Experimental evaluation of a minnow trap for small lotic fish

A minnow trap that operates in various flow regimes in streams and allows sampling of small fish from stream bed microhabitats was developed. In laboratory and field tests, the most efficient trap design for capturing and retaining various species of fish had one funnel oriented downstream, a plexiglass body, and commercial trout food as bait. These lightweight traps can be set in a wide range of current velocities and depths, and can be useful in investigations that examine the microhabitat use, diel activity patterns or population densities of small lotic fish. Guidelines for the trap's use and for quick verification of capture success in new situations are suggested.     

Multiple and alternative adhesive responses on defined substrata of an immortalized dorsal root neuron hybrid cell line

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: modulation of affinity binding to fibronectin

Tissue culture substratum adhesion sites from EGTA-detached Platt human neuroblastoma cells were extracted with a buffer containing ocytlglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4 = -radiolabeled proteoglycans and 3H-leucine-radiolabeled proteins. Of the sulfate-radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE-Sephadex columns under two different buffer elution conditions. Under DEAE-I conditions in low ionic strength acetate buffer, two major peaks of 35SO4 = -radiolabeled material (A,B) and a minor peak (C) could be resolved in the NaCl gradient; however, three-fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE-II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE-I binds to the hydrophobic matrix, octyl-Sepharose, while 80% of the proteoglycan in DEAE-I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor-4 affinity columns. In contrast, peak AB proteoglycan from DEAE-II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor-4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several other proteins. These results demonstrate selectivity and specificity in this modulation and indicate that a previously unidentified protein(s) is responsible.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.     

Reduction of predation risk under the cover of darkness: Avoidance responses of mayfly larvae to a benthic fish

Summary Mayfly larvae of Paraleptophlebia heteronea (McDunnough) had two antipredator responses to a nocturnal fish predator (Rhinichthys cataractae (Valenciennes)): flight into the drift and retreat into interstitial crevices. Drift rates of Paraleptophlebia abruptly increased by 30 fold when fish were actively foraging in the laboratory streams but, even before fish were removed, drift began returning to control levels because larvae settled to the substrate and moved to areas of low risk beneath stones. This drifting response was used as an immediate escape behavior which likely decreases risk of capture from predators which forage actively at night. Surprisingly, drift most often occurred before contact between predator and prey, and we suggest that in darkness this mayfly may use hydrodynamic pressure waves for predator detection, rather than chemical cues, since fish forage in an upstream direction. Although drifting may represent a cost to mayfly larvae in terms of relocation to a new foraging area with unknown food resources, the immediate mortality risk probably out-weighs the importance of staying within a profitable food patch because larvae can survive starvation for at least 2 d. In addition to drifting, mayflies retreated from upper, exposed substrate surfaces to concealed interstitial crevices immediately after a predator encounter, or subsequent to resettlement on the substrate after predator-induced drift. A latency period was associated with this response and mayflies remained in these concealed locations for at least 3 h after dace foraging ceased. Because this mayfly feeds at night and food levels are significantly lower in field refugia under stones, relative to exposed stone surfaces, predator avoidance activity may limit foraging time and, ultimately, reduce the food intake of this stream mayfly.     

Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: aging in vivo or in vitro

Attachment, spreading, and microfilament reorganization have been evaluated in human dermal reticular fibroblasts isolated from the inner, upper aspect of the arm of a newborn male (RET5 cells) and a 78-year-old male (RET8 cells). Substrata were tested using a set of complementary fragments from individual polypeptide chains of human plasma fibronectin (pFN) or cellular FNs (cFN). With both cell classes, fragments containing the C-terminal heparin-binding (HepII) domain only elicited linear bundles of microfilaments in spreading cells but no stress fibers; fragments containing the RGDS-dependent cell-binding (CellI) domain elicited only partial spreading with condensations of F-actin at ruffling membranes and at other regions along the plasma membrane. The minimum sequence required to obtain responses identical to those on intact pFN (broad spreading with extensive stress fiber formation) was found in fragment 155 (F155) from the beta chain of pFN; F155 contains both HepII and CellI domains. In contrast, the analogous fragment from the alpha chain of pFN (F145) was notably less effective for generating stress fibers. This evidence along with the better attachment, spreading, and microfilament bundle formation on the HepII fragment from the beta chain than the analogous fragment from the alpha chain indicates that the extra type III homology unit permits more effective interaction of beta chain fragments with cell-surface heparan sulfate proteoglycan and possibly integrin (binding efficiency to the substratum was similar for fragments from both chains). Therefore, alternatively spliced sequences that neighbor binding domains can play significant roles in the interaction of the domain with cell-surface receptors of dermal fibroblasts. Comparison of RET5 responses with those of RET8 cells has identified changes in adhesive mechanisms as cells undergo "aging" processes. Attachment and microfilament bundle formation were far more effective for RET5 cells than for RET8 cells on any of the HepII fragments. Conversely, RET8 cells were far more sensitive to an RGDS-containing peptide in their medium on CellI fragments than RET5 cells. These results together indicate that in vivo aging leads to greater dependence upon cell-surface integrin binding and less dependence upon heparan sulfate proteoglycan binding for responses on FN matrices. When RET5 cells entered senescence (in vitro aging), they also became much more sensitive to peptide A. On several fragments and on intact pFN, RET8 cells generated very thick stress fibers that were observed only on one fragment with RET5 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ethanol on the binding of 35S-T-butylbicyclophosphorothionate to mouse brain membranes

The effect of in vitro addition of ethanol (0.02-1.0 M) on the binding of 35S-TBPS was examined in brain membranes from cerebellum and cortex of naive or chronically ethanol-treated C57B1 mice. In brain membranes of untreated animals, increasing concentrations of ethanol produced a dose-related inhibition of 35S-TBPS binding in the brain areas investigated. Additional studies showed that this effect of ethanol was due to a decreased affinity of 35S-TBPS for its binding sites. Chronic treatment of the animals with ethanol, which produced tolerance to and dependence on ethanol, did not alter ethanol's ability to inhibit the binding of 35S-TBPS. In naive animals, the in vitro addition of GABA or pentobarbital produced a pronounced inhibition of 35S-TBPS, both drugs being more potent in the cerebellum than in the cortex. Picrotoxin also produced a dose-dependent inhibition at 35S-TBPS, but was equally potent in the brain areas investigated. The inhibition by GABA or pentobarbital was not influenced by in vitro addition of a physiologically relevant concentration of ethanol (100 mM), whereas ethanol produced a significant increase in the IC50 values for picrotoxin both in the cortex and in the cerebellum. Furthermore, the inhibitory effects of GABA or pentobarbital on 35S-TBPS binding remained unchanged in animals chronically treated with ethanol for 7 days. Our data indicate that ethanol may affect the GABA receptor system through a rather specific interaction with the 35S-TBPS recognition site, but that this action of ethanol is not altered by the development of tolerance to and dependence on ethanol.