A Change in Tubulin Synthesis in the Process of Tracheary Element Differentiation and Cell Division of Isolated Zinnia Mesophyll Cells

Changes in tubulin synthesis in the process of cytodifferentiationinto tracheary elements and cell division were investigatedusing a culture of single cells isolated from the mesophyllof Zinnia elegans. The tubulin content was measured by a sensitiveimmunoblotting method using a mouse monoclonal antibody to -or ß-tubulin as a probe and mung bean tubulin as astandard. Freshly isolated mesophyll cells had only small amountsof tubulin, but the content increased rapidly between 24 and48 h of culture before morphological differentiation and celldivision. The content rose more than sixfold during 48 h cultureand then decreased slightly. This pattern of increase closelyresembled that of the increase in cortical microtubules (MTs)estimated by electron microscopic analysis. The - and ß-tubulincontents in the cultured cells were almost the same and changedin coordination during culture. The activity of tubulin synthesis was determined by densitometricscanning of spots corresponding to tubulin subunits on an autoradiogramof a two-dimensional polyacrylamide gel of [³⁵S]-methionine-labeledproteins. Tubulin synthesis began as early as between 4 and8 h of culture and its rate increased similarly to the increasein the tubulin content, with the former always preceding thelatter, indicating that the increase in content resulted fromnew tubulin synthesis. (Received December 16, 1986; Accepted February 25, 1987)     

Characterization of the Inhibitory Effect of Aphidicolin on Transdifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

Inhibition by aphidicolin (APC), an inhibitor specific for -typeDNA polymerase, of trans-differentiation into tracheary elementswas characterized in Zinnia mesophyll cells. APC was effectivewhen given in the first 24 h of culture and exposure continueduntil the 36th hour. This suggests temporal involvement of -typeDNA polymerase in transdifferentiation. ¹Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejiro, Tokyo,112 Japan     

Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells

To clarifyinteractions between the cytoskeleton and activity of L-typeCa²⁺ (Ca_L) channels in vascular smooth muscle(VSM) cells, we investigated the effect of disruption of actinfilaments and microtubules on the L-type Ca²⁺ current[I_Ba(L)] of cultured VSM cells (A7r5 cellline) using whole cell voltage clamp. The cells were exposed to eachdisrupter for 1 h and then examined electrophysiologically andmorphologically. Results of immunostaining using anti--actin andanti--tubulin antibodies showed that colchicine disrupted both actinfilaments and microtubules, cytochalasin D disrupted only actinfilaments, and nocodazole disrupted only microtubules.I_Ba(L) was greatly reduced in cells that wereexposed to colchicine or cytochalasin D but not to nocodazole.Colchicine even inhibited I_Ba(L) by about 40%when the actin filaments were stabilized by phalloidin or when thecells were treated with phalloidin plus taxol to stabilize bothcytoskeletal components. These results suggest that colchicine mustalso cause some inhibition of I_Ba(L) due toanother unknown mechanism, e.g., a direct block of Ca_Lchannels. In summary, actin filament disruption of VSM cells inhibitsCa_L channel activity, whereas disrupting the microtubulesdoes not.

     

Transient and Specific Expression of a Cysteine Endopeptidase Associated with Autolysis during Differentiation of Zinnia Mesophyll Cells into Tracheary Elements

Endopeptidase activities during the differentiation of Zinniacells into tracheary elements (TEs) were examined with severalpeptidyl 4-methyl-7-coumarylamido (MCA) as substrates. The activitythat hydrolysed carbobenzoxy-Phe-Arg-MCA (Z-Phe-Arg-MCA) atpH 5 increased in a differentiation-related manner: this activity,which was not observed in freshly isolated mesophyll cells wasinduced by the combination of -naphthaleneacetic acid (NAA)and 6-benzyladenine (BA) that is necessary for differentiationof TEs, but not by NAA or BA alone. The activity in cells culturedin TE-inductive medium that contained both NAA and BA increasedvery rapidly between the 48th and 60th hours of culture, whenthe number of TE increased rapidly. A protease responsible forthis activity with a molecular mass of 30 kDa was partiallypurified from cells which had been cultured in the TE-inductivemedium and included many immature TEs. Strong inhibition by[L-3-trans-carboxyoxiran-2-carbonyl]-L-Leu-agmatin (E-64), andactivation by dithiothreitol (DTT) indicated that this proteasebelongs to a family of cysteine endopeptidases (EC 3.4.22). ¹Present address: Department of Material and Biological Engineering,Tsuruoka National College of Technology, Inooka-Sawada 104,Tsuruoka, Yamagata, 997 Japan     

