The Effects of Solvent Environment on the Optical Rotatory Dispersion Parameters of Polypeptides: I. Studies on Poly-γ-Benzyl-l-Glutamate

The constancy of the Moffitt optical rotatory dispersion parameters for polypeptides in different solvents was tested by dispersion measurements on poly-γ-benzyl-L-glutamate in fifty-five solvents and solvent mixtures. b₀ was not constant but varied linearly with the refractive index of the solvent according to the equation -b₀ = 1701 - 730.3 n₈. This variation could not be explained by changes in configuration of the polypeptide. a₀ also showed a trend with solvent index but the values were widely scattered. λ₀ did not show a statistically significant dependence on solvent index. The variation in b₀ can be interpreted as an effect of solvent polarizability on the frequencies of optically active transitions.     

Photolysis Accompanying Peptide Absorption in Proteins: Electrophoresis and Optical Rotary Dispersion Studies

Exposure of proteins and polypeptides to ultraviolet radiation below 240 nm produces peptide cleavage which may or may not be accompanied by observable changes in conformation and optical rotary dispersion (ORD) properties, depending on the stability of the secondary and tertiary structure of the macromolecule under the experimental conditions. Helical and coiled forms of poly-L-glutamic acid undergo degradation at similar rates but only the helical form shows a significant change in rotatory properties. The helical form of poly-L-lysine, but neither the coiled nor β forms, shows a change in [α]₂₃₃ on irradiation at 233 nm. β-Lactoglobulin shows essentially no change in [α]₂₃₃ on irradiation in either dilute salt solution or 4 M urea at room temperature; however, in 4 M urea at 56°C a large change occurs. A model is developed which shows that studies of the effect of radiation on ORD properties may be useful in providing information on possible intermediate steps in protein denaturation. The method is illustrated with results on bovine plasma albumin. A quantum yield, 4.3 × 10^-3 moles/einstein, was obtained for peptide cleavage in this protein at 225 nm. These studies, based on gel electrophoresis, also showed that the fragments produced are essentially random, suggesting that transfer of energy from aromatic residues is not an important contributor to the peptide photolysis. Possible errors which could arise in ORD and other studies involving intense ultraviolet radiation are considered.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.     

Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.     

The Effects of Temperature on the Labellar Chemoreceptors of the Blowfly

In the labellar chemosensory hairs of the blowfly, Phormia regina Meigen, stationary amplitudes of the slow potentials induced by salt and sugar stimulations were decreased to 50–80% at 12°C of the values measured at 28°C. The amplitudes induced by water did not show any dependence on temperature change. The maximum rate of rise of the receptor potentials was strongly increased with rising temperature. The value of K_b, the apparent Michaelis constant, was less by a factor of six at 28°C compared to the 12°C value for the sugar receptor.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

Limited heat tolerance in an Arctic passerine: Thermoregulatory implications for cold‐specialized birds in a rapidly warming world

1. Arctic animals inhabit some of the coldest environments on the planet and have evolved physiological mechanisms for minimizing heat loss under extreme cold. However, the Arctic is warming faster than the global average and how well Arctic animals tolerate even moderately high air temperatures (T _a) is unknown.
2. Using flow‐through respirometry, we investigated the heat tolerance and evaporative cooling capacity of snow buntings (Plectrophenax nivalis; ≈31 g, N = 42), a cold specialist, Arctic songbird. We exposed buntings to increasing T _a and measured body temperature (T _b), resting metabolic rate (RMR), rates of evaporative water loss (EWL), and evaporative cooling efficiency (the ratio of evaporative heat loss to metabolic heat production).
3. Buntings had an average (±SD) T _b of 41.3 ± 0.2°C at thermoneutral T _a and increased T _b to a maximum of 43.5 ± 0.3°C. Buntings started panting at T _a of 33.2 ± 1.7°C, with rapid increases in EWL starting at T _a = 34.6°C, meaning they experienced heat stress when air temperatures were well below their body temperature. Maximum rates of EWL were only 2.9× baseline rates at thermoneutral T _a, a markedly lower increase than seen in more heat‐tolerant arid‐zone species (e.g., ≥4.7× baseline rates). Heat‐stressed buntings also had low evaporative cooling efficiencies, with 95% of individuals unable to evaporatively dissipate an amount of heat equivalent to their own metabolic heat production.
4. Our results suggest that buntings’ well‐developed cold tolerance may come at the cost of reduced heat tolerance. As the Arctic warms, and this and other species experience increased periods of heat stress, a limited capacity for evaporative cooling may force birds to increasingly rely on behavioral thermoregulation, such as minimizing activity, at the expense of diminished performance or reproductive investment.

