Impairment of Tonoplast H-ATPase as an Initial Physiological Response of Cells to Chilling in Mung Bean (Vigna radiata [L.] Wilczek)

Biochemical alterations of cellular membranes in chilling-sensitive mung bean (Vigna radiata [L.] Wilczek) hypocotyls were investigated with reference to chilling injury. Reversible decreases in activities of tonoplast H⁺-ATPase and in vivo respiration became manifest within 24 hours of chilling when tissues suffered no permanent injury as assessed by electrolyte leakage and regrowth capacity. These changes were found to be the earliest cellular responses to chilling. A density-shift on a sucrose density gradient was observed in Golgi membranes early in the chilling treatment, suggesting that Golgi function and/or membrane biogenesis via the Golgi may have been altered upon chilling. After chilling more than 2 days, irreversible changes were generally produced in cellular membranes including the plasma membrane, endoplasmic reticulum, and mitochondria. Respiratory functions remained intact in mitochondria isolated from tissues prechilled for 24 hours, but were impaired after prechilling for 3 days. Given the important role of the tonoplast H⁺-ATPase in the active transport of ions and metabolites, the early decline in the tonoplast H⁺-ATPase activity may give rise to an alteration of the cytoplasmic environment and, consequently, trigger a series of degenerative reactions in the cells.     

Differential Inhibition of Tonoplast H-ATPase Activities by Fluorescamine and Its Derivatives

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H⁺-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H⁺ leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.     

Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Binding of 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole to an Essential Cysteine Residue(s) in the Tonoplast H-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls

Vacuolar-type H⁺-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [¹⁴C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of ¹⁴C incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [¹⁴C]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H⁺-ATPase and that the modification closely correlates with the loss of ATPase activity.     

Characterization and Localization of a Phenoloxidase in Mung Bean Hypocotyl Cell Walls

The occurrence of proteins able to oxidize polyphenols even in the absence of H2O2 was recently reported in mung bean (Vigna radiata L.) hypocotyl cell wall extracts (R. Goldberg, A. Chabanet, A.M. Catesson [1993] In K.G. Welinder, S.K. Rasmussen, C. Penel, H. Greppin, eds, Plant Peroxidases: Biochemistry and Physiology, pp. 296-300). Therefore, the possible presence of a laccase in the extracts was investigated using immunocytological and biochemical approaches. An enzyme catalyzing phenol oxidation in the presence of molecular O2 was extracted and purified from the cell walls. This 38-kD cationic protein, like o-diphenoloxidases, was unable to oxidize p-diphenols or p-diamines. However, it crossreacted with an anti-laccase antiserum and, like laccases, its activity was inhibited by N-cetyl-N,N,N-trimethylammonium bromide but not by ferulic acid salts. Immunolabeling data showed that the 38-kD oxidase was absent from all cellulosic cell walls. It was localized only in lignifying and lignified cell walls. This restricted localization suggests that this laccase-like phenoloxidase could participate in the lignification process but not in the primary wall stiffening, which develops in the epidermal and cortical tissues along the mung bean hypocotyl.     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris

Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia-lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors.     

绿豆芽提取物对SDS致红细胞膜和DNA损伤的保护的研究

为探讨绿豆芽提取物(MBSE)对十二烷基硫酸钠SDS致红细胞膜和DNA损伤的保护研究,分别采用红细胞(RBC)溶血试验和彗星试验检测细胞膜和DNA的损伤程度。实验分为三组:阳性对照组;MBSE自溶对照组;MBSE+SDS组。通过测定血红细胞溶血率和致损细胞拖尾率及尾长分别表征细胞膜及DNA的损伤程度。结果显示与阳性对照相比,各剂量MBSE具有抑制SDS致细胞膜损伤的功能,对RBC细胞膜具有较好的保护作用,且呈量效关系;MBSE+SDS各剂量组DNA损伤明显减弱,拖尾率下降,尾长减小。提示MBSE对SDS致红细胞膜和DNA损伤具有保护作用。     

Involvement of Cell Wall Characteristics in Growth Processes along the Mung Bean Hypocotyl

To detect the cell wall characteristics involved in the regulationof growth responses, the composition of wall polysaccharidesand the kinetic behaviour of wall-bound glucanases on mung beanhypocotyls were investigated. In the parts of the hypocotyllocated below the "elongation zone", relationships have beenshown between the characteristics of pectins and the first phaseof auxin- or acid-induced growth responses. Only small pecticmolecules with a low calcium content, are compatible with acid-inducedwall loosening. The breakage of acid-labile bonds between uronide chains andhemicelluloses is believed to be responsible for the early burstof growth. In young tissues, acid-induced modifications in thecell wall structure might then produce changes in the kineticbehaviour of cell wall polysaccharidases that depend on thisstructure for support. In old tissues, the number of glycosyllinkages is not sufficient to permit glucanase activities. Enzymaticrupture of covalent bonds may indeed regulate the supply ofwall material requisite for sustained growth. (Received March 13, 1982; Accepted June 30, 1982)     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

Changes in Peroxidase Activity in Bean Suspension Cultures after B. cinerea and Elicitor Treatment

Treatment of cell suspension cultures of bean ( Phaseolus vulgaris ) c.v. 'Gold Saxa') with Botrytis cinerea resulted in the increase in extra- and intracellular peroxidase activity. A similar effect was obtained by treatment of cell suspension cultures with an elicitor active extract from fungal mycelium.
It was shown by means of electrofocusing that the infection- or elicitor-related increase in total intracellular peroxidase activity was associated with the increase in the activity of a specific isozyme with an isoelectric point of 4.8. This anionic peroxidase did not differ in substrate specificity to guaiacol, syringaldazine and chlorogenic acid.     

Partial Synchronization of Cell Division in Suspension Cultures of Haplopappus gracilis

Four different chemicals were tested in their ability to synchronize cell division in asynchronous cell cultures of Haplopappus gracilis. Twentyfour-hour treatments with 5-amino uracil resulted in a peak in the mitotic index about 14–16 hours after the end of the treatment. The increase in the frequency of mitoses was about three times that of the control. Hydroxyurea, at a concentration of 3 mM, gave after a treatment period of 12–24 hours an increase in the frequency of mitoses which appeared about 10 hours after the treatment. The mitotic index was about 35 per cent, which is 4 times that of the control. 5-Fluorodeoxyuridine (FUdR) at a concentration of 2 × 10^?7M gave a mitotic burst about 16 hours after treatment. At that time about 15 per cent of the cells were dividing which was about twice that of the control. The block was reversed with 4 × 10^?5M thymidine. Thymidine at a high concentration caused a reduction in the frequency of mitoses during the treatment. After 15 to 16 hours in a thymidine free medium a mitotic peak appeared with a doubling of the frequency of mitoses in treated cells. Cytological studies showed that parlicularly hydroxyurea but also 5-aminouracil and 5-fluorodeoxyuridine produced gaps and fragments at the concentrations which gave cell synchronization.     

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinenesis

Taxol production of Taxus chinensis（Pilger） Rehd. var.mairei (Lemeeet Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f.vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡(英)

在真菌 (Fusariumoxysporumf.vasinfectum (Atkinson)SnyderetHansen)寡聚糖诱导悬浮培养南方红豆杉(Taxuschinensis (Pilger)Rehd .var.mairei (LemeeetL啨ｖｌ.)ChengetL .K .Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡 ,次生代谢增强。电镜观察到细胞核质和原生质出现凝集现象 ,液泡内出现大量的高电子致密体。核DNA经琼脂糖凝胶电泳 ,呈 2 0 0bp的整数倍的梯状条带 (ladders) ;而对照组细胞核DNA完整 ,呈大片段 ,细胞完整 ,细胞器发达 ,但紫杉醇合成速率很低。加入寡聚糖后 ,细胞防御系统开启 ,细胞生长停止 ,次生代谢物酚类物质大量积累且次生壁加厚 ,多酚氧化酶活性迅速提高 ,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在 1h后急速提高 ,目的产物紫杉醇在诱导后 72h达到峰值 ,比对照组提高了 6倍 ,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性。     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡

在真菌(Fusarium oxysporum f.vasinfectum (Atkinson) Snyder et Hansen)寡聚糖诱导悬浮培养南方红豆杉(Taxus chinensis (Pilger) Rehd.var.mairei (Lemee et Lévl.) Cheng et L.K.Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡,次生代谢增强.电镜观察到细胞核质和原生质出现凝集现象,液泡内出现大量的高电子致密体.核DNA经琼脂糖凝胶电泳,呈200 bp的整数倍的梯状条带(ladders);而对照组细胞核DNA完整,呈大片段,细胞完整,细胞器发达,但紫杉醇合成速率很低.加入寡聚糖后,细胞防御系统开启,细胞生长停止,次生代谢物酚类物质大量积累且次生壁加厚,多酚氧化酶活性迅速提高,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在1 h后急速提高,目的产物紫杉醇在诱导后72 h达到峰值,比对照组提高了6倍,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性.     

Salt Tolerance in Cell Suspension Cultures of the Halophyte Kosteletzkya virginica

Tolerance to NaCl was studied in cell suspension cultures ofKosteletzkya virginica (L.) Presl. (Malvaceae), a dicotyledonoushalophyte that grows in tidal marshes of the eastern UnitedStates. Growth of salinized cultures was significantly inhibitedat high (255 mol m^–3 NaCl), but not at lower externalsalinities. Adjustment of cell suspensions to Nacl was rapid,with the duration of the normal growth cycle unaffected by salinity.Maximum biomass was attained when cultures were exposed to NaClduring early log growth. Patterns of inorganic ion accumulationreflected the utilization of both Na⁺ and K⁺ as osmotica, withNa⁺ content substantially increasing when cells were grown atan external salinity sufficient to reduce growth. K⁺ uptakeselectivity was high and Na⁺/K⁺ ratios were low in salt-treatedcultures even though K⁺ content was somewhat lower comparedto unsalinized cultures. Free proline and microsomal lipid contentincreased in salt-treated cell cultures. Key words: Kosteletzkya virginica, halophyte, salt tolerance, cell suspension culture     

In Vitro and In Situ Properties of Cell Wall Pectinmethylesterases From Mung Bean Hypocotyls

Pectinmethylesterases (EC 3.1.1.11 [EC] ) have been solubilized fromyoung and mature tissues of mung bean hypocotyls. Whatever theplastic potential of the tissues, most of the pectinmethylesteraseactivity was located in the cell walls. Several active fractionswere obtained after chromatography on CM Sépharose. Equilibriumsedimentation in an analytical ultracentrifuge indicated theMW of the isolated isoforms to be close to 75 000 whereas SDS-PAGEelectrophoresis gave a MW around 32 000, suggesting the possibilityof dimeric structures. Mung bean pectinmethylesterase (PME)showed cross reactivity with soybean antiserum. Experiments carried out with p-nitrophenylacetate and Citruspectin revealed that PME and esterase activities might correspondto different isoforms. It was also noted that the stimulationinduced by cations was stronger when the enzymes were boundto the cell walls. The high ionic sensitivity suggested that,in situ, the ionic environment regulates pectinmethylesteraseactivity principally by modifying the pectin molecules, whichenhances the affinity of the enzymes for their substrate. Thesedata indicate the importance of the calcium content of the cellwalls and might explain the decrease in methylated pectins alongthe mung bean hypocotyl and, in turn, the loss of plasticity. Key words: Cell wall, hypocotyl, pectinmethylesterase, Vigna radiata     

Selection of Copper and Zinc Resistant Nicotiana plumbaginifolia Cell Suspension Cultures

Copper and zinc resistant cells of Nicotiana plumbaginifoliawere selected using unmutagenized cell suspensions in mediumcontaining normally lethal concentrations of CuSO₄ or ZnSO₄.Both resistances were retained for thirty cell doublings withoutselection pressure. The Cu resistant cells were 10-times andthe Zn resistant cells were 6-times as resistant as the wildtype cells. The Zn resistant cells were also somewhat resistantto AlCl₃ in comparison with the wild type cells, while the Curesistant cells were also somewhat resistant to ZnSO₄ and AlCl₃.The uptake of Cu by the Cu resistant cells and Zn by the Znresistant cells was higher than that of the wild type cells. (Received April 21, 1986; Accepted June 30, 1986)