Kinetic analysis of drug-induced G2 block in vitro

The data on cell-cycle effects of two prospective antitumour agents, (+)-1,2,-bis(3,5-dioxopiperazine-1-yl)propane (soluble ICRF; NSC 169780) and 1,4-bis(2'chloroethyl)-1,4-diazabicyclo [2.2.1] heptane diperchlorate (CBH; NSC 57198) were used to determine whether a modified stathmokinetic experiment could predict the effects of continuous, long-term (0-48 hr) drug exposure in an in vitro L1210 murine leukaemia cell system. Generally, continuous drug exposure of exponentially growing cells does not provide sufficient quantitative information concerning cell-cycle-phase-specific mechanisms of drug action. Alternatively, stathmokinetic experiments, which are usually limited to some fraction of one cell doubling time, provide little information about long-term drug effects. By using mathematical models constructed for this purpose, however, stathmokinetic data can predict the overall proportion of cells affected by a drug though failing to discern between various kinds of drug action (e.g. reversible v. irreversible block, blocking v. killing action, etc.), especially when it occurs in G2 phase. In addition, it can be shown that for at least one of the drugs (soluble ICRF) the stathmokinetic experiment fails to predict 'after-effects' of drug treatment which extend into the following cell cycle(s). It also becomes clear that the degradation of exponential growth characteristics of quickly dividing cells during long-term, continuous drug exposure makes prediction of cell-cycle kinetic perturbations uncertain when derived from short-duration stathmokinetic experiments. However, with care, the joint application of 'short term' (e.g. stathmokinesis) and 'long term' (e.g. continuous exposure) techniques allow adequate quantitative insight into drug-perturbed cell-cycle kinetics. The applicability of modelling techniques is discussed: in the present instance it is limited to lower drug concentrations. For higher drug concentrations, effects like increased ploidy, ineffective division, etc., make it impossible in the present study to obtain a clear picture of the kinetics.     

Estimation and prediction of cell cycle specific effects of anticancer drugs

A mathematical model is used to estimate the cell cycle phase specific action of a new anticancer drug, CI-921, based on results of a short term (8 hr) stathmokinetic (blocked divisions) study on Friend leukemia cells. The estimate obtained is in the form of a sequence of fractions of the cell flow blocked in successive subcompartments of the cell cycle. At specific drug concentrations, the information contained in this estimate is sufficient to correctly predict the results of long term (24 hr) continuous drug exposure. The result obtained seems to be of interest, since the ability to predict the cell cycle phase specific effects of anticancer drugs is crucial for any attempt to improve the existing chemotherapeutic treatment schedules for cancer. This study required developing new mathematical techniques, among them a nonparametric method for estimation of the distributions of cell residence times in the G₁ phase of the cell cycle (Appendix). The methods developed are general enough to be applicable, in principle, to any chemotherapeutic agent the action of which is distributed throughout the cell cycle.     

Comparison of the interaction of antineoplastic aminoanthraquinone analogs with DNA using competitive fluorescence polarization

1,4-dihydroxy-5, 8-bis{{2-{(2-hydroxyethyl)amino}ethyl}amino} -9,10-anthracenedione (NSC 287836) and 1,4-bis{{2-{(2-hydroxyethyl) amino}ethyl}amino}-9, 10-anthracenedione diacetate (NSC 287513) have shown activity against solid tumors and are now in Phase I clinical trials. Fluorescence polarization was used to determine the extent of inhibition of the binding of acridine orange to DNA (Richardson, Roboz, Holland, Res. Comm. Chem. Pathol. Pharmac. $\text{27}$, 497, 1980). Displacement of 50% of acridine orange from calf thymus DNA was obtained with 0.18 uM of NSC 287836 while 0.52 uM of NSC 287513 was needed to displace an equivalent amount of acridine orange. NSC 287513 showed preference for polynucleotides of high adenine+thymine content while NSC 287836 did not. Analogs lacking both hydroxyethylaminoethyl-amino side chains did not displace acridine orange.     

