Extracellular matrix allows PC12 neurite elongation in the absence of microtubules

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.     

A cytomechanical investigation of neurite growth on different culture surfaces

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.     

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G+C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome. Correspondence to: C. Diviès     

Biomarker assessment of organic matter sources and degradation in Canadian High Arctic littoral sediments

This paper presents an uncomplicated approach to improve estimates of groundwater nutrient load to a marine embayment. A two-dimensional chemical profile of shallow groundwater was analysed in a sandy beach in three seasons (early summer, late summer and mid winter) and an adjusted estimate of groundwater nutrient discharge was derived that accounts for a complex biogeochemical environment and non-conservative behaviour of nutrients in the pre-discharge beach groundwater. The study was conducted at Cockburn Sound, Western Australia, where there has been significant groundwater contamination and associated marine ecological degradation. Losses in nitrogen and increases in phosphorus were observed along the discharge pathway beyond that expected from mixing with marine water, and the changes were attributed to chemically and biologically mediated reactions. A slow groundwater velocity (0.14–0.18 m day^?1), high organic carbon (TOC = 0.35–4.9 mmol l^?1, DOC = 0.28–4.6 mmol l^?1) and low to sub-oxic conditions (DO = 0.4–24% saturation) were deemed suitable for chemically and biologically mediated reactions to occur and subsequently alter regional estimates of groundwater nutrient concentration. Accounting for this environment, groundwater loads were calculated that were 1–2 orders of magnitude less than previous regional-based estimates: 0.4–13 kg NO _x ^?  day^?1, 0.2–24 kg NH₄ ⁺ day^?1 and 0.004–0.8 kg FRP day^?1. This paper applies knowledge of recent research and presents scope to marine managers or modellers to account for groundwater inputs to the marine environment.     

Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family.     

Hydrological controls on the diatom assemblage of a seasonal arctic river: Boothia Peninsula,Nunavut, Canada

This study characterizes the melt season diatom assemblage of a middle arctic river with respect to hydrological conditions. In addition, the potential to identify species that show a strong affinity for the lotic environment provides an opportunity to interpret stratigraphic changes in these species in the lake sedimentary record in terms of past hydrological change. Understanding long-term hydrological variability is critical for assessing both current and future environmental change. Significantly higher relative abundances of Achnanthes minutissimaKützing, Fragilaria capucina var. vaucheriae (Kützing) Lange-Bertalot, Diatoma tenuisAgardh, Cymbella arctica(Lagerstedt) Schmidt, C. minutaHilse ex. Rabenhorst, C. silesiaca Bleisch and Encyonema fogedii Krammer in the lotic environment throughout the 2001 growing season compared to the lacustrine sedimentary record suggest that these species characterize the Lord Lindsay River diatom assemblage. Comparison of seasonal abundances of these taxa to hydrological parameters including discharge, electrical conductivity, and water temperature reveal key information about the character of this community. The fact that the river diatom assemblage changes very little throughout the sampling period, despite major changes in hydrological conditions, suggests a degree of resilience and inherent structure in the community. However, a decrease in diatom biomass in response to rapid and dramatic changes in hydrological conditions following a major rainfall event suggests that a threshold tolerance may exist, with potentially important implications for interpreting stratigraphic changes in the paleoenvironmental record.