Relationship between DNA synthesis and the increase in the level of tubulin during dedifferentiation of isolatedZinnia mesophyll cells

Summary The level of tubulin in cultured matureZinnia mesophyll cells increases to between 4 and 5 times its initial value when DNA synthesis occurs. The rapid increase in the level of tubulin requires the presence of auxin and cytokinin, as does the induction of DNA synthesis. Inhibitors of DNA synthesis suppress the rapid increase in the level of tubulin. These results imply that the rapid increase in the level of tubulin is dependent on the occurrence of DNA synthesis,i.e., on the reinitiation of the cell cycle. The presence of microtubules is not important for either the increase in the level of tubulin or for the induction of DNA synthesis, because both the increase in the level of tubulin and DNA synthesis occur even when microtubules are depolymerized by colchicine.Abbreviations APM amiprophos-methyl - araC arabinosylcytosine - BA 6-benzyladenine - BSA bovine serum albumin - DMSO dimethylsulfoxide - EGTA ethylene glycol bis(2-aminoethylether)tetraacetic acid - FITC fluorescein isothiocyanate - FUdR fluorodeoxyuridine - GA gibberellin A₃ - IAA indole-3-acetic acid - MES 2-(N-morpholino)ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PBS phosphate buffered saline - PCA perchloric acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - SDS sodium dodecyl sulfate     

Involvement of Poly(ADP-Ribose) Synthesis in Transdifferentiation of Isolated Mesophyll Cells of Zinnia elegans into Tracheary Elements

The relationship between poly(ADP-ribose) synthesis and cytodifferentiationwas studied in the well characterized Zinnia system, in whichisolated mesophyll cells of Zinnia elegans transdifferentiateinto tracheary elements (TE) in a suspension culture in thepresence of both auxin and cytokinin. The rate of poly(ADP-ribose)synthesis was measured in nuclei isolated from cells that hadbeen induced to undergo transdifferentiation, and activationof such synthesis was observed before the appearance of TE duringculture. In cultures without auxin or cytokinin, poly-(ADP-ribose)synthesis appeared to proceed much more slowly. Treatment of cells with a potent inhibitor of poly-(ADP-ribose)polymerase, namely, 6(5H)-phenanthridinone (PT), resulted inthe blockage of TE formation and a decrease in the frequencyof cell division. PT was very effective in interfering withtransdifferentiation, in particular, when supplied between the24th hour and the 36th hour of culture. Repair-type DNA synthesis,which has been proposed to participate in transdifferentiation,was suppressed by the treatment with PT. These results suggestthat poIy(ADP-ribose) synthesis and subsequent repair-type DNAsynthesis might play a critical role in the transdifferentiationof Zinnia cells. ³Present address: Botanical Gardens, Faculty of Science, Universityof Tokyo, Hakusan, Bunkyo-ku, Tokyo, 112 Japan. ⁴Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku,Tokyo, 112 Japan.     

Regulation of phosphatase synthesis by orthophosphate in cultured tobacco cells

Phosphatase activity in cultured tobacco cells XD-6 increasedremarkably under phosphate-deficient culture conditions. Suchan increase did not occur in the activities of -amylase, ß-galactosidase,succinate dehydrogenase and catalase under the same cultureconditions. By replenishment with Pi, the increase in totalphosphatase activity was suppressed and the specific activitywas reduced to a low level. The suppression of increase in theactivity resulted form a repression of de novo synthesis ofthe enzyme. Added Pi also suppressed the release of phosphataseinto the culture medium. The effect of Pi was found to be greaterthan the effect on the increase in the intracellular activity. At least three phosphatases were extracted from XD-6 cells.One of the enzymes increased when the phosphatase synthesiswas increased by phosphate deficiency. ¹ Present address: Laboratory of Applied Microbiology, Facultyof Agriculture, Yamagata University, Tsuruoka, Yamagata 997,Japan. (Received May 18, 1977; )     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

Biosynthesis, processing, and secretion of {alpha}-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