Cell kinetic analysis of a murine macrophage cell line

For the present study, which was performed to find a reliable method suitable for determination of the cell kinetic parameters of a continuous cell line, use was made of the macrophage cell line J774.1. The doubling time of the cell population was approximately 27 h. The continuous labeling curve showed that all the cells divide and almost no quiescent cells occur. The cell-cycle time as determined from the curve of the labeled cells in mitosis, the course of the stathmokinetic index, and time-lapse videorecordings, was about 19 h. The discrepancy between the population doubling time and the cell-cycle time must be due to death and disintegration of cells during culture in vitro. The results indicate that the doubling time of a cell population is not a reliable parameter to determine the kinetics of a population of continuously proliferating cells and that determination of the course of the stathmokinetic index offers a rapid and simple method to establish the cell-cycle time reliably.     

Wheat variety carrying 2NvS chromosomal segment provides yield advantage through lowering terminal heat–induced oxidative stress

A 2N^vS chromosomal segment carrying bread wheat variety, BARI Gom 33 (‘BG33’), showed tolerance to terminal heat stress and higher yield over a heat-tolerant non-2N^vS BARI Gom 26 (‘BG26’) and a heat-susceptible Pavon 76 (‘Pavon’). This study aimed to ascertain the potential of the 2N^vS ‘BG33’ in terminal heat-induced oxidative stress tolerance compared to non-2N^vS ‘BG26’ and heat-susceptible ‘Pavon’ under two heat regimes at the reproductive stages viz. control (optimum sowing time) and heat stress (late sowing). We found that both ‘BG26’ and ‘BG33’ showed significantly higher tolerance to oxidative stress by limiting the generation of reactive oxygen species (ROS), methylglyoxal under heat stress. During terminal heat stress, both ‘BG33’ and ‘BG26’ exhibited greater cellular homeostasis than heat-susceptible ‘Pavon’, which was maintained by the increased accumulation of osmolytes, nonenzymatic antioxidants, and enzymes associated with ROS scavenging, ascorbate–glutathione cycle, and glyoxalase system. Lesser cellular damage in ‘BG26’ and ‘BG33’ was eventually imitated in a smaller reduction in grain yield (15 and 12%, respectively) than in ‘Pavon’, which had a 33% reduction owing to heat stress. Collectively, our findings revealed that the chromosomal segment 2N^vS provides yield advantage to ‘BG33’ under terminal heat stress by lowering oxidative damage. As 2N^vS translocation contains multiple nucleotide-binding domain leucine-rich repeat containing, cytochrome P450, and other gene families associated with plant stress tolerance, further studies are warranted to dissect the underlying molecular mechanisms associated with higher heat stress tolerance of 2N^vS carrying ‘BG33’.

     

Synthesis and chemiluminescence of copolymers of 5-amino-8-vinyl-phthalazine-1, 4(2H,3H)-dione with methyl methacrylate or styrene,and of α, ω-bis[5-amino-phthalazine-1,4 (2H,3H)-dion-]8-yl alkanes [=α, ω-bis(6-luminyl) alkanes]: Investigations on an intramolecular ‘distance effect’

Oligomers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione exhibit about 0.05% of the chemiluminescence quantum yield of the corresponding ‘monomer unit’, i.e. 5-amino-8-ethyl-phthalazine-1,4(2H,3H)-dione which has a similar quantum yield to luminol. The quantum yields of copolymers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione (1a) with methyl methacrylate or with styrene increase up to 1000-fold, relative to the quantum yield of oligomers of (1a). Thus the monomer units of methyl methacrylate or styrene appear to act as ‘spacers’ between the lumigenic groups. α,ω-Bis[(5-amino-phthalazine-1,4(2H,3H)-dion-)8-yl] alkanes show an analogue ‘distance’ effect: the chemiluminescence quantum yield increases with increasing alkane chain length. As the fluorescence of the corresponding amino phthalates (which are intermediates in the synthesis of the phthalazine diones) is only slightly influenced by the distance between the lumigenic groups it is suggested that a mainly chemical ‘distance effect’ is working here: the smaller the intramolecular distance between the hydrazide groups the more inhibition exists in respect of the oxidative reaction producing the luminol-type chemiluminescence.     