     

Rotatory dispersion of the β-form of polypeptides

The rotatory dispersion of the β-form of polypeptides is considered. The parameter b₀ of the Moffit-Yang equation is calculated quantum mechanically. The results obtained b₀ = ?30, agrees with experiment which gives b₀ near zero for the β-form.     

Ditylenchus dipsaci Infestation of Trifolium repens. I. Temperature Effects,Seedling Invasion,and a Field Survey

Rates of development of stem nematode (Ditylenchus dipsaci) in white clover (Trifolium repens) seedlings were found to be linearly related to temperature. Basal developmental temperature (T_b) was 3 °C, and the thermal constant (S) for development of gravid adult females from freshly laid eggs was 270 accumulated day-degrees above the T_b. Only 12% at 20 °C and 4% at 4 °C of the gravid female nematodes inoculated into seedling axils successfully penetrated seedling epidermis. These nematodes slowly migrated within the seedling and after a lag of 5 days at 20 °C started to lay eggs. The maximal rate of egg production was temperature-dependent, being 0.8 and 3.1 eggs female⁻¹ day⁻¹ at 10 and 20 °C, respectively. Nematodes emigrated rapidly from infested stolons when they were immersed in water, with rates being highest at 25 °C and lowest at 4 °C. The sensitivity to temperature of many of the parameters that govern nematode population dynamics indicates that climatic changes will have a marked effect upon this host-parasite system. A study of infested stolons from the field indicated that nematode numbers increased up to 3,000 or more before tissue senesence, triggered by nematode damage, caused a mass emigration of nematodes from the stolon.     

Low temperature development of winter rye leaves alters the detergent solubilization of thylakoid membranes

Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5°C were extracted with the nonionic detergents Triton X-100 and octyl glucoside. Less total chlorophyll was extracted from 5°C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5°C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20°C thylakoids than from 5°C thylakoids. When 20 and 5°C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the ³²Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5°C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20°C membranes. Since 20 and 5°C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X-100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature.     

TEMPERATURE CHARACTERISTICS FOR THE PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rates of production of CO₂ by germinating seeds of Lupinus albus and Zea mays were studied between temperatures 12.5° and 25°C. with the HCl-Ba(OH)₂ titration method. The temperature characteristics found are different from those previously obtained for the oxygen consumption of the same seeds germinated in the same manner. For Lupinus, the temperature characteristics above and below the critical temperature of 20° are 16,100 ± and 24,000 ± calories respectively. For Zea, no evidence of a critical temperature was found in this region, and the temperature characteristic is 20,750 ± calories throughout the range of temperature tested. The possible interpretations of the difference in the values of temperature characteristics for oxygen consumption and for production of CO₂ are noted.     

A study of the effects of reaction with formaldehyde on some optical and physical properties of reticulocyte ribosomes

1. The optical rotatory dispersion and ultraviolet-absorption spectrum of ribosomal RNA in situ appear to be unchanged when the ribosome is dissociated into its RNA and protein moieties. 2. Reaction with 0·05% formaldehyde at 20° for 2hr. `fixes' ribosomes so that they remain intact in 1% sodium dodecyl sulphate. 3. The RNA moiety of the ribosome undergoes a conformational change when ribosomes in 8% formaldehyde are heated at 70° for 10min. and cooled to 20°. After this treatment no double-helical character can be detected, but neither the sedimentation coefficient nor the morphology of the ribosome determined by electron microscopy is altered. 4. It is concluded that the RNA moiety of reticulocyte ribosomes is freely accessible to formaldehyde.     

Propagation Speed in Myelinated Nerve: I. Experimental Dependence on External Na+ and on Temperature

Conduction speed (θ) in single myelinated Rana pipiens sciatic nerve fibers has been precisely measured using intracellular recording and on-line digital computer techniques. The dependence of relative speed on external Na concentration at 15°C has been found to be ln(θ₁/θ₂) = 0.524 (±0.018) ln ([Na⁺]₁/[Na⁺]₂) + 0.003. Thus θ has very close to a square root dependence on [Na⁺]₀ for these fibers. This experimental finding is not in complete agreement with a theoretical prediction based on a solution of the Hodgkin-Huxley (H.H.) equations. The effect of small temperature variations around 15°C on θ has also been measured for Rana fibers in Ringer's solution. θ has close to an exponential dependence on T and a Q₁₀ of 2.95 has been estimated.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

Strategic (adaptive) hypothermia in bull dromedary camels during rut; could it increase reproductive success?

