Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

STUDIES ON THE POPULATION KINETICS OF THE WALKER CARCINOMA BY AUTORADIOGRAPHY AND PULSE CYTOPHOTOMETRY

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. The transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5-6 days after transplantation, a PLM curve was performed, yielding estimates of Tc ? 18.0 hr, Ts ? 6.4 hr, T_G2+M? 4.1 hr. With the double labelling technique in vitro under 2.2 atm oxygen we obtained: Tc ? 18.2hr, Ts ? 8.2 hr, T_G2+M? 2.0hr. From pulse cyto-photometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: f_G1? (47.6 ± 1.1)%, f_s? (34.1 ± 1.0)%, f_G2+M? (18.3 ± 1.5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0.93 on the seventh and eleventh day. The cell loss factor was φ? 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.     

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

The role of lipid and antioxidant exchanges in cell division synchronization (mathematical model)

Cell-cycle synchronization of two diffusecoupled cells has been studied in the framework of the membrane model for the cell division cycle, proposed by Chernavskii et al. (1977). It has been shown semianalytically (using the averaging principle) and by computer stimulation that a) if the duration of theG1-phase (T _G1 ) for two identical cells is comparable with the duration of the remaining cycle (T _S+G2+M ), the lipid (L)-exchange results in a synchronization with phase difference =0. The antioxidant (A)-exchange leads to a phase-locking with =T ₀/2 (whereT ₀ is the cell cycle period; b) ifT _G1 T _S+G2+M (orT _G1 T _S+G2+M ) theL-exchange makes synchronization possible both with =0 and =T ₀/2 while theA-exchange results in phase-locking with confined to the region 0 toT ₀/2; c) for non-identical cells differing in the values of kinetic parameters, the locking band narrows as the population density increases (when some model parameters are close to the bifurcation thresholds). We expect that the cells selected artificially at a definite phase of cycle might maintain the synchronous division for a long time if the lipid exchange between cells were stimulated.     

Determination of DNA base compositions from melting profiles in dilute buffers

Equations were determined for the dependency of the melting temperature (T_m) of DNA upon the logarithm of the sodium ion concentration, for four DNA samples of widely different base compositions (θ_GC). The slopes of these T_m versus log M equations wore found to decrease with increasing θ_{G C}of the samples. An empirical equation relating T_m, log M (Na⁺) and θ_{G C} was derived, which also accounts for differences in T_m versus log M slopes. Data from the literature for some synthetic polynucleotides and for the crab(Cancer pagarus) satellite poly AT are discussed in relation to the above finding. The changes in T_m versus log M slopes with θ_{G C} are interpreted in terms of changes in the thermodynamic parameters ΔS and ΔH with base composition.     

Effects of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordinated to purine nucleosides and DNA

 DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, 4-bnpy) is highly selective for G⁷ with K _G=1.1×10⁴ to 2.8×10⁴, with the more hydrophobic Pyr ligands exhibiting slightly higher binding. A strong dependence on ionic strength indicates that ion-pairing with DNA occurs prior to binding. At μ=0.05, d[Ru^II-DNA]/dt=k[Ru^II][DNA], where k=0.17–0.21 M^–1s^–1 with the various Pyr ligands. The air oxidation of [(py)(NH₃)₄Ru^II] _n -DNA to [(py)(NH₃)₄Ru^III] _n -DNA at pH 6 occurs with a pseudo-first-order rate constant of k _obs=5.6×10^–4s^–1 at μ=0.1, T=25  °C. Strand cleavage of plasmid DNA appears to occur by both Fenton/Haber-Weiss chemistry and by base-catalyzed routes, some of which are independent of oxygen. Base-catalyzed cleavage is more efficient than O₂ activation at neutral pH and involves the disproportionation of covalently bound Ru^III and, in the presence of O₂, Ru-facilitated autoxidation to 8-oxoguanine. Disproportionation of [py(NH₃)₄Ru^III] _n -DNA occurs according to the rate law: d[Ru^II–G_DNA]/dt=k ₀[Ru^III–G_DNA]+k ₁[Ru^III–G_DNA][OH^–], where k ₀=5.4×10^–4s^–1 and k ₁=8.8 M^–1s^–1 at 25  °C, μ=0.1. The appearance of [(Gua)(py)(NH₃)₄Ru^III] under argon, which occurs according to the rate law: d[Ru^III–G]/dt=k ₀[Ru^III–G_DNA]+k ₁[OH^–][Ru^III–G_DNA] (k ₀=5.74×10^–5 s^–1, k ₁=1.93×10^–2M^–1s^–1 at T=25  °C, μ=0.1), is consistent with lysis of the N-glycosidic bond by Ru^IV-induced general acid hydrolysis. In air, the ratio of [Ru-8-OG]/[Ru-G] and their net rates of appearance are 1.7 at pH 11, 25  °C. Small amounts of phosphate glycolate indicate a minor oxidative pathway involving C4′ of the sugar. In air, a dynamic steady-state system arises in which reduction of Ru^IV produces additional Ru^II. Received: 11 November 1998 / Accepted: 3 March 1999     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