Optimal control problems arising in cell-cycle-specific cancer chemotherapy

We explore mathematical properties of models of cancer chemotherapy including cell-cycle dependence. Using the mathematical methods of control theory, we demonstrate two assertions of interest for the biomedical community: 1 Periodic chemotherapy protocols are close to the optimum for a wide class of models and have additional favourable properties. 2 Two possible approaches, (a) to minimize the final count of malignant cells and the cumulative effect of the drug on normal cells, or (b) to maximize the final count of normal cells and the cumulative effect of the drug on malignant cells, lead to similar principles of optimization. From the mathematical viewpoint, the paper provides a catalogue of simplest mathematical models of cell-cycle dependent chemotherapy. They can be classified based on the number of compartments and types of drug action modelled. In all these models the optimal controls are complicated by the singular and periodic trajectories and multiple solutions. However, efficient numerical methods have been developed. In simpler cases, it is also possible to provide an exhaustive classification of solutions. We also discuss developments in estimation of cell cycle parameters and cell-cycle dependent drug action.     

隔离降雨对马尾松针叶非结构性碳水化合物和碳、氮、磷的影响

植物叶片中的非结构性碳水化合物(NSC)和碳(C)、氮(N)、磷(P)含量可反映植物和生态系统对水分亏缺环境的响应及适应程度。以福建省长汀县25a马尾松(Pinus massoniana)为对象,原位观测分析了3年持续100%隔离降雨对成年马尾松针叶NSC与C、N、P的影响。结果表明：(1)持续隔离降雨导致马尾松针叶NSC含量先显著增加后减少,最终导致针叶NSC含量季节变化消失;且针叶可溶性糖含量、可溶性糖/淀粉比值在后期显著增加;(2)持续隔离降雨使马尾松针叶N含量、P含量、N∶P均表现为前期与对照组差异不显著,后期显著高于对照组;(3)马尾松针叶NSC含量、可溶性糖含量、P含量、N∶P与土壤水分含量呈显著或极显著相关,且均与P含量显著或极显著相关,与N素无相关性。以上研究结果表明,随着土壤水分含量的持续减少马尾松可通过调整针叶中NSC含量的积累及分配和提高针叶N、P含量来适应缺水环境,P含量的增加对NSC含量波动及可溶性糖和淀粉的相互转化起促进作用。     

Trubs,but no trianas: filled and empty regions of angiosperm stem length‐diameter‐mechanics space

Discrete plant habit categories such as ‘tree’, ‘shrub’, and ‘liana’ belie continuous variation in nature. To study the evolution of this continuous variation, we gathered data on stem length, diameter and tissue mechanical stiffness across a highly morphologically diverse highland xerophytic scrub on a lava flow in central Mexico. With stem allometric and mechanical data from 1216 segments from 50 species, we examined relationships between stem length–diameter proportions and tissue mechanical stiffness using linear mixed‐effects models. Rather than a series of discrete clouds in stem length–diameter–tissue stiffness space, corresponding to traditional habit categories, the plants of this xerophytic scrub formed a single continuous one. Within this cloud, self‐supporting plants had stems that became predictably longer and tissues that became stiffer for a given diameter increase, and there was no paucity of intermediates between trees and shrubs (‘trubs’). Non self‐supporting plants had a steeper stem length–diameter slope and their tissues did not increase in stiffness with stem size. The area between self‐ and non self‐supporting plants was sparsely occupied as stem size increased. We predict that this ‘empty’ space between lianas and trees is developmentally accessible but of low fitness, meaning that there should be few ‘trianas’ in nature. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 361–373.     

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.     

