Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Effect of osmotic gradient on ADH-induced intramembranous particle aggregates in toad bladder

Summary Paired toad urinary bladders were prepared without or with an osmotic gradient (175 mosm) across them, stimulated for 2.5 (n=6), 5 (n=6), 30 (n=6) or 60 (n=6) min with ADH (20 mU/ml), and studied by freeze-fracture electron microscopy. Water permeability at these times was assessed in additional bladders (n=6 for each case) after tissue fixation according to the technique of Eggena. After both 60 and 30 min of ADH stimulation, the presence of a gradient compared with the absence of one was associated with fewer aggregates (242±35vs. 382±14 ×235 m^–2 at 60 min,P<0.01; 279±36vs. 470±51 ×235 m^–2 at 30 min,P<0.01) and lower water permeability (8.4±1.1vs. 18.8±1.8g×min^–1×cm^–1 ×mosm ^–1 at60min,P<0.005; 9.2±1.0vs. 22.0±2.1 g ×min^–1×cm^–2×mosm ^–1 at 30 min,P<0.001). In addition, with a gradient both maximum water permeability and maximum aggregate frequency were reached nearly together; a similar correspondence occurred without a gradient. We conclude that in the presence of an osmotic gradient both the ADH-associated aggregates and the water permeability response to ADH are prevented from reaching the higher levels observed in bladders not exposed to a gradient.     

Alpha and beta adrenergic agonists stimulate water absorption in the rat proximal tubule

Summary Simultaneous capillary and luminal microperfusion studies were performed in the rat proximal tubule to determine the effects of the beta agonist isoproterenol and the alpha agonist phenylephrine on water absorption. Capillary and luminal perfusion solutions were composed such that organic solutes were not present, no bicarbonate was present in the lumen, and no chloride gradient was imposed. Under such conditions, water absorption (Jv) averaged 0.36±0.11 nl·min^–1·mm^–1. The addition of isoproterenol to the capillary solution in concentrations of 10^–6 and 10^–4 m resulted in significantly higherJv's of 0.68±0.10 and 0.71±0.11 nl·min^–1·mm^–1, respectively. The enhancing effect of isoproterenol was inhibited by the beta blocker propranolol (10^–4 m), but not by the alpha blocker phentolamine (10^–7 m). The addition of phenylephrine (10^–6 m) to the capillary perfusion solution also resulted in a significantly higherJv of 0.84±0.14 nl·min^–1·mm^–1, an effect inhibited by phentolamine (10^–7 m), but not by propranolol (10^–4 m). Neither phentolamine nor propranolol alone in the concentrations indicated had an effect on water absorption. These experiments indicate that both alpha and beta agonists stimulate water absorption in the superficial proximal tubule of the rat. This effect appears to be relatively specific for each class of agonist, as demonstrated by the effects of the specific antagonists.     

Function and regulation of the avian caecal bulb: influence of dietary NaCl and aldosterone on water and electrolyte fluxes in the hen (Gallus domesticus) perfused in vivo

Summary The function of the caecal bulb, and its adaptation to chronic high- or low-Na⁺ intake, was investigated by in vivo perfusion of anaesthetised birds. Effects of acute aldosterone injection (125 g·kg^–1 body mass) were also measured.Evidence was found for primary active net absorption of Na⁺, inducing parallel Na-linked absorption of water and Cl^– and secretion of K⁺. Around 20–35% of total Cl^– absorption and K⁺ secretion were independent of Na⁺ fluxes, and these components appear to be driven by passive processes with apparent conductances of 6.3×10^–3 (G _Cl) and 1.1×10^–3 (G _K) S·cm^–2.Acetate (40mM) stimulated Na⁺ fluxes (8.5–9.9 Eq·cm^–2·h^–1) and Na-linked water fluxes (27–44 l·cm^–2·h^–1). Increased coupling ratios (2.9–4.6 l·Eq^–1) and other data indicate that these effects may be due to increased osmotic permeabilities of barriers involved in the Na-linked water transfer pathway.Low-Na⁺ maintenance enhanced EPD (49–69 mV, serosa positive) and all net fluxes:J _Na (6.8–11.6);J _K (–3.2––4.3);J _Cl (4.3–5.6 Eq·cm serosal area^–2·h^–1);J _v (28–43 l·cm^–2·h^–1) (mucosal-serosal fluxes positive).Acute aldosterone enhancedJ _Na (10.8–14.0 Eq·cm^–2·h^–1) and EPD (54–66 mV) by 3 h after injection, but had no effect on the Na-linked components ofJ _K orJ _Cl.Abbreviations ECPD, EPD Electrochemical or electrical potential difference - G _Cl ,G _K ionic conductances (Cl^–, K⁺) - J _v ,J _ion net volume or ion flux rate, mucosa-serosa positive;P _d (Cl) diffusive permeability coefficient (of Cl^–) - SEDM standard error of difference between means     

