Water and sodium balances and metabolic physiology of house mice (Mus domesticus) and short-tailed mice (Leggadina lakedownensis) under laboratory conditions

A laboratory study investigated the metabolic physiology, and response to variable periods of water and sodium supply, of two arid-zone rodents, the house mouse (Mus domesticus) and the Lakeland Downs short-tailed mouse (Leggadina lakedownensis) under controlled conditions. Fractional water fluxes for M. domesticus (24 ± 0.8%) were significantly higher than those of L. lakedownensis (17 ± 0.7%) when provided with food ad libitum. In addition, the amount of water produced by M. domesticus and by L. lakedownensis from metabolic processes (1.3 ± 0.4 ml · day⁻¹ and 1.2 ± 0.4 ml · day⁻¹, respectively) was insufficient to provide them with their minimum water requirement (1.4 ± 0.2 ml · day⁻¹ and 2.0 ± 0.3 ml · day⁻¹, respectively). For both species of rodent, evaporative water loss was lowest at 25 °C, but remained significantly higher in M. domesticus (1.1 ± 0.1 mg H₂O · g^−0.122 · h⁻¹) than in L. lakedownensis (0.6 ± 0.1 mg H₂O · g^−0.122 · h⁻¹). When deprived of drinking water, mice of both species initially lost body mass, but regained it within 18 days following an increase in the amount of seed consumed. Both species were capable of drinking water of variable saline concentrations up to 1 mol · l⁻¹, and compensated for the increased sodium in the water by excreting more urine to remove the sodium. Basal metabolic rate was significantly higher in M. domesticus (3.3 ± 0.2 mg O₂ · g^−0.75 · h⁻¹) than in L. lakedownensis (2.5 ± 0.1 mg O₂ · g^−0.75 · h⁻¹). The study provides good evidence that water flux differences between M. domesticus and L. lakedownensis in the field are due to a requirement for more water in M. domesticus to meet their physiological and metabolic demands. Sodium fluxes were lower than those observed in free-ranging mice, whose relatively high sodium fluxes may reflect sodium associated with available food. Accepted: 16 August 1999     

The use of amiloride to uncouple branchial sodium and proton fluxes in the brown trout, Salmo trutta

Resting proton, ammonium and sodium fluxes in Salmo trutta were 492.6 ± 19.5 (n = 29); 122.9 ± 34.2 (n = 28) and 277.1 ± 18.5 (n = 50) μmol · kg⁻¹ · h⁻¹, respectively. The resting transepithelial potential was found to be composed of three successive potentials, the outermost averaging −7.36 ± 0.19mV, the second, −14.3 ± 1.4 mV and the third −37 ± 1.7 mV. Amiloride inhibits the proton, ammonium and sodium fluxes in a dose-dependent manner at concentrations of 0.5 mmol · 1⁻¹ and 0.1 mmol · l⁻¹, but at 0.01 mmol · l⁻¹, proton and ammonium fluxes remained at control levels whilst the sodium was reduced to 70.59 ± 7.29 μmol · kg⁻¹ · h⁻¹. The trans-epithelial potential was effected in a bi-phasic manner by 0.5 mmol · l⁻¹ amiloride. An initial hyperpolarisation of ca. 6 mV was followed by a sustained depolarisation of ca. 14 mV (towards zero) which persisted until the amiloride was washed off the gill. The initial hyperpolarisation was thought to reflect a rapid inhibition of a positive inward sodium current and the subsequent depolarisation was due to the inhibition of a positive outward current (proton) which would abolish the transepithelial potential. However, at 0.01 mmol ·  l⁻¹ only the hyperpolarisation was seen, due to the inhibition of only the inward sodium current. Acetazolamide (0.1 mmol · l⁻¹) was found to have no significant effect on the proton, ammonium and sodium fluxes. These results indicate that the proton and sodium fluxes across the gill of the freshwater trout are not tightly linked. While this suggests that the trout gill resembles the model of Ehrenburg et al. (1985) of sodium uptake in frog skin, the apical potentials measured in the pavement epithelial cell(s) are too low to account for sodium uptake unless the activity of the sodium in the cells is very low. Accepted: 8 August 1996     

