Isolation of human fibrinogen by axial and radial flow chromatography from Nitschmann fraction I

An axial column (3×2.6 cm) and a radial flow column (3.5×5 cm) packed with DEAE Sepharose Fast Flow media had been evaluated for the separation of human fibrinogen. Nitschmann fraction I dissolved in buffered saline (0.015 M NaCl buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Under radial flow conditions, sample flow up to 15 ml min^–1 (i.e., 18 bed volumes h^–1) was achieved. The operating pressures were below 0.2 MPa, even though the elution velocity was 30 ml min^–1 (i.e., 36 bed volumes h^–1).     

Antibiotic production by the immobilized cyanobacterium,Scytonema sp. TISTR 8208, in a seaweed-type photobioreactor

A photobioreactor was constructed using either anchored polyurethane foam strips (1 × 1 × 40 cm, PU-strips) fixed on a stainless-steel ring to prevent flotation, or free-floating polyurethane foam blocks (1 × 1 × 1 cm, PU-blocks) as biomass supporting materials (BSM). The cyanobacterium,Scytonema sp. TISTR 8208, which produces an antibiotic, was immobilized onto PU-strips or -blocks. The free-floating PU-blocks could immobilize only about 70% of the total cells, while the anchored PU-strips could immobilize as much as 97%. PU-strips were chosen as the BSM and we named this type of reactor, seaweed-type bioreactor (STB). Optimal physical conditions for antibiotic production were determined in the STB. Inoculum density was 0.4 g l^–1 and cells were sparged with air containing 5% CO₂ circulated at the gas flow rate of 250 ml min^–1 and illuminated at a light intensity of 200 mol photon m^–2 s^–1. The production of antibiotic could be increased 3-fold.Author for correspondence     

Photosynthetic production of the filamentous cyanobacteriumSpirulina platensis in a cone-shaped helical tubular photobioreactor

The photosynthetic productivity of the filamentous cyanobacteriumSpirulina platensis was investigated in a cone-shaped helical tubular photobioreactor. A laboratory-scale photobioreactor was constructed with a 0.255-m² basal area and a conical shape (0.64 m high × 0.57 m top diameter). The photostage comprised transparent reinforced polyvinyl chloride (PVC) tubing with spirally wound, metal-wire reinforcing in the tubing wall (31 m in length and 1.6 cm internal diameter with 0.25 cm wall thickness; total volume = 6.23 l). The inner surface of the photostage (0.651 m²) was illuminated with compact fluorescent cool white lamps; the photosynthetically active radiation (400–700 nm) energy input into the photobioreactor was 1249 KJ day^–1 (12 h day/12 h night). The operation of an air-lift photobioreactor with CO₂-enriched air (4%) at a flow rate of 0.3 l min^–1 showed a maximum daily photosynthetic efficiency of 6.83% under batch-culture conditions. This corresponded to a production rate of 15.9 g dry biomass m^–2(basal area) day^–1 or 0.51 g dry biomass l medium^–1 day^–1.     

Ethanol tolerance in free and sol-gel immobilised Saccharomyces cerevisiae

The tolerance of sol-gel immobilised and free Saccharomyces cerevisiae to ethanol was studied. The effects of ethanol preincubation time showed that the specific death velocity decreased from 2×10⁵ c.f.u. min^–1 for free cells to 2×10⁴ c.f.u. min^–1 for immobilised cells thus indicating that immobilised yeast was far less sensitive to the ethanol damage. The specific glucose consumption of immobilised and free cells on a per cell basis was 3×10^–12 g cell^–1 h^–1 and 9×10^–12 g cell^–1 h^–1, respectively.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7×10^–3 cm s^–1 at 10°C, 1.2×10^–3 cm s^–1 at 15°C, 1.4×10^–3 cm s^–1 at 20°C, 1.8×10^–3 cm s^–1 at 25°C, 2.1×10^–3 cm s^–1 at 30°C and 3.5×10^–3 cm s^–1 at 37°C. The mean value of the activation energy of water diffusion (E_a,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS-inhibited erythrocytes. The values of P and E_a,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.     