Secretion of electrolytes,protein and urea by the mandibular gland of the common wombat (Vombatus ursinus)

Saliva was collected from the mandibular glands of anaesthetized common wombats (Vombatus ursinus) to ascertain maximal flow rates, salivary compostion and possible adaptations, particularly PO₄ ^3- secretion, to assist digestion. After temporary catheterization of the main duct through its oral opening, salivary secretion was evoked at flow rates ranging from 0.02±0.002 (±SEM) ml·min^-1 (0.7±0.07 l·min^-1·kg body weight^-1) to 0.4±0.05 ml·min^-1(14±1.9 l·min^-1·kg body weight^-1) by ipsilateral intracarotid infusion of acetylcholine. The [Na⁺] (15±5.1 to 58±8.6 mmol·l^-1) and [HCO₃ ^-] (35±1.9 to 60±1.9 mmol·l^-1) were positively correlated with salivary flow rate. The [K⁺] (58±5.2 to 30±2.4 mmol·l^-1), [Ca²⁺] (10.4±1.67 to 4.1±0.44 mmol·l^-1), [Mg²⁺] (0.94±0.137 to 0.17±0.032 mmol·l^-1), [Cl^-] (71±9.2 to 45±6.0 mmol·l^-1), [urea] (9.3±0.79 to 5.1±0.54 mmol·l^-1), H⁺ activity (29±1.6 to 17±1.6 nEq·l^-1) and amylase activity (251±57.4 to 92±23.3 kat·l^-1) were negatively correlated with flow. Both concentration and osmolality fell with increasing flow at the lower end of the flow range but osmolality always increased again by maximal flow whereas the relation between protein and flow was not consistent at the higher levels of flow and stimulation. Salivary [PO₄ ³⁺] was not correlated with flow and at 3–14% of the plasma concentration was extremely low. Thus, in contrast to its nearest relative, the koala (Phascolarctos cinereus), the wombat secretes little PO₄ ³⁺ presumably because it does not need high levels of PO₄ ³⁺ in its saliva to facilitate microbial digestion of plant fibre.Abbreviations bw body weight - ww wet weight     

Caecal water and electrolyte absorption and the effects of acetate and glucose, in dehydrated, low-NaCl diet hens

Summary In vivo electrolyte transport and water absorption from the caeca of dehydrated, low-NaCl diet hens are reported. In the absence of luminal glucose or acetate, net electrolyte transport rates and water absorption are small. When physiological concentrations of acetate (40 mM) are included in the perfusate, Na⁺ transport and water absorption increase significantly (P<0.01): 38±7 eqNa⁺/caecum kg·h and 256±33 l H₂O/caecum · kg · h.A similar increase in water absorption occurs with the inclusion of 15 mM glucose in the perfusate (219±30 l H₂O/caecum · kg · h), however both net Na⁺ and Cl^– absorption increase: 28±6 eq Na⁺/caecum · kg · h and 21±5 eq Cl^–/caecum kg · h.These pronounced increases in electrolyte and water absorption are not accompanied by any significant increase in transmural potential difference.The data presented establish caeca as important sites in the recuperation of water and electrolytes in dehydrated, low-NaCl diet hens.Abbreviations ECPD electrochemical potential difference - PD (transmural) potential difference - PEG polyethylene glycol     