Effects of hypercapnia on blood-gas and acid-base status in the white sturgeon, Acipenser transmontanus

The effect of environmental hypercapnia on respiratory and acid-base variables was studied in white sturgeon, Acipenser transmontanus. Blood PCO₂, PO₂, pH, hemoglobin concentration, and plasma lactate, glucose, catecholamines and cortisol were measured first under normocapnia (water PCO₂ < 0.5 Torr, 1 Torr = 133.32 Pa), then under hypercapnia (25–35 Torr) and a final return to normocapnia at 19 ± 0.5 °C. Acute (≤ 2h) hypercapnia significantly increased arterial PCO₂ (8-fold increase), ventilation frequency (2-fold increase), plasma HCO₃ ⁻ (2.3-fold) and decreased arterial pH (to 7.15 ± 0.02). After 24 h, norepinephrine, epinephrine and cortisol, were significantly increased, and arterial pH reached its nadir (7.10 ± 0.03). During the 72- and 96-h-periods, arterial PCO₂ (24 ± 4.4 Torr) and ventilatory frequency (105 ± 5 breaths min⁻¹) stabilized, HCO₃ ⁻ reached its apparent maximum (23.6 ± 0.0 mmol⁻¹), glucose decreased by 32%, and pH increased significantly to 7.31 + 0.03. The return to normocapnia completely restored arterial PCO₂ (2.5 ± 0.14 Torr), HCO₃ ⁻ (7.4 ± 0.59 mmol · l⁻¹), ventilation frequency (71 ± 7 breaths · min⁻¹), and pH (7.75 ± 0.04). Overall, hypercapnia produced a respiratory acidosis, hyperventilation, a transient norepinephrine “spike”, and increased plasma catecholamines, cortisol, and arterial PO₂. The respiratory acidosis was only partially compensated (35% pH restoration) 96 h after the onset of hypercapnia and resulted in a significantly decreased blood-O₂ affinity (Bohr effect), as determined by construction of in vitro blood O₂ equilibrium curves at 15 °C and 20 °C. Prolonged exposure to hypercapnia may lead to acid-base disturbances and negatively affect growth of white sturgeon. Accepted: 17 August 1997     

Gas exchange and heart rate in the harbour porpoise, Phocoena phocoena

The respiratory physiology, heart rates and metabolic rates of two captive juvenile male harbour porpoises (both 28 kg) were measured using a rapid-response respiratory gas analysis system in the laboratory. Breath-hold durations in the laboratory (12 ± 0.3 s, mean ± SEM) were shorter than field observations, although a few breath-holds of over 40 s were recorded. The mean percentage time spent submerged was 89 ± 0.4%. Relative to similarly-sized terrestrial mammals, the respiratory frequency was low (4.9 ± 0.19 breaths · min⁻¹) but with high tidal volumes (1.1 ± 0.01 l), enabling a comparatively high minute rate of gas exchange. Oxygen consumption under these experimental conditions (247 ± 13.8 ml O₂ · min⁻¹) was 1.9-fold higher than predicted by standard scaling relations. These data together with an estimate of the total oxygen stores predicted an aerobic dive limit of 5.4 min. The peak end-tidal O₂ values were related to the length of the previous breath-hold, demonstrating the increased oxygen uptake from the lung for the longer dives. Blood oxygen capacity was 23.5 ± 1.0 ml · 100 ml⁻¹, and the oxygen affinity was high, enabling rapid oxygen loading during ventilation. Accepted: 11 August 1999     

