The Quiescent Centre and Rates of Mitosis in the Root Meristem of Allium sativum

The root apices of Allium sativum have been examined by continuous-and pulse-labelling with tritiated thymidine and by colchicinetreatment to measure the time parameters of the mitotic cyclein various parts of the meristem. There is a quiescent centre of 30–50 cells whose averagerate of mitosis is low because the G₁ period is extended toabout 140 h compared with about 4 h in the othe regions of themeristem. The stele just above the quiescent centre and at 200microns above it and the cap initials just below the quiescentcentre are very similar in their mitotic cycles, the total lengthsof which are about 30 h of which nearly half is taken up byDNA synthesis. Allium thus differs from Zea in having root capinitials whose mitotic cycle is not telescoped by the eliminationof the G₁ phase. These facts are discussed in relation to theradiosensitivity of the meristem.     

Synthesis of DNA during Mitosis

Growing roots of Zea mays in tritiated thymidine for brief periodsconfirms that G₁ may be eliminated from the mitotic cycle andDNA synthesis may be advanced into telophase but no further,in the fastest dividing cells of the cap initials, but not inthe stele.     

Commitment of Mitotic Cells to Meiosis during the G2 Phase of Premeiosis

In the developing anther, archesporial cells that proliferateby mitotic division are converted into meiotic cells duringthe premeiotic interphase. Experiments with explanted microsporocytesof Lilium and Trillium were made to obtain evidence for theconversion of mitotic to meiotic cells during the premeioticperiod. Explanted premeiotic cells were cultured through thedivision cycle at relatively high division frequencies and showeda variety of division types with respect to chromosomal events.The type of division depended on the premeiotic stage at whichthe cells were explanted. Cells in the G₁, S and early G² phasesunderwent mitotic division and formed a diad or binucleate monad.Cells explanted at the late G₂ phase were cultured throughoutthe normal meiotic cycle, which resulted in typical tetrad configuration. In microsporocytes explanted during the main part of the G₂interval, centromere behavior was meiotic, but chromosome pairingand chiasma formation were disturbed. Thus, she G₂ intervalwas shown to be critical for the commitment of mitotic cellsto meiotic division. Detailed analysis showed that the intracellularchanges that commit the cells to meiosis begin shortly aftercompletion of premeiotic DNA synthesis and that these changesare progressive and cumulative. (Received February 2, 1982; Accepted May 24, 1982)     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Effects of the ovarian cycle on sympathetic neural outflow during static exercise

We comparedreflex responses to static handgrip at 30% maximal voluntarycontraction (MVC) in 10 women (mean age 24.1 ± 1.7 yr) during twophases of their ovarian cycle: the menstrual phase (days 1-4) and the follicularphase (days10-12). Changes in muscle sympathetic nerve activity (MSNA; microneurography) in response tostatic exercise were greater during the menstrual compared withfollicular phase (phase effect P = 0.01). Levels of estrogen were less during the menstrual phase(75 ± 5.5 vs. 116 ± 9.6 pg/ml, days 1-4 vs.days 10-12;P = 0.002). Generated tension did not explain differences in MSNA responses (MVC: 29.3 ± 1.3 vs. 28.2 ± 1.5 kg, days 1-4 vs.days 10-12;P = 0.13). In a group of experiments with the use of ³¹P-NMRspectroscopy, no phase effect was observed forH⁺ andH₂PO₄ concentrations(n = 5). During an ischemicrhythmic handgrip paradigm (20% MVC), a phase effect was notobserved for MSNA or H⁺ orH₂PO₄ concentrations,suggesting that blood flow was necessary for the expression of thecycle-related effect. The present studies suggest that, during statichandgrip exercise, MSNA is increased during the menstrual compared withthe follicular phase of the ovarian cycle.

