ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

Inwardlyrectifying K⁺ current(I_Kir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, I_Kir randown completely in <10 min [half time(t_1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustainedI_Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used,I_Kir ran down by~71%. Mg²⁺ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg²⁺(t_1/2 = 1.8 min).I_Kir also randown when the pipette solution contained 4 mMMg²⁺ + 4 mM5'-adenylylimidodiphosphate(t_1/2 = 2.7 min)or 4 mM adenosine 5'-O-(3-thiotriphosphate)(t_1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity ofI_Kirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis.

     

Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol

An efficient vitrification procedure was developed and successfullyapplied to cryopreserve six endangered West Australian species(family Haemodoraceae: Anigozanthos humilis ssp. chrysanthusHopper;A. kalbarriensis Hopper;A. viridis ssp. terraspectansHopper;Conostylis dielsia ssp.teres Hopper;C. micrantha Hopperand C. wonganensis Hopper). Species were initially evaluatedfor cryostorage using a basic vitrification protocol involving:culturing plantlets in vitro for 21 d; excision of shoot apices;preculture of apical tips on 0.4 M sorbitol for 2 d, followedby incubation in PVS2 (plant vitrification solution 2) for 25min at 0 °C, then direct immersion in liquid nitrogen (LN).Warming of retrieved material was for 1 min in a 40 °C waterbath. Using this protocol five of the six species exhibitedlow post-storage survival, while the sixth species, A. viridisssp. terraspectans posted higher survival (61.1%). Using A.viridis ssp. terraspectans as an indicator species, the initialprotocol was modified to include: 3 d preculture on 0.80 M glycerol,loading treatment with 2.0 M glycerol plus 0.4 M sucrose solutionfor 20 min, followed by 25 min exposure to a modified PVS2.Survival was significantly improved in the test species, andin further experiments three other species also showed significantimprovements with the new protocol. Key findings include: effectivenessof glycerol in the preculture medium; the effect of precultureduration; the importance of a loading stage for these species;and the successful use of modified PVS2 solutions with reducedor zero dimethyl sulfoxide (DMSO). Copyright 2001 Annals ofBotany Company A. humilis ssp. chrysanthus, A. kalbarriensis, A. viridis ssp.terraspectans , Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, kangaroo paws, Haemodoraceae, vitrification, cryopreservation, rare and endangered, conservation     

Differences in cold lability of pyruvate, Pi dikinase among C4 species

The cold lability of pyruvate, Pi dikinase in crude leaf extractswas studied in a number of C⁴ plants. The survey included C⁴monocots and dicots and species representingthe three C⁴ subgroups:NADP-malic enzyme, NAD-malic enzyme, and PEP-carboxykinase types. In some species (e.g., Digitaria sanguinalis, Sorghum bicolorand Echinochloa crus-galli), the enzyme was very sensitive tocold treatment (half life of about 8 min at 0?C and 10 to 15min at 10?C). In other species (Panicum miliaceum, Panicum maximumand Panicum texanum), the enzyme was very cold tolerant (retentionof 60 to 85% activity after 60 min at 0?C and 90% activity after60 min at 10?C). Among the plants examined, the most cold sensitivepyruvate,Pi dikinase was found among species of the NADP-malicenzyme subgroup. (Received March 9, 1979; )     

Stomatal responses to changing irradiance in Phaseolus vulgaris L.

The effects of air temperature (T_o), leaf-air vapour pressuredifferences [VPD) and water deficit on stomatal responses tochanging irradiance were studied in Phaseolus vulgaris L. Responseswere approximately sigmoidal, with rates of closure being fasterthan the rates of opening. The mean half-time for closure was5.4 min and for the opening 9.2 min. Under water deficit, bothstomatal opening and closing were faster than in well-wateredconditions. Stomata were more sensitive to VPD and water stressthan to T_o. The higher the VPD or T_o the more rapid was thestomatal response, except in stressed plants where there wasno significant effect of T_o. Under water stress, stomata weremore sensitive to water potential (     