Water influx and efflux in free-flying pigeons

We used tritium-labeled water to measure total body water, water influx (which approximated oxidative water production) and water efflux in free-flying tippler pigeons (Columba livia) during flights that lasted on average 4.2 h. At experimental air temperatures ranging from 18 to 27 °C, mean water efflux by evaporation and excretion [6.3 ± 1.3 (SD) ml · h⁻¹, n = 14] exceeded water influx from oxidative water and inspired air (1.4 ± 0.7 ml · h⁻¹, n = 14), and the birds dehydrated at 4.9 ± 0.9 ml · h⁻¹. This was not significantly different from gravimetrically measured mass loss of 6.2 ± 2.1 g · h⁻¹ (t = 1.902, n = 14, P>0.05). This flight-induced dehydration resulted in an increase in plasma osmolality of 4.3 ± 3.0 mosmol · kg⁻¹ · h⁻¹ during flights of 3–4 h. At 27 °C, the increase in plasma osmolality above pre-flight levels (ΔP _osm = 7.6±4.29 mosmol · kg⁻¹ · h⁻¹, n = 6) was significantly higher than that at 18 °C (ΔP _osm = 0.83±2.23 mosmol · kg⁻¹ · h⁻¹, (t = 3.43, n = 6, P < 0.05). Post-flight haematocrit values were on average 1.1% lower than pre-flight levels, suggesting plasma expansion. Water efflux values during free flight were within 9% of those in the one published field study (Gessaman et al. 1991), and within the range of values for net water loss determined from mass balance during wind tunnel experiments (Biesel and Nachtigall 1987). Our net water loss rates were substantially higher than those estimated by a simulation model (Carmi et al. 1992) suggesting some re-evaluation of the model assumptions is required. Accepted: 8 April 1997     

Catecholamine release in heat-stressed Antarctic fish causes proton extrusion by the red cells

Two species of Antarctic fish were stressed by moving them from seawater at −1 °C to seawater at 10 °C and holding them for a period of 10 min. The active cryopelagic species Pagothenia borchgrevinki maintained heart rate while in the benthic species Trematomus bernacchii there was an increase in heart rate. Blood pressure did not change in either species. Both species released catecholamines into the circulation as a consequence of the stress. P. borchgrevinki released the greater amounts, having mean plasma concentrations of 177 ± 54 nmol · l⁻¹ noradrenaline and 263 ± 131 nmol · l⁻¹ adrenaline at 10 min. Plasma noradrenaline concentrations rose to 47 ± 14 nmol · l⁻¹ and adrenaline to 73 ± 28 nmol · l⁻¹ in T. bernacchii. Blood from P. borchgrevinki was tonometered in the presence of isoprenaline. A fall in extracellular pH suggests the presence of a Na⁺/H⁺ antiporter on the red cell membrane, the first demonstration of this in an Antarctic fish. Treatment with the β-adrenergic antagonist drug sotalol inhibited swelling of red blood cells taken from temperature-stressed P. borchgrevinki, suggesting that the antiporter responds to endogenous catecholamines. Accepted: 22 January 1998     

Field energetics and the estimation of pollen and nectar intake in the marsupial honey possum, Tarsipes rostratus, in heathland habitats of South-Western Australia

A method is described, based on the simultaneous turnover of both stable (¹⁸O) and radioactive isotopes (³H and ²²Na), whereby the daily nectar and pollen intake of free-ranging marsupial honey possums (Tarsipes rostratus) may be estimated. The field metabolic rate is measured using doubly labelled water and nectar intake is estimated independently from the measured water and sodium fluxes. The method assumes that free-water intake is negligible (but may be accounted for if not the case), that virtually all dietary sodium is derived from nectar rather than from pollen, and that the animals are in energetic balance over the period of measurement. These assumptions have been tested and found to be robust, except during periods of heavy rain when significant intakes of free-water were recorded. Leaching experiments with pollen grains suggest that less than 10% of the sodium ingested by honey possums is derived from pollen and calculations thus assumed a 90%:10% split between nectar and pollen. Nectar intake averaged 5.9 ± 0.6 ml · day⁻¹ and regressing nectar intake on daily change in body mass predicts an intake of approximately 7 ml · day⁻¹ nectar to maintain balance for a 9 g honey possum. Estimates of pollen intake averaged 660 ± 156 mg · day⁻¹ and a similar regression analysis of the data predicts that a daily intake of approximately 1 g pollen would be needed to maintain mass balance of honey possums. Estimated nectar and pollen intakes did not differ significantly between males and females, but nectar intake was higher in winter compared with dry periods of the year. The sugar content of nectar falls during winter, however, and the overall energy derived from nectar thus remains roughly constant. Estimates of pollen and nectar intake for individual animals were not significantly correlated, suggesting that honey possums forage selectively for these two food items. Accepted: 19 August 1999     