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells

The binding of the enkephalin dimer [d-Ala², Leu⁵-NH-CH₂-]₂ (DPE₂) is characterized by (1) its high affinity for receptors on NG108-15 hybrid cells, the affinity constantK=4.7×10⁹ M^–1 is up to 8-fold that of monomers (0.6 to 1.0×10⁹ M^–1), and (2) a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE₂ binds with a 2-fold higher rate, k₁=6.3×10⁷ M^–1min^–1, than monomers (2.4 to 3.8×10⁷ M^–1min^–1), and dissociates at a slower rate than monomers. Dissociation of DPE₂ was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE₂ binds by cross-linking two subunits of one receptor.     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.     

Ion and sugar permeabilities of lecithin bilayers: Comparison of curved and planar bilayers

Summary Na⁺ and sugar permeabilities of egg lecithin bilayers were measured using curved bilayers and planar bilayers as represented by single-bilayer vesicles and black lipid films, respectively. The Na⁺ permeability coefficient measured with single-bilayer vesicles at 25°C is (2.1±0.6)×10^–13 cm sec^–1. Because of technical difficulties it has been impossible to measure ionic permeabilities of values lower than about 10^–10 cm sec^–1 in planar (black) lipid bilayers using tracer methods. Thed-glucose andd-fructose permeabilities were measured with both curved and planar bilayers. The permeability coefficients measured with vesicles at 25°C are (0.3±0.2)×10^–10 cm sec^–1 for glucose and (4±1)×10^–10 cm sec^–1 ford-fructose; these are in reasonable agreement with the corresponding values obtained for planar (black) lipid bilayers which are (1.1±0.3)×10^–10 cm sec^–1 ford-glucose and (9.3±0.3)×10^–10 cm sec^–1 ford-fructose, respectively.This paper is dedicated to the memory of Walther Wilbrandt,cuius nomini nullum par elogium.     

Immobilisation of lipases by adsorption and deposition: high protein loading gives lower water activity optimum

Two different immobilisation techniques for lipases were investigated: adsorption on to Accurel EP-100 and deposition on to Celite. The specific activities were in the same order of magnitude, 2.9 (mol min^–1 mg protein) when Celite was used as support and 2.3 (mol min^–1 mg^–1 protein) when Accurel EP-100 was used as support, even if the amount of lipase loaded differed by 2 orders of magnitude. Immobilisation on Accurel EP-100 was the preferred technique since 40–100 times more protein can be loaded/per g carrier, thus yielding a more active catalyst. The water activity profiles in lipase catalysed esterification were influenced by the amount of protein adsorbed to Accurel EP-100. Higher protein loading (40 mg g^–1) resulted in a bell-shaped water activity profile with highest specific activity (6.1 mol min^–1 mg^–1 protein) at a _w=0.11, while an enzyme preparation with low protein loading (4 mg g^–1) showed highest specific activity at a _w=0.75.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

Studies on lithium transport across the red cell membrane

Summary Binding of³H-saxitoxin to Na⁺ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1nm; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30nm. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K⁺ concentrations, or by activation of the Na⁺ channels with batrachotoxin plus a purified polypeptide toxin from the scorpionLeiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70–80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60–70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2nm. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0° of 0.1 min^–1 for membrane bound and 0.2 min^–1 for detergent-solubilized binding sites. The measured association rate constant was 6×10⁸ m ^–1 min^–1 at 0° for membrane-bound saxitoxin binding sites.     

Secretion by the mandibular gland of the red kangaroo (Macropus rufus) during isoprenaline infusion

Summary Intracarotid infusion of isoprenaline, either alone or in combination with acetylcholine infusion was used to stimulate salivation by the mandibular glands of anaesthetized red kangaroos. Isoprenaline alone (0.20–1.25 mol·kg^–1·min^–1) elicited flow rates ranging from 0.014 to 0.239 ml·min^–1 (1.21–28.1 l·g gland^–1·min^–1). Salivary concentrations of sodium, chloride, phosphate and urea were negatively correlated with flow, whereas potassium, calcium, magnesium, hydrogen ion, bicarbonate, protein, and osmolality were poorly correlated with flow. Relative to cholinergic saliva produced at equivalent flow rates, isoprenaline-evoked saliva had higher osmolality, saliva/plasma urea ratios and concentrations of protein, potassium, magnesium, bicarbonate, and phosphate, but lower sodium, chloride and hydrogen ion levels. At a steady salivary flow (0.5 ml·min^–1), superimposition of isoprenaline infusion (0.15 mol·kg^–1·min^–1) on a pre-existing acetylcholine infusion reduced the rate of acetylcholine administration necessary to maintain flow, increased osmolality and the concentrations of protein, urea, potassium, calcium, magnesium, bicarbonate and phosphate and decreased sodium, chloride and hydrogen ion in the saliva. Salivary amylase activity was low and highly variable and the amylase activity/protein ratio fell substantially during isoprenaline stimulation. These results support the conclusion that the enzyme is of extrinsic origin. The response of the kangaroo mandibular gland to isoprenaline stimulation was very similar to that reported for rat mandibular gland, suggesting that the same ion transport phenomena underlie mandibular secretion in both species and probably in therian mammals generally.     

