Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Effects of two common macrophytes on methane dynamics in freshwater sediments

The methane cycle in constructed wetlands without plants and withPhragmites australis (reed) and Scirpus lacustris (bulrush) wasinvestigated. Variations in CH₄production largely determined variations in CH₄ emission among the systems, rather than variations inCH₄ storage and oxidation. Twofoldlower CH₄ production rates in theScirpus system (5.6–13 mmol m^-2 d^-1) relative to the control (16.7–17.6 mmolm^-2 d^-1) were accompanied by a lower contribution ofmethanogenesis to organic carbon metabolism (20% for Scirpus vs.80% for control). Sedimentary iron(II) reservoirs were smallerin the Scirpus than control sediment (300 vs. 485 mmol.m^-2) and a shuttle role for iron asan intermediate between root O₂release and carbon oxidation, attenuating the availability of substrate formethanogens, is suggested. Differences in CH₄ production among the Phragmites and Scirpus systemswere controlled by the interspecific variation in sediment oxidationcapacities of both plant species. Comparatively, in the Phragmites sediment,dissolved iron reservoirs were larger (340 mmol.m^-2) and methanogenesis was a more importantpathway (80%). Methane transport was mainly plant mediated inthe Phragmites and Scirpus systems, but ebullition dominated in thenon-vegetated control systems as well as in the vegetated systems when plantbiomass was low.     

Secretion of Escherichia coli DsbA and DsbC proteins from Brevibacillus choshinensis: stimulation of human epidermal growth factor production

DsbA and DsbC, members of the thioredoxin super-family of redox proteins, which are expressed in the periplasmic space of Escherichia coli, were cloned into and successfully secreted from Brevibacillus choshinensis at 100 g ml^–1. Both proteins were active in exchanging disulfide bonds of bovine insulin in vitro. Furthermore, DsbA secreted by B. choshinensis promoted the conversion of non-native human epidermal growth factor to the native form.     

An analysis of flash-induced electrical transients of a BLM containing chloroplast lamella extracts

Summary The electrical transients produced by chloroplast bilayer lipid membranes (Chl-BLM) from flash excitation are seen to result from three photocurrents and a discharge current. Each of the three photo-initiated charge transports in Chl-BLM (designated as Components A, B and C) exhibits an action spectrum similar to chlorophyll absorption spectra. The fast components (A and B), which are induced by electron acceptors such as Fe⁺³, have rise-times of 3 sec and 20 msec, and occur in TLM (thin lipid membranes, i.e., colored membranes up to 1 thick) as well as in BLM. Component C is induced by a transmembrane pH difference or applied voltage, has a rise-time of 1 sec, and occurs only in BLM. Component C is associated with exciton dissociation and proton transport. The mobility of the Component A current carriers in TLM is estimated to be about 1×10^–2 cm²/volt sec, and are, hence,electronic. The photovoltage waveforms are described by equations developed, which consider Component A as being caused by a direct charge separation proportional to the illumination intensity (within 0.5 sec), and Components B and C being caused by two types of exciton processes which cause charge transport after the illumination period.     

Stable isotope and radiocarbon compositions of methane emitted from tropical rice paddies and swamps in Southern Thailand

Stable isotopes (¹³C, D) and radiocarbon weremeasured in methane bubbles emitted from rice paddies and swamps in southernThailand. Methane emitted from the Thai rice paddies was enriched in¹³C (mean ¹³C; –51.5 ±7.1 and–56.5 ± 4.6 for mineral soil and peat soil paddies,respectively)relative to the reported mean value of methane from temperate rice paddies(– 63 ± 5). Large seasonal variation was observed in¹³C(32) in the rice paddies, whereas variationinD was much more smaller (20), indicating that variation in¹³C is due mainly to changes in methane production pathways.Values of ¹³C were lower in swamps (–66.1 ±5.1)than in rice paddies. The calculated contribution of acetate fermentation from¹³C value was greater in rice paddies (mineral soils:62–81%, peat soils: 57–73%) than in swamps (27–42%). Din methane from Thai rice paddies (–324± 7 (n=46)) isrelativelyhigher than those from 14 stations in Japanese rice paddies ranging from–362 ± 5 (Mito: n=2) to –322 ± 8(Okinawa: n=3), due tohigher D in floodwaters. ¹⁴C content in methane produced fromThai rice paddies (127±1 pMC) show higher ¹⁴Cactivity compared with previous work in paddy fields and those from Thai swamps(110±2 pMC).     

Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml^–1) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml^–1 dropped to 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml^–1 probably reflects a loss of cell division capability rather than cell death.     