Optical fractionation of chlorophyll and primary production for coastal waters of the Southern Ocean

Our objective was to quantify the potential variability in remotely sensed chlorophyll a (Chl a) and primary productivity in coastal waters of the Southern Ocean. From data collected throughout the springs/summers of 1991–1994, we calculated the proportion of water column Chl a and primary productivity within the upper optical attenuation length (K ⁻¹ _par) and the satellite-weighted depth. The temporal variability was resolved every 2–3 days and was observed to be greater within years than between years. Three-year averages (n=223) revealed that 10.2 ± 3.6% of total Chl a and 14.8 ± 6.5% of production occurred within satellite-weighted depth in predominantly Case I waters. The average values were twice as high within K ⁻¹ _par, 24.1 ± 8% of total Chl a and 34 ± 9% of production respectively. Masked in these long-term averages are very large changes occurring on short time scales of seasonal blooms. We observed that the patterns of Chl a vertical distribution within blooms are also subject to taxonomic influence and dependent upon the physiological state of the phytoplankton. Highest proportions of water column Chl a in the first optical depth were measured during the rapid onset of surface cryptophyte blooms each year, i.e. 50% within K ⁻¹ _par and 30% above the satellite-weighted depth. Lowest fractions, 6% and 2% of biomass within K ⁻¹ _par and satellite-weighted depth respectively, were associated with peak bloom conditions independent of taxonomy. Our analyses suggest that satellite-dependent models of Chl a and subsequent chlorophyll-dependent primary production will be challenging to develop for the near-shore Southern Ocean, especially given the potentially high natural variability in the vertical distribution of Chl a driven by physical forcing, the photoadaptive abilities of polar phytoplankton, and taxonomic influences. Accepted: 27 August 1999     

Interprotein metal ion exchange between cadmium-carbonic anhydrase and apo- or zinc-metallothionein

 − 1  s^− 1 at 25 °C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 °C, Zn₇-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 ± 0.02 M^− 1 s^− 1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49–61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd²⁺ into the peptide. At pH 7.4 and 25 °C, Cd²⁺ has higher affinity for apometallothionein than for the apo-peptide. Received: 25 February 1999 / Accepted: 17 September 1999     

Effect of manipulation of the renin-angiotensin system in control of drinking in juvenile Atlantic salmon (Salmosalar L) in fresh water and after transfer to sea water

Drinking in Atlantic salmon (Salmo salar) juveniles was investigated in fresh water and following transfer to sea water. There was a significant effect of fish size on drinking, and smolts (20–30 g) imbibed about ten times less water than alevins of 0.2–0.3 g. Freshwater smolts drank at a rate of 0.15 ± 0.03 ml · kg⁻¹ · h⁻¹ and administration of doses of 10 or 20 mg · kg⁻¹ of papaverine (stimulator of the renin- angiotensin system RAS) or [Asn¹, Val⁵]-Angiotensin II (0.4 μmol · kg⁻¹) resulted in significant increases in drinking, while administration of the angiotensin converting enzyme inhibitor, enalapril (50 mg · kg⁻¹) had no effect on drinking. Transfer of Atlantic salmon smolts to 1/3, 2/3 and full strength sea water resulted in significant increases in drinking to 1.06 ± 0.12, 1.24 ± 0.0.16 and 3.89 ± 0.28 ml · kg⁻¹ · h⁻¹, respectively. In sea water, stimulation of the endogenous RAS by administration of papaverine (20 mg · kg⁻¹) resulted in a 20% increase in drinking, while administration of enalapril to doses of 50 and 200 mg · kg⁻¹ lowered drinking to 1.99 ± 0.48 and 0.32 ± 0.06 ml · kg⁻¹ · h⁻¹, respectively. All treatments were without effect on blood plasma levels of Na⁺ and Cl⁻ in fresh water, while in sea water smolts both stimulation and inhibition of drinking resulted in hemoconcentration of Na⁺ and Cl⁻. The role of the renin angiotensin system in control of drinking and hydromineral balance in Atlantic salmon is discussed. Accepted: 27 February 1997     

