Aspects of the life cycle of marine nematodes

Summary 1. Life cycles of approximately one month are recorded for laboratory maintained cultures of chromadorid, monhysterid and oncholaimid nematodes.2. Amphimictic and parthenogenetic reproduction occurs in the species investigated. Reproduction inMonhystera parelegantula is by parthenogenesis.3. The chromadoridChromadorina epidemos and the oncholaimidViscosia macramphida are able to reproduce by either amphimixis or parthenogenesis.4.Acanthonchus cobbi, Chromadora macrolaimoides andEuchromadora gaulica are amphimitically reproducing species.

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Aspekte des Lebenszyklus mariner Nematoden

Kurzfassung Bei einigen marinen Nematoden, die in Biscayne Bay (Florida) gesammelt und im Labor gezüchtet worden sind, vollzieht sich der gesamte Lebenszyklus ungefähr innerhalb eines Monats. BeiAcanthonchus cobbi, Chromadora macrolaimoides undEuchromadora gaulica ist zweigeschlechtliche Fortpflanzung festgestellt worden.Monohystera parelegantula vermehrt sich parthenogenetisch. Ein möglicherweise durch Umwelteinflüsse bedingter Wechsel von bisexueller und parthenogenetischer Fortpflanzung tritt beiChromadora epidemos undViscosia macramphida auf.

     

Studies on marine fungal-nematode associations and plant degradation

Summary 1. Studies of the broad-leafed turtle grass,Thalassia testudinum König, have revealed a diverse range of fungal infestation different in generic composition and dynamics of attack from that found on submerged wood. Certain of the fungi, notably the AscomyceteLindra thalassiae, initiate considerable degradation of leaf tissue and show a developmental cycle in nature related to the physiological state of the host plant.2. Use of fungal-cellulose mats as a trapping substrate has been extremely effective for discernment of ecologically significant shifts in nematode concentrations, especially those of the omnivorous species,Metoncholaimus scissus.3. Patterns of activity ofM. scissus, as well as those of various foliicolous nematodes, suggest that loci of organic material, such as fungal infested leaves and decaying plant tissue, significantly affect biological activity of these animals.4. Laboratory analysis of degraded cotton cellulose filters show a striking incidence of fungal reproduction of the ascomycetous fungusLulworthia, along with development of a considerable associated nematode fauna, especially species ofViscosia (V. macramphida) andLeptolaimus (L. plectoides). Successional patterns in nematode development are noted with continued degradation of the cotton cellulose matrix.

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Studien über marine Pilz-Nematoden-Assoziationen und Pflanzendegradation

Kurzfassung Untersuchungen am SeegrasThalassia testudinum König haben ergeben, daß sich hier Pilzinfektionen hinsichtlich der Komposition der beteiligten Gattungen und der Dynamik des Befalls von den am untergetauchten Holz festgestellten Infektionen unterscheiden. Bestimmte Pilze, insbesondere der AscomycetLindra thalassiae, leiten eine erhebliche Degradation des Blattgewebes ein und zeigen einen Entwicklungszyklus, welcher in Beziehung steht zum physiologischen Zustand der Wirtspflanze. Die Anwendung von Pilz-Zellulose-Matten als Einfangsubstrat war außerordentlich erfolgreich für das Erkennen ökologisch signifikanter Verschiebungen in den Nematodenkonzentrationen, insbesondere bei der omnivoren ArtMetoncholaimus scissus. Die Aktivitätsmuster vonM. scissus — ebenso wie die verschiedener foliicolöser Nematoden — deuten darauf hin, daß pilzinfizierte und zerfallende Pflanzenteile in entscheidendem Maße die biologische Aktivität dieser Tiere beeinflussen. Laboratoriumsanalysen degradierter Wollzellulosefilter lassen eine überraschend starke Vermehrung des AscomycetenLulworthia erkennen und gleichzeitig die Entwicklung einer beachtlichen Fauna assoziierter Nematodenarten, insbesondere vonViscosia macramphida undLeptolaimus plectoides. Im Verlaufe der weiteren Degradation der Wollzellulosematrix kommt es bei der Nematodenfauna zu entsprechenden Sukzessionen.