Conditions for high frequency embryogenesis from orange (Citrus sinensis Osb.) protoplasts

Using Trovita orange (Citrus sinensis Osb.) protoplasts isolated from 6-year-old nucellar callus, the effects of protoplast density and mannitol concentration on cell divisions and embryoid formation were examined.Somatic embryogenesis in nearly direct manner was observed only at a combination of low cell densities (4×10⁴/ml) and low mannitol concentrations (0.4 M). Two alternatives to achieve high frequency embryogenesis (70%) were to either dilute the cells to lower densities, or to do serial transfers of cells to fresh medium.Orange protoplasts (cells) showed embryogenic potential, and repression of embryogenesis occurred when protoplasts were cultured at a high density and/or under high osmotic pressure.     

Characterization of a chloride channel reconstituted from cardiac sarcoplasmic reticulum

We have characterized a voltage-sensitive chloride channel from cardiac sarcoplasmic reticulum (SR) following reconstitution of porcine heart SR into planar lipid bilayers. In 250 mm KCl, the channel had a main conductance level of 130 pS and exhibited two substrates of 61 and 154 pS. The channel was very selective for Cl^– over K⁺ or Na⁺ ( and ). It was permeable to several anions and displayed the following sequence of anion permeability: SCN^– > I^– > NO ₃ ^– Br^– > Cl^– > f^– > HCOO^–. Single-channel conductance saturated with increasing Cl^– concentrations (K _m= 900 mm and _max = 488 pS). Channel activity was voltage dependent, with an open probability ranging from 1.0 around 0 mV to 0.5 at +80 mV. From –20 to +80 mV, channel gating was time-independent. However, at voltages below –40 mV the channel entered a long-lasting closed state. Mean open times varied with voltage, from 340 msec at –20 mV to 6 msec at +80 mV, whereas closed times were unaffected. The channel was not Ca²⁺-dependent. Channel activity was blocked by disulfonic stilbenes, arylaminobenzoates, zinc, and cadmium. Single-channel conductance was sensitive to trans pH, ranging from 190 pS at pH 5.5 to 60 pS at pH 9.0. These characteristics are different from those previously described for Cl^– channels from skeletal or cardiac muscle SR.We thank Dr. Barry Pallotta for help with open and closed intervals analysis and Dr. Gerhard Meissner for his suggestions for the preparation of cardiac sarcoplasmic reticulum membranes. This work was supported by a grant from the National Institutes of Health to R.L.R. and a Student Grant-in-Aid from the American Heart Association, North Carolina affiliate to C.T. R.L.R. is an Established Investigator of the American Heart Association.     

Release of carboxylic anions and protons by tomato roots in response to ammonium nitrate ratio and pH in nutrient solution

The exudation of certain organic anions and protons by roots which may affect solubility of metals and P and uptake by plants, is affected by nitrogen form and pH. The objective of this work was to study exudation of carboxylates and H⁺/OH^– by tomato plants in response to NH₄/NO₃ ratio and pH in nutrient solution. Four NH₄/(NH₄+NO₃) ratios (R= 0, 0.33, 0.67 and 1) and constant vs. variable solution pH treatments were investigated. The sum of the exudation rates of all carboxylates tended to decline with increasing R, particularly tri- and dicarboxylates. The molar fraction of the exuded tri- and dicarboxylates, averaged over all treatments and plant ages, increased in the order tartarate 2%), malate (6%), succinate (15%), citrate (26%) and fumarate (46%). At R=1 the solution pH dropped from 5.2 to 3 and at R=0 increased to 8. The R corresponding to the pH stat of tomato plant was 0.3. For the constant solution pH treatment, the effect of solution pH on carboxylate exudation rate was small as compared to the effect of R. The exudation of citrate and H⁺ efflux which were initiated when NO₃ and NH₄ uptake rates per plant exceeded certain threshold values, increased with plant age.     

Comparative studies on cellular resistance of bivalves from marine and brackish waters

Summary 1. The cellular resistance of representatives from four euryhaline poikilosmotic bivalves caught in Büsum (North Sea, 30 S), Kieler Förde (Belt Sea, 15 S), and Tvärminne (Gulf of Finland, 6 S) has been tested. All experiments were performed in October.2. According to their cellular salinity rangesMacoma baltica andMytilus edulis are more euryhaline thanMya arenaria andCardium edule.3. The cellular heat resistance decreases in specimens from the North Sea in the following order:Cardium, Mya, Macoma, Mytilus. Among the representatives collected near Tvärminne,Macoma has the highest heat resistance.4. The cellular freezing resistance in specimens from the North Sea is highest inMytilus. The bivalves of the Gulf of Finland practically lack resistance to freezing.5. In localities of decreased salinity the cellular resistance of these bivalves to a dilution of the external medium is somewhat increased, but simultaneously the cellular resistance to other environmental factors, such as heat, freezing and high salinity, decreases unspecifically.