Effect of temperature on the duration of egg development, and moulting and growth in juveniles of Crangonyx pseudogracilis (Crustacea: Amphipoda) in the laboratory

SUMMARY. The interval between moults is an extension of egg development time, increasing from birth to sexual maturity which is probably reached at instar 6 or 7. The duration of each instar increased with the animal's age. Incubation time for eggs and the intermoult interval have the same curvilinear inverse relationship with water temperature in the range 3.5–25°C. Results are expressed as degree-days above predicted threshold temperatures of 3.8°C for eggs and 3.2°C for instar 1 after birth, but inverse power-law relationships were a better fit to the results, with exponents of - 1.355 for eggs, - 1.263 for instar 1 and - 1.37 to - 1.92 for instars 2–4. Temperature — dependence apparently altered in instars 5 and 6 at 15–25°C. From a multiple regression of geometric mean moult interval (M_i, days) against mean age (A) and temperature (T, °C), M_i= 56.4 T^?0.7 e^0.016A, with mean ages of 106 days at 15°C and 85 days at 25°C after six moults. The mean number of primary flagellar segments on the antennules increased from 4.0 in instar 1 to 6.0 in instar 2 and 8.0 in instar 3. Thereafter, segments were added less regularly to give a mean of 13.2 in instar 7. In a natural population, when the sexes became distinctive they had 11–13 flagellar segments. From birth at c. 0.05 mg wet wt, individual growth rates were highly variable; mean growth rates (G_s, % wet wt day^?1) were similar in animals fed on dried, leached elm leaves and living, green leaves of Callitriche; there was a power-law relationship with temperature in the range 3.5–25°C, (G_s= 0.27 T^0.59). Faster growth rates were obtained on living leaves of Elodea. Sexual maturity is reached at c. 0.4–0.5 mg wet wt. A brief comparison is made with Gammarus pulex; C. pseudogracilis may be better adapted to warm-water habitats.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (K_s = 3.2 ± 1.6 μM, K_i = 38 ± 1.4 μM) and delF508 CFTR (K_s = 3 ± 1.8 μM, K_i = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (K_s = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (K_i = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

ANALYSIS OF THE KINETICS OF GRANULOSA CELL POPULATIONS IN THE MOUSE OVARY

The kinetics of granulosa cell populations in two types of follicles in ovaries of 28-day-old Bagg mice are investigated. The analysis includes estimations of mean values and standard deviations of the transit times (T_G1, T_S, T_G2 and T_C), the doubling time T_D, and the proliferative fraction p. First the percentage of labelled mitosis curve (PLM-curve) and the continuous labelling curve (CL-curve) are estimated. Then a hypothesis concerning the cell kinetics of the granulosa cells in the two follicle types is set up. The normal distribution is chosen to simulate the probability density functions of the transit times. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are worked out. By fitting the calculated PLM-curve to the experimental one it is possible to estimate mean values and standard deviations of T_G1, T_S>, and T_G2. As a test of the hypothesis the CL-curve is calculated by means of the estimated parameter values and compared to the experimental one. The calculated PLM- and CL-curves are found to be in good agreement with the experimental data as far as both follicle types are concerned. It is concluded that the method is a useful procedure. The choice of a normal distribution does not imply a significant limitation of the method in these investigations. Moreover it is concluded that the hypothesis is plausible. This means, e.g., that the proliferative fraction is unity in the two follicle types and that there is no cell loss from the cell systems.     