A variant in ANKK1 modulates acute subjective effects of cocaine: a preliminary study

This study aimed to evaluate whether functional variants in the ankyrin repeat and kinase domain‐containing 1 (ANKK1) gene and/or the dopamine receptor D2 (DRD2) gene modulate the subjective effects (reward or non‐reward response to a stimulus) produced by cocaine administration. Cocaine‐dependent participants (N = 47) were administered 40 mg of cocaine or placebo at time 0, and a subjective effects questionnaire (visual analog scale) was administered 15 min prior to cocaine administration, and at 5, 10, 15 and 20 min following administration. The influence of polymorphisms in the ANKK1 and DRD2 genes on subjective experience of cocaine in the laboratory was tested. Participants with a T allele of ANKK1 rs1800497 experienced greater subjective ‘high’ (P = 0.00006), ‘any drug effect’ (P = 0.0003) and ‘like’ (P = 0.0004) relative to the CC genotype group. Although the variant in the DRD2 gene was shown to be associated with subjective effects, linkage disequilibrium analysis revealed that this association was driven by the ANKK1 rs1800497 variant. A participant's ANKK1 genotype may identify individuals who are likely to experience greater positive subjective effects following cocaine exposure, including greater ‘high’ and ‘like’, and these individuals may have increased vulnerability to continue using cocaine or they may be at greater risk to relapse during periods of abstinence. However, these results are preliminary and replication is necessary to confirm these findings.     

'Photoinhibition' of Heliconia under natural tropical conditions: the importance of leaf orientation for light interception and leaf temperature

The influence of irradiance on photosynthesis under natural conditions was studied in aseasonal Singapore using three Heliconia taxa: H. rostrata, H. psittacorum × H. spathocircinata cv. Golden Torch and H. psittacorum cv. Tay. When grown under full sunlight, all three heliconias exhibited reduced phatosynthetic capacities and lowered chlorophyll content per leaf area as compared with those grown under intermediate and deep shade. A marked decrease in the chlorophyll fluorescence F_v/Fm ratio and an increase in photochemical quenching (1- q_p) and non-photochemical quenching (q_N) were observed in upper leaves of plants grown under full sunlight. Increases in q_N suggest that ‘photoinhibition’ (decreases in F_v/F_m) in Heliconia grown under natural tropical conditions are probably due to photoprotective energy dissipation processes. The quantum yield, the maximum photosynthetic rate, F_v/F_m and the chlorophyll content of upper leaves were lower than those of lower leaves on the same plants grown under full sunlight. Similarly, lower values were obtained for the tip (sun) portion than for the base (shaded) portion of the leaves. The changes in F_v/F_m and in the levels of (1 –q_p) in leaves grown under intermediate and deep shade were negligible in plants during the course of day. However, there was a steep decrease in F_v/F_m and an increase in the levels of (1 –q_p), along with an increase in incident light in the sun leaves. The lowest F_v/F_m and the highest level of (1 –q_p) indicated minimum PSII efficiency at midday in full sun. These results indicate that, in Heliconia, the top leaves (particularly leaf tips) experienced sustained decreases in PSII efficiency upon exposure to full sunlight. Although all three taxa exhibited sustained decreases in photosynthetic capacity in full sunlight, the sun leaves of ‘Tay’ showed higher photosynthetic capacity than those of the other two taxa. This could be due, at least in part, to the vertical leaf angle and smaller lamina area. When the upright leaves of ‘Tay’ were constrained to a horizontal angle, they exhibited lower PSII efficiency (F_vIF_m ratio), while horizontal leaves of ‘Rostrata’ and ‘Golden Torch’ inclined lo near-vertical angles showed increased efficiency. Thus, an increase in leaf angle helps to achieve a reduction in the sustained decrease in PSII efficiency by decreasing the levels of incident sunlight and subsequently the leaf temperature.     

Crystal structure of nonaquasamarium(III)bromide-l,4-dioxan adduct (1:3:2), Sm(H2O)9·Br3(C4H8O2)2

The title compound has been found to consist of tricapped trigonal prismatic Sm(H₂O)₉³⁺ ions in C_2v symmetry, sandwiched between expanded ‘close packed’ layers of bromide ions, with 1,4-dioxan molecules in chair conformation hydrogen bonding between the equatorial water molecules of adjacent cations.Orthorhombic, Amm2, a = 8.010(3), b = 19.848(4), c = 7.388(3) Å, R = 0.022 for 992 unique reflexions.     