     

Studies on microspore development in liliaceous plants II. The behavior of explanted microspores of the lily, Lilium longiflorum

Uninucleate microspores of Lilium longiflorum from differentlengths of buds were explanted under various culture conditions,and their behavior was studied during subsequent cultures. Thenutritional conditions permitting survival of microspores wererelatively simple, but most of the living cells showed cytologicalabnormalities. A typical type of cell division could only beinduced in explanted microspores at the late G₁ phase of thecell cycle. For the normal development of microspores in culture,the microspore environment with respect to moisture was an importantfactor. The rate of the mitotic cycle of the cultured microsporeswas essentially the same as that for microspores grown undergreenhouse conditions. ¹Department of Medicine, Kochi Medical School, Nangoku, Kochi781-51, Japan. (Received March 25, 1980; )     

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line

Our aim was to determinewhether the expression of K⁺ currents is related to thecell cycle in the excitable GH3 pituitary cell line. K⁺currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cellcycle. We show that the peak density of the transient outward K⁺ current (I_to) was 33% lower incells in S phase (BrdU+) than in cells in other phases of the cellcycle (BrdU). The voltage-dependence of I_towas not modified. However, of the two kinetic components ofI_to inactivation, the characteristics of thefast component differed significantly between BrdU+ and BrdU cells.Recovery from inactivation of I_to showedbiexponential and monoexponential function in BrdU and BrdU+ cells,respectively. This suggests that the molecular basis of this currentvaries during the cell cycle. We further demonstrated that4-aminopyridine, which blocks I_to, inhibited GH3cell proliferation without altering the membrane potential. These datasuggest that I_to may play a role in GH3 cellproliferation processes.

     

Mitotic Activities and Levels of Nuclear DNA in the Apical Meristem of Silene armeria (strain SI.2) Following Application of Gibberellin A3

Strain S1.2 of Silene armeria was grown under an 8h-photoperiodand treated with GA₃ every day for 20 days. This growth substancecaused stem elongation, but no flowering in this long-day plant.Changes in the mitotic index and DNA content of cells in thevarious zones of the apical meristem before, during and afterGA₃ treatment were described. Mitotic activity and increasein the proportion of nuclei at the 4C level (S+G₂ phase of thecell cycle) were strongly stimulated in the rib-meristem, andto a lesser extent in the lateral zone, but not in the axialzone. This stimulation of apical activity reached a peak aftertwo GA₃ treatments and then declined gradually, so that after20 days the activity in GA₃-treated meristems was lower thanthat in untreated controls; at this point most cells were inthe G₁ phase. When the GA₃ treatment was discontinued, there was a gradualincrease in the mitotic activity which ultimately reached thesame level as that in controls. Stem elongation ceased and leavesformed aerial rosettes. It is concluded that in vegetative plants of strain S1.2 ofSilene armeria GA₃ acts mainly on the rib-meristem cells whichresults in stem elongation. Lack of response in the axial cellsexplains why GA₃ fails to induce flowering in this strain ofSilene armeria. (Received June 18, 1983; Accepted August 3, 1983)     

Relation between Water Stress and DNA Synthesis during Germination of Zea mays L.

Mitotic index, percent nuclei in DNA synthesis and the relativeDNA content per nucleus were determined from cells of the Zeamays radicle at various times after the beginning of germination.Nuclear DNA synthesis was initiated after 45 h and mitosis wasfirst observed after 74 h from sowing. Most of the dormant nucleiwere in the pre-synthetic or G₁ phase of the mitotic cycle.By 72 h most cells were in S and 77 h after the beginning ofgermination, the cells of the primary root were observed inall phases of the mitotic cycle. Dehydration of karyopses after45–74 h of imbibition progressively reduced the percentof germination to zero upon dehydration and subsequent replantingdemonstrating that drought sensitivity was related to the onsetof nuclear DNA synthesis and genome duplication.     