In this study of body temperatures (T_b) in free ranging dromedary camels, we found that bulls in rut start the days cooler. Daily minima during rut averaged 0.6°C lower than at other times (95% CI 0.27–0.94°C) and daily maxima averaged 0.45°C higher (95% CI −0.01 to –0.91°C), increasing the daily T_b cycle. Knut Schmidt-Nielsen described a similar pattern in captive dromedaries deprived of water in hot conditions, which he interpreted as a strategy to conserve water. Our observations were made in winter and with water freely available. Dromedaries can apparently employ heterothermy for more than just water conservation. In the strenuous daily contests between rival bulls in rut, a lower T_b early in the day should extend the time for which a contestant can challenge or defend before heat stress becomes a problem. Calculations show that lowering T_b by even 0.6°C extends that time by more than 30 min, and many daily minima during rut were lower than that. Because the eventual winner of contests gains or retains a herd of females, we speculate that cooler T_b at the start of daily contests confers an advantage which translates directly into increased reproductive success.     

Temperature-dependent optical rotatory dispersion properties of helical muscle proteins and homopolymers

Thermally induced helix–coil transitions of myosin rod, light meromyosin, and tropomyosin were studied by optical rotatory dispersion (ORD). Fractional helicity was calculated from both the Moffitt-Yang parameter, b₀, and the corrected mean residue rotation [m′] at 231.4 nm. Between 3 and 30°C, [m′] increases linearly with a slope of 59/°C, whereas b₀ is virtually constant, indicating apparently different thermal melting behavior. Poly(L -lysine) and poly(L -glutamic acid) in their helical forms and myoglobin also show a nearly linear temperature dependence of [m′]_231.4. Muscle proteins in 6M guanidine hydrochloride and the random-coil forms of the homopolymers exhibit temperature-dependent values of [m′]_231.4 and b₀. We conclude from these observations that ORD properties of both α-helices and random-coil polypeptides have significant intrinsic temperature dependencies. A new method of estimating fractional helicity as a function of temperature is proposed.     

The Effect of Double – Blind Carbohydrate Ingestion during 60 km of Self-Paced Exercise in Warm Ambient Conditions

This study evaluated double blind ingestions of placebo (PLA) versus 6% carbohydrate (CHO) either as capsules (c) or beverage (b) during 60 km self-paced cycling in the heat (32°C and 50% relative humidity). Ten well-trained males (mean ± SD: 26±3 years; 64.5±7.7 kg and 70.7±8.8 ml.kg⁻¹.min⁻¹ maximal oxygen consumption) completed four separate 60 km time trials (TT) punctuated by 1 km sprints (14, 29, 44, 59 km) whilst ingesting either PLA_b or PLA_c or CHO_b or CHO_c. The TT was not different among treatments (PLA_b 130.2±11.2 min, CHO_b 140.5±18.1 min, PLA_c 143.1±29.2 min, CHO_c 137.3±20.1 min; P>0.05). Effect size (Cohen’s d) for time was only moderate when comparing CHO_b – PLA_b (d = 0.68) and PLA_b – PLA_c (d = 0.57) whereas all other ES were ‘trivial’ to ‘small’. Mean speed throughout the trial was significantly higher for PLA_b only (P<0.05). Power output was only different (P<0.05) between the sprints and low intensity efforts within and across conditions. Core and mean skin temperatures were similar among trials. We conclude that CHO ingestion is of little or no benefit as a beverage compared with placebo during 60 km TT in the heat.     

STUDIES ON THE CHLOROPHYLLS AND PHOTOSYNTHESIS OF THERMAL ALGAE FROM YELLOWSTONE NATIONAL PARK,CALIFORNIA, AND NEVADA

1. Myxophyceae normally growing at 65°C. evolved oxygen upon irradiation and showed evidence of retaining the power to carry on the process of photosynthesis at 20°C. This indicates that extra thermal energy is not essential for photosynthesis at least over a short period of time. 2. Chlorophyll a and b found in several species of Myxophyceae growing in waters ranging in temperature from 37–72°C. are essentially the same as found in plants growing all over the world. Certain standard chemical tests and spectroscopic examination of the chlorophylls were used as the criteria for these comparisons. The ratio of chlorophyll a to b often varied considerably but in general chlorophyll a showed an increase over the percentage found in most plants. 3. Green algae (Chlorella sp.?) were the only forms found at The Geysers, California. The temperature of the waters from which collections were made varied from 49–66°C. 4. Collections from Beowawe, Nevada were from waters ranging from 60–71°C. The algae belonged to the Myxophyceae and the species were like some of those found in Yellowstone National Park. 5. In some of the calcareous regions of Yellowstone National Park spectroscopic study of the chlorophylls revealed an unidentified absorption band at 548 mµ.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]_act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]_plaq., [extracellular phage]_act., and [extracellular phage]_plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück''s objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.