桂西南岩溶区八种适生植物光合性状的变异与关联

为探究岩溶植物的光合生理适应机制，采用Li-6400XT便携式光合作用测量系统，对广西平果市岩溶区8种适生植物的叶片净光合速率(P_n)、气孔导度(G_s)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)、水分利用效率(WUE)和气孔限制值(L_s)等光合特征参数进行了测定分析。结果表明：(1)6个光合特征参数在种内和种间均存在不同程度的变异，并且种内变异均大于种间变异。(2)G_s和T_r的变化主要来源于种间变异(46.72%～49.76%),而P_n、C_i、WUE和L_s变化主要来源于种内变异(48.66%～64.50%)。在生活型水平上，P_n、G_s和T_r的种内变异表现为常绿植物小于落叶植物，而C_i、WUE和L_s则相反。(3)各参数的种间变异均表现为落叶植...     

Inference for an age-dependent,multitype branching-process model of mast cells

We consider an age-dependent, multitype model for the growth of mast cells in culture. After a colony of cells is established by an initiator type, the two possible types of cells are resting and proliferative. Using novel inferential procedures, we estimate the generation-time distribution and the offspring distribution of proliferative cells, and the waiting-time distribution of resting cells.List of Notations B _i cumulative distribution function for the time until branching of a cell of type i - b _i probability density function for the time until branching of a cell of type i - b _i b _i (1–D _i ) - D _i cumulative distribution function for the time until death of a cell of type i - d _i probability density function for the time until death of a cell of type i - probability density function of a gamma distribution - G _i cumulative distribution function for the lifetime of a cell of type i - G _1*2 Convolution of G ₁ and G ₂ - ¯G _i 1–G _i - g _i probability density function for the lifetime of a cell of type i - L _i likelihood of a history of type i - m average number of proliferative daughters produced by dividing cells - M _ij (t) the expected number of type-j cells in a colony at time t if that colony began at time 0 with one type-i cell - M _i+ (t) M _i0 (t) + M _i 1(t) + M _i 2(t) - p _rs probability that a dividing cell produces r proliferative and s resting daughters - t _i times defining colony histories. See IV.2.1 - T ₀ time to division of an initiator cell - T ₁, T ₂ times from birth to division of the two daughters of an initiator cell - T ₍₁₎, T ₍₂₎ order statistics of T ₁ and T ₂ - minimum value of a gamma distribution - scale parameter of a gamma distribution or of an exponential distribution - probability per unit time of death for proliferative and resting cells - _rs expected value of p _rs when there is heterogeneity - shape parameter of a gamma distribution     

Photosynthetic glow peaks and their relationship with the free energy changes

This paper is concerned with relating thermoluminescence to the total free-energy change, G, involved in detrapping a particular electron-hole pair as a photosynthetic sample is warmed from an initial low temperature. It extends a mathematical discussion of four possible mechanisms introduced in an earlier paper [DeVault, Govindjee and Arnold, Proc Nat'l Acad Sci USA 80: 983–987 (1983)]; here, particular attention is paid to the dependence of the absolute temperature of the maximum of a glow-peak, T _m , on the total free-energy change, G. The conclusion from the cases studied is that T _m =G/(k _B W) where G is evaluated at T _m , W is a complicated function of temperature and of thermodynamic parameters in the steps of the mechanism, and k _B is the Boltzmann constant. If the rate limiting step in the mechanism of detrapping is not preceded by any step in which G is appreciably negative, W is likely to have a value of about 33 and T _m is approximately proportional to G. Otherwise W can become much smaller and more strongly dependent on temperature and T _m is no longer proportional to G. These conclusions are of significance in lending theoretical support to the practice of inferring redox midpoint potential changes from shifts in T _m .     