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia

Activation ofprotein kinase C with phorbol 12-myristate 13-acetate (PMA) causedcomplex transient perturbations of amiloride-sensitive short-circuitNa⁺ currents(I_Na) in A6epithelia and frog skins that were tissue and concentration dependent.A noninvasive channel blocker pulse method of noise analysis (18) wasused to investigate how PMA caused time-dependent changes of apicalmembrane epithelial Na⁺ channel(ENaC) single-channel currents, channel open probabilities (P_o), andchannel densities(N_T). In A6epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependentsustained decreases ofP_o (~55% belowcontrol, 50 nM) and rapid compensatory transient increases ofN_T within 7 min(~220% above control, 50 nM), resulting in either small transientincreases of I_Naat 5 nM PMA or small biphasic decreases ofI_Na at 50 nM PMA.In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin causedafter a delay of at least 10 min transient increases ofN_T to~60-70% above control at 30-60 min. Unlike A6 epithelia,P_o was increased~15% above control within 7 min and remained within±10-15% of control for the duration of the 2-h experiments.Despite differences in the time courses of secondary inhibition oftransport in A6 epithelia and frog skin, the delayed downregulation oftransport was due to time-dependent decreases ofN_T from theirpreelevated levels in both tissues. WhereasP_o is decreasedwithin minutes in A6 epithelia as measured by noise analysis or bypatch clamp (8), the discrepancy in regulation ofN_T in A6epithelia as measured by noise analysis and patch clamp is most likelyexplained by the inability of on-cell patches formed before treatmentof tissues with PMA to respond to regulation of their channeldensities.

     

Water Transport during Acid-Induced Growth of Excised Hypocotyl Sections of Vigna unguiculata under Xylem Perfusion

Elongation growth of abraded hypocotyl sections of Vigna unguiculataunder xylem perfusion was markedly promoted a few minutes afterthe application of an acid aerosol generated from a solutionof HCl. At the beginning of the acid-induced growth, intracellularpressure (Pⁱ) began to decrease and the membrane potential betweenthe symplast and the xylem apoplast (V^px) began to depolarize.Subsequently, Pⁱ and V^px remained at a reduced level and a depolarizedlevel, respectively, while the promotion of elongation growthcontinued for more than 4 hours. The electrogenic componentof the xylem membrane potential (V^px_act) gradually increasedto about twice that before acid treatment. There was a closecorrelation between the enhanced growth and the decrease inintracellular pressure within 30 min after application of acidbut little correltion after 60 min. By contrast, there was littlecorrelation between the promotion of growth and the activityof the xylem pump after 30 min while a close correlation wasobserved after 60 min. It is inferred that the acid-induced activation of water uptakeconsists of two major processes, in series, that are drivenby different forces: the rapid uptake of water for more than30 min, driven by hydrostatic force generated by loosening ofcell walls; and a long-lasting enhancement of water uptake forat least 4 h, which is driven by osmotic force that is generatedby the canal system within the xylem. (Received October 17, 1994; Accepted January 23, 1995)     

Mechanism of mosaic attenuation of the lungs on computed tomography in induced bronchospasm

The purpose of this study was to investigatewhether hypoxic pulmonary vasoconstriction is the major determinant ofthe computed tomography (CT) pattern of mosaic attenuation in asthmaticpatients with induced bronchoconstriction. Thin-section CT wasperformed at suspended full inspiration immediately and 30 min aftermethacholine bronchoprovocation in 22 asthmatic subjects, who wererandomly assigned to breathe room air (group A,n = 8), oxygen via nasal prongs at 5 l/min (group B,n = 8), and oxygen via face mask at 12 l/min (group C,n = 6). CT changes were quantified interms of global lung density and density in hypodense and hyperdense areas. Lung parenchymal density increases were greatest ingroup C and greater ingroup B than in groupA, globally (P = 0.03) and in hypodense regions (P = 0.01).On bivariate analysis, the only change in cross-sectional area wasrelated to change in global density. In hypodense regions, densitychange was related both to reduction in cross-sectional area(P < 0.0005) and to oxygen administration (P = 0.01). Aftercorrection for changes in global lung density, only oxygen wasindependently related to density increase in hypodense areas(P = 0.02). In inducedbronchoconstriction, the CT appearance of mosaic attenuation can belargely ascribed to hypoxic vasoconstriction rather than to changes inlung inflation.     