Thermoregulatory responses to cold transients: effects of menstrual cycle in resting women

Effects of themenstrual cycle on heat loss and heat production(M) and core and skin temperatureresponses to cold were studied in six unacclimatized female nonsmokers(18-29 yr of age). Each woman, resting supine, was exposed to acold transient (ambient temperature = mean radiant temperature = 20 to5°C at 0.32°C/min, relative humidity = 50 ± 2%, wind speed = 1 m/s) in the follicular (F) phase(days 2-6) and midluteal (L)phase (days 19-23) of her menstrual cycle. Clothed in each of two ensembles with different thermal resistances, women performed multiple experiments in the F andL phases. Thermal resistance was 0.2 and 0.4 m² · K · W¹for ensembles A andB, respectively. Esophagealtemperature (T_es), mean weightedskin temperature(_sk),finger temperature (T_fing), andarea-weighted heat flux were recorded continuously. Rate of heat debt(S) and integrated mean bodytemperature(_b,i)were calculated by partitional calorimetry throughout the cold ramp. Extensive peripheral vasoconstriction in the F phase during early periods of the ramp elevated T_esabove thermoneutral levels. Shivering thermogenesis(M = M  M_basal,W /m²) was highly correlated withdeclines in_sk andT_fing(P <0.0001). There was a reducedslope in M as a function of_b,i inthe L phase with ensembles A(P < 0.02) andB (P < 0.01). Heat flux was higher andS was less in the L phases withensemble A(P < 0.05). An analytic modelrevealed that_sk andT_es contribute as additive inputsand T_fing has a multiplicativeeffect on the total control of Mduring cold transients(R² = 0.9).Endogenous hormonal levels at each menstrual cycle phase, coretemperature and_skinputs, vascular responses, and variations in body heat balance must beconsidered in quantifying thermoregulatory responses in women duringcold stress.

     

Duration of the Mitotic Cycle in a Meristem

The length of the mitotic cycle and the time spent in mitosisare calculated for six regions of the apical meristem of rootsof Zea. The two methods used are the measurement of the ratesof labelling nuclei with radioactive thymidine and the ratesof accumulation of metaphases in roots grown in colchicine.The mitotic cycle lasts from 12 hours in the cap initials toabout 200 hours in the quiescent centre of the same roots. Somepoints about the organization of apices are discussed in thelight of these estimates.     

Micronuclei and Radiosensitiviy in the Root Meristem of Vicia faba

Percentages of cells with micronuclei in four regions of theroot meristems of Vicia faba are used as measures of sensitivityto acute X-irradiation. There are two peaks in these percentages,occurring at about four and eight days after 360 rads and twoand six days after 180 rads. Two peaks exist, probably becausethe radiation delays cells that were in G₁ much more than cellsthat were in G₂ in reaching the first post-irradiation mitosisand consequently in displaying micronuclei in the followinginterphase. The relative heights of the two peaks thereforereflect the relative numbers of cells in G₁ and G₂ as well asthe relative sensitivity of the two phases to chromosomal damage. The cells of the quiescent centre are injured least by the radiationas they are mostly held at Gt. The meristem thus obeys the lawof Bergonié and Tribondeau, but differs from that ofZea in that the meristematic cells of the cap initials and steleimmediately adjacent to the quiescent centre resemble the quiescentcentre much more closely than the stele 250 µ away inthe numbers of micronuclei produced. This is consistent withthe differences already known between the two species concerningrates of division in the different regions of the meristem andthe behaviour of the meristem after severe radiation injury.     

The Effect of High Concentrations of Heavy Water on Root Morphogenesis in Zea mays

The effect of high concentrations of heavy water on young rootsof Zea mays was investigated. Submersion for 24 hrs. in solutionsof 80–90 per cent. D₂O will temporarily stop growth ofthe primary root. During the treatment period the root-tip regionswill swell as the result of radial cell enlargement, primarilyin the cortical region. Upon removal from D₂O growth will resume,initially at a slower rate than controls but ultimately at similarmaximum rates. Lateral root production is inhibited in thatportion of the primary root which is the region of elongationat time of treatment. However, in the swollen region lateralroot formation is accentuated. The mitotic index drops sharplyupon introduction of the primary roots into 80 per cent. D₂O.Within 12 hrs. the index drops to zero and remains at this leveluntil the roots are removed from the heavy water. The mitoticindex then rises again, reaching control values within 24 hrs.It is suggested that the gentle, temporary inhibitory actionof D₂O makes this substance a useful tool in morphogenetic investigations.     