Surface receptors and immune activity of purified human circulating mononuclear cells: IV. The demonstration of seven subclasses of T cells in the circulation of the normal individual: The cytotoxic activities of these cells

T lymphocytes were isolated from monocyte-depleted mononuclear cells of normal individuals by rosetting them with sheep erythrocytes. These purified T cells were preferentially depleted of cells with receptors for FcG (T_G cells), FcM (T_M cells), or C3 (T_C cells) by rosette formation with EA(G), EA(M), and EAC, respectively, before or after incubation for 24 hr in medium 199 fortified with fetal calf serum (20%). The unfractionated lymphocytes and the purified and the depleted T cells were analyzed for receptors to FcG, FcM, and C3 and for cytotoxic activity in the natural killer (NK), antibody dependent cell-mediated cytotoxicity (ADCC), and mitogen-induced cell-mediated cytotoxicity (MICC) assays. The T_G and T_C cells were detected among the freshly isolated T cells, whereas the T_M cells were detected only following 24 hr of incubation. Removal of T_C cells from the 24-hr-cultured T cells resulted in removal of all the T_C cells and in the concomitant removal of the majority of T_M cells. Similarly, removal of T_M cells from the 24-hr-cultured T cells resulted in the elimination of all T_M cells as well as the majority of T_C cells. These results demonstrate the in vitro generation of T cells with receptors for both FcM and C3 (T_M+C cells). Ten percent of the freshly isolated T_G cells possessed detectable receptors for C3 and/or FcM. These cells constitute the T_G+C and T_G+M lymphocytes. Support for consideration of these receptor-bearing cells as unique and stable cells is provided by the finding that T_M and T_C cells maintained in culture for up to 72 hr do not generate other receptors but retain the single receptor which characterizes each of these cells. Only a small percentage of cultured T_G cells generate receptors for C3 and FcM. It may therefore be concluded that the T_G, T_M, and T_C cells are stable unireceptor-bearing cells. The T_G, T_M, T_C, T_G+C, T_G+M, and T_M+C lymphocytes account for approximately 50% of the circulating lymphocytes. Whether the remaining cells, the T null or T_N cells, constitute the precursors for any or all of the receptor-bearing T cells remains to be determined. Unfractionated freshly isolated T cells were highly cytotoxic in the NK and PWM-mediated MICC assays but were relatively inactive in the ADCC, naturally occurring cell-mediated cytotoxicity (NOCC), and PHA- and Con-A-mediated MICC assays. In contradistinction, T cells incubated for 24 hr displayed marked cytotoxic activity in the ADCC and PHA-mediated MICC assays; they were inactive in the NOCC and Con-Amediated MICC assays. The T_G cells were the predominant cytotoxic cells in the ADCC, NK, and MICC cytotoxic assays since their selective elimination from either the freshly isolated or 24-hr-incubated T cells resulted in almost total loss of cytotoxic activity of the remaining cells. Removal of the T_G+C cells from the freshly isolated or 24-hr-incubated T cells resulted in a significant decrease in PHA- and PWM-mediated MICC cytotoxic activity. T cells depleted of T_M, T_M+C, and T_C cells exhibited the same cytotoxic activity as did the unfractionated T cells. These results suggest that the predominant cytotoxic T cells in all the assays investigated are the T_G cells, that limited cytotoxic activity is also displayed by the T_G+C cells, and that the T_M, T_M+C, T_C, and T_N cells display no cytotoxic activity in the assays utilized in this investigation.     

Development, validation and field application of an RNA-based growth index in juvenile plaice Pleuronectes platessa

A general mechanism relating RNA concentration and growth rate is derived from four physiological assumptions and developed into a growth index for juvenile plaice Pleuronectes platessa. The index describing instantaneous growth rates (G, day^?1) in the laboratory with the lowest Akaike information criterion with small‐sample bias adjustment was a function of RNA concentration (R, ), temperature (T, ° K), body mass (M, g) and DNA concentration (D, ): G = β₀ + β_RR + β_TT + β_T2T² + β_MM + β_DD + β_RTRT. RNA concentration began to respond to changes in feeding conditions within 8 days, suggesting that the index reflects growth rate in the short‐term. Furthermore, the index distinguished between rapid growth and negative growth of juvenile P. platessa measured directly in laboratory and field enclosures, respectively. An application of the RNA‐based growth index at two beaches on the west coast of Scotland suggested that the growth of juvenile P. platessa varies considerably in space and time and is submaximum in late summer.