ cell (PGC) marker genes and thereby sterilizing channel catfish, Ictalurus punctatus, by delivering knockdown constructs driven by a constitutive promoter from yeast and a copper transport protein gene into fish embryos by electroporation. Two PGC marker genes, nanos and dead end, were the target knockdown genes, and their expressions, along with that of an off-target gene, vasa, were evaluated temporally using real-time polymerase chain reaction. Copper sulfate was evaluated as a repressor compound. Some of the constructs knocked down PGC marker gene expression, and some of the constructs were partially repressed by application of 0.1-ppm copper sulfate. When the rate of sexual maturity was compared for three-year-old broodfish that had been exposed to the sterilizing constructs during embryologic development and controls that had not been exposed, several treatments had reduced sexual maturity for the exposed fish. Of two promoter systems evaluated, the one which had been designed to be less sensitive to copper generally was more effective at achieving sterilization and more responsive to repression. Knockdown constructs based on 3′ nanos short hairpin RNA interference appeared to result in the best repression and restoration of normal sexual maturity. We conclude that these copper-based systems exhibited good potential for repressible transgenic sterilization. Optimization of this system could allow environmentally safe application of transgenic technology and might be applicable to other applications for aquatic organisms.     

A simple and inexpensive method for investigating microbiological, enzymatic, or inorganic catalysis using standard histology and microbiology laboratory equipment: assembly, mass transfer properties, hydrodynamic conditions and evaluation

We introduce a generic, simple, and inexpensive method for performing microbiological, enzymatic, or inorganic catalysis with solids using standard histology and microbiology laboratory equipment. Histology cassettes were used to standardize hydrodynamic conditions and to protect the catalysts and their solid supports. Histology cassettes have the following advantages: they are readily available, inexpensive, solvent and acid resistant, automatable, and the slots in the cassette walls allow liquid to circulate freely. Standard Erlenmeyer flasks were used as reaction vessels. We developed a new camera to observe the movement and position of the histology cassettes as well as the liquid in the Erlenmeyer flasks. The camera produces a stable image of the rotating liquid in the Erlenmeyer flask. This visualization method revealed that in a 250 ml Erlenmeyer flask, stable operating conditions are achieved at a shaking frequency of 300 rpm and a fill volume of 30 ml. In vessels with vertical walls, such as beakers or laboratory bottles, the movement of the histology cassette is not reproducible. Mass transfer characterization using a biological model system and the chemical sulfite-oxidation method revealed that the histology cassette does not influence gas-liquid mass transfer.