N-Acetylglucosamine 1-phosphotransferase is a key enzyme requiredfor synthesis of the mannose 6-phosphate recognition markerthat is used by many newly made acid hydrolases for their transportto lysosomes. It has previously been found that lymphoid cellsfrom patients with I-cell disease and pseudo-Hurler polydystrophyhave nearly normal intracellular and intralysosomal activitiesof several lysosomal acid hydrolases, despite a deficiency ofN-acetylglucosamine 1-phosphotransferase. These results suggestthat lymphoid cells may provide an important system to investigatealternate mechanisms for targeting newly made acid hydrolasesto lysosomes. In the present study, the biosynthesis, processingand secretion of -L-fucosidase in I-cell and pseudoHurler lymphoidcells was used as a model system to study the existence of suchmechanisms. The level of intracellular -L-fucosidase proteinin exponentially growing I-cell or pseudo-Hurler lymphoid cultureswas statistically indistinguishable from the mean of 19 controlcultures. A 1.5 h [³⁵S]methionine pulse experiment showed that-L-fucosidase is initially sythesized by I-cell, pseudo-Hurlerand control cultures as an intracellular form (M_r = 58 000).Companion cultures chased with methionine from 2 to 21 h processedthe enzyme to an intracellular form (M_r = 60 000) and an extracellularform (M_r = 62 000). All enzyme forms were glycoproteins withpolypeptide chains of Mr 52 000. In control cells incubatedwith radioactive inorganic phosphate (³²Pi), <1% of the ³²Piincorporated into -L-fucosidase was associated with carbohydratechains and >99% with polypeptide chains. In I-cell diseaselymphoid cells, the ³²Pi incorporated into -L-fucosidase wasassociated solely with polypeptide chains. A qualitative analysisof phosphorylated residues identified phosphoserine in -L-fucosidasefrom control and I-cell lymphoid cells. Only -L-fucosidase fromcontrol cells contained mannose 6-phosphate. These results areconsistent with the proposal that I-cell lymphoid cells mayuse a mannose 6-phosphate-independent mechanism for routing-L-fucosidase. Additional metabolic labelling experiments demonstratedthe presence of ³²P-labelled -L-fucosidase in both cells andmedium of a control lymphoid culture, but only in cells of anI-cell lymphoid culture. In contrast, -L-fucosidase labelledwith [³⁵S]methionine was found in cells and medium of controland I-cell lymphoid cultures. Since phosphoserine was only foundto occur in intracellular, but not in extracellular -L-fucosidaseof the I-cell culture, we speculate that phosphoserine may beinvolved in intracellular retention of -L-fucosidase in I-celllymphoid cells. -L-fucosidase I-cell disease lymphoid cells phos-phorylation pseudo-Hurler polydystrophy     

Hormonal Induction of Vascular Differentiation in Cultured Zinnia Leaf Disks

Mesophyll cells differentiated into tracheary elements whenZinnia leaf disks were cultured in media containing sufficientauxin and cytokinin. Moderate increases in the levels of phytohormonesinduced the recruitment of adjacent cells into the preexistingvasculature, resulting in the "expansion" of leaf veins. Higherhormonal concentrations induced unpatterned differentiationinto tracheary elements throughout the mesophyll and epidermis.This novel system should facilitate the study of organized vasculardifferentiation. (Received July 30, 1988; Accepted October 31, 1988)     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Physiological Responses of Phytoflagellates to Dissolved Organic Substrate Additions. 1. Dominant Role of Heterotrophic Nutrition in Poterioochromonas malhamensis (Chrysophyceae)

Algal heterotrophy is a potentially important considerationin the flow of carbon through aquatic food webs. The physiologicalresponses to organic compound additions under various lightintensities were examined with Poterioochromonas malhamensis,a freshwater chrysophyte with an exceptionally high heterotrophiccapability. P. malhamensis demonstrated a much greater potentialfor heterotrophic growth than for photoautotrophic growth. Whenorganic substrates (glucose, glycerol, or ethanol) were addedto the culture medium, the growth rate of P. malhamensis significantlyincreased while the chlorophyll content cell ^–1 decreased,even at light intensities saturating for photoautotrophic growth.After an initial decline in chlorophyll production caused byorganic substrate uptake, chlorophyll cell¹ increased and theuptake rate of organic substrates decreased, despite the persistenceof a relatively high substrate concentration in the medium.The results are consistent with the production of substance(s)by P. malhamensis that conditioned the culture medium, leadingto a relief of the inhibitory effect of organic substrates onchlorophyll production. (Received October 29, 1990; Accepted May 8, 1991)     