Effect of luteal phase elevation in core temperature on forearm blood flow during exercise

Kolka, Margaret A., and Lou A. Stephenson. Effect ofluteal phase elevation in core temperature on forearm blood flow duringexercise. J. Appl. Physiol. 82(4):1079-1083, 1997.Forearm blood flow (FBF) as an index of skinblood flow in the forearm was measured in five healthy women by venousocclusion plethysmography during leg exercise at 80% peak aerobicpower and ambient temperature of 35°C (relative humidity 22%;dew-point temperature 10°C). Resting esophagealtemperature (T_es) was 0.3 ± 0.1°C higher in the midluteal than in the early follicular phase ofthe menstrual cycle (P < 0.05).Resting FBF was not different between menstrual cycle phases. TheT_es threshold for onset of skinvasodilation was higher (37.4 ± 0.2°C) in midluteal than inearly follicular phase (37.0 ± 0.1°C; P < 0.05). The slope of the FBF toT_es relationship was not different between menstrual cycle phases (14.0 ± 4.2 ml · 100 ml¹ · min¹ · °C¹for early follicular and 16.3 ± 3.2 ml · 100 ml¹ · min¹ · °C¹for midluteal phase). Plateau FBF was higher during exercise inmidluteal (14.6 ± 2.2 ml · 100 ml¹ · min¹ · °C¹)compared with early follicular phase (10.9 ± 2.4 ml · 100 ml¹ · min¹ · °C¹;P < 0.05). The attenuation of theincrease in FBF to T_es occurred when T_es was 0.6°C higher andat higher FBF in midluteal than in early follicular experiments(P < 0.05). In summary, the FBF response is different during exercise in the two menstrual cycle phasesstudied. After the attenuation of the increase in FBF and whileT_es was still increasing, thegreater FBF in the midluteal phase may have been due to the effects ofincreased endogenous reproductive endocrines on the cutaneousvasculature.

     

Adenylyl cyclase is involved in desensitization and recovery of ATP-stimulated Cl- secretion in MDCK cells

We investigated the process of and recovery from desensitizationof the P₂ receptor-mediatedstimulation of Cl secretionin Madin-Darby canine kidney (MDCK) cell monolayers by assaying theresponse of short-circuit current(I_sc). When thecells were exposed to repeated 3-min challenges of ATP or UTPinterspersed with 5-min washes, the response ofI_sc desensitized rapidly followed by spontaneous recovery. The pattern of inhibition byvarious channel blockers or enzyme inhibitors revealed that both theinitial and recovered responses ofI_sc have the same ionic and signaling mechanisms. The desensitization and recovery processes were confined to the membrane exposed to the repeated challenges. When added during the desensitized phase, 8-bromoadenosine 3',5'-cyclic monophosphate enhanced the ATP-stimulatedI_sc response, whereas it did not during the initial or recovered phases. ATP-induced increases of intracellular adenosine 3',5'-cyclicmonophosphate showed similar desensitization and recovery in parallelwith the changes in the responses ofI_sc. Thedesensitization process was attenuated by pretreatment with choleratoxin or pertussis toxin. Taken together, our results suggest that theadenylyl cyclase system plays a role in the desensitization andrecovery mechanism of the ATP-stimulatedCl secretion in MDCK cells.

     

Cardiovascular responses to treadmill and cycle ergometer exercise in children and adults

Turley, Kenneth R., and Jack H. Wilmore. Cardiovascularresponses to treadmill and cycle ergometer exercise in children andadults. J. Appl. Physiol. 83(3):948-957, 1997.This study was conducted to determine whethersubmaximal cardiovascular responses at a given rate of work aredifferent in children and adults, and, if different, what mechanismsare involved and whether the differences are exercise-modalitydependent. A total of 24 children, 7 to 9 yr old, and 24 adults, 18 to26 yr old (12 males and 12 females in each group), participated in bothsubmaximal and maximal exercise tests on both the treadmill and cycleergometer. With the use of regression analysis, it was determined thatcardiac output () was significantly lower(P  0.05) at a givenO₂ consumption level(O₂, l/min) in boys vs. menand in girls vs. women on both the treadmill and cycle ergometer. Thelower in the children was compensated for by asignificantly higher (P  0.05)arterial-mixed venous O₂difference to achieve the same or similarO₂. Furthermore, heart rateand total peripheral resistance were higher and stroke volume was lowerin the children vs. in the adult groups on both exercise modalities.Stroke volume at a given rate of work was closely related to leftventricular mass, with correlation coefficients ranging fromr = 0.89-0.92 andr = 0.88-0.93 in the males and females, respectively. It was concluded that submaximal cardiovascular responses are different in children and adults and that these differences are related to smaller hearts and a smaller absolute amountof muscle doing a given rate of work in the children. The differenceswere not exercise-modality dependent.