Sodium influx in isolated gills of the freshwater mussel,Ligumia subrostrata

Summary Isolated gills of the freshwater mussel,Ligumia subrostrata, accumulate Na from a pondwater bathing medium. The rate of Na transport by the isolated gill is 13.2±1.1 mol (g dry gill·10 min)^–1 which equals or exceeds the estimated Na transport rate of intact animals. Sodium influx is saturable with aV _max of 13.6±1.2 mol (g dry gill·10 min)^–1 and an affinity (K _s) of 0.17 mM Na/l. The isolated gills survive prolonged exposure to pondwater with a constant of 890 l O₂ (g dry gill·h)^–1 over a 4 h period. Sodium transport in the isolated gills is stimulated 80% above control values by 10^–4 M serotonin, 60% by 0.5 mM cAMP and 60% by 12.5 g/ml nystatin. Sodium influx is inhibited by 0.5 mM amiloride and 1 mM lithium.     

Down-regulation of blood-brain glucose transport in the hyperglycemic nonobese diabetic mouse

The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in hyperglycemic (4–6 days) mice. In anesthetized mice, Brain Uptake Indices were measured over a range of glucose concentrations from 0.010–50 mmol/l; glucose uptake was found to be saturable and kinetically characterized. The maximal velocity (V_max) for glucose transport was 989±214 nmol·min^–1·g^–1· and the half-saturation constant estimated to be 5.80±1.38 mmol/l. The unsaturated Permeability Surface are product (PS) is=171+8 l·min.^–1·g^–1. A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter immunocytochemically confirmed the presence of the GLUT1 isoform in non-obese diabetic (NOD) mouse brain capillary endothelia. These studies indicate that a down-regulation of BBB glucose transport occurs in these spontaneously hyperglycemic mice; both BBB glucose permeability (as indicated by PS product) and transporter maximal velocity are reduced (in comparison to normoglycemic CD-1 mice), but the half-saturation constant remains unchanged.     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Cardiovascular and metabolic responses to noradrenaline in men acclimatized to cold baths

The purpose of this study was to see whether artificial acclimatization to cold would reduce the pressor response to noradrenaline (NA) as natural acclimatization has been shown to do, and whether it would induce nonshivering thermogenesis. Three white men were infused with NA at four dosage levels between 0.038 and 0.300 g·kg^–1·min^–1 (2–23 g·min^–1), before and after artificial acclimatization to cold and again 4 months later when acclimatization had decayed. Acclimatization was induced by ten daily cold (15°Q baths of 30–60 min followed by rapid rewarming in hot (38–42°C) water, and was confirmed by tests of the subjects responses to whole-body cooling in air. Three control subjects also underwent the first and third tests. Acclimatization substantially reduced the pressor response to NA at 0.150 and 0.300 g·kg^–1·min^–1, confirming earlier findings by the same technique in naturally acclimatized men, and its decay increased this response to beyond its initial levels (P<0.05 for both changes). Acclimatization did not change the response to NA of heart rate, subjective impressions, skin temperature of finger and toe, pulmonary ventilation, or plasma free fatty acids and ketone bodies. At no time did NA increase oxygen consumption, or increase skin temperature or heat flow over reported sites of brown fat. These findings would seem to show that acclimatization to cold reduces sensitivity to the pressor effect of NA but does not induce nonshivering thermogenesis, and that the reduced sensitivity is replaced by a hypersensitivity to NA when acclimatization decays.     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

Sodium excretion rates and renal responses to acute salt loading in the European starling

Summary Renal clearance studies were performed in European starlings (Sturnus vulgaris) in order to determine the extent of ureteral sodium excretion under control conditions and during an acute, hyperosmotic salt stress. These experiments also estimated the contribution of the lower intestine (colon and cloaca) to postrenal solute reabsorption by making both cloacal and ureteral urine collections in the same birds. A comparison of ureteral vs cloacal excretion rates found significantly higher sodium (9.09±1.30 vs 1.03±0.38 Eq·kg^–1·min^–1) and chloride (4.15±0.56 vs 1.00±0.38 Eq·kg^–1·min^–1) excretion rates during the ureteral collections. Fractional excretion of sodium was also significantly higher during ureteral collections, but this value did not exceed 1% of the filtered sodium load during either collection series. Urine flow rate was significantly higher during cloacal collections, suggesting osmotic back-flux of water across the cloacal wall. Infusion of a 1M NaCl solution resulted in rapid increases in glomerular filtration rate (GFR), urine flow rate, and urine osmolality. Fractional sodium and water reabsorption decreased by 11% and 4%, respectively. Glomerular counts and size distribution profiles, measured by in vivo alcian blue labelling, provided no evidence for a reduction in the number of filtering glomeruli during hyperosmotic saline loading. We conclude that renal sodium excretion rates for the starling are similar to those seen in other avian species and in mammals. These studies also provide direct evidence for postrenal modification of urine in this species, even under conditions of continuous flow. Acute hyperosmotic salt stress can, under some conditions, cause increased rather than decreased GFR, indicating multiple regulatory pathways. Finally, there was no evidence in these studies for glomerular shutdown in response to salt loading.     