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Vergleichende Untersuchungen über zelluläre Resistenz bei Muscheln aus Meer- und Brackwasser

Kurzfassung An vier euryhalinen poikilosmotischen Muschelarten aus Büsum (Nord-seeküste, 30 S), der Kieler Förde Beltsee, 15 S) und Tvärminne (Finnischer Meerbusen, 6 S), wurden zur gleichen Jahreszeit (Herbst) vergleichende Untersuchungen über die zellulären Resistenzgrenzen durchgeführt. Auf Grund ihrer zellulären Salzgehaltsbereiche sindMacoma baltica undMytilus edulis euryhaliner alsMya arenaria undCardium edule. Die zelluläre Hitzeresistenz nimmt bei den Individuen aus der Nordsee in der Reihenfolge:Cardium, Mya, Macoma, Mytilus ab. Bei Exemplaren aus Tvärminne ist die zelluläre Hitzeresistenz vonMacoma am größten. Die zelluläre Gefrierresistenz ist unter den Muscheln aus der Nordsee am größten, fehlt dagegen praktisch bei den Tieren aus dem Finnischen Meerbusen. Bei verringertem Salzgehalt des Fundortwassers ist die zelluläre Resistenz der untersuchten Muscheln gegenüber Verdünnung des Außenmediums jeweils etwas erhöht, gleichzeitig nimmt die zelluläre Resistenz gegenüuber anderen Außenfaktoren wie Hitze, Frost und hohen Salzkonzentrationen unspezifisch ab.

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Dedicated to Professor Dr.Friedrich Krüger on his 65^th birthday, August 18, 1967.     

Geochemical controls for Al, Fe, Mn, Cd, Cu, Pb, and Zn during experimental acidification and recovery of Little Rock Lake, WI, USA

Concentrations of Al, Fe, Mn, Cd, Cu, Pb, and Zn were measured in thereference and treatment basins of Little Rock Lake (Vilas County, Wisconsin), alow-alkalinity, seepage system (pH 6.1, alkalinity25eq/L) during six years of a whole-basinacidificationand the first four years of the lake's recovery. The treatment basin wasacidified with H₂SO₄ in three two-year steps to pH5.6, 5.1, and 4.7. By the end of year 4 of recovery, treatmentbasin pH increased to 5.3 as a result of internal alkalinity generation.During acidification, dissolved Mn and Fe (0.4mpore-size filters) increased at pH 5.6; dissolved Al, Cd, and Zn becameelevated at pH 5.1; and dissolved Pb at pH 4.7. Dissolved Cu remainedsimilar in both basins to pH 4.7. Al, Fe and Mn levels declinedsignificantly during the recovery period, approaching values at pH 5.3intermediate between the concentrations at pH 5.6 and 5.1 during acidification.Dissolved Al and Fe in the reference basin were near the equilibrium levels forsolubility of gibbsite (Al(OH)₃) and amorphousFe(OH)₃(s).The acidified basin was undersaturated relative to gibbsite, and dissolved Alwas limited by pH disequilibrium between the water column and sediments andpossibly by Al-DOC precipitation. Dissolved Fe apparently was controlled bysolubility of amorphous Fe(OH)₃(s) and Fe-DOC precipitation.Dissolved Mn levels in both basins were consistent with manganite[-MnOOH(s)] solubility. Elevated levels of Cd, Pb, and Zn in thetreatment basin during acidification probably resulted from less efficientscavenging of atmospherically-deposited Cd, Pb, and Zn by settling particles.     

Oscillatory luminescence from lux-gene engineered bacteria

The experimental conditions for oscillatoryluminescence from Alcaligenes eutrophus harbouring luxCDABE genes inducible by copper were developed. The incubation of optimised bacterial suspensions (concentration of copper sulphate – 2 mM, concentration of biomass – 0.15–0.20 mg ml^–1) for 4–6 h under weak visible light followed by sudden transition of the cells to the dark resulted in pronounced oscillations with an average period of 10 min.     