     

Basal chloride currents in murine airway epithelial cells: modulation by CFTR

We have isolatedciliated respiratory cells from the nasal epithelium of wild-type andcystic fibrosis (CF) null mice and used the patch-clamp technique toinvestigate their basal conductances. Current-clamp experiments onunstimulated cells indicated the presence ofK⁺ andCl conductances and, undercertain conditions, a small Na⁺conductance. Voltage-clamp experiments revealed three distinct Cl conductances.I_tv-indep wastime and voltage independent with a linear current-voltage(I-V)plot; I_v-actexhibited activation at potentials greater than ±50 mV, giving anS-shapedI-Vplot; andI_hyp-act wasactivated by hyperpolarizing potentials and had an inwardly rectifiedI-Vplot. The current density sequence was I_hyp-act = I_v-act  I_tv-indep. Theseconductances hadCl-to-N-methyl-D-glucaminecation permeability ratios of between 2.8 and 10.3 and were unaffectedby tamoxifen, flufenamate, glibenclamide, DIDS, and5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited byZn²⁺ andGd³⁺.I_tv-indep andI_v-act werepresent in wild-type and CF cells at equal density and frequency.However, I_hyp-actwas detected in only 3% of CF cells compared with 26% of wild-typecells, suggesting that this conductance may be modulated by cysticfibrosis transmembrane conductance regulator (CFTR).

     

Use of nonlinear methods to assess effects of clonidine on blood pressure in spontaneously hypertensive rats

In spontaneously hypertensive rats (SHR),chronic infusion of clonidine failed to decrease blood pressure andblood pressure variability. We used nonlinear methods to get a deeperinsight on the effects of clonidine on blood pressure dynamics. For 24 h and 4 wk, clonidine (0.1 mg · kg¹ · day¹sc) was infused by minipumps in the conscious SHRs, and, for comparison, a vehicle was infused in SHRs and in Wistar-Kyoto rats.Blood pressure was recorded for 30 min before and after treatments. Weused the Lyapunov exponent, approximated by the inverse of thel_max indexderived from the recurrence plot method, to characterize nonlineardynamics. Before treatment,l_max index ofblood pressure was lower (P < 0.01)in the SHRs than in the Wistar-Kyoto rats. Clonidine significantlyincreased l_max(P < 0.01) to the level observed innormotensive rats, at 24 h and up to 4 wk after infusion. We concludethat clonidine has a significant chronic effect on blood pressuredynamics, as evidenced by nonlinear methods. Our study also suggeststhat the mechanisms governing blood pressure variations are nonlinear.

     

Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms

Micromolar concentrations ofATP stimulate biphasic change in transepithelial conductance acrossCaSki cultures, an acute increase (phase I response) followed by aslower decrease (phase II response). Phase I andphase II responses involve two distinct calcium-dependentpathways, calcium mobilization and calcium influx. To test thehypothesis that phase I and phase II responsesare mediated by distinct P2 purinergic receptors, changes inpermeability were uncoupled by blocking calcium mobilization with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA) or by lowering extracellular calcium, respectively. Under theseconditions ATP EC₅₀ was 25 µM for phase Iresponse and 2 µM for phase II response. The respectiveagonist profiles were ATP > UTP > adenosine5'-O-(3-thiotriphosphate) (ATP-S) = N⁶-([6-aminohexyl]carbamoylmethyl)adenosine5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-S. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course andpharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and asaturable concentration-dependent effect (EC₅₀ of 2-3µM). RT-PCR experiments revealed expression of mRNA for both theP2Y₂ and P2X₄ receptors. These results suggestthat the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.