A quantitative histochemical technique for the characterisation of α-glucosidases in the brush-border membrane of rat jejunum

Summary A quantitative histochemical method to determine the K_m and V_max of -glucosidases in the intestinal epithelium without disruption of the cellular structure is described. 2-Naphthyl--D-glucoside was used as substrate and hexazonium-p-rosaniline as coupling agent. Using a Leitz MPV2 microdensitometer and a field measuring 4×4 m, and reading the test samples against a blank focused on the lamina propria, we observed that the intensity of the colour was a linear function of both the incubation time up to 20 min, and the thickness of the slice up to 20 m. The ratio between the extinction at the absorption maximum and at a second wavelength was constant, whatever the intensity of the colour.By determining the relationship between the extinction and the substrate concentration under standard conditions (slice thickness of 10 m and incubation time of 10 min), we obtained a saturation curve described by a K_m of 0.68±0.038 mM and a V_max of 1.41±0.039 A₄₈₀·10^–2·m^–1·min^–1. When the hydrolysis of the same substrate by a homogenate of jejunal mucosa was examined biochemically under comparable conditions, a K_m of 0.64±0.012 mM and a V_max of 57.3±0.70 mU/mg protein were obtained. When the natural substrate, sucrose, was used in the biochemical study, a K_m of 15±3.5 mM and a V_max of 149±24.7 mU/mg protein were obtained.These experiments demonstrate that the kinetic constants of enzyme reactions can be assessed with equal accuracy on histochemical sections as in tissue homogenates.     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Age and segmental differences in 5-hydroxytryptamine-induced hypersecretion in the pig small intestine

5-Hydroxytryptamine is a mediator in cholera toxin-induced hypersecretion in the small intestine. Our hypothesis is that the hypersecretion induced by 5-hydroxytryptamine in the small intestine decreases with increasing age and in an aboral direction in the small intestine. In vivo, measuring accumulated fluid in ligated loops, the apparent maximal efficacy of the 5-hydroxytryptamine-induced jejunal secretion in pig neonates was 4.8±1.1 mg·mg^–1 dry loop·45 min^–1. The apparent maximal efficacy decreased by 23% and 63% in young and adult pigs, respectively, compared with neonates. In vitro, measuring changes in short-circuit current in Ussing chambers, the apparent maximal efficacy was 66.7±4.8 A·cm^–2 in neonates and was reduced by 30% and 57% in young and adult pigs, respectively. Young pigs were used in the segmental study. The apparent maximal efficacy in vivo was 3.7±0.5 mg·mg^–1 dry loop and decreased by 22% and 56% in the mid and distal small intestine, respectively. By contrast, in vitro the apparent maximal efficacy was elevated by 56% to 72.0±5.0 A·cm^–2 in the distal compared with the proximal part. In conclusion, the secretory response to 5-hydroxytryptamine in pig small intestine decreases with increasing age and in the aboral direction according to in vivo results. We suggest that the decrease in sensitivity to 5-hydroxytryptamine can explain a part of the reduced secretory response to cholera toxin with age and in the aboral direction of the small intestine.Abbreviations ASF pituitary peptide antisecretory factor - AMP cyclic adenosine monophosphate - CT cholera toxin - E _max apparent maximal efficacy - EC enterochromaffine ENS enteric nerve system - 5-HT 5-hydroxytryptamine serotonin - LT heat-labile - E. coli enterotoxin - PD electrical potential difference - R tissue resistance - SCC short-circuit current - VIP vasoactive intestinal peptide     