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Contribution No. 768 from the Institute of Marine Science, University of Miami, Miami, Florida, and from the Canada Department of Agriculture, Ottawa, Canada. This work was supported at the IMS by grant GM 12842 from the National Institutes of Health, USA.     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Oxidant inhibition of epithelial active sodium transport

The purpose of this study was to quantify the effects of extracellularly generated partially reduced oxygen species on active sodium (NA⁺) transport across the ventral toad skin, a well-studied epithelium. Sections of skin from decapitated toads were mounted in an Ussing chamber, bathed on both sides with electrolyte solution containing 500 μM xanthine and bubbled continuously with room air. The tissues were short-circuited, and short circuit current (Isc) and tissue resistance (R_t were monitored continuously with an automatic voltage clamp apparatus. Fifteen mU/ml of xanthine oxidase (XO), either purchased from Calbiochem or purified from cream, were instilled in either the apical (mucosal) or basolateral (serosal) baths at t = 0 and T = 10 min. Hydrogen peroxide (H₂O₂) concentrations increased to 200 μM within the first 20 min and then decreased, reaching a value of 40 μM by 60 min. Mean [H₂O₂] was 90 μM. Instillation of XO in the apical bath resulted in a large decrease in Isc and an increase in R_t, their values being 43% and 160% of their corresponding controls 85 min after the first instillation. Addition of superoxide dismutase and catalase completely prevented these changes. Instillation of XO in the basolateral bath had no effect. Similar physiological responses were obtained using the Calbiochem XO or the purified XO, which contained no measurable protease activity. It was concluded that extracellularly generated partially reduced oxygen species may interfere with active Na⁺ transport by possibly damaging apical Na⁺ channel proteins.     

Coordinated regulation of Na/H exchange and [K-Cl] cotransport in dog red cells

Swelling-activated [K-Cl] cotransport and shrinkage-activated Na/H exchange were studied in dog red cells with altered internal Mg or Li content. The two pathways responded in a coordinated fashion. When cells were depleted of Mg, [K-Cl] cotransport was stimulated and Na/H exchange was inhibited. Raising internal Mg had the opposite effect: [K-Cl] cotransport was inhibited and Na/H exchange was stimulated. Li loading, previously shown to stimulate Na/H exchange, inhibited [K-Cl] cotransport. From these reciprocal effects and from other evidence, we surmise that the regulation of Na/H exchange and [K-Cl] cotransport is conducted and coordinated by a discrete mechanism that responds to changes in cell volume and is sensitive to cytoplasmic Mg and Li concentrations.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties

Summary Two different³H-saxitoxin-binding proteins, with distinct biochemical and functional properties, were isolated from rat brain using a combination of anion exchange and lectin affinity chromatography as well as high resolution size exclusion and anion exchange HPLC. The alpha subunits of the binding proteins had different apparent molecular weights on SDS-PAGE (Type A: 235,000; Type B: 260,000). When reconstituted into planar lipid bilayers, the two saxitoxin-binding proteins formed sodium channels with different apparent single-channel conductances in the presence of batrachotoxin (Type A: 22 pS; Type B: 12 pS) and veratridine (Type A: 9 pS; Type B: 5 pS). The subtypes were further distinguished by scorpion (Leiurus quinquestriatus) venom which had different effects on single-channel conductance and gating of veratridine-activated Type A and Type B channels. Scorpion venom caused a 19% increase in single-channel conductance of Type A channels and a 35-mV hyperpolarizing shift in activation. Scropion venom double the single-channel conductance of Type B channels and shifted activation by at least 85 mV.     

Voltage-independent barium-permeable channel activated inLymnaea neurons by internal perfusion or patch excision

Summary Isolated nerve cells fromLymnaea stagnalis were studied using the internal-perfusion and patch-clamp techniques. Patch excision frequently activated a voltage-independent Ba²⁺-permeable channel with a slope conductance of 27 pS at negative potentials (50mm Ba²⁺). This channel is not seen in patches on healthy cells and, unlike the voltage-dependent Ca channel, is not labile in isolated patches. The activity of the channel in inside-out patches is unaffected by intracellular ATP, Ca²⁺ below 1mm or the catalytic subunit of cAMP-dependent protein kinase but is reversibly blocked by millimolar intracellular Ca²⁺ or Ba²⁺. The channel can be activated in on-cell patches by either internal perfusion with high Ca²⁺ or the long-term internal perfusion of low Ca²⁺ solutions not containing ATP. These channels may carry the inward Ca²⁺ current which causes a regenerative increase in intracellular Ca⁺ when snail neurons are perfused with high Ca²⁺ solutions. High internal Ca²⁺, or long periods of internal perfusion with ATP-free solutions, induces an increase in a resting (–50 mV) whole-cell Ba²⁺ conductance. This conductance can be turned off by returning the intracellular perfusate to a low Ca²⁺ solution containing ATP and Mg²⁺. The activity of this channel appears to have an opposite dependence on intracellular conditions to that of the voltage-dependent Ca channel.