Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

Na and Cl transport across the isolated turtle colon: Parallel pathways for transmural ion movement

Summary Transmural fluxes of³H-mannitol and²²Na or³⁶Cl were measured simultaneously in portions of isolated turtle colon stripped of serosal musculature. The relationships between mannitol flux and the flux of Na or Cl are characteristic of simple diffusion and suggest that transmural mannitol flow is largely confined to a paracellular pathway where Na, Cl and mannitol move much as in free solution. The contribution of edge damage to the transmural mannitol flow appears to be minimal. Mucosal hyperosmolarity causes blisters in epithelial tight junctions and increases the diffusional permeability to Na and mannitol, suggesting that the rate-limiting barrier in the shunt path is the tight junction. If the total mucosa to serosa flux of Na is corrected for the portion traversing the shunt pathway it is apparent that changes in the short-circuit current are completely accounted for by the mucosa to serosal movement of Na through a cellular path. In addition, the serosa to mucosa flux of Na appears to be restricted to the shunt. These observations suggest that there is no appreciable backflux of Na through the active, cellular path. In the presence of 10^–4 m amiloride the short-circuit current is markedly reduced and the mucosa to serosa Na flux is restricted to the shunt, so that the net Na flux is abolished. The small amiloride-insensitive short-circuit current is consistent with HCO₃ secretion. Mucosa to serosa and serosa to mucosa fluxes of Cl appear to be largely restricted to the paracellular shunt path and there is no evidence for any net flow of Cl under short-circuit conditions. The total tissue conductance can be described as the sum of three components: a shunt conductance which is linearly related to the transmural mannitol flow, an active conductance which is linearly related to the short-circuit current and a small residual conductance. The shunt conductance is attributable to the diffusive movements of Na and Cl through the paracellular path. Variations in the active Na transport from tissue to tissue are largely attributable to variations in the apparent conductance of the active Na transport path. The driving force for active Na transport can be described as an apparent emf of approximately 130 mV. These results suggest that transmural mannitol flux provides a quantitative estimate of the ion permeability and electrical conductance of a paracellular shunt path across the isolated turtle colon and thereby facilitates the study of the transport characteristics and electrical properties of cellular paths for transepithelial solute movement.     

The conventional short-circuiting technique under-short-circuits most epithelia

Summary The relationships among ion current, membrane potential difference, and resistance of an epithelium are studied. The short-circuit technique introduced by Ussing and Zerahn does not completely short circuit the epithelium if the series resistance parallel to the cell layer between the voltage electrodes is not properly compensated. The residual potential difference across the epithelial cell layer in the short-circuit state is proportional to both the measured short-circuit current and the resistance of the diffusion barriers not compensated. In the conventionally short-circuited small intestinal mucosa the villus and crypt areas are hypo-polarized to different degrees rather than simultaneously hyper- and hypo-polarized. Short-circuiting the whole tissue reduces but does not abolish the passive net ion movement across the tissue. Measurements of the electrical properties of the whole and denuded rat distal small intestine in HCO₃-Ringer solution containing 10mm glucose reveal that the measured short-circuit current has under-estimated approximately 33% of the true short-circuit current and that the passive net Na flux from serosa to mucosa and Cl flux from mucosa to serosa are not negligible in the short-circuit state.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

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

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

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

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).     

Summer forage biomass and the importance of litterfall for a high-density sika deer population

The biomass of summer forage and their contributions were surveyed to show that litterfall supported a high-density population of sika deer (Cervus nippon Temminck) in summer on Nakanoshima Island, Toya Lake, Japan. In July 1974, the grassland had the highest productivity among understory vegetations (228±55kgha^–1: mean±SE). In deciduous forests, palatable plants occupied only 0.1% of the biomass of 0.872±0.366kgha^–1, and deciduous leaves within the reach of deer (=220cm at height) produced 0.208±0.070kgha^–1. However, litterfall during this period had the highest productivity, 28.7± 5.3kgha^–1. The deer consumed litterfall (75.6% in dry weight), short grasses (17.2%), deciduous forest understory (4.1%), deciduous leaves within the reach of deer (3.0%) and conifer plantation under story (0.1%). It is suggested that the high-density deer population would be maintained by litterfall through the year instead of browsing in deciduous forests, which has been overlooked.     

Thermophilic and mesophilic bacteria in biofilms associated with corrosion in a heat exchanger

Biofilm development on AISI-1020 carbon steel coupons installed at the outlet of a heat exchanger was evaluated at the thirtieth and the sixtieth days of exposure. Water temperature varied between 41 and 60°C. The most probable number technique (MPN) was applied to quantify mesophilic and thermophilic species of aerobic, anaerobic, and sulphate-reducing bacteria (SRB) in planktonic and sessile phases. The results showed predominance of thermophilic aerobic bacteria in both phases, corresponding to 9.5±0.8×10⁶cells/ml in the planktonic phase. In biofilms, maximal aerobic cell concentration, 7.8±0.6×10⁸cells/cm2, was registered at the sixtieth day. An increase in the number of thermophilic anaerobic and SRB with elapsed time was also observed. The results obtained after 60 days were 5.8±0.4×10⁷ and 8.9±0.9×10⁴cells/cm² for anaerobic and sulphate-reducing bacteria, respectively. Scanning electron microscopy showed a varied composition of species in the biofilms and corrosion on the carbon steel surfaces after biofilm removal.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Metabolic cost of sodium transport in toad urinary bladder