Irradiance-dependent regulation of gravitropism by red light in protonemata of the moss Ceratodon purpureus

Apical cells of protonemata of the moss Ceratodon purpureus (Hedw.) Brid. are negatively gravitropic in the dark and positively phototropic in red light. Various fluence rates of unilateral red light were tested to determine whether both tropisms operate simultaneously. At irradiances ≥140 nmol m⁻² s⁻¹ no gravitropism could be detected and phototropism predominated, despite the presence of amyloplast sedimentation. Gravitropism occurred at irradiances lower than 140 nmol m⁻² s⁻¹ with most cells oriented above the horizontal but not upright. At these low fluence rates, phototropism was indistinct at 1 g but apparent in microgravity, indicating that gravitropism and phototropism compete at 1 g. The frequency of protonemata that were negatively phototropic varied with the fluence rate and the duration of illumination, as well as with the position of the apical cell before illumination. These data show that the fluence rate of red light regulates whether gravitropism is allowed or completely repressed, and that it influences the polarity of phototropism and the extent to which apical cells are aligned in the light path. Received: 19 January 1999 / Accepted: 19 March 1999     

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 10⁵ cells ml⁻¹, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l⁻¹) KNO₃ 0.41, Na₂HPO₄ 0.03, MgSO₄ · 7H₂O 0.246, CaCl₂ · 2H₂O 0.11, (in mg l⁻¹) Fe(III)citrate · H₂O 2.62, CoCl₂ · 6H₂O 0.011, CuSO₄ · 5H₂O 0.012, Cr₂O₃ 0.075, MnCl₂ · 4H₂O 0.98, Na₂MoO₄ · 2H₂O 0.12, SeO₂ 0.005 and (in μg l⁻¹]) biotin 25, thiamine 17.5 and B₁₂ 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis. Received: 10 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Characteristics of dipeptide transport in pig jejunum in vitro

 Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-l-glutamine and glycyl-l-sarcosine (20 mmol · l⁻¹) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 ± 0.15 and 1.57 ± 0.20 μeq · cm⁻² · h⁻¹, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-l-sarcosine, a net flux rate for glycyl-l-sarcosine of 49.8 ± 6.7 nmol · cm⁻² · h⁻¹ was calculated. In mucosal uptake experiments, the apical influx of ¹⁴C-labelled glycyl-l-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-l-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut. Accepted: 30 June 1999     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

RNA turnover and protein synthesis in fish cells

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from ³H-phenylalanine and ³H-uridine incorporation, respectively, and expressed as % · day⁻¹ and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (∼175 μg RNA · mg⁻¹ cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 ± 1.8)>RTP (25.1 ± 1.7)>CHSE 214 (17.6 ± 1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised · μg⁻¹ RNA · day⁻¹, corresponded to the lowest RNA half-life, 75.4 ± 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5–6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis. Accepted: 12 November 1999     

Equilibrium dialysis study and mechanistic implications of coenzyme A binding to acetyl-CoA synthase/carbon monoxide dehydrogenase from Clostridium thermoaceticum