Continuous culture of the hyperthermophilic archaeum Pyrococcus furiosus

Summary A system for continuous culture of the hyperthermophilic archaeum Pyrococcus furiosus in the absence of elemental sulphur has been developed. An all-glass gas-lift bioreactor was used to provide high mass transfer at low shear forces, whilst eliminating the potential for corrosion. Steady-state cell densities of P. furiosus were found to increase with higher inert gas flow rates, reaching a maximum in this system with 0.5 vol. vol^–1 min^–1 of nitrogen (N₂). N₂ permitted higher cell densities than the other inert gases tested (argon, helium and sulphur hexafluoride) under equivalent conditions. At 0.5 vol. vol^–1 min^–1 of N₂ a cell density in excess of 3 × 10⁹ ml^–1 could be maintained indefinitely at a dilution rate of 0.2 h^–1. Higher dilution rates gave progressively lower steady-state cell densities. Teh biomass production was maximal, however, at a dilution rate of 0.4 h^–1. At this dilution rate the bioreactor was able to generate more than 1.5 g wet weight of cells h^–1 l^–1 culture volume.Correspondence to: N. Raven     

Lactate content and lactate dehydrogenase activity inPalaemon serratus abdominal muscle during temperature changes

Summary Lactate concentration was measured in the abdominal muscle of the shrimpPalaemon serratus. Rapid and seasonal temperature changes result in an increase of the lactate content of approximately 3–4 fold.Lactate dehydrogenase from the abdominal muscle exhibits a temperature dependent pyruvate inhibition with pyruvate as substrate.The kinetic parameters of lactate dehydrogenase fromPalaemon serratus are found to vary during rapid temperature changes: V_max increases with temperature from 0.06 mol min^–1 (mg protein)^–1 at 10°C to 0.28 mol min^–1 (mg protein)^–1 at 30°C with lactate as substrate, and from 5.5 mol min^–1 (mg protein)^–1 at 10°C to 26.2 mol min^–1 (mg protein)^–1 at 30°C, with pyruvate (Table 1). The Hill coefficientn _H, decreases with temperature from 2.2 to 1.2 when the pyruvate reduction is examined, but remains near 1.2 when the activity is measured with lactate as substrate (Table 1). The S_0.5 values for lactate show a tendency to increase below 30 °C (18.9 mM l^–1 at 20 °C) whereas the S_0.5 for pyruvate is found to increase greatly with temperature (0.004 mM l^–1 at 10 °C and 0.06 mM l^–1 at 20 °C).Long term temperature changes involve variations of lactate dehydrogenase activity leading to inverse thermal compensation (Table 2).Activation energy (about 56 kJ both with pyruvate and lactate) does not vary during the year, suggesting that temperature adaptation does not induce important catalytic changes (Table 3).Abbreviation LDH lactate dehydrogenase     

Estimation of cardiac output and stroke volume from thermal equilibration and heartbeat rates in fish

Cardiac output and stroke volume were estimated for a 200 g largemouth blackbass (Micropterus salmoides) by a modified whole-body thermodilution method using the relation between thermal equilibration rates and heartbeat frequencies. The reciprocal of the thermal time constant, k (min^–1), was related to the heartbeat frequency, F (beats min^–1), by the equation k=0.00146 F + 0.309; the slope is the weight-specific stroke volume (ml g^–1) and the intercept is the weight-specific heat transfer constant (cal °C^–1 min^–1 g^–1). Stroke volume was 0.292 ml (0.00146 ml/g body weight), yielding cardiac output values ranging from 44 ml kg^–1 min^–1 (at 30 beats min) to 158 ml kg^–1 min^–1 (at 108 beats min^–1), or 4.4 to 15.8% of body weight. Active (convective) heat transfer due to blood flow constituted an estimated 11 to 34% (mean 22.5%) of total heat transfer, depending on heartbeat frequency; this variability constitutes physiological thermoregulation.     