Changes in respiration of mitochondria isolated from cotyledons of ethylene-treated pea seedlings

Treatment of intact, germinating pea (Pisum sativum L. cv. Homesteader) seedlings with ethylene enhanced the cyanide-resistant respiration of mitochondria isolated from the cotyledons. The level of enhancement depended on the concentration of ethylene. Thus, exposure to 0.9 l·l^-1 of ethylene in air for days 4–6 of germination had little effect on cyanide-resistant respiration, while exposure to 130 l·l^-1 increased it from 10 to 50 nmol O₂·min^-1·(mg protein)^-1. The length of exposure to ethylene also affected the degree of enhancement. According to some literature data, lipoxygenase (EC 1.13.11.12) activity can be mistaken for cyanide-resistant respiration, but in our preparations of purified pea mitochondria ethylene had no effect on lipoxygenase activity, nor did the gas disrupt the outer mitochondrial membrane. Bahr and Bonner plots of respiration in the presence of salicylhydroxamic acid (SHAM) indicated that ethylene did not affect respiration proceeding via the cytochrome pathway. Thus, increases in total respiration in mitochondria from cotyledons of ethylene-treated pea seedlings reflect increases in cyanide-resistant respiration.Abbreviations Cyt c cytochrome c - SHAM salicylhydroxamic acid     

Water chemistry changes in the gill micro-environment of rainbow trout: experimental observations and theory

Summary Soft water of low buffer capacity was drawn from near the branchial surface of rainbow trout (Salmo gairdneri) at 15°C, using opercular catheters, to determine pH changes in water passing over the gills. Latex masks allowed measurement of ventilation volume, and concentrations of carbon dioxide, oxygen, ammonia, and titratable base in expired water were compared to concentrations in inspired water. Water passing over the gills was more basic than inspired water if the inspired water was pH 4–6 (maximum increase: +0.7 pH units near pH 5). Expired water was more acidic than inspired water if the inspired water was pH 6–10 (maximum decrease: –1.7 pH units near pH 9). Ventilation volume (0.37 l·kg^–1·min^–1) and oxygen consumption (1.7 mmol·kg^–1·h^–1) were constant in the pH range 4.6–10.1, but both increased by 1.6–2.4× near pH 4. Carbon dioxide transfer near the gills was about 100 M, ammonia transfer about 15 M, and titratable base added at the gills was about 30 M. A theoretical model using CO₂, titratable base, and ammonia added at the gills, the titration characteristics of the defined soft water medium, and aquatic equilibria for CO₂ and ammonia, adequately explained the experimentally observed changes in pH near trout gills. Our observations and predictive model indicate that any gill contaminant whose toxicity varies with pH may be more or less toxic at the gills than predicted from bulk water chemistry alone.Abbreviations pH _ex expired pH - pH _in inspired pH     

Physical restraints underlying short-term inhibition by auxin of root elongation in intact maize seedlings

This report investigates physical changes associated with the short-term inhibition of root elongation in intact maize seedlings (Zea mays L. vs. Halamish) by exogenous auxin. Movement of root tips was assayed by video microscopy in control roots, roots grown for 45 min in 10^–6 M indole3-acetic acid (IAA), or roots chilled for 3 min at 11°C. IAA and chilling treatments similarly reduced root elongation rates (from 29 ± 6 m min^–1 to 6 ± 2 m min^–1). Initial rates of root tip contraction induced by 300 mOsmol mannitol were used to calculate tissue contractibility values. These allowed a comparison of effects of IAA and chilling treatments on apparent rates of water transport out of the root tip tissues. Chilling treatment reduced root tip contractibility by 66%, whereas IAA had much less effect (26% reduction). Roots were also exposed to an osmotic jump treatment; the initial osmotically induced increase in elongation rate was used to determine root tip extensibility values. Both IAA and chilling treatments reduced root tip extensibilities by 57%. Inhibition of wall-yielding properties, rather than hydraulic limitations, appeared to be primarily associated with inhibition of intact root tip elongation by exogenous IAA.     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase