Protection of olfactory responses from inhibition by ethyl bromoacetate, diethylamine, and other chemically active odorants by certain esters and other compounds

Certain vaporous chemicals (chemically active odorants) arecapable of both stimulating olfactory responses and reactingwith receptors, ion channels, or receptor/ionophore macromoleculesto inhibit olfactory responses. We have studied the physiologicaleffects of several chemically active odorants using electrophysiologicaltechniques to record electroolfactogram (EOG) responses fromthe frog's olfactory mucosa. So far, the most studied agentsare ethyl bromoacetate (EBA), an alkylating agent, and diethylamine(DEA), a compound which is one of the strongest neutral organicbases. Certain odorants, or ‘protectants’, whenpresent before, during, and after exposure of the olfactorymucosa to either EBA or DEA have the property of maintainingolfactory responses which would otherwise be inhibited by exposureto the chemically active odorant alone. Protection from inhibitionby EBA is conferred by the presence of isoamyl acetate and afew closely-related esters, while protection from inhibitionby DEA is produced by the presence of p-dichlorobenzene. Protectionfrom inhibition by DEA is also achieved by lowering the pH ofthe olfactory mucosa through the simultaneous delivery of CO₂which produces carbonic acid. The mechanism of protection byesters and p-dichlorobenzene is unknown, but it seems likelythat these odorants somehow interfere with the access of thechemically active odorant to a site where it would normallyreact. ¹Present address: PSC Box 511, Peterson AFB, Colorado Springs,Colorado 80914, USA. ²Permanent address: Department of Chemistry, New Mexico Instituteof Mining and Technology, Socorro, New Mexico 87801, USA.     

Myr-Ric-8A enhances G(alpha15)-mediated Ca2+ response of vertebrate olfactory receptors

The determination of ligand specificities of odorant receptorswill contribute to the understanding of how odorants are discriminatedby the olfactory system. To date, the ways in which some olfactoryreceptors (ORs) pair with their cognate ligands has been studiedusing a Ca²⁺ imaging technique. This approach has been usedto investigate orphan G protein–coupled receptors expressedin heterologous cells; however, most attempts to functionallyexpress ORs on the cell surface of heterologous cells have failed.Recently, receptor-transporting protein 1 and Ric-8B have beenidentified as proteins involved in targeting receptors to thecell membrane and amplifying receptor signals, and thus, theyare able to facilitate cellular responses via ORs in a heterologouscell system. Here, we describe a technique in which we employeda myristoylation sequence–conjugated mutant of Ric-8A(Myr-Ric-8A) as a signal amplifier and show Myr-Ric-8A greatlyenhances G₁₅-mediated Ca²⁺ responses of ORs in HEK293 cells.Coexpression of Myr-Ric-8A enabled us to deorphanize a mouseOR and to determine its molecular receptive range. Our resultssuggest that Myr-Ric-8A should be helpful in functional characterizationof ORs in heterologous cells using Ca²⁺ imaging.     

Role of divalent cations in EOG generation

Underwater EOGs in response to 10^–4 M isoamyl acetatewere recorded in vivo in frogs. The olfactory mucosa was superfusedby sodium-free sucrose solutions, and the effect of the additionof millimolar concentrations of sodium, calcium, cobalt, bariumor cadmium was studied. All voltage responses were standardizedaccording to the solution resistivities. Barium and calciumefficiently restored the response; sodium and cobalt were lessefficient. Cobalt did not alter the restoring effect of calcium.Cadmium induced an irreversible blockage irrespective of thepresence of calcium. These results are discussed in terms ofpossible ionic mechanisms mediating olfactory responses.     

Odour discrimination by frog olfactory receptors: a second study

Single unit activity of olfactory neuroreceptors was recordedin frogs. Stimulations with 20 pure chemicals delivered at knownconcentrations elicited excitatory and/or inhibitory responsesin 60 of the 76 recorded units. The responses exhibited varioustime patterns, partly depending on stimulus intensity. Longlasting after-effects were observed. Out of a total of 1520odour trials, 317 excited and 33 inhibited the cells, leadingto a receptor overall responsiveness of 23%. Various degreesof individual selectivity were encountered in the receptors;the greatest number responded to seven of the 20 odorants. Amarked tendency to stimulate the same receptors was observedfor several odorants. Three groups could be evidenced: benzene,anisole, dichlorobenzene and bromobenzene; camphor and cineole;tert-butyl alcohol, cyclohexanone and cyclohexanol. Fatty acidstended to be grouped. Sulphurous compounds elicited few responses,except tiophenol. Most of the neuroreceptors responded to odorantsbelonging to more than one odour group.     

Olfactory studies using ethyl bromoacetate and other chemically active odorants

Eleven chemically active odorants were tested to determine theireffectiveness and specificity in inhibiting electroolfactogram(EOG) responses in the frog olfactory mucosa. These inhibitoryagents probably act by several different mechanisms, but theyall produced a comparable degree of inhibition when approximatelythe same amount of inhibitor had been applied. One agent, ethylbromoacetate, produced a specific pattern of inhibition in whichresponses to all odorants tested were inhibited except responsesto certain amines. A related agent, ethyl chloroacetate, produceda similar, but less well-defined specific effect. Non-specificinhibitory effects were produced by seven of the agents tested.Two agents produced no inhibition, presumably because theirlow vapor pressure prevented the application of sufficient reagentin the vapor phase. The majority of the effective inhibitorsare alkylating agents or substrates for nucleophilic additionwhich react with sulfhydryl or amino groups of proteins. Bycontrast, the inhibitory effect of diethylamine is probablythe result of its basicity. The basicity would enable neutralizationreactions with acidic groups of proteins or other substancesin the microenvironment of the receptors. Several lines of evidencelead to the conclusion that the inhibitory action of the chemicallyactive odorants is principally the result of disruption of molecularolfactory receptors in the membranes of olfactory neurons, andthat sulfhydryl, amino, and carboxyl groups are of importanceto the function of olfactory receptors, ion channels, or receptor/ionophoremacromolecules. ¹Present address: PSC Box 511, Peterson AFB, Colorado Springs,Colorado 80914, USA. ²Permanent address: Department of Chemistry, New Mexico Instituteof Mining and Technology, Socorro, New Mexico 87801, USA.     

Difference in Behavior Between Responses to Forskolin and General Odorants in Turtle Vomeronasal Organ

To elucidate the signal transduction mechanisms in the turtlevomeronasal receptor neurons, the effects of forskolin, changesin mucosal Ca²⁺ concentrations and ruthenium red on the responsesof the accessory olfactory bulb to general odorants were examined.Forskolin elicited a large response, suggesting that there arecAMP-gated channels in the vomeronasal neurons. On the otherhand, the dependence of the responses to general odorants onCa²⁺ concentrations was different from that of the responseto forskolin. A large response to an odorant (n-amyl acetate)appeared after the cAMP-mediated pathway was fully desensitizedby application of 50 µM forskolin. These results suggestthat the cAMP-mediated pathway does not contribute significantlyto generation of the response to general odorants. A concentrationof 50 µM ruthenium red significantly reduced the responsesto n-amyl acetate alone and after 50 µM forskolin desensitization,suggesting that the inositol triphosphate-mediated pathway contributespartly to generation of the responses to general odorants inthe vomeronasal neurons. Chem Senses 21: 763–771, 1996.     

Behavioral and Electrophysiological Studies on Chemoreception in Aplysia

Using the typical mouth opening response (MOR) as the indexof chemore-ception, several amino acid constituents of naturalfood for Aplysia were effective in eliciting MOR in concentrationsas low is 10^–6 10^–7 M (glutamic acid) and 10^–510^–6 M (aspartic acid). It was suggested that these substancesmay serve as food attractants for this animal. Combined resultsof behavioral and electrophysiological experiments indicatedthat the most sensitive food receptors of Aplysia are predominantlylocated in the anterior tentacular groove area of the oral veil.These receptors appeared to strongly project to the caudal darkcell clusters of the cerebral ganglion. Extracellular recordingof single afferent units following oral veil stimulation withvarious chemicals showed that the chemoreceptors in the anteriortentacular groove area were 100 to 1000 times more sensitiveto food substances than other stimuli tested. It was suggestedthat these receptors are relatively "specific" to food attractants.There was a correlation between behavioral and electrophysiologicalthreshold data for the individual compounds. The electrophysiologicalthresholds however were 5–15 times higher than the behavioralthresholds.     

Egg production and hatching success in the peri-Antarctic copepod Calanus simillimus

In situ rates of egg production and hatching success are reportedfor Calanus simillimus, one of the most abundant calanoid speciesin peri-Antarctic regions, during the Italian ‘Italics’cruise in the Straits of Magellan in March-April 1995. Low fecundity(8.6 eggs female^–1 day^–1) and fecal pellet production(0.8 fecal pellets female^–1 day^–1) in this periodindicate that the species was feeding very poorly. Sixty-sixof the 126 females sampled did not produce eggs and 80 of thesedid not produce any fecal pellets during the 24 h period ofincubation. Striking abnormal naupliar and embryonic developmentwas recorded in 81.8% of the eggs spawned. Aberrant eggs didnot undergo normal cleavage, and failed to develop to hatching.Deformed nauplii were asymmetrical and presented strong anatomicalanomalies in the total body length and number of swimming appendages.These results are discussed in the light of recent findingson the causes of low hatching success in copepods.     

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.     

Response of Seed Germination in Three Genera of Compositae to White Light of Varying Photon Flux Density and Photoperiod

Various photon doses (net number of photons per unit area perday), provided by varying both photon flux density and photoperiod,were applied to imbibing seeds of seven lots of four speciesof Compositae in various germination test regimes. In all fourspecies germination was dependent upon photon dose, the productof photon flux density and daily duration of exposure. The responsewas quantified by linear relations between the probit of percentagegermination and the logarithm of photon dose. In general, photonflux density and photoperiod only influenced the stimulationof germination by the low energy reaction indirectly (as factorsof daily photon dose), whereas there was a tendency for photoperiodto have a direct influence on the inhibition of germinationby the high irradiance reaction. Reducing the germination testtemperature from 25?C to 20?C and 15?C not only increased thedark germination of L. sativa L., but also broadened the photondose range at which full germination occurred by reducing theminimum value necessary for the germination of the most dormantseeds, and increasing the maximum value which failed to inhibitthe germination of any seeds. Differences between L. sativaand L. serriola L. in the response of germination to white lightwere only quantitative, rather than qualitative. The singlemost promotory dose for all four species was 3 ? 10^–3mol m^–2 d^–1, although the inhibitory action of dosesup to 10– mol m– d– was generally only slight. Key words: Light, seed germination, seed dormancy, Compositae     

Laboratory and field observations on the scuticociliate Histiobalantium from the pelagic zone of Lake Constance, FRG

Histiobalantium sp. was found regularly in the pelagic zoneof Lake Constance, FRG, over five annual cycles. Maxima of upto 6400 cells l^–1 were recorded in late summer, with similarnumbers in the 0–8 and 8–20 m depth intervals. Onan annual average, the population accounted for 10–17%of the total biomass of planktonic ciliates. In the laboratory,Histiobalantium grew well on a diet of the cryptophyte Rhodomonassp. Maximum growth rates obtained in batch cultures were 0.21and 0.33 day^–11 at 9 and 18°C, respectively. In situexperiments using diffusion chambers yielded positive growthrates in autumn and winter. The highest values recorded at theambient temperatures 5, 14 and 17°C were 0.17, 0.32 and0.40 day^–1, respectively. Comparing these results withthe different seasonal distributions and higher measured growthrates of other ciliates from Lake Constance, we conclude thatHistiobalantium is a superior competitor at relatively low algalfood concentrations. ²Present address: Fisheries & Oceans Canada, 4160 MarineDrive, West Vancouver, BC, V7V 1N6, Canada     

Mechanoreception in marine copepods: electrophysiological studies on the first antennae

Neural activity was recorded extracellularly at the base ofthe first antenna in 15 marine copepods. Controlled mechanicalstimuli were delivered with a vibrator driven by a waveformgenerator. Many species exhibited responses characterized bya large number of small spikes, while others were characterizedby the presence of a small number of large units. Two bay species,Labidocera madurae and Acartia fossae, exhibited large unitsthat could be easily distinguished from the background activityof smaller units. In these species, the antennal receptors firedshort latency (>5 ms) trains of one to several impulses inresponse to a brief mechanical stimulus and sustained trainsto a prolonged sinusoidal stimulus. They were extremely sensitiveto small displacements and sensitivity increased with stimulusfrequency. The receptors responded to stimuli between 40 and1000 Hz and receptors required displacement velocities of 20µm s^–1 or more to fire. Displacements as small as10 nm were capable of triggering spikes. With an increase inthe amplitude of the displacement, a decrease in the latencyand an increase in the number of units recruited and/or firingfrequency was recorded. Phase-locking to oscillatory stimuliwas observed over a frequency range of 80–500 Hz. Neuralactivity increased in response to bending of individual setae.Setae appear innervated and structurally constrained to movementsin specific directions. These experiments suggest that (i) somecopepod setal receptors may be more nearly velocity detectorsthan purely displacement sensors, (ii) they may be capable ofsensing closely spaced stimuli, (iii) the patterns of responsemay code for intensity and duration of the stimulus, and (iv)receptors may be capable of supplying directional information.     

Evidence for Ca(2+)-permeable AMPA receptors in the olfactory bulb

-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), a subtype of glutamate receptor, contribute to olfactory processing in the olfactory bulb (OB). These ion channels consist of various combinations of the subunits GluR1–GluR4, which bestow certain properties. For example, AMPARs that lack GluR2 are highly permeable to Ca²⁺ and generate inwardly rectifying currents. Because increased intracellular Ca²⁺ could trigger a host of Ca²⁺-dependent odor-encoding processes, we used whole cell recording as well as histological and immunocytochemical (ICC) techniques to investigate whether AMPARs on rat OB neurons flux Ca²⁺. Application of 1-naphthylacetyl spermine (NAS), a selective antagonist of Ca²⁺-permeable AMPARs (CP-AMPARs), inhibited AMPAR-mediated currents in subsets of interneurons and principal cells in cultures and slices. The addition of spermine to the electrode yielded inwardly rectifying current-voltage plots in some cells. In OB slices, olfactory nerve stimulation elicited excitatory responses in juxtaglomerular and mitral cells. Bath application of NAS with D,L-2-amino-5-phosphonovaleric acid (AP5) to isolate AMPARs suppressed the amplitudes of these synaptic responses compared with responses obtained using AP5 alone. Co²⁺ staining, which involves the kainate-stimulated influx of Co²⁺ through CP-AMPARs, produced diverse patterns of labeling in cultures and slices as did ICC techniques used with a GluR2-selective antibody. These results suggest that subsets of OB neurons express CP-AMPARs, including functional CP-AMPARs at synapses. Ca²⁺ entry into cells via these receptors could influence odor encoding by modulating K⁺ channels, N-methyl-D-aspartate receptors, and Ca²⁺-binding proteins, or it could facilitate synaptic vesicle fusion. GluR2; polyamines; cobalt; glutamate receptor; olfaction     

New studies on odour discrimination in the frog's olfactory receptor cells. I. Experimental results

New experiments were performed within a systematic investigationof the discriminative properties of olfactory receptor cellsin the frog. Extracellular spike responses of receptor unitswere recorded during stimulation with 39 different odorants.Several general properties of the olfactory receptors, alreadydescribed, were confirmed. The overall selectivity was foundto be lower than in previous studies. Enantiomeric forms ofcitronellol and carvone were discriminated by several receptorcells, mainly in quantitative terms. In a series of eight cycloketones,each compound was the best stimulus for at least one receptorcell and some receptor cells had several ‘best stimuli’.Five odorants were delivered at two concentrations. All thereceptors responding to the lowest concentration also respondedto the highest one.     

Effect of antibody to anisole binding protein on odorant perturbation of Na+-K+ ATPase activity

Odorant perturbation of Na⁺-K⁺ ATPase activity from cow olfactorytissue was strongly affected by ng quantities of antibodiesto anisole binding protein from dog olfactory mucosa. Antibodyprotein (80 ng per ml reaction mixture) prevented odorant perturbationof Na⁺-K⁺ ATPase activity. Antibody effect on odorant perturbationshowed concentration dependence and was active against a numberof different odorous chemicals. Electrophysiological studies(Goldberg et al, 1979) showed that mouse EOG responses due toodorants were inhibited 50% by previous exposure to 0.8 ng antibodies.Thus electrophysiological and biochemical responses showed sensitivityto the antibodies from the anisole binding protein from dogolfactory tissue. It is proposed that NA⁺-K⁺ ATPase may participatein the initiation of nerve signals caused by odorant-enzymecomplex interactions.     

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

We previously showed that plasma membrane Ca²⁺-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca²⁺ ([Ca²⁺]_i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1^+/–, Pmca4^+/–, Pmca4^–/–, and Pmca1^+/–Pmca4^–/– mice. There were no differences in basal [Ca²⁺]_i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca²⁺]_i (130–180%) in smooth muscle from Pmca1^+/– and Pmca1^+/–Pmca4^–/– bladders than those in WT or Pmca4^–/–. The responses to carbachol (CCh: 10 µM) were also greater in Pmca1^+/– (120–150%) than in WT bladders. In contrast, the responses in Pmca4^–/– and Pmca1^+/–Pmca4^–/– bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca²⁺]_i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4^–/– (130–190%) and Pmca1^+/–Pmca4^–/– (120–250%) bladders, but not in Pmca1^+/– bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca²⁺ clearance, while PMCA4 is essential for the [Ca²⁺]_i increase and contractile response to the CCh receptor-mediated signal transduction pathway. PMCA; bladder smooth muscle; gene-altered mice     

Differences in the Interacting Effects of Light and Temperature on Growth of Four Species in the Vegetative Phase

A comparative study, employing the concepts of growth analysis,has been made of the varying responses in the early vegetativephase of Gossypium hirsutum, Helianthus annuus, Phaseolus vulgaris,and Zea mays to combinations of light intensity (1.08, 2.16,3.24, 4.32, and 5.4 x 10⁴ lx—photoperiod 14 h) and constantdiurnal air temperatures (10, 15, 20, 25, 30, and 35 °C).Depending on the combination of treatments, the temperatureof the internal tissues departed from air temperature by 6.9to 1.4 °C: so only the internal temperatures are cited here. For each species there are complex interactions between theeffects of light and temperature on the net assimilation rate,the leaf-area ratio, and the relative growth-rates of plantweight and leaf area. The magnitude of the changes induced bythe two factors vary both with the growth component and thespecies. The temperature responses are maximal up to 20–5°C while at the highest temperatures they may be negative.The temperature coefficients for leaf-area ratio are consistentlyless than those of the other three components: here betweenspecies the coefficients over 10–20 °C vary by a factorof 9.6, 5.4, and 5.1 for the rates of gain in plant weight andleaf area and the net assimilation rate, while the orderingwithin each growth component is species dependent. Under conditions of optimal temperature the relative growth-rateand net assimilation rate progressively increase, accordingto the species, up to either 4.32 or 5.4x 10⁴ lx. The leaf-arearatio is always largest at the lowest intensity. The level oflight at which the rate of gain in leaf area reaches a maximumranges from 2.16x 10⁴ lx for Phaseolus to between 4.32 and 5.40x10⁴ lx for Gossypium. The highest relative growth-rate and net assimilation rate ofHelianthus exceed those of Zea substantially. Indeed the maximalassimilation rate for Helianthus of 2.10 g dm^–2 week^–1is the highest ever recorded under field or controlled conditions.Possible reasons for this reversal of the photosynthetic potentialsof the two species observed by previous workers are discussed.     