Clostridium thermoaceticum were determined by equilibrium dialysis. CoA bound to as-isolated native α ₂ β ₂ enzyme with K _D = 10 ± 8 μM and n = 0.2 ± 0.1 moles per αβ dimer, where K _D is the thermodynamic dissociation constant and n is the number of CoAs bound per αβ dimer of the enzyme. The enzyme is heterogeneous; for example, only  ∼ 30% of α subunits contain A-clusters with labile Ni ions (the remainder have nonlabile Ni ions and are nonfunctional). The observed n value suggests that CoA binds only to αβ units with Ni-labile A-clusters. The CoA binding properties of enzyme lacking labile Ni was essentially the same, indicating that CoA does not bind directly to the Ni of the A-cluster. This was further evidenced by the observation that bound CoA did not inhibit removal of the labile Ni by 1,10-phenanthroline. CoA did not bind CO-reduced enzyme, and the EPR signal exhibited by the one-electron reduced and CO-bound form of the A-cluster was unaffected by the presence of up to 200 μM CoA. In contrast, CoA did bind Ti(III)-citrate-reduced enzyme (K _D = 36 ± 16 μM, n = 0.16 ± 0.08). Implications of these results for the mechanism of catalysis are discussed. Received: 29 March 1999 / Accepted: 8 September 1999     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Kinetic studies on the reduction of the R2 subunit of mouse ribonucleotide reductase with hydroxyurea, hydrazine, phenylhydrazine and hydroxylamine

 Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe₂ ^II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction of radical Tyr ^˙ and Fe₂ ^II I components to tyrosine and Fe₂ ^II respectively. The rate constants, determined at 370 nm (emphasising Fe^III decay) and 417 nm (emphasising Tyr ^˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe₂ ^II I → Fe₂ ^II change and is followed by fast intramolecular Fe₂ ^II reduction of the higher potential Tyr ^˙. The latter changes are believed to hold also for (active) mouse R2. The Fe^IIFe^III semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of Fe^IIFe^III occurs at a comparable rate to account for the transient behaviour of Fe^IIFe^III. For mouse R2 the combined Fe^III decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line (FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ^˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M^–1 s^–1) at 25  °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ^˙ as the only redox step (rate constant 27 M^–1 s^–1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of Fe^III. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors affecting the reactivity of R2. Received: 11 October 1996 / Accepted: 11 February 1997     

Ca2+ regulation of heart contractility in Octopus

Isometric force development of electrically paced preparations isolated from the systemic heart of Octopus vulgaris were utilized to examine the regulation of contractility by Ca²⁺. Increases in extracellular Ca²⁺, to the physiological level, resulted in enhancement of twitch force. For instance, at 36 beats · min⁻¹ an increase in Ca²⁺ from 3 to 9 mmol · l⁻¹ resulted in a threefold increase in twitch force development. When steady-state contraction at 12 beats · min⁻¹ was followed by a rest period of either 5 or 10 min, the first contraction always exhibited either an increase in twitch force or stayed unchanged such that post-rest twitch force was about 133% of the last value in the steady-state train. Ryanodine (12.5 μmol · l⁻¹), which is considered to be a specific inhibitor of the Ca²⁺ storage and release capabilities of the sarcoplasmic reticulum (SR), was applied to further assess Ca²⁺ handling. Twitch force fell to about 22% of the preteatment level in preparations paced at either 12 or 36 beats · min⁻¹. In all preparations the frequency transition from 12 to 36 beats · min⁻¹ was associated with an increase in resting tension. The␣increase␣was 37 ± 14% prior to ryanodine treatment and was significantly elevated to 127 ± 33% following treatment. When steady-state contraction at 36 beats · min⁻¹ was followed by a rest period of 10 s, the first contraction was not significantly different from the last beat in the train prior to ryanodine; however, with ryanodine treatment, post-rest twitch force development significantly decreased. Twitch force development was regular at pacing rates of up to 300 beats · min⁻¹. Twitch force was maintained up to rates of 84 beats · min⁻¹ but␣decreased thereafter and reached a value of about 10% at 300 beats · min⁻¹. Resting tension increased substantially as frequency was elevated from 12 to 36 beats · min⁻¹ and then gradually increased as frequency was further elevated to 180 beats · min⁻¹. In conclusion, the Octopus ventricle is dependent upon extracellular Ca²⁺ for contraction. A post-rest potentiation of force development, the negative impact of ryanodine, and the ability to respond regularly at high pacing rates imply a strong reliance on the SR in Ca²⁺ cycling based on criteria established for vertebrate hearts. Accepted: 19 January 1997     

Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999