The effects of brefeldin A upon the Golgi apparatus of the green algal flagellate, Gloeomonas kupfferi

The fungal-derived derivative, brefeldin A, was used to disruptthe Golgi apparatus (GA) of the green alga, Gloeomonas kupfferi.Upon short treatments (10 µg ml^–1 for 10 min orless), the Golgi bodies maintain their perinuclear positioning.However, the medial locus transforms from a tight stack of elongatecisternae to a network of swollen tubules. Upon longer treatments(60 min), swelling and vesiculation of cis face cisternae becomeapparent. Likewise, the edges of several trans face cisternaemay fuse with those of adjacent Golgi bodies leading to theformation of multiGolgi complexes. Key words: Brefeldin A, Golgi apparatus, Gloeomonas kupfferi     

我国新疆和湖南棉铃虫种群的遗传学研究

采自湖南衡阳(HY)和新疆哈密(HM)的棉铃虫Helicoverpa armigera(Hubner)种群被室内杂交和回交,杂交F₁代和回交F₂代雌成虫的产卵量、卵孵化率和产卵时间等繁殖力参数均无明显的变化。在22℃下,HM、HY、HM♀×HY♂、HY♀×HM♂和(HY♀×HM♂)×HY♂后代蛹滞育的临界光周期分别为13h 35 min,11 h,11 h 45 min,11 h 43min和11 h9min。在一15℃下,HM、HY、HM♀×HY♂、HY♀×HM♂、HY♀×(HY♀×HM♂)♂和(HY♀×HM♂)♀×HM♂后代滞育蛹的致死中时间(LT”)分别是159.7h、34.27h、100.65 h、116.75h、90.78h和135.58h,亲本F₁代显性度D(HM♀×HY♂)和D(HY♀XHM♂)分别为0.3998和0.5926。遗传分析表明棉铃虫滞育蛹的抗寒性由多个不完全显性的基因控制。     

Variable G2 Duration in Root-Meristem Cells of Vicia faba

Scintillation autoradiography was utilized to demonstrate thevariable duration of G₂ in Vicia faba root meristems. The ratioof the longest (max G) to the shortest (min ) duration was 5.4,with min being approximately 2.5 h.     

Grazing rates of the freshwater ciliate Balanion planctonicum determined by flow cytometry

Feeding of Balanion planctonicum on the cryptomonad Rhodomonassp. was recorded in vivo at 2–3 min intervals by flowcytometry. Ingestion rates were 1.6–1.7 algal cells ciliate^–1h^–1. On average, 20–30 min elapsed between ingestionand egestion.     

INHIBITION OF OLFACTION IN THE MOTH HELIOTHIS VIRESCENS BY THE SULFHYDRYL REAGENT FLUORESCEIN MERCURIC ACETATE

A combined electrophysiological, behavioral, and biochemicalstudy was initiated to determine the effects of the sulfhydryl-specificreagent fluorescein mercuric acetate (FMA) on olfaction in thetobacco budworm moth Heliothis virescens. The electroantennogram(EAG) response to the standard odorant n-pentyl acetate showedboth a time and concentration dependent inhibition by FMA. Treatmentof insect antennae with 2.52 x 10^–5 M FMA for 2 min reducedthe EAG by 50%, while treatment for 17 min reduced the EAG by80%. Incubation of antennae for 7 min with 2.52 x 10^–6M FMA resulted in 30% inhibition, while incubation with 2.52x 10^–6 M FMA for 7 min resulted in 65% inhibition. Antennalgrooming behavior was inhibited by FMA in a similar time andconcentration dependent manner as the EAG. Regeneration of previouslyinhibited behavioral and EAG responses has been observed withina 24-hr period. The interaction of protein, obtained by sonicatingintact antennae in phosphate buffer, with FMA was monitoredfluorometrically. Successive additions of antennal sonicateto FMA resulted in stepwise decreases in fluorescence. Partialrecovery of fluorescence was obtained by addition of cysteineto the FMA-antennal sonicate solution. The polarization of theFMA-antennal sonicate fluorescence decreased upon addition ofcysteine. These data indicate that FMA is reacting with a relativelylarge antennal protein (s) by mercaptide linkage and blockingthe olfactory transduction process.     