Pattern of cadmium accumulation and essential cations during growth of cadmium-tolerant fungi

The present study evaluates the growth response of two strains of filamentous fungi; a Fusarium sp. and Alternaria tenuis, grown on both solid and liquid Czapek Dox medium amended with different concentrations of CdCl₂. Colony extension and the mycelial dry weight of both fungi were significantly inhibited by high concentrations of cadmium. Extended lag phases and low growth rates resulted from cadmium administration. Cadmium drastically affected fungal morphogenesis by the production of stunted sterile thick mycelial filaments of the Fusarium sp. and chains of uncharacterized swellings instead of conidia in A. tenuis. Experiments showed that cadmium accumulation by the Fusarium sp. grown in liquid medium was a concentration dependent, and over the incubation time it displayed a plateau pattern. The cells grown on medium containing 0.25 mmol l^–1 CdCl₂ accumulated up to 89 ± 12 mol Cd (gm dw)^–1 after two days, falling to 29 ± 10 mol Cd (gm dw)^–1 after five days. At 0.5 mmol l^–1 CdCl₂ treatment the maximum cellular cadmium content was 132 ± 14 mol (gm dw)^–1, attained after 3 days, and decreased to 98 ± 9 mol (gm dw)^–1 at the end of the incubation time. There was a simultaneous marked drop in cadmium content and pH of the growth medium during the first few days. The presence of cadmium markedly altered the cellular essential cations; K⁺ and Mg²⁺ being decreased while Na⁺ increased during the growth period. Such findings resulted a reverse pattern of cellular Na⁺/K⁺ ratio for cells grown on cadmium-containing medium in respect to the control treatment. The results are discussed in relation to a further dimension of cadmium effects that might reflect its toxicity, as well as the implication of cadmium extrusion for tolerance during fungal growth.     

Electrical properties of parenchymal cell membranes in the oat coleoptile

Summary Parenchymal cells of oat (Avena sativa) coleoptiles had an osmotic concentration of 410 mM (determined by plasmolysis); of this only 22 mM was K⁺ and 1 mM Na⁺ (flame photometry). Cells were impaled with micropipette electrodes. Iontophoretic injection of the dye Niagara sky-blue from the micropipette showed that the tip of the electrode penetrated the vacuole. When sections of tissue were immersed in a solution of 22 mM KCl, 1 mM CaCl₂, and 50 mM glucose, average membrane potential was found to be 38.5 mV inside negative specific membrane resistance was 510 cm², and specific membrane capacitance, 2 f cm^-2. The cell membranes showed <25% retification and no electrical excitability. Electrotonic coupling of adjacent cells could not be demonstrated.     

Methane and oxygen dynamics in a shallow floodplain lake: the significance of periodic stratification

Temperature, dissolved oxygen and dissolved methane profiles were measured during autumn and summer, in a shallow floodplain lake in south-eastern Australia to determine the effects of water-column stability on methane and oxygen dynamics. The water column was well mixed in autumn. Strong thermal stratification developed in the late afternoon in summer, with top-to-bottom temperature differences of up to 6 °C. Methane concentrations in surface waters varied over a daily cycle by an 18-fold range in summer, but only by a 2-fold range in autumn. The implication of short-term temporal variation is that static chambers deployed on the water surface for short times (less than a day) in summer will significantly underestimate the diffusive component of methane emissions across the water–atmosphere interface. There was a marked diel variation in dissolved oxygen concentrations in summer, with the highest oxygen values (commonly 5–8 mg l^–1) occurring in the surface waters in late afternoon; the bottom waters were then devoid of oxygen (< 0.2 mg l^–1). Because of high respiratory demands, even the surface water layers could be nearly anoxic by morning in summer. The concentration of dissolved oxygen in the surface waters was always less than the equilibrium value. When the water column became thermally stratified in summer, the dissolved oxygen and methane maxima were spatially separated, and planktonic methanotrophy would be limited to a moving zone, at variable depth, in the water column. In summer the whole-wetland rates of oxygen production and respiration, calculated from long-term (5 h) shifts in dissolved oxygen concentrations over a diel period, were approximately 6–10 and 3–6 mmol m^–3 h^–1, respectively. These values correspond to net and gross primary production rates of 0.7–1.2 and 1.0–1.9 g C m^–3 day^–1, respectively.     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin

The giant, 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca²⁺ and all the Cu and Zn, the globin subassembly had 0.4 to 3 Ca²⁺, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH 2.2 and at neutral pH demonstrated avid binding of 1 Ca²⁺ and additional weaker binding of 7 Ca²⁺ in the presence of added Ca²⁺. Based on these and previous results which document the heterogeneous nature of the Ca²⁺-binding sites in Lumbricus Hb, we propose three classes of Ca²⁺-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.