Response to Waterlogging and Differing Sensitivity to Divalent Iron and Manganese in Clones of Dactylis glomerata L. derived from Well-drained and Poorly-drained Soils

Clonally propagated plants of Dactylis glomerata derived froma well-drained, heavily grazed cliff habitat (clone L) and froman undergrazed poorly-drained soil (clone A) were tested forwaterlogging tolerance in soil-culture. Water-logging did notaffect the two clones differentially, a result, which contrastedstrongly with that of a previous experiment in which simulatedgrazing (clipping to 20 cm) unexpectedly caused clone A to beless tolerant of waterlogging than clone L. Maximum leaf andleaf sheath length was reduced more by water-logging in cloneL than in clone A (P < 0.05). In solution-culture when providedwith factorial combinations of 0.5, 5 and 50 mg dm^–2 ofFe²⁺ and Mn²⁺ the shoot dry weight yield of the dry-soil clonewas reduced more than that of the wet-soil clone by 50 mg Fedm^–3 irrespective of Mn²⁺ concentration (P < 0.01)but the reduction of growth was less at higher Mn²⁺ concentrations.Fifty milligrams of Mn²⁺ dm^–3 reduced the growth of thedry soil clone but increased the growth of the wet soil clonewith Fe²⁺ at 5 mg dm^–2 (P < 0.05). Iron at 0.5 mg dm^–2was suboptimal for shoot growth of both clones at any levelof Mn²⁺ and caused more severe leaf chlorosis in the wet soilclone. Leaf tissue of clone L contained more iron than thatof clone A after waterlogging (P < 0.01) but in solutionculture, though increasing iron from 0.5 to 50 mg dm^–3almost doubled leaf iron content (P < 0.001), the interactionClones x Mn x Fe just failed to reach significance at P <0.05. The manganese content of leaf tissue from the two clonesvaried differently in response to solution manganese (Clonesx Mn P < 0.01), clone A showing a slightly greater increaseof manganese content at high solution concentration. Iron at50 mg dm^–3 suppressed Mn uptake (Mn x Fe, P < 0.001)in both clones. The two clones thus show marked environmentaladaptation to the chemistry of wet and dry soils. Dactylis glomerata, Cocksfoot grass, Orchard grass, waterlogging, iron, manganese, toxicity, deficiency, ecotypes     

Electrophysiological Evidence for the Broad Distribution of Specific Odorant Receptor Molecules across the Olfactory Organ of the Channel Catfish

To determine if there is a spatial segregation of responsivenessto odorants within the olfactory epithelium, microelectroderecordings were obtained from small populations of olfactoryreceptor neurons located across different lamellar sensory regionsof the olfactory organ of the channel catfish, lctalurus punctatus.Stimuli included L-alanine, L-methionine, L-arginine hydrochloride,L-glutamic acid, ATP and a mixture of bile salts—odorantspreviously reported to stimulate independent receptor sitesin aquatic species. The peak integrated olfactory receptor responsesat each recording site were standardized to the response toL-alanine. The relative stimulatory effectiveness of the stimuliwas preserved across the 10 olfactory lamellae recording sites.These data support previous molecular biological results ofa broad distribution of receptor neurons that express specificreceptor genes across the olfactory organ of the channel catfish.Chem. Senses 21: 519-527, 1996. ¹Present address: Institute of Cognitive and Computational SciencesGeorgetown University Medical Center, Washington, DC 20007,USA     

The Kinetics of Tracheid Development in Tsuga canadensis Carr. and its Relation to Tree Vigour

Cell counts from samples taken at weekly intervals, from 14May to 22 October 1969, in a T. canadensis stand in Massachusetts,U.S.A., showed that the width of the annual ring was correlatedwith the rate of cell production and that only the least vigoroustrees (c. 20 tracheids year^–1) had a shorter growing season.The time required for completion of a cell-division cycle inthe cambial zone decreased during the course of the season,from 35 to 20 days for the less vigorous trees (25–45tracheids year^–1) and from 28 to 10 days for the morevigorous trees (45–100 tracheids year^–1). The timerequired for the completion of radial growth of the tracheidsdecreased from 18 to 9 days, with no evidence of any changeswith tree vigour. The actual radial growth-rate of the tracheidswas constant within the range 1.5–3 um day^–1. Thetime required for deposition of the secondary cell wall increasedfrom 10 to 50 days, with little evidence of any changes withtree vigour. The actual rate of deposition of cell wall materialwas about 0.15 µ² µ^–1 day^–1 and seemedto show little change during the course of the season. The timeperiod required for lysis of the cytoplasm was about 4 days,with no evidence of any changes with tree vigour and littleevidence of any changes during the course of the season.