Load compensation as a function of state during sleep onset

Ventilation decreases and airway resistanceincreases with the loss of electroencephalogram alpha activity at sleeponset. The aim of this study was to determine whether reflexive load compensation is lost immediately on the loss of alpha activity. Sixhealthy male subjects were studied under two conditions (load andcontrol-no load), in three states (continuous alpha, continuous theta,and immediately after a transition from alpha to theta), and in twophases (early and late sleep onset). Ventilation and respiratory timingwere measured. A comparison of loaded with control conditions indicatedthat loading had no effect on inspiratory minute ventilation duringcontinuous alpha (differential effect of 0.00 l/min) and only a small,nonsignificant effect in theta immediately after phase2 transitions (0.31 l/min), indicating a preservationof load compensation at these times. However, there were significantdecreases in inspiratory minute ventilation on loaded trials duringcontinuous theta in phase 2 (0.77 l/min) and phase 3 (1.15 l/min) andduring theta immediately after a transition in phase3 (0.87 l/min), indicating a lack of reflexive loadcompensation. The results indicate that, because reflex load compensation is state dependent, state-related changes in airway resistance contribute to state-related changes in ventilation duringsleep onset. However, this effect was slightly delayed with transitionsinto theta early in sleep.

     

Feeding of planktonic rotifers on ciliates: a method using natural ciliate assemblages labelled with fluorescent microparticles

A method was developed to allow direct measurements of predationexerted by metazooplankton on ciliates. The method relied onthe use of ciliates labelled with fluorescent microparticles(FMP). Optimal labelling conditions were determined with ciliatesfrom cultures (Tetrahymena pyriformis) and with natural ciliateassemblages sampled in a river. Labelled T. pyriformis wereused as tracer food to determine gut passage time (GPT) andingestion rates of the rotifer Brachionus calyciflorus in thelaboratory. Predation of metazooplankton from the lowland riverMeuse (Belgium) was determined by labelling natural assemblagesof ciliates and using them as tracer food for metazooplankterssampled in the river. Optimal labels of ciliates, i.e. sharpdistribution of FMP in cells, were obtained with short incubations(10 min) and low FMP concentrations (1 x 10⁵ mL^–1). GPTvaried between 30 and 45 min for B. calyciflorus and from 25up to >35 min for rotifers from the river. The ingestionrate of B. calyciflorus fed with T. pyriformis was 3.3 ±0.6 ciliate rot^–1 h^–1, i.e. 1.4 ± 0.3 ngCrot^–1 h^–1. Metazooplankton species for which theingestion of ciliates could be measured were the rotifers Keratellacochlearis, Euchlanis dilatata and Synchaeta spp. Ingestionrates measured ranged from 0.4 to 12.5 ngC rot^–1 h^–1.The method proposed proved to be useful in estimating the predationof microplankton on ciliates in semi- in situ conditions; infurther developments, labelled natural assemblages of ciliatescould be used for in situ incubations with the Haney chamber.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Calcification in the Green Alga Halimeda: II. THE EXCHANGE OF CA2+ AND THE OCCURRENCE OF AGE GRADIENTS IN CALCIFICATION AND PHOTOSYNTHESIS