Intertidal territoriality and time-budget of the surgeonfish, Acanthurus lineatus, in American Samoa

Synopsis The herbivorous surgeonfish Acanthurus lineatus aggressively maintained feeding territories in the surf zone of the outer reef flat in American Samoa. Intertidal territories were re-established each morning, as well as after displacement by low tides or rough surf. Day-to-day site fidelity of recognizable individuals was high: 99.9% return rate per day for adults (15–20 cm), 99.6% for juveniles (8–13 cm), and 97.2% for recruits (2.5–5 cm). Fish fed on turf algae primarily in the afternoon (80% of available time), and spent 10% of their time on active territorial defense and 2–13% of their time on forays from their territory. On average, a fish defended its territory 1900 times daily and took 17 000 bites (= 7400 bites m^–2 d^–1), but rough surf reduced feeding by 60% and defense by 75%. High territorial defense requirements significantly reduced feeding rates. Although the distribution and behavior of this species in Samoa was in large part similar to that reported for it elsewhere (Australia, Indian Ocean), there were notable differences: in Samoa A. lineatus densities within colonies were greater (0.4 fish m^–2), territory size was smaller (2.3 m²), and defense rate against intruders was greater (2.5 attacks min^–1). These differences in Samoa may be related to their smaller body size, greater abundance or increased food supply caused by hurricane damage to reefs which has enhanced the algal turfs that A. lineatus feeds upon.     

Collagen Fractions Obtained by Water–Salt Extraction from Raw Materials of Animal Origin

Collagen fractions have been isolated by water–salt extraction from raw materials of animal origin (various tendon types or subcutaneous tissues of cattle or porcine skin). Collagen fractions with maximum capacity for water and fat retention were isolated with high efficiency by water–salt solutions containing 1–10% sodium chloride at temperatures below 50°C. The values of the effective constant of extraction rate (min^–1) at pH 6.5, 9.0, and 12.0 were equal to (2.7 ± 0.1) × 10^–3, (6.2 ± 0.5) × 10^–3, and (15.4 ± 0.7) × 10^–3, respectively. The optimum conditions found made it possible to isolate from collagen those proteinaceous fractions that are of practical use in food industry.     

Continuous culture with deep seawater of a benthic food diatom Nitzschia sp.

A continuous culture system for a benthic food diatom Nitzschia sp. wasestablished by using properties of high nutrient and clean of deep seawater(DSW). DSW collected from 320 m depth in Muroto City, Japan, was introducedinto a glass-pipe bioreactor (14 cm length, 3 cm diam.) containing glassbeads of 0.5 cm diam. as substrata for the alga, and it was incubated at18°C · 80Em^–2sec^–1 · L:D=14:10. The chlorophyll a yield of benthicdiatoms in a reactor as a unit of surface area of the substratum was only0.001–0.003 g cm^–2 when the flow rate of DSW was 0(batch culture conditions). However, when DSW was supplied continuously to areactor, the yield increased to 1.4 g-chl.a cm^–2 alongwith the increase in flow rate of DSW. Moreover, amounts of chl.a washed outof the system were negligible, 0.0014 to 0.0045%, even though theflow rate of DSW was as much as 25 times h^–1, suggesting thatsloughing of benthic diatoms from the substratum was minimized. Although theyield of diatoms fluctuated significantly at the time that the DSW wascollected, the variation could be minimized by increasing the flow rate ofDSW. These results indicate that the continuous culturing system with DSWsupports the stable and effective mass culture of benthic food diatom.     

Compartment analysis of plant cells by means of turgor pressure relaxation: II. Experimental results onChara corallina

Summary The three-compartment model (paper I) described turgor pressure relaxations as a sum of two exponential functions. The predicted shape of the curves could be confirmed inChara corallina by improving the recording and processing of data. An evaluation on the basis of the three-compartment model provided values for the hydraulic conductivity of the plasmalemma ofLp _p=2×10^–5 to 4×10^–5 cm sec^–1 bar^–1 and ofLp _i=3×10^–5 to 1×10^–4 cm sec^–1 bar^–1 for the tonoplast (assuming the area to be 90% of the plasmalemma area). The mean proportion of the total volume occupied by the cytoplasm was calculated to be 9%. This value is consistent with the concept of a highly vacuolated cell. Other explanations for the biphasic relaxation curves are discussed.