Summary The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential, , by continuous and simultaneous measurements of CO₂ production and of transepithelial electric current. Amiloride was used to block active sodium transport in order to assess the nontransport-linked, basal, production of CO₂ and the passive permeability of the tissue. From these determinations active sodium transport,J _Na, and suprabasal CO₂ production, , were calculated. Since large transients inJ _Na and frequently accompanied any abrupt change in , steady state conditions were carefully defined.Some 20 to 40 min were required after a change in before steady state of transport activity and of CO₂ production were achieved. The metabolic cost of sodium transport proved to be the same whether the bladder expended energy moving sodium against a transepithelial electrical potential grandient of +50 mV or whether sodium was being pulled through the active transport pathway by an electrical gradient of –50 mV. In both cases the value of the ratio averaged some 20 sodium ions transported per molecule of CO₂ produced.When the Na pump was blocked by 10^–2 m ouabain, the perturbations of the transepithelial electrical potential did not elicit changes ofJ _Na nor, consequently, of .The independence of the ratio from over the range ±50 mV indicates a high degree of coupling between active sodium transport and metabolism.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

Stable isotope ratio as a tracer of mangrove carbon in Malaysian ecosystems

Summary The ratio of stable carbon isotopes (¹³C) in plants and animals from Malaysian mangrove swamps, coastal inlets, and offshore waters was determined. Vascular plants of the swamps were isotopically distinct ( x±s.d.=-27.1±1.2) from plankton (-21.0±0.3) and other algae (-18.7±2.2). Animals from the swamps (-20.9±4.1) and inlets (-19.8±2.5) had a wide range of isotope ratios (-28.6 to-15.4), indicating consumption of both mangrove and algal carbon. Several commercially important species of bivalves, shrimp, crabs, and fish obtained carbon from mangrove trees. Mangrove carbon was carried offshore as detritus and was isotopically distinguishable in suspended particulate matter and sediments. Animals collected from 2 to 18 km offshore, however, showed no isotopic evidence of mangrove carbon assimilation, with ratios (-16.5±1.1, range-19.1 to-13.1) virtually identical to those reported for similar animals from other plankton-based ecosystems. Within groups of animals, isotope ratios reflected intergencric and interspecific differences in feeding and trophic position. In particular, there was a trend to less negative ratios with increasing trophic level.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Root traits, nutrient uptake, multi-location grain yield and benefit–cost ratio of two lentil (Lens culinaris, Medikus.) varieties

Lentil is a protein-rich pulse, grown mainly in developing countries as a rain-fed crop in nutrient-poor soils. Hence, the importance of root traits for efficient capture of soil nutrients and water can be crucial to its economical yield. Little is known about the lentil root system and even less about its relationship to grain yield. We compared the root system of two Bangladeshi lentil varieties, Barimasur-3 (BM-3) and Barimasur-4 (BM-4), in a pot experiment and related it to their multi-location grain yield in the fields. BM-4 maintained faster root development both at an early growth stage (20days after sowing) and at flowering (60days) compared to BM-3. The roots of BM-4 penetrated the 25cm depth of the soil profile after 19±1days and while those of BM-3 took 24±2days to reach the same depth. The roots of BM-4 were covered with denser (26±3mm^–1) and longer (0.48±0.11) root hairs than BM-3 (density 17±2mm^–1, length 0.32±0.09mm). The differential presence of root hairs increased the effective length of root system of BM-4 by 12 times and that of BM-3 by five times. The lentil varieties did not differ in their ability to induce pH change and acid phosphatase activity in rhizosphere. In the pot experiment, the uptake of macro-nutrients (K, P, Ca, and Mg) as well as micro-nutrients (Fe, Mn, Zn, Cu, B and Mo) by BM-4 was significantly higher, compared to BM-3. The varieties produced the same amount of shoot biomass. At five of six agro-ecological distinct field locations in Bangladesh, BM-4 gave significantly higher (10–20%) grain yield than BM-3. Linked with the higher grain yield, the benefit-cost ratio (BCR) of BM-4 was 3.14 and that of BM-3 were 2.62, indicating that BM-4 provided better return per unit investment, compared to BM-3, supported by the better root morphology and higher nutrient uptake. This may be one of the reasons supporting the popularity and preferred adoption of BM-4 among the Bangladeshi farmers, who grow lentil mainly on nutrient-poor soils. The results indicate the benefits of selection and breeding for superior root traits for better agro-economics.     

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.)

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m -naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.Abbreviations BA Benzyl adenine - IBA Indole-3-butyric acid - 2-ip N⁶-(-dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - NAA -Naphthaleneacetic acid