The fate of recently fixed carbon after drought release: towards unravelling C storage regulation in Tilia platyphyllos and Pinus sylvestris

Carbon reserves are important for maintaining tree function during and after stress. Increasing tree mortality driven by drought globally has renewed the interest in how plants regulate allocation of recently fixed C to reserve formation. Three‐year‐old seedlings of two species (Tilia platyphyllos and Pinus sylvestris) were exposed to two intensities of experimental drought during ~10 weeks, and ¹³C pulse labelling was subsequently applied with rewetting. Tracking the ¹³C label across different organs and C compounds (soluble sugars, starch, myo‐inositol, lipids and cellulose), together with the monitoring of gas exchange and C mass balances over time, allowed for the identification of variations in C allocation priorities and tree C balances that are associated with drought effects and subsequent drought release. The results demonstrate that soluble sugars accumulated in P. sylvestris under drought conditions independently of growth trends; thus, non‐structural carbohydrates (NSC) formation cannot be simply considered a passive overflow process in this species. Once drought ceased, C allocation to storage was still prioritized at the expense of growth, which suggested the presence of ‘drought memory effects’, possibly to ensure future growth and survival. On the contrary, NSC and growth dynamics in T. platyphyllos were consistent with a passive (overflow) view of NSC formation.     

Hypoglycemic activity of curcumin synthetic analogues in alloxan-induced diabetic rats

The currently available therapies for type 2 diabetes have been unable to achieve normoglycemic status in the majority of patients. The reason may be attributed to the limitations of the drug itself or its side effects. In an effort to develop potent and safe oral antidiabetic agents, we evaluated the in vitro and in vivo hypoglycemic effects of 10 synthetic polyphenolic curcumin analogues on alloxan-induced male diabetic albino rats. In vitro studies showed 7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (4) to be the most potential hypoglycemic agent followed by 1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one (10). Structure activity relationship (SAR) of the tested compounds was elucidated and the results were interpreted in terms of in vitro hypoglycemic activities. Furthermore, oral glucose tolerance test (OGTT) with compounds 4, 10 and reference hypoglycemic drug glipizide showed that compound 4 and glipizide had relatively similar effects on the reduction of blood glucose levels within 2?h. Thus, compound 4 might be regarded as a potential hypoglycemic agent being able to reduce glucose concentration both in vitro and in vivo.     

Windkanalversuche zur Abh?ngigkeit der Fluggeschwindigkeit einer Wei?scheitelammer (Zonotrichia leucophrys) von der Windgeschwindigkeit

Zusammenfassung In einem Windkanal wurde für eine Weißscheitelammer (Zonotrichia leucophrys) die Abhängigkeit der Fluggeschwindigkeit in Luft (v_F) und der Geschwindigkeit über Grund (v_G) von der Windgeschwindigkeit (v_W) bestimmt. Das Versuchstier flog bei Windstille mit einer mittleren v_F von 5,15 m/sec. Bei zunehmenden Gegenwinden wurde v_F so stark erhöht, daß sich v_G nur langsam verringerte. Bei v_W=16,2 m/sec wurde noch v_G=1,4 m/sec erreicht. Bei zunehmenden Rückenwinden wurde v_F immer mehr herabgesetzt, so daß sich v_G kaum erhöhte. Bei v_W=–7 m/sec flog das Versuchstier sogar langsam rückwärts in der Luft, um seine Manövrierfähigkeit im engen Kanal zu erhalten. Der Gesamtgeschwindigkeitsbereich für v_F lag zwischen –0,9 bis 17,6 m/sec.

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Flight speed of a White-Crowned Sparrow in a Wind Tunnel at different Wind Speeds

Summary In an experimental wind tunnel air speed (v_F) and ground speed (v_G) of a White-Crowned Sparrow (Zonotrichia leucophrys) were measured as a function of wind speed (v_W). The bird had an average v_F of 5,15 m/sec in still air. With rising head winds it increased its v_F. At v_W=16,2 m/sec v_F was 17,6 m/sec and v_G was still 1,4 m/sec. With tailwinds it tended to maintain constant v_G and lowered v_F. At v_W=–7 m/sec it even flew slowly backward in the air in order to maintain its maneuvrability. The complete speed range for v_F was from –0,9 to + 17,6 m/sec.