In Halimeda cylindracea and H. tuna segments, the concentrationof CaCO₃, MgCO₃, protein, and chlorophyll, as well as segmentvolume and wet and dry weight, increase with ‘age’i.e. from the apex of a branch downwards. Photosynthetic andcalcification rates decrease with age as does the degree oflight stimulation of calcification. Studies of the exchange of ⁴⁵Ca between the Halimeda thallusand the sea water under various conditions showed that mostof the Ca exchange is between the cell walls, the aragonitecrystals, and the intercellular space. The cell wall has twodistinguishable phases with half-times (t_0?5) of 200 and 35min while the CaCO₃ has a rapidly exchanging phase with a t_0?5of approximately 6 min. The t_0?5 of the exchange of Ca betweenthe intercellular space and the external medium is estimatedat about 6 min, on the basis of uptake studies. If the integrityof the barrier between the intercellular space and the externalsea water, created by the adpressed peripheral utricles is destroyedthe t_0?5 is smaller (<<3 min). These kinetic studies as well as comparative measurements ofcalcification rates by both isotopic and chemical methods showthat the ⁴⁵Ca method for measuring calcification rates overestimatesthe calcification rate, due to binding of ⁴⁵Ca in the cell wallsand retention of ⁴⁵Ca in the intercellular space. The ¹⁴C methodgives more accurate results and has the further advantage ofallowing simultaneous measurement of the photosynthetic andcalcification rate on the same segment.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Production and absorption of nitric oxide gas in the nose

Some nitric oxide gas (NO) produced in thesinuses and nasal cavity is absorbed before leaving the nose. Tomeasure production and absorption, we introduced NO at differentconcentrations into one nostril while sampling the NO leaving theopposite nostril with the soft palate closed. The quantity of NO gasproduced in six normal subjects (amount leaving plus the amountabsorbed) averaged 352 nl/min and was the same at gas flows rangingfrom 8 to 347 ml/min and at 10 l/min. An absorption coefficientA was calculated by dividing theamount of NO absorbed by the concentration leaving the nose.A ranged from 17 ml/min at a nasal gasflow of 8 ml/min to an A of 24 ml/minat a nasal gas flow of 347 ml/min. The calculated rates of productionand absorption did not change when gas flow rate was increased,suggesting diffusion equilibrium. The amount of uptake of NO in thenasal mucosa can be explained by its solubility coupled with tissue andblood reactivity.

     

Transpiration Stream of Desert Species: Resistances and Capacitances for a C3, a C4, and a CAM Plant

Transpiration rates and water potentials of three sympatricdesert perennials, a C3 subshrub (Encelia farinosa), a C₄ bunchgrass(Hilaria rigida), and a CAM succulent (Agave deserti), wereanalysed using an electrical circuit analogue that includedresistances and capacitances for the leaves, stems, and roots.The water storage capability of the organs differed considerably,capacitance ranging over 1000-fold from the thin leaves of H,rigida to the massive leaves of A. deserti, although the capacitanceper unit volume varied only 1.9-fold. The diurnal changes inwater storage could support maximum transpiration rates of H.rigida for 4 min, E. farinosa for 7 min, and A. deserti for16 h. The time constant for equilibration of water from storageto the xylem ranged from 29 s for roots of H. rigida to 52 minfor leaves of A. deserti. Resistances for such movement wererelatively low for the succulent leaves of A. deserti and wereup to about 50-fold higher for the three organs of E. farinosa.Xylem resistances calculated using the Hagen-Poiseuille lawand measured xylem dimensions were 2.1- to 2.1-fold lower thanresistances estimated from observed water potential drops, adiscrepancy which is in agreement with other published data.Contrary to data on other plants, the xylem resistances in theroots and leaves of E. farinosa and H. rigida averaged only15% of the stem xylem resistance. Key words: Capacitance, Xylem resistance, Transpiration stream, Desert     

ERRATUM

In Table 2 (page 626), in the paragraph beginning with the words‘10 min before zero time ....’, lines 2 and 4: for containing 50 µc ³H-acetate read containing 200 µc ³H-acetate