A voltage-gated K(+) current in renal inner medullary collecting duct cells

We studied the K⁺-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above –20 mV induced a time-dependent outward K⁺ current (I_vto) similar to a delayed rectifier. I_vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K⁺/Na⁺ selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba²⁺ and was not Ca²⁺ dependent. The delayed rectifying characteristics of I_vto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv -subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv -subunit antibodies for Kv1.1 and Kv1.3 showed labeling of 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na⁺-K⁺-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells. kidney; Kv1.3; potassium channel; potassium transport; whole cell clamp; immunocytochemistry; confocal microscopy     

Establishment of a Sensitized Immunoblotting Method for Measuring Plant Tubulin Content

A sensitized immunoblotting method was established for measuringsmall amounts of plant tubulin. The method involves electrophoretictransfer of protein including tubulin from SDS-polyacrylamidegels onto nitrocellulose paper, successive incubation of thenitrocellulose paper with a mouse monoclonal antibody to - orß-tubulin of chicken brain, an antibody to mouse IgGas the second antibody and the radioactive iodinated proteinA, and determination of the radioactivities of the bands onthe nitrocellulose paper thus probed. The radioactivities werelinearly proportional to the amounts of - or ß-tubulinfrom dark-grown Vigna mungo seedlings within a range of 4 to56 ng or of 4 to 32 ng, respectively. This method was used to estimate the tubulin contents of severalplant species using Vigna tubulin as a standard. -Tubulin contentsthus estimated were 25, 9, 19 and 11 µg-equivalents ofVigna tubulin per mg protein for Vigna seedlings, Daucus suspensioncells, Catharanthus suspension cells and Mougeolia cells, respectively.ß-Tubulin contents of Vigna, Daucus, Catharanthusand Mougeotia cells were 29, 10, 13 and 5 µg-equivalentsof Vigna tubulin per mg protein, respectively. (Received August 6, 1985; Accepted December 5, 1985)     

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated theeffects of epidermal growth factor (EGF) on activeNa⁺ absorption by alveolarepithelium. Rat alveolar epithelial cells (AEC) were isolated andcultivated in serum-free medium on tissue culture-treated polycarbonatefilters. mRNA for rat epithelial Na⁺ channel (rENaC) -, -,and -subunits and Na⁺ pump₁- and₁-subunits were detected inday 4 monolayers by Northern analysisand were unchanged in abundance in day5 monolayers in the absence of EGF. Monolayerscultivated in the presence of EGF (20 ng/ml) for 24 h fromday 4 to day5 showed an increase in both₁ and₁Na⁺ pump subunit mRNA but noincrease in rENaC subunit mRNA. EGF-treated monolayers showed parallelincreases in Na⁺ pump₁- and₁-subunit protein by immunoblotrelative to untreated monolayers. Fixed AEC monolayers demonstratedpredominantly membrane-associated immunofluorescent labeling withanti-Na⁺ pump₁- and₁-subunit antibodies, withincreased intensity of cell labeling for both subunits seen at 24 hfollowing exposure to EGF. These changes inNa⁺ pump mRNA and protein precededa delayed (>12 h) increase in short-current circuit (measure ofactive transepithelial Na⁺transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases activeNa⁺ resorption across AECmonolayers primarily via direct effects onNa⁺ pump subunit mRNA expressionand protein synthesis, leading to increased numbers of functionalNa⁺ pumps in the basolateralmembranes.

     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Uptake and Accumulation of {alpha}-Naphthalene Acetic Acid by Cell Suspensions of Galium mollugo L.

Fidgeon, C. and Wilson, G. 1988. Uptake and accumulation ofa-naphthalene acetic acid by cell suspensions of Galium mollugoL.—J. exp. Bot. 39: 241-249. Galium mollugo cell suspensions require -NAA for continued growthand cell division. The kinetics of -NAA uptake from the medium(B₅) by Galium cells was assessed using 1-¹⁴C -NAA in a standardratio of cells to medium (0.25 g: 2.5 cm³). It was found thatthe uptake of -NAA was rapid, over 90% being taken up within4 h. Cells which had accumulated -NAA for 4 h or more did notrelease it back into the medium. It was found that Galium cellsaccumulated -NAA against a significant concentration gradient;suggesting the participation of an active component in the uptakemechanism. The effect of free-space and surface adsorption onthe uptake of -NAA was determined by means of a repeated washtechnique. These two factors were found to be of importanceonly during the first hour of uptake. Neither dead cells norplasmolysed cells absorbed -NAA. It is clear that, in the normal growth cycle, Galium cells cantake up the available -NAA within 3 or 4 h of inoculation andthat this can stimulate a cell division response of 3-4 generationsover the subsequent 14 d. Key words: Galium, cell suspension, -naphthalene acetic acid