     

Changes in energy metabolism due to anthelmintics in Fasciola hepatica maintained in vitro

The effects of rafoxanide (RFX), nitroscanate (NSC) and mebendazole (MBZ) on oxidative pathways in whole F. hepatica maintained in a simple salt solution have been examined. The anthelmintics did not alter glucose uptake or glycogen mobilization. NSC and RFX depressed ATP and increased AMP levels. MBZ behaved similarly at first, but later depressed the total adenine nucleotides. All three drugs influenced end product formation, increasing it initially, although by different mechanisms. With NSC, early increases in lactate and acetate excretion were later abolished. With RFX, there was an initial increased production of acetate and propionate. Later, excretion of propionate was reduced and that of succinate was increased. MBZ also increased succinate excretion, but to a much greater extent. In addition, it inhibited lactate production. A number of effects of the drugs on the internal concentrations of metabolic intermediates are described. The mechanisms of action of the drugs are discussed.     

Photoperiodic time measurement in the aphid Megoura viciae

Megoura produces parthenogenetic virginoparae in long day conditions, gamic oviparae in short days. The nature of this photoperiodic response has been analysed by rearing parent apterae in a wide range of circadian and non-circadian light cycles. By varying the light and dark components independently in a two-component cycle it has been established that the time measuring function is associated primarily with the dark period. There is no evidence that an endogenous circadian oscillation is implicated: thus (a) the ‘short day’ response is abolished by ‘night interruptions’ positioned in the early or late night. But this bimodal response pattern remains unchanged when the duration of the ‘main’ photoperiod is varied from ca. 6 hr to at least 25·5 hr. The stability of the maxima within the scotophase is inconsistent with the ‘coincidence’ models of photoperiodic timing that have been proposed. It is suggested that the essential timing process operates on the hour-glass principle, beginning anew with the onset of each period of darkness; (b) night interruption experiments employing very long (up to 72 hr) scanned dark periods yielded response maxima explicable in terms of the hour-glass hypothesis but did not reveal any circadian relationship between the maxima.The ‘dark reaction’ comprises a sequence of four stages, definable by the effects of light. Stage 1, extending from dark hr 0 to ca. 2·5, is fully photoreversible: at the next dark period the entire timing sequence is repeated up to the 9·5 hr critical night length. Towards the end of stage 1 reversibility is gradually lost and after a light interruption the reaction is resumed from a later time equivalent than dark hr 0; the subsequent critical night length is therefore reduced. The extent of the photoreversal is related to light duration. The period of maximum light insensitivity (stage 2) is attained at the end of the fourth hour. From ca. dark hr 5 to just short of the critical night length light exerts an increasingly promotive action which favours the production of virginoparae. This dark process is not photoreversible. Stage 4, which begins at hr 9·5, marks the end of the timing sequence. Light will not then annul the non-promotive action of the previous long night.Light has three effects which are determined by its duration and position within the cycle. The two terminal effects, mentioned above, are associated with the interception of dark stages 1 and 3 by either short (1 hr) or longer photoperiods. Light also prepares or primes the dark period timer. Thus the critical length is increased, and timing accuracy lost, if the preceding photoperiod is less than ca. 6 hr. Light during stage 4 has a priming action but no terminal function. Repeated cycles are ‘read’ in various ways, depending on the cycle structure. For example, if light intercepts stage 3, a two-component cycle is interpreted as the overlapping sequence light/dark/light. One and the same photoperiod then acts terminally in respect of the preceding dark period and as a primer for the next dark period.There is also a mechanism for summing the promotive effects produced by repeated interruption of dark stage 3. With complex (four-component) cycles both halves of the same cycle may contribute. ‘Product accumulation’ falls below threshold if the frequency of presentation of a given promotive cycle is too low. This occurs if there are very long, relatively non-promotive dark components. Such cycles are accepted as ‘continuous darkness’.